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Marine and Freshwater Research Marine and Freshwater Research Society
Advances in the aquatic sciences
RESEARCH ARTICLE

Horseshoe crabs as potential sentinel species for coastal health: juvenile haemolymph quality and relationship to habitat conditions

Billy K. Y. Kwan A B C , Virginia K. Y. Un C , S. G. Cheung C D and Paul K. S. Shin C D E
+ Author Affiliations
- Author Affiliations

A Guangxi Key Laboratory of Beibu Gulf Marine Biodiversity Conservation, Qinzhou University, 535011, P.R. China

B College of Ocean, Qinzhou University, 535011, P.R. China

C Department of Biology and Chemistry, City University of Hong Kong, Hong Kong, Hong Kong S.A.R., P.R. China.

D State Key Laboratory in Marine Pollution, City University of Hong Kong, Hong Kong, Hong Kong S.A.R., P.R. China.

E Corresponding author. Email: paulksshin@gmail.com

Marine and Freshwater Research 69(6) 894-905 https://doi.org/10.1071/MF17210
Submitted: 11 July 2017  Accepted: 11 November 2017   Published: 19 February 2018

Abstract

As an interface between terrestrial and marine environments, coastal and estuarine areas are particularly prone to various pollution stresses. Identification of sentinel species is, therefore, essential to provide precautionary information on coastal health conditions. Given their significant ecological roles in estuarine ecosystems, horseshoe crabs are a potential species to indicate the general health status of coastal habitats. The present study demonstrated that the changes in haemolymph composition pattern of two Asian juvenile horseshoe crab species (Tachypleus tridentatus and Carcinoscorpius rotundicauda) are sensitive to reflect heavy metal (lead, nickel and iron) and nutrient (nitrate and ammonia) concentrations of intertidal sediments. In particular, a significant negative correlation was found for the amebocyte viability and ratio of granular–spherical to granular–flattened and degranulated dendritic-like morphological states of amebocytes in juvenile haemolymph with nitrate and lead concentrations respectively. There were also significantly higher concentrations of haemolymph haemocyanin and plasma protein in juvenile C. rotundicauda than in T. tridentatus on the same shore, possibly being related to the difference in foraging habitats of these two horseshoe crab species. Such non-lethal sampling of haemolymph from juvenile horseshoe crabs can be useful for a further development of the monitoring program in assessing potential environmental impacts by anthropogenic activities.

Additional keywords: benthic zone, biomonitoring, disturbance, heavy metals, invertebrates, marine.


References

Adachi, K., Endo, H., Watanabe, T., Nishioka, T., and Hirata, T. (2005). Hemocyanin in the exoskeleton of crustaceans: enzymatic properties and immunolocalization. Pigment Cell Research 18, 136–143.
Hemocyanin in the exoskeleton of crustaceans: enzymatic properties and immunolocalization.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXjt1Clur4%3D&md5=f319f2d20c879281bfd5c6861d6c8fdeCAS |

Alongi, D. M. (1988). Bacterial productivity and microbial biomass in tropical mangrove sediments. Microbial Ecology 15, 59–79.
Bacterial productivity and microbial biomass in tropical mangrove sediments.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BC2c7ktVelsg%3D%3D&md5=3f449d69586e3de4ea039f19cec3f18dCAS |

Alpuche, J., Pereyra, A., Mendoza-Hernández, G., Agundis, C., Rosas, C., and Zenteno, E. (2010). Purification and partial characterization of an agglutinin from Octopus maya serum. Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology 156, 1–5.
Purification and partial characterization of an agglutinin from Octopus maya serum.Crossref | GoogleScholarGoogle Scholar |

Bai, J., Xiao, R., Cui, B., Zhang, K., Wang, Q., Liu, X., Gao, H., and Huang, L. (2011). Assessment of heavy metal pollution in wetland soils from the young and old reclaimed regions in the Pearl River Estuary, South China. Environmental Pollution 159, 817–824.
Assessment of heavy metal pollution in wetland soils from the young and old reclaimed regions in the Pearl River Estuary, South China.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXnsFWqsw%3D%3D&md5=d72b8ddde5d5fd8f244cf3fd39fdc504CAS |

Barrett, T. J., and Munkittrick, K. R. (2010). Seasonal reproductive patterns and recommended sampling times for sentinel fish species used in environmental effects monitoring programs in Canada. Environmental Reviews 18, 115–135.
Seasonal reproductive patterns and recommended sampling times for sentinel fish species used in environmental effects monitoring programs in Canada.Crossref | GoogleScholarGoogle Scholar |

Basu, N., Scheuhammer, A. M., Bursian, S. J., Elliott, J., Rouvinen-Watt, K., and Chan, H. M. (2007). Mink as a sentinel species in environmental health. Environmental Research 103, 130–144.
Mink as a sentinel species in environmental health.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XhtleisbvI&md5=6b09eb023d0181f05682a3d52fa8a611CAS |

Basuyaux, O., and Mathieu, M. (1999). Inorganic nitrogen and its effect on growth of the abalone Haliotis tuberculata Linnaeus and the sea urchin Paracentrotus lividus Lamarck. Aquaculture 174, 95–107.
Inorganic nitrogen and its effect on growth of the abalone Haliotis tuberculata Linnaeus and the sea urchin Paracentrotus lividus Lamarck.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1MXhs1Omtrw%3D&md5=e0ce9742f810fc66224bb8a7d2df49c4CAS |

Botton, M. L., Hodge, M., and Gonzalez, T. I. (1998a). High tolerance to tributyltin in embryos and larvae of the horseshoe crab, Limulus polyphemus. Estuaries 21, 340–346.
High tolerance to tributyltin in embryos and larvae of the horseshoe crab, Limulus polyphemus.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXlslWiuro%3D&md5=6179be699a8a5ec3e27f5a1fdbc25ff9CAS |

Botton, M. L., Johnson, K., and Helleby, L. (1998b). Effects of copper and zinc on embryos and larvae of the horseshoe crab, Limulus polyphemus. Archives of Environmental Contamination and Toxicology 35, 25–32.
Effects of copper and zinc on embryos and larvae of the horseshoe crab, Limulus polyphemus.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXjvFektb8%3D&md5=248b09ec8f5d7f4573a434075de94ca1CAS |

Bradford, M. M. (1976). Rapid and sensitive method for quantification of microgram quantities of protein utilizing principle of protein-dye binding. Analytical Biochemistry 72, 248–254.
Rapid and sensitive method for quantification of microgram quantities of protein utilizing principle of protein-dye binding.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaE28XksVehtrY%3D&md5=8e40a094816fe271d3d8d44c49f62377CAS |

Camargo, J. A., Alonso, A., and Salamanca, A. (2005). Nitrate toxicity to aquatic animals: a review with new data for freshwater invertebrates. Chemosphere 58, 1255–1267.
Nitrate toxicity to aquatic animals: a review with new data for freshwater invertebrates.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXmvFaktQ%3D%3D&md5=303c95fcb625bbfca13f0afd26ddd7deCAS |

Carmichael, R. H., Rutecki, D., and Valiela, I. (2003). Abundance and population structure of the Atlantic horseshoe crab Limulus polyphemus in Pleasant Bay, Cape Cod. Marine Ecology Progress Series 246, 225–239.
Abundance and population structure of the Atlantic horseshoe crab Limulus polyphemus in Pleasant Bay, Cape Cod.Crossref | GoogleScholarGoogle Scholar |

Chen, C. P., Yeh, H. Y., and Lin, P. F. (2004). Conservation of the horseshoe crab at Kinmen, Taiwan: strategies and practices. Biodiversity and Conservation 13, 1889–1904.
Conservation of the horseshoe crab at Kinmen, Taiwan: strategies and practices.Crossref | GoogleScholarGoogle Scholar |

Chen, C. P., Yang, M. C., Fan, L. F., Qiu, G. L., Liao, Y. Y., and Hsieh, H. L. (2015). Co-occurrence of juvenile horseshoe crabs Tachypleus tridentatus and Carcinoscorpius rotundicauda in an estuarine bay, southwestern China. Aquatic Biology 24, 117–126.
Co-occurrence of juvenile horseshoe crabs Tachypleus tridentatus and Carcinoscorpius rotundicauda in an estuarine bay, southwestern China.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2sXptlCguw%3D%3D&md5=66f3d4a70a4394accddc5fda54da230bCAS |

Chiu, H. M. C., and Morton, B. (2003). The sediment and hydrographic characteristics of three horseshoe crab nursery beaches in Hong Kong. Journal of the Ocean University of Qingdao 2, 35–43.
| 1:CAS:528:DC%2BD3sXksFOhu78%3D&md5=07cbb1ab47eb4689729d3cdb158dbb3fCAS |

Clarke, K. R., and Gorley, R. N. (2006). PRIMER v6: user manual/tutorial. PRIMER-E (version 6). Available at http://chemsrv0.pph.univie.ac.at/scripten/EDV/Software/Primer6/Getting%20started%20with%20v6.doc [Verified 6 February 2017].

Coates, C. J., and Nairn, J. (2014). Diverse immune functions of hemocyanins. Developmental and Comparative Immunology 45, 43–55.
Diverse immune functions of hemocyanins.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2cXmtlSks7o%3D&md5=b05570d340f4a0d75d82e5656a236eecCAS |

Coates, C. J., Bradford, E., Krome, C. A., and Nairn, J. (2012). Effect of temperature on biochemical and cellular properties of captive Limulus polyphemus. Aquaculture 334–337, 30–38.
Effect of temperature on biochemical and cellular properties of captive Limulus polyphemus.Crossref | GoogleScholarGoogle Scholar |

Fortey, R. (Eds) (2011). ‘Survivors: the Animals and Plants that Time has Left Behind.’ (HarperPress: London, UK.)

Gao, A. A., Chen, Q. Z., Zeng, J. N., Liao, Y. B., and Yang, J. Y. (2005). Macrofauna community in the mangrove area of Ximen Island, Zhejiang. Journal of Marine Science 23, 33–40.

Gray, M. A., Curry, A. R., and Munkittrick, K. R. (2002). Non-lethal sampling methods for assessing environmental impacts using a small-bodied sentinel fish species. Water Quality Research Journal of Canada 37, 195–211.
| 1:CAS:528:DC%2BD38XhsFCns74%3D&md5=db9488d98618630297d151939fb0c014CAS |

Hu, M., Kwan, B. K. Y., Wang, Y., Cheung, S. G., and Shin, P. K. S. (2015). Population structure and growth of juvenile horseshoe crabs Tachypleus tridentatus and Carcinoscorpius rotundicauda (Xiphosura) in southern China. In ‘Changing Global Perspectives on Horseshoe Crab Biology, Conservation and Management’. (Eds R. H. Carmichael, M. L. Botton, P. K. S. Shin, and S. G. Cheung.) pp. 167–180. (Springer International Publishing: Cham, Switzerland.)

Hurton, L., Berkson, J. M., and Smith, S. A. (2005). Selection of a standard culture medium for primary culture of Limulus polyphemus amebocytes. In Vitro Cellular & Developmental Biology. Animal 41, 325–329.
Selection of a standard culture medium for primary culture of Limulus polyphemus amebocytes.Crossref | GoogleScholarGoogle Scholar |

Hurvich, C. M., and Tsai, C. L. (1989). Regression and time series model selection in small samples. Biometrika 76, 297–307.
Regression and time series model selection in small samples.Crossref | GoogleScholarGoogle Scholar |

Itow, T., Igarashi, T., Botton, M. L., and Loveland, R. E. (1998a). Heavy metals inhibit limb regeneration in horseshoe crab larvae. Archives of Environmental Contamination and Toxicology 35, 457–463.
Heavy metals inhibit limb regeneration in horseshoe crab larvae.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXlvFGmt7g%3D&md5=7ba5ec40e07080f8d5df0ee9b574fb31CAS |

Itow, T., Loveland, R. E., and Botton, M. L. (1998b). Developmental abnormalities in horseshoe crab embryos caused by exposure to heavy metals. Archives of Environmental Contamination and Toxicology 35, 33–40.
Developmental abnormalities in horseshoe crab embryos caused by exposure to heavy metals.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXjvFekur0%3D&md5=b9404404a15395f6c08c22040afd6605CAS |

Jaenicke, E., Foll, R., and Decker, H. (1999). Spider hemocyanin binds ecdysone and 20-OH-ecdysone. The Journal of Biological Chemistry 274, 34267–34271.
Spider hemocyanin binds ecdysone and 20-OH-ecdysone.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1MXnslOksbc%3D&md5=4852f02b6566db1ef1b1df778337618dCAS |

James-Pirri, M. J., Tuxbury, K., and Marino, 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, 296–313.
Spawning densities, egg densities, size structure, and movement patterns of spawning horseshoe crabs, Limulus polyphemus, within four coastal embayments on Cape Cod, Massachusetts.Crossref | GoogleScholarGoogle Scholar |

Jiang, N., Tan, N. S., Ho, B., and Ding, J. L. (2007). Respiratory protein generated reactive oxygen species as an antimicrobial strategy. Nature Immunology 8, 1114–1122.
Respiratory protein generated reactive oxygen species as an antimicrobial strategy.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXhtVCkurnK&md5=40ca8876bccd8049e792606e0f9614deCAS |

Kannan, K., Yasunaga, Y., Iwata, H., Ichihashi, H., Tanabe, S., and Tatsukawa, R. (1995). Concentrations of heavy metals, organochlorines, and organotins in horseshoe crab, Tachypleus tridentatus, from Japanese coastal waters. Archives of Environmental Contamination and Toxicology 28, 40–47.
Concentrations of heavy metals, organochlorines, and organotins in horseshoe crab, Tachypleus tridentatus, from Japanese coastal waters.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2MXltFGlt74%3D&md5=23f30ada14a11b90d473d42d06075e3bCAS |

Kwan, B. K. Y., Chan, A. K. Y., Cheung, S. G., and Shin, P. K. S. (2014). Hemolymph quality as indicator of health status in juvenile Chinese horseshoe crab Tachypleus tridentatus (Xiphosura) under laboratory culture. Journal of Experimental Marine Biology and Ecology 457, 135–142.
Hemolymph quality as indicator of health status in juvenile Chinese horseshoe crab Tachypleus tridentatus (Xiphosura) under laboratory culture.Crossref | GoogleScholarGoogle Scholar |

Kwan, B. K. Y., Chan, A. K. Y., Cheung, S. G., and Shin, P. K. S. (2015a). A dual stable isotope study for diet composition of juvenile Chinese horseshoe crab Tachypleus tridentatus (Xiphosura) on a seagrass-covered intertidal mudflat. Marine Biology 162, 1137–1143.
A dual stable isotope study for diet composition of juvenile Chinese horseshoe crab Tachypleus tridentatus (Xiphosura) on a seagrass-covered intertidal mudflat.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2MXkvFars7Y%3D&md5=da4b59d4bfbdad4fa01d81c13c14c49fCAS |

Kwan, B. K. Y., Chan, A. K. Y., Cheung, S. G., and Shin, P. K. S. (2015b). Responses of growth and hemolymph quality in juvenile Chinese horseshoe crab Tachypleus tridentatus (Xiphosura) to sublethal tributyltin and cadmium. Ecotoxicology 24, 1880–1895.
Responses of growth and hemolymph quality in juvenile Chinese horseshoe crab Tachypleus tridentatus (Xiphosura) to sublethal tributyltin and cadmium.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2MXht12mu7nI&md5=0acbf2364a0e6410f913e9cbb965dc99CAS |

Kwan, B. K. Y., Hsieh, H. L., Cheung, S. G., and Shin, P. K. S. (2016). Present population and habitat status of potentially threatened Asian horseshoe crabs Tachypleus tridentatus and Carcinoscorpius rotundicauda in Hong Kong: a proposal for marine protected areas. Biodiversity and Conservation 25, 673–692.
Present population and habitat status of potentially threatened Asian horseshoe crabs Tachypleus tridentatus and Carcinoscorpius rotundicauda in Hong Kong: a proposal for marine protected areas.Crossref | GoogleScholarGoogle Scholar |

Kwan, B. K. Y., Chan, A. K. Y., Cheung, S. G., and Shin, P. K. S. (2017). Marine microalgae as dietary supplements in the culture of juvenile Chinese horseshoe crabs, Tachypleus tridentatus (Xiphosura). Aquaculture Research 48, 3910–3924.
Marine microalgae as dietary supplements in the culture of juvenile Chinese horseshoe crabs, Tachypleus tridentatus (Xiphosura).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2sXhtVWjsrrK&md5=fe3984c7d56a81ee6579da136bc46696CAS |

Lachat Instruments (2008). Data pack: Lachat applications in standard methods (edition 21). Available at http://www.lachatinstruments.com/download/Std-Methods-Datapack-v1_1-09.pdf [Verified 6 February 2017].

Lau, S. S. S., and Chu, L. M. (1999). Contaminant release from sediments in a coastal wetland. Water Research 33, 909–918.
Contaminant release from sediments in a coastal wetland.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1MXht1aqsb0%3D&md5=0d5daf288e498cbdd4f06e195dc51a0bCAS |

Lau, P. S., Wong, H. L., and Garrigues, P. (2004). Seasonal variation in antioxidative responses and acetylcholinesterase activity in Perna viridis in eastern oceanic and western estuarine waters of Hong Kong. Continental Shelf Research 24, 1969–1987.
Seasonal variation in antioxidative responses and acetylcholinesterase activity in Perna viridis in eastern oceanic and western estuarine waters of Hong Kong.Crossref | GoogleScholarGoogle Scholar |

Lee, S. Y. (2008). Mangrove macrobenthos: assemblages, services, and linkages. Journal of Sea Research 59, 16–29.
Mangrove macrobenthos: assemblages, services, and linkages.Crossref | GoogleScholarGoogle Scholar |

Lei, K., Li, F., Zhang, M., Yang, H., Luo, T., and Xu, X. (2008). Difference between hemocyanin subunits from shrimp Penaeus japonicus in anti-WSSV defense. Developmental and Comparative Immunology 32, 808–813.
Difference between hemocyanin subunits from shrimp Penaeus japonicus in anti-WSSV defense.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXjtlKgsr8%3D&md5=457420976595116aafb884308f03ab3fCAS |

Levin, L. A., Boesch, D. F., Covich, A., Dahm, C., Erséus, C., Ewel, K. C., Kneib, R. T., Moldenke, A., Palmer, M. A., Snelgrove, P., Strayer, D., and Weslawski, J. M. (2001). The function of marine critical transition zones and the importance of sediment biodiversity. Ecosystems 4, 430–451.
The function of marine critical transition zones and the importance of sediment biodiversity.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXntVKitbc%3D&md5=5ff7ba3c84ec6288de5ca7eb81d15699CAS |

McArdle, B. H., and Anderson, M. J. (2001). Fitting multivariate models to community data: a comment on distance-based redundancy analysis. Ecology 82, 290–297.
Fitting multivariate models to community data: a comment on distance-based redundancy analysis.Crossref | GoogleScholarGoogle Scholar |

Nagai, T., Osaki, T., and Kawabata, S. (2001). Functional conversion of hemocyanin to phenoloxidase by horseshoe crab antimicrobial peptides. The Journal of Biological Chemistry 276, 27166–27170.
Functional conversion of hemocyanin to phenoloxidase by horseshoe crab antimicrobial peptides.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXlsV2jur4%3D&md5=e57e37317d83334748f5915d68d78159CAS |

Pascual, C., Arena, L., Cuzon, G., Gaxiola, G., Taboada, G., Valenzuela, M., and Rosas, C. (2004). Effect of size-based selection program on blood metabolites and immune response of Litopenaeus vannamei juveniles fed different dietary carbohydrate levels. Aquaculture 230, 405–416.
Effect of size-based selection program on blood metabolites and immune response of Litopenaeus vannamei juveniles fed different dietary carbohydrate levels.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXos1Gg&md5=592a3ccfe3111fc39d71ae31c65a7717CAS |

Pascual, C., Sánchez, A., Zenteno, E., Cuzon, G., Gabriela, G., Brito, R., Gelabert, R., Hidalgo, E., and Rosas, C. (2006). Biochemical, physiological, and immunological changes during starvation in juveniles of Litopenaeus vannamei. Aquaculture 251, 416–429.
Biochemical, physiological, and immunological changes during starvation in juveniles of Litopenaeus vannamei.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XosFKitw%3D%3D&md5=7281fddc755059f3d8a5af8cb4673a4eCAS |

Paul, R. J., and Pirow, R. (1998). The physiological significance of respiratory proteins in invertebrates. Zoology 100, 298–306.
| 1:CAS:528:DyaK1cXit1Oks7Y%3D&md5=b7cf06c07eaab3665c18f2250535b47dCAS |

Penn, D., and Brockmann, H. J. (1994). Nest-site selection in the horseshoe crab, Limulus polyphemus. The Biological Bulletin 187, 373–384.
Nest-site selection in the horseshoe crab, Limulus polyphemus.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BC1MzlsFSntw%3D%3D&md5=da2532405ee9144953bbd3f941e32b99CAS |

Rudkin, D. M., and Young, G. A. (2009). Horseshoe crabs–an ancient ancestry revealed. In ‘Biology and Conservation of Horseshoe Crabs’. (Eds. J. T. Tanacredi, M. L. Botton, and D. R. Smith.) pp. 25–44. (Springer: New York, NY, USA.)

Sánchez, A., Pascual, C., Sánchez, A., Vargas-Albores, F., Moullac, G. L., and Rosas, C. (2001). Hemolymph metabolic variables and immune response in Litopenaeus setiferus adult males: the effect of acclimation. Aquaculture 198, 13–28.
Hemolymph metabolic variables and immune response in Litopenaeus setiferus adult males: the effect of acclimation.Crossref | GoogleScholarGoogle Scholar |

Seino, S., Uda, T., Tsuchiya, Y., and Tsuchiya, K. (2004). Conservation history of horseshoe crab Tachypleus tridentatus and its spawning ground, a designated natural monument in Kasaoka Bay in Okayama Prefecture. In ‘Asian and Pacific Coasts 2003 – Proceedings of the 2nd International Conference’, 29 February–4 March 2004, Makuhari, Japan. (Eds Y. Goda, W. Kioka, and K. Nadaoka.) pp. 551–556. (World Scientific Publishing Co. Pte. Ltd: Singapore.)

Sekiguchi, K. (Ed.) (1988). ‘Biology of Horseshoe Crabs.’ (Science House Co. Ltd: Tokyo, Japan.)

Sullivan, B., Bonaventura, J., and Bonaventura, C. (1974). Functional differences in the multiple hemocyanins of the horseshoe crab, Limulus polyphemus L. Proceedings of the National Academy of Sciences of the United States of America 71, 2558–2562.
Functional differences in the multiple hemocyanins of the horseshoe crab, Limulus polyphemus L.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaE2cXltFKht70%3D&md5=34054bce1781fc881d9e624b65837d7aCAS |

Tam, N. F. Y., and Yao, M. W. Y. (1999). Three digestion methods to determine concentrations of Cu, Zn, Cd, Ni, Pb, Cr, Mn, and Fe in mangrove sediments from Sai Keng, Chek Keng, and Sha Tau Kok, Hong Kong. Bulletin of Environmental Contamination and Toxicology 62, 708–716.
Three digestion methods to determine concentrations of Cu, Zn, Cd, Ni, Pb, Cr, Mn, and Fe in mangrove sediments from Sai Keng, Chek Keng, and Sha Tau Kok, Hong Kong.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1MXjvVGls7c%3D&md5=fc213c9fb15500d86073f5e029334dffCAS |

Taylor, A., and Maher, W. (2016). Developing a sentinel mollusc species for toxicity assessment: metal exposure, dose and response: laboratory v. field exposures and resident organisms. Environmental Chemistry 13, 434–446.
| 1:CAS:528:DC%2BC28Xos1Wjtrw%3D&md5=138a1266391472b49c3f1a382713eea7CAS |

Van Roy, P., Orr, P. J., Botting, J. P., Muir, L. A., Vinther, J., Lefebvre, B. E. L., Hariri, K., and Briggs, D. E. G. (2010). Ordovician faunas of Burgess Shale type. Nature 465, 215–218.
Ordovician faunas of Burgess Shale type.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXlvFOkt7s%3D&md5=3d5090408f4ce377c6f8558390ba74eaCAS |

Vasquez, M. C., Johnson, S. L., Brockmann, H. J., and Julian, D. (2015). Nest site selection minimizes environmental stressor exposure in the American horseshoe crab, Limulus polyphemus (L.). Journal of Experimental Marine Biology and Ecology 463, 105–114.
Nest site selection minimizes environmental stressor exposure in the American horseshoe crab, Limulus polyphemus (L.).Crossref | GoogleScholarGoogle Scholar |

Wada, T., Mitsushio, T., Inoue, S., Koike, H., and Kawabe, R. (2016). Movement patterns and residency of the critically endangered horseshoe crab Tachypleus tridentatus in a semi-enclosed bay determined using acoustic telemetry. PLoS One 11, e0147429.
Movement patterns and residency of the critically endangered horseshoe crab Tachypleus tridentatus in a semi-enclosed bay determined using acoustic telemetry.Crossref | GoogleScholarGoogle Scholar |

Williams, T. P., Bubb, J. M., and Lester, J. N. (1994). Metal accumulation within salt marsh environments: a review. Marine Pollution Bulletin 28, 277–290.
Metal accumulation within salt marsh environments: a review.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2cXltFKrtLw%3D&md5=12e176356efffd86c7ea6fd6608fc827CAS |

Wilson, A. P. (1986). Cytotoxicity and viability assays. In ‘Animal Cell Culture’. (Ed. R. I. Freshney.) pp. 192–193. (IRL Press: Oxford, UK.)

Wu, F. L., Huang, Z. X., Li, Q. Z., Hu, M. H., Lu, W. Q., and Wang, Y. J. (2015). Classification and characterization of hemocytes between Tachypleus tridentatus and Carcinoscorpius rotundicauda. Acta Hydrobiologica Sinica 39, 1169–1176.

Yin, K., Qian, P. Y., Chen, J. C., Hsieh, D. P. H., and Harrison, P. J. (2000). Dynamics of nutrients and phytoplankton biomass in the Pearl River estuary and adjacent waters of Hong Kong during summer: preliminary evidence for phosphorus and silicon limitation. Marine Ecology Progress Series 194, 295–305.
Dynamics of nutrients and phytoplankton biomass in the Pearl River estuary and adjacent waters of Hong Kong during summer: preliminary evidence for phosphorus and silicon limitation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXjvVWiurg%3D&md5=3354d7219445476a34e559e7aaf1c007CAS |

Zhou, H., and Morton, B. (2004). The diets of juvenile horseshoe crabs, Tachypleus tridentatus and Carcinoscorpius rotundicauda (Xiphosura), from nursery beaches proposed for conservation in Hong Kong. Journal of Natural History 38, 1915–1925.
The diets of juvenile horseshoe crabs, Tachypleus tridentatus and Carcinoscorpius rotundicauda (Xiphosura), from nursery beaches proposed for conservation in Hong Kong.Crossref | GoogleScholarGoogle Scholar |

Zimbro, M. J., Power, D. A., Miller, S. M., Wilson, G. E., and Johnson, J. (2009). Manual of microbiological culture media. Available at http://www.bd.com/resource.aspx?IDX=9572 [Verified 15 March 2016].

Zorita, I., Apraiz, I., Ortiz-Zarragoitia, M., Orbea, A., Cancio, I., Soto, M., Marigómez, I., and Cajaraville, M. P. (2007). Assessment of biological effects of environmental pollution along the NW Mediterranean Sea using mussels as sentinel organisms. Environmental Pollution 148, 236–250.
Assessment of biological effects of environmental pollution along the NW Mediterranean Sea using mussels as sentinel organisms.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXlt1Chtr0%3D&md5=00f66be05963e4046703ea8ed8b98a14CAS |