Larval hatching in the horseshoe crab, Limulus polyphemus: facilitation by environmental cues

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Abstract

Female horseshoe crabs, Limulus polyphemus (Linnaeus), lay their eggs in nests on sandy beaches near the high water line. Embryos develop within the sand, hatch into trilobite larvae, and enter the water column when the nest is inundated. Given the diversity of tidal and shoreline inundation patterns that populations of L. polyphemus experience throughout their range (semidiurnal and diurnal tides, microtidal, and nontidal), hatching may also be facilitated by environmental triggers that serve to synchronize hatching and larval emergence with periods of high water. The objective of this study was to determine if larval hatching in L. polyphemus is triggered or facilitated by environmental cues. Stage 21 embryos were subjected to one of seven different treatments that simulated conditions experienced during inundation: (1) hydration, (2) agitation, (3) hydration and agitation, (4) hydration and agitation with sand, (5) osmotic shock, (6) terrestrial hypoxia, and (7) aquatic hypoxia. Hatching rates increased significantly under all simulated tidal conditions compared to controls and were highest (96%) for eggs simultaneously exposed to both hydration and agitation with sand. Measurements of the osmolarity of the perivitelline fluid of developing eggs collected from the field indicated that it is hyperosmotic to the ambient seawater and porewater. Thus, when inundated, eggs also experience a hypoosmotic shock, which would likely facilitate hatching by causing the eggs to swell, rupturing the egg membrane and thereby increasing the likelihood that larvae would hatch and enter the water column during periods of high water.

Introduction

Sandy beaches serve as nesting sites for a diversity of marine species including grunions Leuresthes spp., sea turtles, and the horseshoe crabs Limulus polyphemus, Tachypleus tridentatus, Tachypleus gigas, and Carcinoscorpius rotundicauda. In aquatic species that lay eggs in terrestrial and semiterrestrial habitats, hatching is often triggered by environmental cues that time hatching to coincide with conditions favorable for larval survival. Most eggs laid terrestrially by aquatic fish do not hatch at a set developmental stage. Rather, development is delayed until the eggs are stimulated to hatch by an environmental trigger associated with the return of aquatic conditions (Martin, 1999). For example, eggs of the grunion Leuresthes tenuis complete development within the period between the new and full moon, yet they typically hatch during the highest spring tide (Moffatt and Thompson, 1978). If the water does not rise high enough to release the larvae after one spring tide, the eggs remain in the sand until the subsequent spring tide.

Studies of hatching mechanisms in aquatic invertebrates have focused on mechanical and osmotic processes of eclosion (see Davis, 1968, Davis, 1981, Pandian, 1970 for reviews). Mechanical eclosion occurs when egg membranes become weak and rupture as a consequence of the movements of the embryos. Hatching can also be aided by external mechanical stimulation and agitation Moffatt and Thompson, 1978, Griem and Martin, 2000. For example, in crustaceans that carry their eggs until hatching, such as crabs and lobsters, hatching is facilitated by abdominal pumping by the female (Forward, 1987). Similarly, Moffatt and Thompson (1978) suggested that erosion of the chorion by sand may stimulate hatching in grunion eggs. Internal and external mechanical processes may be linked. Griem and Martin (2000) found that movements of the embryos of the California grunion are triggered by external agitation. Once stimulated, the embryo breaks the chorion and emerges by thrashing about within the egg. Osmotic hatching is believed to occur as a consequence of an increase in the osmotic concentration within the egg during development. When subjected to hypoosmotic conditions, the subsequent flow of water into the egg causes it to swell, inducing one or more of the membranes to rupture (Davis, 1981). Osmotic hatching can also be aided by chemical means, with embryos releasing enzymes that weaken and/or dissolve egg membranes several hours before larval release Rittschof et al., 1985, De Vries and Forward, 1991. Finally, hypoxia, which results when aquatic conditions return in the form of rainfall or high spring tides, has also been suggested as the universal trigger for hatching in anamniotic eggs DiMichele and Taylor, 1980, Petranka et al., 1982.

The American horseshoe crab L. polyphemus (Linnaeus) nest near the waterline in the mid- to upper-intertidal zone. Up to 20,000 eggs per clutch are laid 10–25 cm below the sediment surface Shuster and Botton, 1985, Brockmann, 1990, Penn and Brockmann, 1994. During spawning, eggs are fertilized by sperm released by an attached male and one or more satellite males (Rudloe, 1980). Peak spawning generally occurs near the time of high tide during new and full moons and appears to be under endogenous control Rudloe, 1980, Cohen and Brockmann, 1983, Barlow et al., 1986.

The use of the sandy beaches by L. polyphemus for nesting affords a great deal of protection for developing eggs from aquatic predators, with most embryos surviving to hatching (Rudloe, 1979). However, this nesting pattern creates problems for the emergence and dispersal of larvae into the aquatic environment. Embryos hatch into trilobite larvae in approximately 28 days and enter the water when inundated during nocturnal high tides near the time of full moon Rudloe, 1979, Penn and Brockmann, 1994. However, because of their wide geographic distribution, tidal conditions experienced by developing L. polyphemus embryos vary significantly. Populations in some areas experience two high and two low tides per day (i.e., semidiurnal tides, Shuster, 1982, Rudloe, 1985, Barlow et al., 1986), while others experience only one high and one low tide per day (i.e., diurnal tides, Rudloe, 1979, Rudloe, 1980, Rudloe, 1985, Cohen and Brockmann, 1983). L. polyphemus also inhabits shallow water coastal lagoons and embayments that lack significant tidal changes, with wind forcing and freshwater input determining changes in water level and patterns of shoreline inundation Rudloe, 1985, Ehlinger et al., 2003. Therefore, inundation of the nest varies throughout the species range and is not always predictable. Given the diverse range of shoreline inundation patterns that occur throughout its range, the hatching of L. polyphemus eggs may be facilitated by environmental triggers that help to synchronize larval hatching with water levels on the beach, especially in microtidal areas. If an environmental cue associated with inundation triggers hatching of L. polyphemus larvae, then larvae will hatch and emerge when conditions are favorable for entry into the water column.

The objective of this study was to determine if hatching of L. polyphemus embryos is triggered or facilitated by environmental conditions that occur when nests are submerged during spring high tides. Possible triggers for hatching include hydration, agitation, osmotic shock, and hypoxia, all of which are experienced by developing embryos during periods of nest inundation.

Section snippets

Collection of gametes and artificial fertilization of eggs

Adult L. polyphemus were collected by hand during the spawning season (February–May 2002) from the Indian River Lagoon, a microtidal system along the east coast of Florida, USA. Adults were collected from two sites, Pineda Causeway, Banana River (28°12′33.9ʺN, 80°38′12.9ʺW) and Peacock Pocket, Indian River (28°39′41.8ʺN, 80°43′45.6ʺW). Crabs were maintained in a recirculating fiberglass tank (2.7 m×1.7 m×1 m) containing natural seawater with a salinity of 30 (salinity is presented as a pure

Hydration and agitation

When exposed to hydration and agitation conditions that simulated submergence at high tide, hatching levels of Stage 21 embryos increased significantly relative to levels in control treatments (Fig. 1, Table 1). In all experimental treatments, the majority of hatching occurred within the first 2 h following exposure (Fig. 1). Most (>65%) embryos in the treatments involving agitation hatched during the exposure period (Fig. 1). Hatching levels in the hydration-only treatment increased rapidly

Discussion

Larval hatching in marine organisms can be stimulated by chemical substances Pandian, 1970, Rittschof et al., 1985, Poulin et al., 1990, De Vries and Forward, 1991, De Vries et al., 1991, mechanical disturbance Pandian, 1970, Frank and Leggett, 1981, Saigusa, 1992, Griem and Martin, 2000, osmotic shock Pandian, 1970, Newton and Mitchell, 1999, or changes in oxygen levels Petranka et al., 1982, Lutz et al., 1992, Helvik and Walther, 1993. These cues help time larval hatching and emergence with

Acknowledgments

This research is supported in part by the National Park Service Grant no. CA518099049. We thank the Canaveral National Seashore, the Merritt Island National Wildlife Refuge, and the NASA/Kennedy Space Center for access to the collection sites. We thank Drs. M. Botton, M. Bush, E. Irlandi, and J. Lin for their comments on an earlier draft of this manuscript. We are grateful to M. Mota, E. Reyier, and D. Scheidt for their assistance in collecting adults and Drs. D. Carroll and J. Grimwade for use

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