Natural History of the American Horseshoe Crab: in Retrospect/Prospect

Natural History of the American Horseshoe Crab: in Retrospect/Prospect H. Jane Brockmann Department of Biology University of Florida Gainesville, FL 32611-8525 HJB@ufl.edu 4 June 2016 Carl Shuster Celebration Limuli Labs, Eldora, NJ

Carl s Questions & Hypotheses Keenly observing, asking questions, proposing explanations and hypotheses about horseshoe crab natural history Retrospect: Identify some of Carl s ideas that are still open questions, mention a little of the work that has been done Prospect: suggest what still needs to be done to address these issues (+ a few interesting new puzzles) Horseshoe crab natural history

Differences between Populations South Carolina Yucatan

Discrete populations Shuster 1955, 1979 King et al. 2015

What keeps the populations apart? Distance? Habitat? Mate choice? New Smyrna Beach Mosquito Lagoon Brockmann et al. 2015

Frequency of Size Category in each Sample 0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.00 Proportion of the population in each size category Males Indian River Lagoon (N=52) New Smyrna Beach (N=79) Cumberland Island (N=62) 0.60 0.50 0.40 0.30 0.20 0.10 0.00 Females Prosomal width (cm) Patrick Norby, in preparation 6

How do populations differ? Why? Size differences among populations Proximate: differ in number of molts? Incremental growth? Ultimate: what is advantage/disadvantage of larger size in some populations? Base color of the carapace differs Differences in eye pigmentation Differences in spines Shuster 2015 DE GA DE GA

Polymorphism within the population Eye color Site light eyes WFL 32% Spines Site with spines Sapelo: 63% Skidaway: 42% Shell color variation at IRL How and why is this variation maintained in the population? Genetic differences? Developmental differences? Advantage to each morph?

Physiological differences between populations Size is a characteristic of discrete populations and is related to latitude and salinity. Regardless of latitude, those individuals occurring in populations frequenting low salinity environments are smaller than those at similar latitudes living in higher salinities. (Shuster 1955/58)

Physiological differences between populations In a comparative study (submitted for publication) Vasquez, Brockmann and Julian tested the effects of 3 environmental conditions on the development of embryos from DE and FL. Interestingly, they found that FL eggs developed faster than DE eggs under low salinity conditions. In contrast, the DE embryos developed faster under most temperature and oxygen conditions at moderate salinity. At the highest temperatures, DE developed faster when oxygen was low and salinities high, FL developed faster when oxygen was high. This suggests that there may be physiological and developmental differences between horseshoe crabs from different populations.

I wish to sound a call for more attention to comparative studies, anatomical, biochemical, physiological, behavioral and ecological. - Shuster 1988

Aging horseshoe crabs Yearling 10 years as a juvenile Adult condition deteriorates with age (terminal molt; aid in estimating recruitment) Young 1-3 years Mid-age 3-6 years Old age 6-10 years 1955/1958, 1993, Shuster 1999

Aging horseshoe crabs Young Old Condition of exoskeleton declines with age including the viscous secretion (slime) from pores through the prosoma: anti-fouling (physical barrier) and antibiotic (cytolytic) production declines with age Stagner & Redmond 1975; Harrington et al. 2008 13

Aging horseshoe crabs Old Why do these changes occur? Too costly to maintain? Are these changes associated with changes in the immune system? Or with the energetic reserves of the animal? Does aging change with the behavior of the animal? 14

Correlates of age categories Younger Males more likely to be/have Lighter in color More slime Less fouling Eyes in better condition Carapace in better condition (no pits, scratches or mating scars) More active Right themselves quickly Attach more quickly in pool Stay attached better Sperm concentration higher Older Males more likely to be/have Darker in color Less slime More fouling Eyes in worse condition Carapace in worse condition (pits, scratches, mating scars) Less active Right themselves slowly Unlikely to attach in pool Let go more easily Lower sperm concentration Brockmann & Penn 1992; Penn & Brockmann 1995; Brockmann 2003; Brockmann et al. 2015

Pairing and Spawning behavior Unpublished 1994

Male Mating Tactics Attached male Unattached male Attached male 100% paternity Female Attached male Satellite males in #1 position 40% paternity each on average older 17

Female Mating Tactics Monandrous: Polyandrous: Attracts satellites Multi-male paternity Does not attract satellites Single male paternity Lay fewer eggs Attached male has higher sperm concentration Brockmann et al. 2000; Johnson & Brockmann 2012; Brockmann et al. 2015 18

Males use visual and chemical cues Unpaired male approaches cement models of nesting pairs Finding mates Caged pairs attract satellites Polyandrous females re-attract satellites when removed Is a pheromone involved? If so, what is it and when and where is it produced? Hassler & Brockmann 2001; Schwab & Brockmann 2007; Saunders et al. 2010 19

Male Female Mechanism of Pairing (amplexus) Male clasps female s posterior opisthosomal spines with his modified first pair of legs Effectiveness of clasping organ may be due to a locking mechanism not unlike that seen in molluscs where catch muscles enable the bivalve to keep its shell closed with little or not effort Does the strength of attachment vary with male age? 20

Advantages of amplexus Allows males to remain with the female when waves are present Allows pairing prior to mating in a sparse population So, are there differences between populations in the time males are attached (amplexus is costly: attached males don t feed)? strength of the attachment? (high energy beaches) structure of the claspers? Cumberland Island, GA Florida Botton et al. 1996

Female mating scars What are the conditions that produce mating scars? Are they indicative of mating struggles? Are monandrous and polyandrous females equally likely to develop mating scars? Does population density or sex ratio affect mating scars? Carmichael et al. 2015

Egg laying She thrusts her legs up and down in the sand creating a slurry of sediment and water. The eggs are extruded from the oviducts in masses and dropped down into the slurry The female uses her last pair of legs to push the eggs down into the slurry of sand and water beneath her The depth of the clump is dependent on the size of the female and how deeply she burrows into the sand. Eggs sticky and adhere to sand grains. What is this glue? What affects the number of eggs laid? Shuster 2003 Presence of male (do females have receptors on spines?) Female size Seasonal changes An unattached female on the shore with a satellite male she does not lay eggs

Fertilizing eggs How are sperm actually getting to the eggs? Fertilization occurs when the spermatozoa are washed into the nests by the waves Shuster 1960 Sperm must travel in currents to the eggs Sperm swim only when close to the egg - How could there be a current when nesting out of the water? - Nearly all eggs are fertilized whether satellites are present or not. - So who sets up the currents?

Fertilizing eggs How are sperm actually getting to the eggs? Fertilization occurs when the spermatozoa are washed into the nests by the waves Shuster 1960 - Satellites are using female s respiratory currents - Who sets up the currents? How could there be a current when nesting out of the water? How do environmental conditions affect paternity?

Fertilizing eggs experimental evidence How are sperm actually getting to the eggs? Experimentally removed satellites When all the satellites are removed, 78% of the eggs were fertilized by the satellites that had been removed - even 20 min later! So, sperm are being retained or held by the female. How? Where? Johnson & Brockmann 2013

Mating groups Best group sizes for satellite based on paternity: 1 or 2 Males join at random Numbers affected by density and environmental conditions Shuster 1955

Carl s Questions & Hypotheses Retrospect / Prospect Genetically distinct populations What keeps these populations separate? Distance? How do the populations differ? Comparative studies Genetic, morphological, physiological, behavioral What are the advantages of those differences? How and why is variation maintained within a population? (e.g. eye color, spines) Aging correlated changes in physiology and behavior Pairing and spawning Finding mates Mechanism and advantage of pairing and amplexus Egg laying and variation in number laid Fertilizing eggs Mating group sizes

Thanks for pulling us along!

My research is funded by the Animal Behavior Program, National Science Foundation Cape Henlopen, DE 30

Many thanks to: Post-docs Sheri Johnson Mary Hart Grad Students Daniel Sasson Matthew Smith Katie Saunders Rachel Schwab Cynthia Hassler Dustin Penn Undergraduates & Field Assistants many have helped us over the years Funding National Science Foundation Animal Behavior Program Facilities UF Marine Lab at Seahorse Key Permits & Collaborations US Fish & Wildlife (Cedar Keys National Wildlife Refuge) Cumberland Island National Seashore Marine Discovery Center, New Smyrna Beach Green Eggs & Sand 31

More questions about spawning What is the process of ovogenesis? Does she actually mature and spawn another full clutch of eggs in the same season? What about spermatogenesis? Are sperm viable all year? How long does a female remain in the vicinity? How to explain erratic spawning behavior associated with specific sites, e.g. trying to spawn in a pile of oyster shells How are the crabs guided back to their natal beach may be dependent upon where they enter the bay Shuster 2015 Males with claspers may be restricted in their foraging which is why they develop only at maturity Shuster 1994 How did ancestral horseshoe crabs breed? Did they have amplexus or did males simply swarm around females? Why do they nest in the intertidal?

References Botton, M. L., C. N. Shuster, K. Sekiguchi, and H. Sugita. 1996. Amplexus and mating behavior in the Japanese horseshoe crab, Tachypleus tridentatus. Zool. Sci. 13:151-159. Brockmann, H. J. 1996. Satellite male groups in horseshoe crabs, Limulus polyphemus. Ethology 102:1-21. Brockmann, H. J. 2001. The evolution of alternative strategies and tactics. Advances in the Study of Behavior 30:1-51. Brockmann, H. J. 2002. An experimental approach to altering mating tactics in male horseshoe crabs (Limulus polyphemus). Behavioral Ecology 13:232-238. Brockmann, H. J. 2003. Male competition and satellite behavior. Pages 50-82 in C. N. Shuster, R. B. Barlow, and H. J. Brockmann, editors. The American Horseshoe Crab. Harvard University Press, Cambridge, MA. Brockmann, H. J. 2003. Nesting behavior: A shoreline phenomenon. Pages 33-49 in C. N. Shuster, R. B. Barlow, and H. J. Brockmann, editors. The American Horseshoe Crab. Harvard University Press, Cambridge, MA. Brockmann, H. J., T. Black, and T. L. King. 2015. Florida horseshoe crabs: Populations, genetics and the marine-life harvest. Pages 97-127 in R. H. Carmichael, M. L. Botton, P. K. S. Shin, and S. G. Cheung, editors. Changing Global Perspectives on Biology, Conservation and Management of Horseshoe Crabs. Springer Scientific, New York. Brockmann, H. J., T. Colson, and W. Potts. 1994. Sperm competition in horseshoe crabs (Limulus polyphemus). Behavioral Ecology and Sociobiology 35:153-160. Brockmann, H. J. and S. L. Johnson. 2011. A long-term study of spawning activity in a Florida Gulf coast population of horseshoe crabs (Limulus polyphemus). Estuaries and Coasts 34:1049-1067. Brockmann, H. J., S. L. Johnson, D. A. Sasson, and M. D. Smith. 2013. Seasonal patterns of horseshoe crab spawning (Limulus polyphemus). Page 26 Coastal & Estuarine Research Federation, San Diego, CA. Brockmann, H. J., S. L. Johnson, D. A. Sasson, and M. D. Smith. in preparation. Seasonal variation in horseshoe crab reproduction (Limulus polyphemus). Brockmann, H. J., S. L. Johnson, M. D. Smith, and D. A. Sasson. 2015. Mating tactics of the American Horseshoe crab..in R. H. Carmichael, M. L. Botton, P. K. S. Shin, and S. G. Cheung, editors. Changing Global Perspectives on Biology, Conservation and Management of Horseshoe Crabs. Springer, New York. Brockmann, H. J., C. Nguyen, and W. Potts. 2000. Paternity in horseshoe crabs when spawning in multiple-male groups. Animal Behaviour 60:837-849. Brockmann, H. J. and D. Penn. 1992. Male mating tactics in the horseshoe crab, Limulus polyphemus. Animal Behaviour 44:653-665. Brockmann, H. J. and M. D. Smith. 2009. Reproductive competition and sexual selection in horseshoe crabs. Pages 199-221 in J. T. Tanacredi, M. Botton, and D. R. Smith, editors. Biology and Conservation of Horseshoe Crabs. Springer, New York. Carmichael, R., E. E. Hieb, G. Gauvry, and C. N. Shuster. 2015. Examination of large exuviae with mating scars: do female American horseshoe crabs, Limulus polyphemus, molt after sexual maturity? Pages 353-365 in R. Carmichael, M. Botton, P. K. S. Shin, and S. G. Cheung, editors. Changing Global Perspectives on Horseshoe Crab Biology, Conservation and Management. Springer, New York. Duffy, E. E., D. J. Penn, M. L. Botton, H. J. Brockmann, and R. E. Loveland. 2006. Eye and clasper damage influence male mating tactics in the horseshoe crab, Limulus polyphemus. Journal of Ethology 24:67-74. Harrington, J. M., M. Leippe, and P. B. Armstrong. 2008. Epithelial immunity in a marine invertebrate: a cytolytic activity from a cuticular secretion of the American horseshoe crab, Limulus polyphemus. Marine Biology 153:1165-1171. Hassler, C. and H. J. Brockmann. 2001. Evidence for use of chemical cues by male horseshoe crabs when locating nesting females (Limulus polyphemus). Journal of Chemical Ecology 27:2319-2335. Johnson, S. L. and H. J. Brockmann. 2010. Costs of multiple mates: an experimental study in horseshoe crabs. Animal Behaviour 80:773-782. Johnson, S. L. and H. J. Brockmann. 2012. Alternative reproductive tactics in female horseshoe crabs. Behavioral Ecology 23:999-1008. Johnson, S. L. and H. J. Brockmann. 2013. Parental effects on early development: testing for indirect benefits of polyandry. Behavioral Ecology 24:1218-1228.

References continued King, T. L., M. S. Eackles, A. W. Aunins, H. J. Brockmann, E. M. Hallerman, and B. B. Brown. 2015. Conservation genetics of the American horseshoe crab (Limulus polyphemus): Allelic diversity, zones of genetic discontinuity, and regional differentiation. Pages 65-96 in R. H. Carmichael, M. L. Botton, P. K. S. Shin, and S. G. Cheung, editors. Changing Global Perspectives on Biology, Conservation and Management of Horseshoe Crabs. Springer, New York. King, T. L., M. S. Eackles, A. P. Spidle, and H. J. Brockmann. 2005. Regional differentiation and sex-biased dispersal among populations of the horseshoe crab Limulus polyphemus. Transactions of the American Fisheries Society 134:441-465. Penn, D. and H. J. Brockmann. 1994. Nest-site selection in the horseshoe crab, Limulus polyphemus. Biological Bulletin 187:373-384. Penn, D. and H. J. Brockmann. 1995. Age-biased stranding and righting in horseshoe crabs, Limulus polyphemus. Animal Behaviour 49:1531-1539. Sasson, D. A. and H. J. Brockmann. 2016. Variation in sperm and ejaculate quantity and quality across populations of horseshoe crabs. Behavioral Ecology and Sociobiology. (in press) Sasson, D. A., S. L. Johnson, and H. J. Brockmann. 2012. The role of age on sperm traits in the American horseshoe crab, Limulus polyphemus. Animal Behaviour 84:975-981. Sasson, D. A., S. L. Johnson, and H. J. Brockmann. 2015. Reproductive tactics and mating contexts affect sperm traits in horseshoe crabs (Limulus polyphemus). Behavioral Ecology and Sociobiology 69:1769-1778. Saunders, K. M., H. J. Brockmann, W. H. Watson, and S. H. Jury. 2010. Male horseshoe crabs Limulus polyphemus use multiple sensory cues to locate mates. Current Zoology 56:485-498. Schwab, R. L. and H. J. Brockmann. 2007. The role of visual and chemical cues in the mating decisions of satellite male horseshoe crabs, Limulus polyphemus. Animal Behaviour 74:837-846. Shuster, C. N. 2015. The Delaware Bay area, U.S.A.: A unique habitat of the American horseshoe crab, Limulus polyphemus. Pages 15-39 in R. Carmichael, M. Botton, P. K. S. Shin, and S. G. Cheung, editors. Changing Global Perspectives on Horseshoe Crab Biology, Conservation and Management. Springer, New York. Shuster, C. N., Jr. 1955. On morphometric and serological relationships within the Limulidae, with particular reference to Limulus polyphemus L. Ph.D. New York University, New York. Shuster, C. N., Jr. 1979. Biology of Limulus polyphemus. Pages 1-2. in E. Cohen, editor. Biomedical Applications of the Horseshoe Crab (Limulidae). Alan R. Liss, Inc., New York. Shuster, C. N., Jr. 1979. Distribution of the American horseshoe "crab", Limulus polyphemus (L.). Pages 3-26 in E. Cohen, editor. Biomedical Applications of the Horseshoe Crab (Limulidae). Alan R. Liss, Inc., New York. Shuster, C. N., Jr. 1982. A pictorial review of the natural history and ecology of the horseshoe crab Limulus polyphemus, with reference to other Limulidae. Pages 1-52 in J. Bonaventura, C. Bonaventura, and S. Tesh, editors. Physiology and Biology of Horseshoe Crabs: Studies on Normal and Environmentally Stressed Animals. Alan R. Liss, Inc., New York. Shuster, C. N., Jr. 1990. The American horseshoe crab, Limulus polyphemus. Pages 15-25 in R. B. Prior, editor. Clinical Applications of the Limulus Amoebocyte Lysate Test. CRC Press, Boca Raton.

References continued Shuster, C. N., Jr. 2001. Two perspectives: horseshoe crabs during 420 million years worldwide, and the past 150 years in Delaware Bay. Pages 17-40 in J. T. Tanacredi, editor. Limulus in the Limelight. Kluwer Academic, New York. Shuster, C. N., Jr. and K. Sekiguchi. 2003. Growing up takes about ten years and eighteen stages. Pages 103-132 in C. N. Shuster, R. B. Barlow, and H. J. Brockmann, editors. The American Horseshoe Crab. Harvard University Press, Cambridge, MA. Shuster, C. N. and K. Sekiguchi. 2009. Basic habitat requirements of the extant species of horseshoe crabs (Limulacea). Pages 115-129 in J. T. Tanacredi, M. Botton, and D. R. Smith, editors. Biology and Conservation of Horseshoe Crabs. Springer, New York. Smith, M. D. and H. J. Brockmann. 2014. The evolution and maintenance of sexual size dimorphism in horseshoe crabs: an evaluation of six functional hypotheses Animal Behaviour 96:127-139. Smith, M. D., H. E. Schrank, and H. J. Brockmann. 2013. Measuring the costs of alternative reproductive tactics in horseshoe crabs, Limulus polyphemus. Animal Behaviour 85:165-173. Stagner, J. I. and J. R. Redmond. 1975. The immunological mechanisms of the horseshoe crab, Limulus polyphemus. Marine Fisheries Review 37:11-19. Vasquez, M. C., S. L. Johnson, H. J. Brockmann, and D. Julian. 2015. Nest site selection minimizes environmental stressor exposure in the American horseshoe crab, Limulus polyphemus. Journal of Experimental Marine Biology and Ecology 463:105-114. Vasquez, M. C., A. Murillo, H. J. Brockmann, and D. Julian. 2015. Multiple-stressor interactions influence embryo development rate in the American horseshoe crab, Limulus polyphemus. Journal of Experimental Biology 218:2355-2364. Vasquez, M.C., D. Julian, H.J. Brockmann. 2016. Latitudinal differences in multi-stressor tolerance during embryo development in the American horseshoe crab, Limulus polyphemus. Estuaries and Coasts (in press).