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Marine and Freshwater Behaviour and Physiology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gmfw20 Selected hemolymph constituents of captive, biomedically bled, and wild caught adult female American horseshoe crabs (Limulus polyphemus) Mary-Jane James-Pirri a , Philip A. Veillette a & Alison S. Leschen b c
a Graduate School of Oceanography, University of Rhode Island, Narragansett, RI, USA b Massachusetts Division of Marine Fisheries, 1213 Purchase Street, New Bedford, MA, USA c Waquoit Bay National Estuarine Research Reserve, PO Box 3092, 149 Waquoit Highway, Waquoit, MA, USA Version of record first published: 16 Oct 2012.
To cite this article: Mary-Jane James-Pirri, Philip A. Veillette & Alison S. Leschen (2012): Selected hemolymph constituents of captive, biomedically bled, and wild caught adult female American horseshoe crabs (Limulus polyphemus), Marine and Freshwater Behaviour and Physiology, 45:4, 281-289 To link to this article: http://dx.doi.org/10.1080/10236244.2012.730216
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Selected hemolymph constituents of captive, biomedically bled, and wild caught adult female American horseshoe crabs (Limulus polyphemus) Mary-Jane James-Pirria*, Philip A. Veillettea and Alison S. Leschenb,c
aGraduate School of Oceanography, University of Rhode Island, Narragansett, RI, USA; bMassachusetts Division of Marine Fisheries, 1213 Purchase Street, New Bedford, MA, USA; cWaquoit Bay National Estuarine Research Reserve, PO Box 3092, 149 Waquoit Highway, Waquoit, MA, USA (Received 11 May 2012; final version received 3 September 2012)
Hemolymph from adult female American horseshoe crabs (Limulus polyphemus) was analyzed from wild caught and three treatments from a biomedical bleeding experiment: captive control, captive bled, and handled according to Best Management Practices (BMP). A total of 10 constituents were measured: blood urea nitrogen, creatinine, glucose, lactate, protein, and ionic concentrations of calcium, chloride, magnesium, potassium, and sodium. Protein concentration was positively correlated with size (prosomal width), while sodium and potassium were negatively correlated with size. Only protein concentration differed among groups, with the captive bled BMP group having significantly lower protein values than either captive control or wild crabs. Wild crabs had higher creatinine, glucose, and potassium values compared to all captive groups. Chloride, calcium, magnesium, and sodium concentrations were lower for wild crabs compared to the captive groups. Lower protein values in the captive bled BMP group suggest that prolonged biomedical bleeding may impact crab physiology. Keywords: Limulus; biomedical bleeding; captivity; hemolymph; protein
Introduction The American horseshoe crab (Limulus polyphemus) is an ecologically and economically important species along the Atlantic Coast of the USA. In Delaware Downloaded by [University Of Rhode Island] at 05:36 17 October 2012 Bay (USA), horseshoe crab eggs are a critical nutritional resource for shorebirds (e.g., red knot, Calidris canutus rufa) during the northward spring migration (Botton et al. 1994; Clark 1996; Karpanty et al. 2006). They are commonly held in captivity at aquariums. Commercially, they are harvested as bait for eel (Anguilla rostrata) and whelk (Busycon spp.), and by the biomedical industry. The biomedical industry obtains Limulus Amebocyte Lysate (LAL) from horseshoe crab hemolymph. LAL is the worldwide standard for detection of pyrogenic endotoxins in all injectable and intravenous drugs and implantable devices (Novitsky 1984, 2009). There is no commercially available synthetic substitute for LAL currently.
*Corresponding author. Email: [email protected]
ISSN 1023–6244 print/ISSN 1029–0362 online ß 2012 Taylor & Francis http://dx.doi.org/10.1080/10236244.2012.730216 http://www.tandfonline.com 282 M.-J. James-Pirri et al.
There are four LAL producing facilities in the USA – three in the mid-Atlantic states and one in Massachusetts. Hemolymph is collected from live horseshoe crabs by puncture of the cardiac sinus with a trocar (via the arthrodial membrane) and is allowed to free-flow until it clots. A greater volume of hemolymph is collected from females, due to their larger, sexually dimorphic body size. Horseshoe crabs are not killed during the bleeding process, but are returned to the water from where they were collected, usually within 72 h (Walls et al. 2002). However, mortality due to bleeding and handling does occur. Early estimates of mortality due to biomedical bleeding were 15% (Rudloe 1983), but recent studies that take handling stress into account place mortality closer to 30% (Hurton and Berkson 2006; Hurton et al. 2009; Leschen and Correia 2010). While there is a vast amount of literature on the properties of the horseshoe crab’s blue hemolymph and the composition of LAL, there are relatively few studies on the biochemical constituents of horseshoe crab hemolymph (Smith et al. 2002; Allender et al. 2010; Hu et al. 2010), and no studies on the impact of biomedical bleeding on the physiology of horseshoe crabs. This study was undertaken as a follow-up to Leschen and Correia’s (2010) recent mortality study to evaluate if there were any long lasting effects of biomedical bleeding and handling stress on horseshoe crab physiology. We examined several ionic and metabolic constituents of the hemolymph from horseshoe crabs in Leschen and Correia’s (2010) study and from a group of wild caught horseshoe crabs.
Methods Experimental design Hemolymph was collected from horseshoe crabs at the termination of the bleeding mortality study of Leschen and Correia (2010). Briefly, Leschen and Correia (2010) maintained three captive treatment groups of adult female horseshoe crabs (refer to descriptions 1–3 below and Figure 1) in randomly assigned flow-through seawater tanks at the Marine Biological Laboratory (MBL) at Woods Hole, MA, for 17 days (2 June–18 June 2009) to estimate mortality related to biomedical bleeding and handling. Holding conditions in Leschen and Correia’s (2010) ambient seawater flow-through system over the 17-day period were: a natural light-dark cycle, mean dissolved oxygen of 8.95 ppm (range 5.91–9.91 ppm), mean water temperature of 15.5 C (range 14.8–16.4 C), salinity range from 30 to 32 ppt, with crabs fed three Downloaded by [University Of Rhode Island] at 05:36 17 October 2012 times per week. At the termination of Leschen and Correia’s (2010) mortality study, hemolymph was collected from the three captive treatment groups for later biochemical analyses. A fourth group composed of wild caught adult female crabs, which was neither bled nor held in captivity, was added for the current study. All crabs originated from Pleasant Bay, MA. The four groups were (Figure 1): (1) Captive control: Adult female crabs that were not bled were transported directly to MBL after collection for a total time out of water of 4 h and held in captivity for 17 days. We refer to this treatment as the captive control group. (2) Captive, bled, and handled according to Best Management Practices (BMP) and returned to water that same day: Adult female crabs were transported to the biomedical facility (Associates of Cape Cod), bled according to standard procedures, and immediately transported to the MBL for a total time out of Marine and Freshwater Behaviour and Physiology 283
Figure 1. Schematic showing differences in handling and transportation of horseshoe crabs from the four groups. Groups 1–3 were captive crabs from Leschen and Correia’s (2010) study on the influence of handling practices on mortality. Crabs in group 4 were wild caught.
water of 6 h and held in captivity for 17 days. We refer to this treatment as Downloaded by [University Of Rhode Island] at 05:36 17 October 2012 the captive bled BMP group. (3) Captive, bled, and handled but returned to water the next morning (as is the current biomedical practice): Adult female crabs were transported to the biomedical facility, bled according to standard procedures, transferred into a refrigerated truck (8.9 C) where they remained overnight. These crabs were driven to the MBL the next morning, for a time out of water of 25 h and held in captivity for 17 days. We refer to this treatment as the captive bled group. (4) Wild caught group: Adult female crabs were collected (24 June 2009), transported to Atlantic Coastal Laboratory at Cape Cod National Seashore (45 min out of water) where hemolymph samples were collected for later analyses. This group was our best effort to get natural, ambient values for hemolymph parameters, although we acknowledge that these individuals may 284 M.-J. James-Pirri et al.
have experienced some stress during transport to the laboratory. We refer to these crabs as the wild caught group. Crabs bled at the biomedical facility (groups 2 and 3 above) were bled according to standard biomedical bleeding procedures that included placing the crab upside down in a rack, puncturing the arthrodial membrane with a syringe to access the cardiac sinus, and allowing hemolymph to drain into a collecting vessel until the bleeding stopped on its own. The volume of hemolymph drawn from individual crabs in the biomedical facility was not standardized since the objective of this study was to evaluate current biomedical practices.
Hemolymph analyses At the termination of the bleeding mortality study of Leschen and Correia (2010), 25 crabs were arbitrarily selected from each treatment group (groups 1–3 above and Figure 1) to collect hemolymph for later analyses. Hemolymph for the fourth group, the wild caught crabs was collected immediately upon arrival at the North Atlantic Coastal Laboratory. Prosomal width (mm) was measured for all crabs. Hemolymph was collected by puncturing the arthrodial membrane with a syringe and withdraw- ing 5–10 mL of hemolymph from the cardiac sinus. Hemolymph was centrifuged at 500 g for 5 min. Supernatant was saved and protein concentration was measured using a calibrated automatic temperature compensated clinical refractometer (Reichert model VET360Õ). Aliquots were frozen and stored for later biochemical analyses (approximately 6 months later). Hemolymph biochemistry analyses were conducted at the Animal Health Department, Animal Medical Center at the New England Aquarium, Boston, MA using a blood gas analyzer with a clot catcher (Stat Profile Critical Care XpresssÕ, NOVA Biomedical). After thawing to room temperature, 0.2 mL of hemolymph was injected into the analyzer. Hemolymph constituents analysed were blood urea nitrogen (BUN), creatinine, glucose, lactate, and ionic concentrations of calcium (Caþþ), chloride (Cl ), magnesium (Mgþþ), potassium (Kþ), and sodium (Naþ). The concentration for Mgþþ was initially above the reporting limits for the analyzer. To obtain an estimate of [Mgþþ], samples were diluted by adding 0.2 mL of distilled water to 0.2 mL of sample and re-analyzed.
Statistical analyses Downloaded by [University Of Rhode Island] at 05:36 17 October 2012 Analysis of variance (ANOVA) was used to evaluate differences in prosomal width among the four groups. An analyses of covariance (ANCOVA) using size (prosomal width) as the covariate was conducted to test for differences of hemolymph constituents among the four groups. If assumptions of normality and homogeneity were not met then analyses were conducted on the ranked data.
Results Size There were no differences in mean prosomal width among the four groups (ANOVA, p ¼ 0.2573; Table 1). Marine and Freshwater Behaviour and Physiology 285
Table 1. Mean values for prosomal width and concentration of ions and metabolites in hemolymph of horseshoe crabs.
Mean value SE (n)
Captive Captive bled Captive bled Wild caught Parameter control (group 1) BMP (group 2) (group 3) (group 4)
Prosomal 230.8 3.8 (25) 231 3.2 (25) 229 2.7 (23) 221.9 4.1 (20) width (mm) BUN (mg dL 1) 5.1 0.1 (25) 5.4 0.3 (25) 5.2 0.2 (23) 5.3 0.3(20) Caþþ (mmol L 1) 6.4 0.1 (25) 6.3 0.2 (25) 6.2 0.1 (23) 4.9[0.2 (20) Cl (mmol L 1) 455.1 13.7 (25) 445.2 16.9 (24) 450.3 17.8 (22) 320.0[11.5 (20) Creatinine 0.09 0.03 (25) 0.05 0.03 (25) 0.08 0.02 (23) 0.22[0.02 (20) (mg dL 1) Glucose (mg dL 1) 7.8 0.4 (25) 8.1 0.3 (25) 9.0 0.5 (23) 11.3[1.2 (20) Kþ (mmol L 1) 10.7 0.08 (25) 10.8 0.09 (25) 10.7 0.1 (23) 12.1[0.5 (20) Mgþþ (mmol L 1) 15.1 0.2 (13) 15.2 0.2 (13) 15.2 0.2 (16) 12.4[0.4 (21) (diluted) Naþ (mmol L 1) 485.7 2.2 (25) 486.0 2.3 (25) 482.5 2.4 (23) 369.2[11.2 (20)
Note: Values given in boldface were significantly different from other groups for each parameter.
Ion concentrations Ionic concentrations of Caþþ,Cl , and Mgþþ were not correlated with prosomal width (ANCOVA, p 4 0.05). The wild caught group had significantly lower values for these three ions compared to all three captive groups (ANCOVA, least squares mean, p 5 0.001 for all comparisons). No differences in [Caþþ], [Cl ], or [Mgþþ] were observed among the three captive groups (least squares mean, p 4 0.05, Table 1). Ionic concentrations of Kþ and Naþ were negatively correlated with prosomal width (ANCOVA, p ¼ 0.0010 and p ¼ 0.0046, respectively). The wild caught group had significantly higher [Kþ] and significantly lower [Naþ] compared to all three captive groups (ANCOVA, least squares mean, p 5 0.0050 for all comparisons). No differences in [Kþ] or [Naþ] were observed among the three captive groups (least squares mean, p 4 0.05, Table 1). Downloaded by [University Of Rhode Island] at 05:36 17 October 2012
Metabolite concentrations Creatinine, glucose, and BUN concentrations were not correlated with prosomal width (ANCOVA, p 4 0.05). The wild caught group had significantly higher creatinine and glucose levels compared to all three captive groups (ANCOVA, least squares mean, p 5 0.05). No differences in creatinine or glucose were observed among three captive groups (least squares mean, p 4 0.05) and BUN concentrations were equivalent among all four groups (ANCOVA, p ¼ 0.6211, Table 1). Lactate concentrations were below the detectable limit of the analyzer (50.2 mmol L 1) for most samples and are therefore not reported. 286 M.-J. James-Pirri et al.
Figure 2. Horseshoe crab hemolymph protein concentration (mean þ SE) for the four groups (group number in parentheses). Bars that do not share the same letters indicate significant differences.
Protein concentration Protein concentration was positively correlated with prosomal width (ANCOVA, p ¼ 0.0091) and was significantly lower in the captive bled BMP group compared to the captive control group (ANCOVA, least squares mean, p ¼ 0.0020). There was marginal significance (p ¼ 0.0830) between the captive bled BMP group and the wild caught group, with the wild group having a higher protein concentration. No other differences in protein concentration were observed among the other groups (least squares mean, p 4 0.05, Figure 2).
Discussion The blue blood of horseshoe crabs is due to hemocyanin, a copper-containing protein that is the extracellular oxygen carrier in hemolymph. Approximately 90% of
Downloaded by [University Of Rhode Island] at 05:36 17 October 2012 the total plasma protein of horseshoe crabs is hemocyanin and the measurement of protein concentration has been used as an indicator of health in horseshoe crabs (Ding et al. 2005; Nolan and Smith 2009; Coates et al. 2012). Only protein concentration showed significant differences among the captive groups as well as between captive groups and the wild caught group. The significantly lower protein concentration in the captive bled BMP group may be attributable to a lingering impact of biomedical bleeding on horseshoe crab physiology. During the biomedical bleeding process, a standard volume of hemolymph is not extracted from crabs, but rather the cardiac sinus is punctured and hemolymph is collected until the blood clots. The impacts of low protein concentration on horseshoe crab physiology (e.g., egg production and maturation) and/or behavior (e.g., spawning or foraging) are unknown, although low protein levels have been associated with starvation and mortality (Alsberg 1914). Severe hypoproteinemic deficiency or panhypoproteinemia Marine and Freshwater Behaviour and Physiology 287
(Nolan and Smith 2009), possibly from nutritional imbalance/deficiency, protein- losing enteropathy, hepatic insufficiency, and/or protein-losing nephropathy (Nolan and Smith 2009), may be responsible for chronic mortality of horseshoe crabs maintained in captivity for long periods ( 6 months) in laboratory and public aquaria (Smith and Berkson 2005). This syndrome results in a decline in protein concentration in the hemolymph after 3–4 weeks in captivity, with levels dropping below the reference interval (3.4–11.7 g dL 1) after 3–4 months (Nolan and Smith 2009). It is unlikely that the lower protein concentration observed in the captive bled BMP group was related to panhypoproteinemia since the time in captivity was comparatively short (53 weeks), protein values were within the reference interval, and all three captive groups were maintained under the same feeding regime. Protein concentration did not differ between the captive bled and the captive control groups. We collected hemolymph from the ‘‘survivors’’ of Leschen and Correia’s (2010) mortality study, where differences in mortality were observed among the three captive groups, with the bled group and the bled BMP group having significantly higher mortality (29% mortality and 23% mortality, respectively) than the control group (3% mortality). There was a tendency for protein levels to be higher in the captive bled group compared to the captive bled BMP group. It was possible that since the bled group had a higher mortality rate, individuals that were less robust had already died, leaving the ‘‘survivors’’ that may have been more robust. Smith et al. (2002) reported mean protein concentration from wild caught male and female horseshoe crabs in Delaware Bay at 8.2 g dL 1, which is higher than the mean concentration observed in our captive control group (6.6 g dL 1) and wild caught group (mean 5.9 g dL 1), but within the range of values observed in this study (3.2–9.7 g dL 1). We observed a positive correlation between protein concentration and prosomal width, with larger females having higher protein values. Horseshoe crabs from Delaware Bay are larger than crabs from New England waters (James- Pirri et al. 2005; Smith et al. 2009; James-Pirri 2012) and this size difference may account for the slight discrepancy in mean protein values. The American horseshoe crab behaves as an osmotic conformer in salinities above 23 ppt (Towle and Henry 2003). Hemolymph from horseshoe crabs in the wild caught group had lower sodium and chloride concentrations compared to the captive groups. This is consistent with the ambient salinities of where the crabs were collected and/or maintained. The smaller bays of Pleasant Bay, such as Pochet Inlet where the wild caught group was collected, have salinities characteristic of estuarine environments ( 26 ppt, K. Medeiros, National Park Service, personal communica-
Downloaded by [University Of Rhode Island] at 05:36 17 October 2012 tion; Pleasant Bay Resource Alliance 1998); whereas, the ambient salinity at MBL, where the captive groups were maintained, was higher at 30–32 ppt. Previously reported means for hemolymph constituents collected from horseshoe crabs originating from Delaware Bay were within the ranges observed in this study (chloride: 445.1 mmol L 1; creatinine: 0.7 mg dL l; sodium: 389.5 mmol L 1; Smith et al. 2002). The value reported by Smith et al. (2002) for glucose was lower (3.25 mmol L 1) and calcium was higher (9.75 mmol L 1). Allender et al. (2010) similarly reported that lactate levels were below detectable limits of an iSTATÕ point-of-care analyzer, and it appears that neither the iSTATÕ nor the Stat Profile Critical Care XpresssÕ (this study) analyzers are suitable for determining lactate levels in horseshoe crabs. These clinical analyzers are designed for human or veterinary diagnostics (generally mammalian), although they are commonly used by aquarium staff to analyse blood from other animals (e.g., sea turtles; C. Innis, New 288 M.-J. James-Pirri et al.
England Aquarium, personal communication) and have been previously used to analyse Limulus hemolymph (Smith et al. 2002; Nolan and Smith 2009; Allender et al. 2010). This study presents the first published values for several hemolymph constituents for the American horseshoe crab and provides baseline information (from the wild caught group) for researchers, veterinarians, and aquaria personnel who maintain horseshoe crabs in captivity. Previous studies have focused on mortality associated with biomedical bleeding of horseshoe crabs, but no research has been conducted on behavioral or physiological impacts associated with bleeding and handling processes. This study observed lower protein concentration in the captive bled BMP group, 17 days post-bleeding. There are therefore measurable physiological effects of biomed- ical bleeding and handling that persist for an extended period ( 2 weeks) post- bleeding. It is not known if lower protein levels lead to further mortality after crabs are released from biomedical facilities or if there are any associated behavioral implications with lower protein levels. This result highlights the need for further study of the non-lethal effects of biomedical bleeding on horseshoe crab physiology and behavior.
Acknowledgments Thanks are extended to National Park Service Bio-technicians Chris Keon, Matt Holt, and Student Conservation Association interns Kelly Bowman, Nikki Kirkton, Stacey Ng, Meg Swecker, and the Massachusetts Department of Marine Fisheries intern Zach Boudreau for field and laboratory assistance. Scott Lindell and Krista Lee kindly provided laboratory space at the Marine Biological Laboratory, Woods Hole, MA, and at the Atlantic Coastal Laboratory at Cape Cod National Seashore, Truro, MA, for collecting hemolymph samples. Charlie Innis, D.M.V., graciously allowed access to laboratory facilities and equipment at the Animal Health Department, Animal Medical Center at the New England Aquarium, Boston, MA. Rossie Ennis and two anonymous reviewers provided helpful suggestions on the draft manuscript. This study was supported by the National Park Service under Cooperative Agreement number CA452099007 with the University of Rhode Island.
References
Allender MC, Schumacher J, George R, Milam J, Odoi A. 2010. The effects of short- and long-term hypoxia on hemolymph gas values in the American horseshoe crab (Limulus polyphemus) using a point-of-care analyzer. J Zoo Wildl Med. 41(2):193–200. Downloaded by [University Of Rhode Island] at 05:36 17 October 2012 Alsberg CL. 1914. Note on the proteins of the blood of Limulus polyphemus. J Bio Chem. 19:77–82. Botton ML, Loveland RE, Jacobsen TR. 1994. Site selection by migratory shorebirds in Delaware Bay, and its relationship to beach characteristics and abundance of horseshoe crab (Limulus polyphemus) eggs. Auk. 111(3):605–616. Clark KE. 1996. Proceedings of the Horseshoe Crab Forum; Status of the Resource Publication No. DEL-SG-05-97. Lewes (DE): University of Delaware Sea Grant College Program. Horseshoe crabs and the shorebird connection. p. 23–25. Coates CJ, Bradford EL, Krome CA, Nairn J. 2012. Effect of temperature on biochemical and cellular properties of captive Limulus polyphemus. Aquaculture. 334–337:30–38. Ding JL, Tan KC, Thangamani S, Kusuma N, Seow WK, Bui THH, Wang J, Ho B. 2005. Spatial and temporal coordination of expression of immune response genes during Pseudomonas infection of horseshoe crab, Carcinoscorpius rotundicauda. Genes Immun. 6:557–574. Marine and Freshwater Behaviour and Physiology 289
Hu M, Wang Y, Tsang ST, Cheung SG, Shin PKS. 2010. Effect of prolonged starvation on body weight and blood-chemistry in two horseshoe crab species Tachypleus tridentatus and Carcinoscorpius rotundicauda (Chelicerate: Xiphosura). J Exp Mar Biol Ecol. 395:112–119. Hurton L, Berkson J. 2006. Potential causes of mortality for horseshoe crabs (Limulus polyphemus) during the biomedical bleeding process. Fish Bull. 104(2):293–298. Hurton L, Berkson J, Smith S. 2009. The effect of hemolymph extraction volume and handling stress on horseshoe crab mortality. In: Botton M., Smith D., and Tanadcredi J, editors. Biology and conservation of horseshoe crabs. New York (NY): Springer. p. 331–346. James-Pirri, MJ. 2012. Assessment of spawning horseshoe crabs (Limulus polyphemus) at Cape Cod National Seashore, 2008–2009. Natural Resource Technical Report NPS/CACO/ NRTR—2012/573. National Park Service, Fort Collins, Colorado. James-Pirri MJ, Tuxbury K, Marino S, Koch S. 2005. Spawning densities, egg densities, size structure, and movement patterns of spawning horseshoe crabs Limulus polyphemus within four coastal embayments on Cape Cod, Massachusetts. Estuaries. 28(2):296–313. Karpanty SM, Fraser JD, Berkson J, Niles LJ, Dey A, Smith EP. 2006. Horseshoe crab eggs determine red knot distribution in Delaware Bay. J Wildl Manage. 70(6):1704–1710. Leschen AS, Correia SJ. 2010. Mortality in female horseshoe crabs Limulus polyphemus from biomedical bleeding and handling: implications for fisheries management. Mar Freshwat Behav Physiol. 43(2):135–147. Nolan MW, Smith SA. 2009. Clinical evaluation, common diseases, and veterinary care of the horseshoe crab, Limulus polyphemus. In: Botton M., Smith D., and Tanadcredi J, editors. Biology and conservation of horseshoe crabs. New York (NY): Springer. p. 479–499. Novitsky TJ. 1984. Discovery to commercialization: the blood of the horseshoe crab. Oceanus. 27:19–26. Novitsky TJ. 2009. Biomedical applications of Limulus amebocyte lysate. In: Botton M., Smith D., and Tanadcredi J, editors. Biology and conservation of horseshoe crabs. New York (NY): Springer. p. 315–329. Pleasant Bay Resource Alliance. 1998. Pleasant Bay Resource Management Plan [Internet]. Harwich (MA): Pleasant Bay Resource Alliance; [cited 2012 March 19] Available from: http://www.pleasantbay.org/ Rudloe A. 1983. The effect of heavy bleeding on mortality of the horseshoe crab, Limulus polyphemus, in the natural environment. J Invertebr Pathol. 42:167–176. Smith SA, Berkson JM. 2005. Laboratory culture and maintenance of the horseshoe crab (Limulus polyphemus). Lab Anim. 34(7):27–34. Smith SA, Berkson JM, Barratt RA. 2002. Horseshoe crab (Limulus polyphemus) hemolymph biochemical and immunological parameters. Proc Int Assoc Aquat Anim Med. 33:101–102. Smith DR, Mandt MT, MacDonald PDM. 2009. Proximate causes of sexual size dimorphism in horseshoe crabs (Limulus polyphemus) of the Delaware Bay. J Shellfish Res. 28(2):405–417.
Downloaded by [University Of Rhode Island] at 05:36 17 October 2012 Towle DW, Henry RP. 2003. Coping with environmental changes: physiological challenges. In: Shuster Jr CN, Barlow RB, and Brockmann HJ, editors. The American horseshoe crab. Cambridge (MA): Harvard University Press. p. 224–244. Walls EA, Berkson JM, Smith SA. 2002. The horseshoe crab Limulus polyphemus: 200 million years of existence, 100 years of study. Rev Fish Sci. 10(1):39–73.