Patterns of larval release by the Asian shore crab Hemigrapsus sanguineus (De Haan): periodicity at diel and tidal frequencies.

ABSTRACT The Asian shore crab, Hemigrapsus sanguineus, is an invasive species along the Atlantic coast of North America. It has spread rapidly since its discovery in the USA in 1988 and has become the dominant crab species in rocky intertidal habitats at many locations in the Middle Atlantic Bight and in the southern Gulf of Maine. The reproductive biology of Hsanguineus has received only cursory study in native east-Asian waters. Available information consists mainly of descriptions of the larval stages and general characterizations of the natural history of the species. This study examined the periodicity of larval release in an invasive population of H. sanguineus near its original site of introduction at the mouth of Delaware Bay. We investigated larval release at diel and tidal frequencies. Analysis of laboratory data showed that larval release always occurs near the time of nocturnal high tide. Field sampling confirmed the presence of stage I zoeae in waters near adult habitat at the expected time of larval release. This pattern of release minimizes the impact of visual predators on newly hatched zoeae and takes advantage of circulation at tidal frequency to transport zoeae away from intertidal habitats where conditions may not be favorable for larval development. However, H. sanguineus is not unique among intertidal crabs in releasing larvae near the time of nocturnal high fide. This pattern has been reported for a large number of species with wide representation among families. Thus, the pattern of release observed in H. sanguineus does not provide any special competitive advantages over co-occurring native species, and the remarkable success of the species in North American habitats may be attributable to competitive interactions occurring after settlement and metamorphosis.

KEY WORDS: invasive species, Hemigrapsus, larval release, diel, tidal, Asian shore crab

INTRODUCTION

Reproductive activity of marine fish and invertebrates is often synchronized with seasonal cycles. Within these seasonal periods of reproduction, the actual release of larvae may occur in phase with lunar, semilunar, tidal, or diel cycles (e.g., Knowlton 1979, Rutberg 1987, Ims 1990). Many species of intertidal crab display a rhythm of larval release wherein hatching occurs during springtide periods near the time of nocturnal high tide (Forward 1987). This minimizes the impact of visual predators on newly hatched zoeae and takes advantage of tidal circulation to transport zoeae away from intertidal habitats where environmental conditions may not be conducive to larval development (Morgan & Christy 1996, Petrone et al. 2005).

The grapsid crab Hemigrapsus sanguineus is a recent invader of rocky intertidal habitat along the east coast of North America. H. sanguineus has spread rapidly since it was first discovered near the mouth of Delaware Bay in 1988, and the role of reproductive biology in promoting this spread is poorly known. The species currently ranges from Maine to North Carolina (ca. 36[degrees]44[degrees]N) in the United States and has been reported along the Atlantic and Mediterranean coasts of Europe (McDermott 1998a, Park et al. 2004, Breton et al. 2002, Schubart 2003). The reproductive biology of H. sanguineus has received only cursory study in native east-Asian waters. Available information consists mainly of descriptions of the larval stages and general characterizations of the natural history of the species (Kurata 1968, Gamo & Muraoka 1977, Hwang & Kim 1995). Earlier investigations of invasive populations have shown that H. sanguineus has an unusually long reproductive season (April to October) in North American habitats (McDermott 1998b, Epifanio et al. 1998). However, patterns of larval release within the reproductive season are not well known, and factors such as semilunar, diel and tidal rhythms of release could have important effects on the invasive potential of the species. An earlier study conducted with native Asian crabs indicated that H. sanguineus females release all of the larvae within a given brood as a single hatching event near the time of high tide and that larval release probably occurs throughout the spring/neap cycle (Saigusa & Kawagoye 1997). However, that investigation was conducted mainly in the laboratory, and results were complicated by the long periods of isolation from natural tidal and light conditions. Subsequent work with an invasive North American population investigated patterns of larval release on a semilunar scale and again found no clear evidence for spring/neap periodicity (Epifanio et al. 1998). In the current work, we present results of a study that used a combined field and laboratory approach to investigate tidal and diel patterns of larval release by H. sanguineus females in a North American habitat. The study addressed two research questions: (a) is there adiel cycle to larval release and (b) is tidal periodicity related to the putative diel cycle?

MATERIALS AND METHODS

General Aspect

Our study had two components. The first component investigated patterns of larval release as related to diel and tidal cycles. This study consisted of laboratory-based observations of larval release by recently collected H. sanguineus females. The second component consisted of plankton sampling in waters adjacent to adult habitat to confirm the timing of larval release in the field. In the laboratory experiment, we used ovigerous females collected from the natural environment during peak spawning season (June and July) in 2001. In an earlier study, we developed an index relating the color of an egg mass to the developmental state of embryos within the eggs (Epifanio et al. 1998). We used this index to select hatch-imminent females for use in the experiment. Females were collected during the diurnal low tide immediately preceding the start of the trials. The majority of crabs used in the study released larvae within 30 h of collection, and none were held in the laboratory for more than 78 h.

Laboratory Investigation of Diel and Tidal Periodicity

This investigation consisted of 3 experiments. The first experiment was designed to determine the diel pattern of larval release. In this study, 35 females were maintained individually in 23-cm diameter glass fingerbowls under constant conditions (25[degrees]C, 30[per thousand]). To simplify the experimental design, we held the crabs in complete darkness. This assured that any observed rhythm was either endogenous or already entrained. The crabs were observed using a far-red light (incandescent light passed through a Kodak red filter with less than 1% transmission of wavelengths <600 nm), which cannot he detected by the crabs (Shukoliukov et al. 1980). Crabs were placed in experimental containers <1 h after collection, and initial observations were made immediately before sunset. Observations then continued on the following sunrises/sunsets until all females had released their larvae (<3 d). Larval release was confirmed by the presence of zoeae in the bowls. By making observations twice a day, we could determine whether release occurred diurnally (between sunrise and sunset) or nocturnally (between sunset and sunrise).

A second experiment was designed to determine the tidal pattern of larval release. This study also used 35 ovigerous females collected within 4 h of initiation of the experiment and held under conditions similar to those described earlier. Crabs were observed every 15 min under far-red light starting approximately 2 h before sunset (18:00 EDT) and continuing until 06:00 the following morning. The initial occurrence of intense flapping of the abdomen, the initial occurrence of zoeae in the water, and the occurrence of full larval release were noted for each female. Observations continued for 3 consecutive nights. A third experiment, again designed to determine tidal periodicity and this time using 28 females, was conducted approximately 2 weeks later.

Pooled data from the 2 tidal-periodicity experiments were analyzed using circular statistics (see later). To conduct this analysis, the observed times of larval release were converted to degrees relative to the occurrence of high tide in the field (i.e., the time of high tide = 0[degrees]; a complete semidiurnal tidal cycle = 360[degrees]; and 1 h = 29[degrees]). Thus, larval release occurring 1 h before high tide in the field would convert to 331[degrees] (360[degrees]-29[degrees] = 331[degrees]). All tidal information was obtained from verified water-level data collected at the NOAA/NOS water level observation station at Lewes, Delaware.

Descriptive circular statistics were used to determine a mean angle and r-vector of dispersion relative to the time of high tide (Batschelet 1981, Kovach 1994). The r-vector in this technique ranges from 0 to 1; larger values indicate greater clustering of observations around the mean. Rayleigh's uniformity test was used to calculate distribution in a uniform manner; a significant level (P < 0.05) is evidence of a preferred release time.

Pattern of Larval Release in the Field

We conducted high-frequency plankton sampling to confirm the presence of stage I zoea larvae (Z-I) at predicted times of larval release. Sampling was carried out in a harbor adjacent to the mouth of the Broadkill River, a secondary estuary that empties into Delaware Bay near Cape Henlopen (Fig. 1). We collected larvae on two separate nights, one in early June and the other in mid-June 2001. The harbor was bordered on three sides by rocky intertidal habitat that contained a well-established breeding population of H. sanguineus (Epifanio et al. 1998, Kopin et al. 2001); the fourth side was open to the Broadkill River. Water depth at the sampling site was approximately 2 m. Each sampling effort consisted of 2-min surface tows (0.5-m net; 253-[micro]m mesh; General Oceanics flow meter) taken every 15 min. Sampling began approximately 1 h before the predicted time of high tide in the harbor and extended over an entire nocturnal tidal cycle; this resulted in a time series of 51 plankton samples for each effort. Samples were preserved in 70% ethanol and analyzed using standard microscopy techniques (Dittel & Epifanio 1982).

[FIGURE 1 OMITTED]

For both sampling efforts, we estimated time of larval release events relative to the time of high tide by visual inspection of the respective time series of larval densities. In addition, we used autocorrelation analysis to determine the duration of each larval release event (Natunewicz & Epifanio 2001). The temporal scale of the respective larval release events was defined as the point in time at which the distribution of zoeal densities first became decorrelated (Rowe & Epifanio 1994, Jones & Epifanio 2005). Decorrelation was defined as the number of lags of the time series at which the correlation coefficient first assumed a value [less than or equal to] 0 (Richerson et al. 1978). Each lag represented a time-step of 15 min, which corresponded to the time between successive plankton tows. The temporal scale of the larval release event on each night was determined by summing the number of lags required to achieve de-correlation and multiplying by the 15-min duration of each time step.

RESULTS

Tidal and Diel Periodicity in Larval Release

Results from the diel periodicity study were unambiguous; all 35 females released their larvae during the nocturnal phase of the diel period. Results from the laboratory investigation of tidal periodicity were also clear-cut and indicate that larval release for all crabs in the study occurred near the time of the nocturnal high tide. Application of circular statistical techniques showed a significant relationship between the occurrence of all 3 indicators of larval release (i.e., time of first abdominal flap, initial observation of zoeae and complete larval release) and the occurrence of high tide in the field (Table 1).

Periodicity of Larval Release in the Field

Results of high-frequency plankton sampling adjacent to an adult population of H. sanguineus generally supported predictions based on laboratory results. Peak abundance of Z-I H. sanguineus occurred near the time of high tide on both nights of sampling (Fig. 2a,b). However, in early June we also observed a second peak of Z-I H. sanguineus beginning near the time of low tide and broadly extending through much of the subsequent flood phase. This second peak was coincident with the appearance of large numbers of Z-I fiddler crab larvae (Uca spp.), indicating an upstream source for the water mass (see below). Autocorrelation analysis revealed similar time scales for the duration of both larval release events for H. sanguineus (Fig. 3a,b). On the first sampling night, decorrelation occurred after 5 lags (75 min); on the second night, decorrelation occurred after 6 lags (90 min).

[FIGURE 2 OMITTED]

DISCUSSION

Overall results of our investigation indicate that H. sanguineus females release larvae near the time of nocturnal high tide. Ovigerous females in the diel-periodicity experiment released larvae only during the nocturnal phase. Likewise, females in the tidal-periodicity experiments always released larvae near the time of high tide. Field sampling confirmed the diel and tidal periodicity in release of larvae. On both sampling nights, the initial appearance of zoeae was manifested as a discrete event with a duration of approximately 1 h. This is comparable to the time scale for release of larvae by individual females in the laboratory (Saigusa & Kawagoye 1997) and supports the notion of synchronized release of larvae within the population of H. sanguineus living in the harbor. In the early-June study, release events began about an hour after sunset and almost coincident with high tide. In the mid-June study, sunset occurred around 15 min after predicted high tide and was approximately coincident with the onset of a larval release event.

In the early-June sampling, we observed a second occurrence of Z-I H. sanguineus beginning about 6 h after nocturnal high tide, near the time of the subsequent low tide. These larvae remained in the water column through much of the ensuing flood phase. This occurrence of H. sanguineus zoeae coincided with the appearance of large numbers of Z-I fiddler crab larvae in the harbor. Adult fiddler crabs do not occur at the sampling site, but are common in marsh habitats that border the Broadkill River upstream of the harbor. Thus, the water mass containing the assemblage of H. sanguineus and fiddler crab zoeae must have originated upstream of our sampling site and must have been transported to the harbor as part of the ebb circulation of the Broadkill River. It is well known that fiddler crabs release larvae near the time of nocturnal high tide (e.g., Wheeler 1978, Christy 1978, Zucker 1978, DeCoursey 1981), and laboratory results from the present study imply a similar pattern of release for H. sanguineus. Thus, the co occurrence of H. sanguineus and fiddler crab zoeae in this water mass was likely the result of coincident release events upstream of the sampling site. These events may have occurred in adjacent adult habitats (e.g., marsh and adjacent shoreline rip-rap) at high tide during the night preceding their appearance in the harbor. The continued presence of H. sanguineus and fiddler crab zoeae during the subsequent flood phase is coherent with results of recent hydrodynamic-modeling studies that encompass the sampling site (Petrone et al. 2005). The second night of plankton sampling, approximately 2 wk later, showed a similar pattern of occurrence of fiddler crab larvae, but with only a single peak of Z-I H. sanguineus near the time of nocturnal high tide. This observation supports the idea of nocturnal-high-tide release of zoeae by the adult population of H. sanguineus in the harbor and also supports the hypothesis that fiddler crab larvae are transported to the harbor from release sites upstream in the Broadkill River. But in this case, there appeared to be no detectable larval-release event in the upstream population of H. sanguineus. Reasons for this are not clear, but may have been a simple function of the lack of semilunar periodicity in release of larvae by H. sanguineus (Saigusa & Kawagoye 1997).

H. sanguineus is not unique among intertidal crabs in releasing larvae near the time of nocturnal high tide. This pattern has been reported for a large number of species with wide representation among families (see Morgan & Christy 1996 for review). However, many of these species also show semilunar rhythms in larval release, and the inability of previous investigations (Saigusa & Kawagoye 1997, Epifanio et al. 1998) to demonstrate this rhythm for H. sanguineus is surprising. Nevertheless, the nocturnal-high-tide pattern that was observed in our investigation serves to minimize the impact of visual predators on female H. sanguineus as they release larvae, as well as on the newly released zoeae themselves. This pattern of release also takes advantage of circulation at tidal frequency to transport zoeae away from intertidal habitats where conditions may not be conducive to larval development. For example, H. sanguineus larvae are quite sensitive to the range of salinity and temperature variation typical of intertidal habitat during the spawning season (Epifanio et al. 1998), and rapid transport to adjacent open water may lead to the avoidance of these physiologic stressors. Subsequent transport of larvae in open waters of the main estuary and the adjacent coastal ocean is dependent on circulation at subtidal frequency and draws no special advantage from nocturnal-high-tide release of larvae (Epifanio & Garvine 2001).

Overall, the early life-history characteristics of invasive populations of H. sanguineus along the east coast of North America are typical of those observed for co-occurring intertidal crabs. As shown in this investigation, H. sanguineus releases larvae on nocturnal high tides. As demonstrated in earlier studies, the various larval stages have a typical range of salinity and temperature tolerance (Epifanio et al. 1998). In addition, the larvae exhibit a suite of behaviors that mitigate patterns of larval transport (Park et al. 2004) and the megalopal stage responds to metamorphosis-inducing cues indicative of suitable intertidal habitat (Andrews et al. 2001, Kopin et al. 2001). Many of these same characteristics are found in the common mud crab Panopeus herbstii (e.g., Christy & Stancyk 1982, Costlow & Bookhout 1961, Weber & Epifanio 1996), which is the most obvious competitor of H. sanguineus in intertidal habitat in the southern Middle Atlantic Bight (Kopin et al. 2001). In fact, there are only 2 clear differences in the early life histories of the respective species. The first concerns the behavior of the zoeae, where P. herbstii is more likely to be retained in the estuary (Epifanio 1988, Park et al. 2004). The second involves the duration of the spawning period, which is restricted to midsummer in P. herbstii (Dittel & Epifanio 1982, Jones & Epifanio 1995), but extends from late spring to early fall in H. sanguineus (McDermott 1998b, Epifanio et al. 1998). This latter difference explains the absence of P. herbstii larvae in plankton collections made in June in our investigation and may provide some competitive advantage in that H. sanguineus that settle in juvenile habitat early in the season will have reached a relatively large size by the time P. herbstii begins settling in midsummer. With that exception, the early life history of H. sanguineus, beginning at larval release and ending at settlement, does not provide any special competitive advantages over co-occurring native species. Thus, the remarkable success of H. sanguineus in North American habitats may be attributable to competitive interactions occurring after settlement and metamorphosis.

ACKNOWLEDGMENTS

The authors to thank C. Tallamy, C. Kopin, E. Grey, M. B. Jones, and C. Petrone for their help with field research. This research was funded by the National Science Foundation's Research Experience for Undergraduates program.

LITERATURE CITED

Andrews, W. R., N. M. Targett & C. E. Epifanio. 2001. Isolation and characterization of the metamorphic inducer of the common mud crab, Panopeus herbstii. J. Exp. Mar. Biol. Ecol. 261:121-134.

Batschelet, E. 1981. Circular statistics in biology. London: Academic Press. 371 pp.

Breton, G., M. Faase, P. Noel & P. Vincent. 2002. A new alien crab in Europe: Hemigrapsus sanguineus (Decapoda: Brachyuran: Grapsidae). J. Crust. Biol. 22:184-189.

Christy, J.H. 1978. Adaptive significance of reproductive cycles in the fiddler crab Uca pugilator: a hypothesis. Science 199:453-455.

Christy, J. H. & S. E. Stancyk. 1982. Movement of larvae from North Inlet estuary, S.C. with special reference to crab zoeae. In: V. Kennedy, editor. Estuarine comparisons. New York: Academic Press. pp. 489-502.

Costlow, J.D. & C. G. Bookhout. 1961. The larval stages of Panopeus herbstii Milne-Edwards reared in the laboratory. Journal of the Elisha Mitchell Scientific Society 77:33-42.

DeCoursey, P. J. 1981. Cyclic reproduction of fiddler crabs. Uca: a model for estuarine adaptation. Estuaries 4:263-264.

Dittel, A. I. & C. E. Epifanio. 1982. Seasonal abundance and vertical distribution of crab larvae in Delaware Bay. Estuaries 5:197-202.

Epifanio, C.E. 1988. Transport of invertebrate larvae between estuaries and the continental shelf. Trans. Am. Fish. Soc. Syrup. Ser. 3:104-114.

Epifanio, C.E. & R. W. Garvine. 2001. Larval transport on the Atlantic continental shelf of North America: a review. Est. Coast. Shelf Sci. 52:51-77.

Epifanio, C. E., A. I. Dittel, S. Park, S. Schwalm & A. Fours. 1998. Early life history of Hemigrapsus sanguineus, a non-indigenous crab in the Middle Atlantic Bight (USA). Mar. Ecol. Prog. Ser. 170:231-238.

Forward, R. B., Jr. 1987. Larval release rhythms of decapod crustaceans: an overview. Bull. Mar. Sci 41:165-176.

Gamo, S. & Y. Muraoka. 1977. Preliminary observations on megalopa larvae of brachyuran and porcelain crab-shaped anomuran Crustacea collected from the drifting seaweeds in Suruga Bay. Science Reports of the Yokohama National University  Sec II 24:1-7.

Hwang, S. G. & C. H. Kim. 1995. Zoeal stages and megalopa of Hemigrapsus penicillatus (De Haan, 1835) (Decapoda, Brachyura, Grapsidae) reared in the laboratory. Korean J. System. Zool. 11:389-408.

Ims, R.A. 1990. The ecology and evolution of reproductive synchrony. Trends Ecol. Evol. 5:135-140.

Jones, M. B. & C. E. Epifanio. 1995. Settlement of brachyuran megalopae in Delaware Bay: a time series analysis. Mar. Ecol. Prog. Ser. 125: 67-76.

Jones, M. B. & C.E. Epifanio. 2005. Patches of crab megalopae in the mouth of Delaware Bay: an analysis of spatial scales. J. Shellfish Res. 24:2

Knowlton, N. 1979. Reproductive synchrony, parental investment, and the evolutionary dynamics of sexual selection. Anim. Behav. 27:1022-1033.

Kopin, C. Y., C. E. Epifanio. S. Nelson & M. Stratton. 2001. Effects of chemical cues on metamorphosis of the Asian shore crab Hemigrapsus sangumeus, an invasive species on the Atlantic coast of North America. J. Exp. Mar. Biol. Ecol 265:141-151.

Kovach. W.L. 1994. Oriana for Windows, ver 1.0. Kovach Computing Services, Pentraeth, Wales, UK.

Kurata, H. 1968. Larvae of Decapoda Brachyura of Arasaki. Sagami Bay-II. Bull Tokai Reg. Fish. Res. Lab. 56:161-165.

McDermott. J.J. 1998a. The western Pacific brachyuran (Hemigrapsus sanguineus: Grapsidae), in its new habitat along the Atlantic coast of the United States: geographic distribution and ecology. ICES Mar. Sci. 55:289-298.

McDermott, J. J. 1998b. The western Pacific brachyuran Hemigrapsus sanguineus (Grapsidae) in its new habitat along the Atlantic coast of the United States: reproduction. J. Crust. Biol. 55:289-298.

Morgan, S. G. & J. H. Christy. 1996. Survival of marine larvae under the countervailing selective pressures of photo-damage and predation. Limnol. Oceanogr. 41:498-504.

Natunewicz, C. C. & C. E. Epifanio. 2001. Spatial and temporal scales of zoeal patches in coastal waters. Mar. Ecol. Prog. Ser. 212:217-222.

Park, S., C. E. Epifanio & E. K. Grey. 2004. Behavior of larval Hemigrapsus sanguineus (de Haan) in response to gravity and pressure. J. Mar. Biol. Exp. Ecol. 307:197-206.

Petrone, C. J., L. B. Jancaitis. M. B. Jones. C. C. Natunewicz. C. E. Tilburg & C. E. Epifanio. 2005. Dynamics of larval patches: spatial distribution of fiddler crab larvae in Delaware Bay and adjacent waters. Mar. Ecol. Prog. Ser in press.

Richerson. P.J., T.M. Powell. M.R. Leigh-Abbott & J. A. Coil. 1978. Spatial heterogeneity in closed basins. In: J.H. Steele, editor. Spatial patterns in plankton communities. New York, NY: Plenum Press. pp. 239-276.

Rowe, P. M. & C. E. Epifanio. 1994. Flux and transport of weakfish larvae in Delaware Bay. Mar. Ecol. Prog. Ser. 110:115-120.

Rutberg, A. T. 1987. Adaptive hypotheses of birth synchrony in ruminants: an interspecific test. Am. Nat. 130:692-710.

Saigusa, M. & O. Kawagoye. 1997. Circatidal rhythm of an intertidal crab, Hemigrapsus sanguineus: synchrony with unequal tide height and involvement of a light-response mechanism. Mar. Biol. 129:87-96.

Schubart, C. D. 2003. The East Asian shore crab Hemigrapsus sanguineus (Brachyura: Varunidae) in the Mediterranean Sea: an independent human-mediated introduction. Scientia Marina (Barcelona) 67:195-200.

Shukoliukov, S.A., P.P. Zak, G.R. Kalamkarov, O.O. Kalishevich & M.A. Ostrovskii. 1980. Spectral sensitivity and visual pigments of coastal crab Hemigrapsus sanguineus. Biofizika 25:510-514.

Weber, J. C. & C. E. Epifanio. 1996. Response of mud crab megalopae to cues from adult habitat. Mar. Biol. 126:655-661.

Wheeler, D. 1978. Semi-lunar hatching periodicity in the mud fiddler crab Uca pugnax (Smith). Estuaries 1:268-269.

Zucker, N. 1978. Monthly reproductive cycles in three sympatric hoodbuilding tropical fiddler crabs (genus Uca). Biol. Bull 155:410-424.

* Corresponding author. E-mail: [e-mail]

SUSAN PARK, CHARLES E. EPIFANIO * AND RAYMOND B. IGLAY Graduate College of Marine Studies, University of Delaware, 700 Pilottown Rd., Lewes, Delaware 19958

TABLE 1.
Results of circular statistics analysis of larval release time relative
to the high tide. Zero degrees represents the time of high tide;
29[degrees] = 1 h past high tide; 58[degrees] = 2 h past high tide;
etc.

                                        First Zoea          Full
                       First Flap         Present         Release

n                    57                57              57
Mean vector          332.95[degrees]   3.11[degrees]   30.86[degrees]
Corresponding time
  from high tide     -0:13             +0:02           +0:15
r-vector             0.94              0.92            0.91
Standard error of
  mean               2.72[degrees]     3.10[degrees]   3.36[degrees]
Rayleigh's test of
  uniformity (p)     <0.01             <0.01           <0.01