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Larval release behaviors in the blue crab Callinectes sapidus: role of chemical cues
Journal of Experimental Marine Biology and Ecology 273 (2002) 1 – 14 www.elsevier.com/locate/jembe
Larval release behaviors in the blue crab Callinectes sapidus: role of chemical cues R.A. Tankersley a,*, T.M. Bullock b, R.B. Forward Jr. c, D. Rittschof c b
a Department of Biological Sciences, Florida Institute of Technology, Melbourne, FL 32901, USA Department of Biological Sciences, University of Maryland Baltimore County, Baltimore, MD 21250, USA c Duke University Nicholas School of Earth and Environmental Sciences, 135 Duke Marine Lab Rd., Beaufort, NC 28516, USA
Received 24 August 2001; received in revised form 20 January 2002; accepted 16 March 2002
Abstract Egg hatching by brachyuran crabs is often precisely timed relative to environmental cycles and may be controlled by the female, the developing embryos, or both. The current conceptual model for larval release in subtidal brachyuran crabs is that the exact time of release is controlled by the developing embryos. At the time of hatching, the eggs release pheromones that induce stereotypic larval release behaviors in ovigerous females consisting of rapid abdominal pumping. This behavior breaks open the eggs and results in synchronized hatching. To test this model, we examined the role of pheromone substances released by developing and hatching eggs in initiating this pumping behavior in ovigerous blue crabs Callinectes sapidus. Pumping behavior was used as a bioassay to determine if pumping activity changes with the developmental state of the eggs and to test the response of ovigerous crabs to (1) substances released by hatching eggs (hatch water), (2) substances present in homogenized eggs containing early- and late-stage embryos (homogenized egg water), and (3) substances released by developing eggs containing early- and late-stage embryos (egg conditioned water). Pumping activity associated with egg maintenance increased with embryo development. Pumping activity increased with increasing concentration of hatch water and the threshold concentrations for females possessing early- and late-stage eggs were similar. Water containing homogenized eggs also evoked larval release behaviors and response thresholds were the same for females exposed to early- and late-stage egg treatments. Egg conditioned water prepared from eggs containing late-stage embryos was more potent than water prepared from eggs with earlystage embryos. Collectively, these results support the model that larval release in C. sapidus is controlled by pheromones released from hatching eggs and indicate that (1) the responsiveness of ovigerous C. sapidus to the pheromones is relatively independent of the stage of embryo development, (2) homogenates of both early- and late-stage eggs contain similar pheromone
*
Corresponding author. Tel.: +1-321-674-8195; fax: +1-321-674-7238. E-mail address: [email protected] (R.A. Tankersley).
0022-0981/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 0 9 8 1 ( 0 2 ) 0 0 1 3 5 - 1
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concentrations, (3) pheromones are released during development and at the time of hatching, and (4) the concentration of pheromones released from developing eggs increases as the embryos mature. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Callinectes sapidus; Blue crab; Larval release; Chemical cues; Pheromone; Larval hatching
1. Introduction Larval release in brachyuran crabs frequently corresponds with lunar, diel, or tidal cycles (reviewed by DeCoursey, 1983; Forward, 1987). The timing of larval release is critical to the reproductive success of the female crab since it insures that larvae are released at times and locations which minimize the threat of predation and adverse environmental factors and maximize the chances of larvae reaching appropriate nursery grounds (reviewed by Morgan, 1995a,b). In crabs that respond to one or more of these environmental cycles, release usually occurs near the time of nocturnal high tide and during new or full moon (e.g., Saigusa and Hidaka, 1978; Saigusa, 1981, 1982; Forward et al., 1982; De Vries et al., 1983; Morgan, 1987, 1994; De Vries and Forward, 1989; Morgan and Christy, 1995). For most crabs, larval release is a brief event, lasting only a few minutes. Eggs hatch while the female vigorously pumps her abdomen in a stereotypic larval release behavior in which she rises on her walking legs, probes her egg mass with the tips of her legs, and rapidly flexes her abdomen (Saigusa and Hidaka, 1978; DeCoursey, 1979; Forward et al., 1982; De Vries and Forward, 1991). During embryo development, this fanning behavior helps to ventilate the eggs, whereas during larval release, it acts to synchronize hatching by mechanically stimulating and rupturing the egg membrane (Davis, 1968, 1981). For most brachyuran crabs, larval release is under endogenous control. However, the actual timing of hatching may be controlled by the female, the developing embryos, or both (reviewed by Forward, 1987). Although in the terrestrial crab Sesarma haematocheir the female initiates the hatching process and influences the hatching synchrony of the embryos (Saigusa, 1992, 1993), in the subtidal xanthid crabs Rhithropanopeus harrisii and Neopanope sayi, egg hatching is mediated by chemical communication between the larvae and the female (Forward et al., 1982, 1987; Forward and Lohmann, 1983; Rittschof et al., 1985, 1989; De Vries et al., 1991). Near the time of hatching, pheromones associated with the developing embryos induce ovigerous females to exhibit stereotypic larval release behaviors. In R. harrisii, the pheromones responsible for triggering this pumping response have been examined and consist of a heterogeneous group of small peptides ( < 500 Da) each containing arginine (Rittschof et al., 1985, 1989; Forward et al., 1987; Rittschof, 1993). Based on these results, Forward et al. (1987) and De Vries et al. (1991) developed a conceptual model for the chemical control of hatching time in subtidal brachyuran crabs. According to the model, embryos are incubated by the female for a period of time before hatching. During this period, an unknown interaction between the embryos and the female results in synchronized development of the eggs. At the time of egg hatching, peptide
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pheromones are released from the eggs that stimulate abdominal pumping. As eggs hatch, the concentration of the pheromones increases, thereby stimulating the female to initiate more vigorous pumping. This pumping activity of the female physically disrupts the eggs, resulting in synchronized release of larvae. The goal of the present study was to investigate the generality of the conceptual model for the control of hatching time in subtidal brachyuran crabs. Specifically, we examined whether the eggs of the blue crab Callinectes sapidus contain substances that stimulate ovigerous females to initiate the pumping response associated with larval release. After mating in low salinity regions of estuaries, newly inseminated female blue crabs migrate seaward to the inlets (Churchill, 1921; Millikin and Williams, 1984; Tankersley et al., 1998). Once near the mouth of the estuary, newly fertilized eggs are extruded and attached to the pleopods of the female’s abdomen forming a large egg mass or ‘‘sponge’’. The attached eggs are carried by the female for c 2 weeks and as embryonic development proceeds the color of the egg mass changes sequentially from a yellow/orange color to dark brown/black (Churchill, 1921; Millikin and Williams, 1984). Larvae are released synchronously near the time of morning high tide (Tankersley, unpublished data) and are transported in ebb currents to shelf waters where they undergo development (reviewed by Epifanio, 1995; Epifanio and Garvine, 2001). We used the stereotypic larval release behavior of ovigerous crabs (abdomen pumping) as a biological assay to determine if female blue crabs respond to (1) pheromone substances released by hatching eggs, (2) substances present in egg homogenates containing early- and late-stage embryos, and (3) pheromones released by developing eggs prior to hatching. Our results suggest that the hatching model developed for R. harrisii (Forward and Lohmann, 1983) and N. sayi (De Vries et al., 1991) applies to C. sapidus.
2. Materials and methods 2.1. Collection and maintenance of ovigerous crabs Ovigerous blue crabs, C. sapidus, were collected using dip nets and commercial crab traps during June –August 1998 from the Newport River Estuary near the Duke University Marine Laboratory, Beaufort, NC, USA (34j43VN; 76j40VW). Those collected with dip nets were captured near the surface during ebb tides at night as they migrated toward Beaufort Inlet, NC, using ebb-tidal transport (Tankersley et al., 1998). Crabs were maintained in the laboratory in plastic aquaria (30 18 10 cm) containing c 3 l of seawater (32 – 34 psu; 24– 26 jC) that was filtered to remove particles that were > 5 Am. The water in the aquaria was changed daily and the crabs were fed ribbed mussels (Geukensia demissa) every other day. A photoperiod that approximated the natural light/ dark cycle at the time of collection was employed. Following collection, ovigerous females were classified according to the developmental stage of their egg masses (De Vries et al., 1983) by removing small groups of eggs (100 – 200) from the sponge and examining them under dissecting microscope. For most assays, egg masses were grouped into two categories based upon yolk content and embryo eye
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development. Early-stage eggs were typically yellow/orange in color, contained embryos that lacked eyespots, and were >6 days from hatching (Stages 1 –5 of De Vries et al., 1983). Late-stage eggs were darker in color (brown/black), contained little yolk ( < 25%), possessed embryos with developing eyespots, and were V 6 days from hatching (Stages 6– 9 of De Vries et al., 1983). 2.2. Pumping activity To determine if the abdominal pumping activity of female C. sapidus changed with the developmental state of the eggs, the pumping rate of 124 crabs carrying eggs of different developmental stages was determined by placing each crab in a circular glass aquarium (23 cm diameter 14 cm) containing 2 l of filtered ( < 5 Am) seawater and recording the number of pumps in a 5-min period. All crabs were tested within 3 h of collection and each crab was tested only once. Observations were made under dim red light ( c 650 nm). Since crabs are very insensitive to this wavelength (Forward and Cronin, 1979), this procedure helped to minimize disturbance and potential artifacts caused by the presence of the observer. Crabs were separated into four groups based upon egg stage (i.e., Stages 1– 2, 3 –4, 5 – 6, and 7 –9; De Vries et al., 1983) and the pumping frequencies of crabs were compared using a Kruskal – Wallis test and nonparametric multiple comparisons based on rank sums (Zar, 1996). The proportion of crabs that exhibited some degree pumping activity during the 5-min observation period was also compared using v2-contingency analysis (Zar, 1996). 2.3. Biological assays We used an abdominal-pumping assay similar to the one described by Forward and Lohmann (1983) and De Vries et al. (1991) to determine if pheromones present in developing eggs of female C. sapidus and released at the time of hatching stimulate larval release behaviors. The assay consisted of placing an ovigerous crab in a circular glass aquarium (23 cm diameter 14 cm) containing 2 l of filtered ( < 0.45 Am) seawater (34 psu) and recording the number of abdomen pumps in a 2.5-min period. The crab was then transferred to a second bowl containing 2 l of a test solution and the count was repeated. Crabs were considered to respond ‘‘positively’’ if their pumping rate increased upon exposure to the test solution and were scored as ‘‘unresponsive’’ if pumping activity declined or stayed the same. The percentage of crabs that responded positively served as a relative measure of the biological activity of the test solution. Although preliminary experiments indicated that the responsiveness of female crabs to chemical stimulation was independent of the time of day, this two-bowl protocol controlled for possible variability (i.e., tidal or diel) in pumping behavior during the testing period since it was based on relative rather than absolute levels of activity (Forward et al., 1987; De Vries et al., 1991). For each test solution, 20– 38 ovigerous crabs were tested in a graded series of seven concentrations. The first set of bowls in the series contained filter seawater (i.e., concentration = 0) and served as a control for the potential effects of the assay procedure on crab pumping activity. To minimize the chances of adaptation, testing in the remaining six concentrations proceeded from lowest to highest and at least 30 min elapsed before a
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crab was retested in the next concentration in the series (Forward et al., 1987). Between trials, crabs were moved from test solutions to a container of clean filtered seawater. Preliminary experiments indicated that the responsiveness of the crabs remained consistent using this procedure. Crabs were tested only once in each concentration and test solutions were changed after 10 crabs to minimize changes in water composition. Responses of crabs to test solutions (i.e., % responding) were compared to control solutions (i.e., filtered seawater) using a v2-test for multiple proportions and an associated Dunnett-type multiple comparison test (Zar, 1996). 2.4. Preparation of test solutions 2.4.1. Larval-hatch water To determine if substances released at the time of hatching stimulate larval release behaviors in ovigerous C. sapidus, we exposed crabs with intact egg masses to water in which C. sapidus had released larvae and monitored the abdominal-pumping activity using the assay described above. Larval-hatch water was prepared by placing ovigerous crabs in clean holding tanks containing 2 l of 0.45 Am filtered seawater approximately 2 –3 h before the predicted time of larval release (i.e., morning high tides; Tankersley, unpublished data). Immediately following egg hatching, the female crab was removed from the container. The titer of the pheromones in hatch water was estimated by counting the number of larvae in subsamples of hatch water and the concentration was expressed in larvae ml 1. The water was then filtered through a 100-Am filter to remove the larvae, egg membranes, and any unhatched embryos. Eggs were assumed to contain equal concentrations of the pheromones. Hatch water was then diluted with clean 0.45 Am filtered seawater to produce the appropriate target concentrations. We compared the minimum concentration that induced a pumping response (threshold concentration) in crabs possessing early- (N = 25) and late-stage eggs (N = 30) to determine whether the sensitivity of ovigerous females to chemical cues in hatch water varies with the developmental state of the female’s embryos. 2.4.2. Homogenized-egg water To determine if the substances that stimulate pumping behavior are present in eggs prior to larval release and whether their effectiveness or concentration varies with the developmental stage of the embryos, we measured the pumping response of female crabs with intact egg masses to water containing crushed early- and late-stage eggs. Sponges were carefully removed from the abdomen of an ovigerous female, homogenized, and diluted in 0.45 Am filtered seawater to obtain the appropriate test concentration. The concentration of eggs in the stock solution was estimated by weighing and counting the number of eggs in subsamples removed from the sponge prior to homogenization. Thus, concentrations are expressed in eggs ml 1. As in the hatch-water assays, we compared the sensitivities of females with early- and late-stage eggs to test waters containing homogenized late-stage eggs to determine whether sensitivity changed with embryo development. Since threshold concentrations were similar for the two groups of females (see Results), the responsiveness of ovigerous crabs to homogenized early-stage eggs was only determined for crabs possessing late-stage embryos.
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2.4.3. Egg-conditioned water To determine if pheromones responsible for stimulating larval release behaviors in crabs are released from developing eggs prior to hatching, we examined the pumping response of ovigerous females with late-stage eggs to water conditioned with eggs containing developing embryos. Egg-conditioned water was prepared by gently removing an egg mass from an ovigerous crab and placing it in a flask containing 1.5 l of 0.45 Am filtered seawater. The excised egg mass was incubated under gentle aeration for 18 h before it was removed and the water was checked for the presence of newly hatched zoeae larvae or broken eggs. Egg-conditioned water that contained larvae or broken eggs was discarded since it was assumed to contain substances from broken eggs or pheromones that were released during hatching. Egg counts, based on subsamples of the egg mass, were used to estimate the concentration of chemical substances present in conditioned water. Aliquots of the stock solution were diluted with 0.45 Am filtered seawater to produce the desired test concentrations (eggs ml 1). To determine if the concentration of stimulatory substances leaching from the eggs varied with the developmental state of the embryos, we compared the pumping response of ovigerous crabs possessing late-stage embryos to water condition with either early- or late-stage eggs.
3. Results 3.1. Pumping activity Background pumping activity of ovigerous C. sapidus varied with the developmental stage of the embryos (H = 18.06, df = 3, P < 0.001), yet pumping rates were similar for crabs possessing eggs V Stage 6 and only increased significantly in crabs with late-stage embryos (Stages 7– 9) (Fig. 1A; Nonparametric multiple contrasts). The percentage of crabs that pumped during the 5-min observation period differed significantly with embryo age (v2 = 18.65, df = 3, P < 0.001). Pumping was relatively rare (10%) in females with very young embryos (Stages 1– 2) and increased sharply to c 40 –60% in crabs with embryos z Stage 3 (Fig. 1B). The percentage of crabs that pumped did not differ significantly among females with later-stage embryos (Stages z 3; Fig. 1B; Tukey multiple comparison test). 3.2. Response to test solutions 3.2.1. Larval-hatch water Pumping activity of ovigerous females with both early- and late-stage embryos increased significantly when exposed to increasing concentrations of hatch water (Fig. 2; early-stage females: v2 = 13.15, df = 6, P < 0.05; N = 25; late-stage females: v2 = 26.09, df = 6, P < 0.001; N = 30), indicating that pheromones stimulating abdominal pumping are released from the eggs at the time of hatching. The responsiveness of female crabs did not vary significantly with embryo development. The lowest concentrations that induced responses that were significantly different from the controls (i.e., a threshold concen-
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Fig. 1. Frequency of spontaneous pumping activity in ovigerous C. sapidus (X¯ F S.E.) (A) and the percentage of crabs that displayed some pumping activity during a 5-min observation period (B) as a function of the developmental stage of the eggs. Egg stage was determined using the classification scheme of De Vries et al. (1983). Values with similar letters are not significantly different at P < 0.05. Numbers in parenthesis in panel B represent the sample size (N) for each group.
tration) were 40 and 30 larvae ml 1 for early- and late-stage females, respectively (Fig. 2, Dunnett test, P < 0.05). Although the responsiveness of females with early- and late-stage eggs was similar at 30 larvae ml 1 (Fig. 2), differences in sample sizes between the two groups (N = 25 vs. 30) enabled this value to be statistically different ( P < 0.05) from controls for females with late-stage eggs but not for those with early-stage eggs. Consequently, these differences in thresholds were the result of slight differences in the
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Fig. 2. Mean percentage of ovigerous C. sapidus with egg masses containing early- (Stages 1 – 5; solid line) and late-stage (Stages 6 – 9; dashed line) embryos that responded to different concentrations of hatch water. For each group of crabs, the lowest concentration at which the pumping response was significantly different from the controls is indicated by an asterisk.
statistical power of the analyses and were not considered to be biologically relevant. At concentrations above 30 larvae ml 1, the responsiveness of both groups of crabs plateaued, yet the percentages of crabs with early-stage embryos that responded were consistently lower than those with late-stage embryos. This difference is probably a consequence of differences in the background pumping activity of the two groups of crabs (Fig. 1) rather than an indication that females with mature eggs are more sensitive or responsive to chemicals present in hatch water. 3.2.2. Homogenized-egg water Increased concentrations of homogenized late-stage egg water resulted in a significant increase in the responsiveness of females with both early- (v2 = 19.21, df = 6, P < 0.01; N = 28) and late-stage embryos (v2 = 49.44, df = 6, P < 0.001; N = 38) (Fig. 3). Females with late-stage eggs had a slightly lower response threshold than those with early-stage eggs (20 vs. 30 eggs ml 1; Dunnett test, P < 0.05), yet the overall pattern of the dose – response curves and the maximum percent responding to concentrations z 60 eggs ml 1 were similar. To determine if the concentration of the pheromone varied with the developmental stage of the embryos, we repeated the assay with water containing homogenized earlystage eggs. Since the responsiveness of females to substances in late-stage egg water (Fig. 3) only varied slightly with the developmental stage of the embryos in the egg mass, we only examined the responsiveness of females with late-stage eggs (N = 27). As with late-
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Fig. 3. Percentage of ovigerous C. sapidus with egg masses containing early- (Stages 1 – 5; solid line) and latestage (Stages 6 – 9; dashed line) embryos that responded to water containing homogenized eggs containing latestage embryos. For each group of crabs, the lowest concentration at which the pumping response was significantly different from the controls is indicated by an asterisk.
Fig. 4. Percentage of ovigerous C. sapidus with egg masses containing late-stage (Stages 6 – 9) embryos that responded to water containing homogenized eggs containing early-stage embryos. The lowest concentration at which the pumping response was significantly different from the controls is indicated by an asterisk.
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stage egg water, the percent pumping response increased sharply with increasing concentrations of early-stage egg water (v2 = 18.20, df = 6, P < 0.01; N = 27, Fig. 4) and plateaued between 40% and 50% at concentrations z 20 eggs ml 1. The concentration necessary to elicit a significant response was equal for crabs exposed to early- and late-stage egg water (20 eggs ml 1; Figs. 3 and 4), indicating that the concentration of substances in eggs does not vary significantly with embryo development. 3.2.3. Egg-conditioned water Ovigerous C. sapidus with late embryos responded significantly to water conditioned with both early- (v2 = 16.49, df = 6, P < 0.05; N = 20) and late-stage eggs (v2 = 28.58, df = 6, P < 0.001; N = 20) (Fig. 5), indicating that pheromones are released by developing eggs prior to hatching. For both types of water, pumping response increased significantly with increasing concentration, but the concentration necessary to elicit a significant response was lower for late-stage water (50 eggs ml 1) than for early-stage water (400 eggs ml 1) (Dunnett test, P < 0.05). At concentrations z 400 egg ml 1, the responsiveness of crabs leveled off but at a consistently higher level for crabs exposed to late-stage water (65%) than for crabs placed in water conditioned with early-stage eggs (45 –50%). Since both solutions were tested using late-stage females, differences in the dose – response curves suggest that the concentration of pheromones released from eggs prior to hatching increases as the embryos mature or as eggs age.
Fig. 5. Percentage of ovigerous C. sapidus with egg masses containing late-stage (Stages 6 – 9) embryos that responded to water that had been conditioned for 18 h. with eggs containing early- (Stages 1 – 5; solid line) or late-stage (Stages 6 – 9; dashed line) embryos. For each group of crabs, the lowest concentration at which the pumping response was significantly different from the controls is indicated by an asterisk.
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4. Discussion Substances associated with hatching and developing eggs of C. sapidus induced sterotypic larval release behaviors. Many of the responses of C. sapidus in the present study are consistent with the model for larval release for the subtidal crabs R. harrisii (Forward and Lohmann, 1983) and N. sayi (De Vries et al., 1991). In these species, the developing embryos control the exact time of hatching, while the female controls the synchrony. When the eggs become ready to hatch, several eggs hatch spontaneously, liberating pheromones into the water that stimulate the female to undergo stereotypic larval release behavior consisting of rapid abdomen pumping. This action breaks open additional eggs, releasing more of the pheromone, which induces addition pumping and egg hatching. This positive-feedback system results in the synchronized release of larvae over a short period of time (seconds to minutes). Demonstrating that this model applies to C. sapidus depends upon establishing that the embryos release pheromones at the time of hatching that induce the stereotypic abdominal pumping behavior associated with larval release. Abdomen pumping not only occurs during larval release but is observed throughout embryonic development. The level of spontaneous pumping increased with embryo age in C. sapidus (Fig. 1A), which is consistent with results for the crab N. sayi (De Vries et al., 1991), but contrary to those for R. harrisii (Forward and Lohmann, 1983). The increase in pumping activity may serve to enhance the synchronized development of the eggs, especially as hatching approaches. Alternatively, De Vries et al. (1991) provided a physiological explanation for this behavior, suggesting that crabs also pump their abdomens to increase water transport around the eggs, thus supplying oxygen to or removing waste from the developing embryos. Sponges of C. sapidus contain up to 2 million eggs (Millikin and Williams, 1984) and are considerably larger than those of N. sayi (2000 – 4000 eggs; Forward, unpublished data) and R. harrisii (1000 eggs; Forward, unpublished data). Thus, the observed relationship between embryonic development and spontaneous pumping behavior may be a consequence of the combined effects of the slow diffusion rate of water through the egg mass and the greater oxygen demand and waste production of more developed embryos (De Vries et al., 1991). If the embryos release pheromones that induce the female to undergo larval release behavior, then the water in which eggs hatch should contain these pheromones. Clearly, increasing concentrations of hatch water induced increased abdomen pumping by C. sapidus (Fig. 2). The lowest concentration to induce a significant increase in pumping was similar for blue crabs with young and mature embryos, but the level of responsiveness was consistently lower for females with early-stage eggs. Studies of R. harrisii (Forward and Lohmann, 1983) and N. sayi (De Vries et al., 1991) also found that the pumping response increased as hatch water concentration increased and that crabs with young embryos were less responsive than those with mature embryos. Since abdomen pumping is not evoked in non-ovigerous crabs (e.g., Forward and Lohmann, 1983), female blue crabs probably become sensitive to the pheromone at the time of egg deposition. Homogenized early- and late-stage embryos were equally effective at inducing the pumping response (Figs. 3 and 4), which indicates that the concentration of pheromones in the eggs is constant throughout embryonic development. This result conflicts with that for
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R. harrisii (Forward and Lohmann, 1983), which had a greater pumping response upon exposure to homogenized late-stage egg water than to early-stage egg water. Forward and Lohmann (1983) concluded that the concentration of the active chemical in the eggs increased as the embryos matured, suggesting that the pheromone may be produced and stored by the embryo during development. Although the concentration of pheromones present in C. sapidus eggs did not change with egg stage, the amount leaching from the eggs varied significantly with embryo development (Fig. 5). Eggs with both early- and late-stage embryos released the pheromones but release increased significantly as the embryos matured, which suggests that the egg membrane becomes progressively more permeable to the substances as development progresses. This result conflicts with the study of N. sayi. De Vries et al. (1991) demonstrated that the pheromones involved in stimulating pumping are not released from developing eggs because substances associate with ovigerous N. sayi females did not produce a significant pumping response in the absence of hatching. Yet, embryos of several brachyuran crabs, including N. sayi, Uca pugilator, S. cinereum, and S. haematocheir, are known to release proteolytic enzymes near the time of hatching which aid larval release by digesting the layers of the egg case (De Vries and Forward, 1991; Saigusa, 1996). Similar enzymes secreted during development may alter the permeability of the egg membranes to pheromones responsible for inducing abdomen pumping. Collectively, the results of the present study support the model for subtidal brachyuran crabs that pheromones from hatching eggs induce larval release behavior in ovigerous females that results in the synchronized release of larvae. However, the chemical identity of the pheromones is unknown for C. sapidus. Since De Vries et al. (1991) demonstrated that hatching water from different species induced the pumping response in N. sayi, the identity of the chemical cue may be similar among subtidal crabs species. The most extensive chemical identification studies have been done with R. harrisii. The initial study found that the pheromones were a group of small peptides with arginine composing approximately 50% of the amino acids (Rittschof et al., 1985). Further studies demonstrated that they were composed of short peptides with a neutral amino acid at the amino-terminus and arginine at the carboxyl-terminus (Forward et al., 1987; Rittschof et al., 1989). Rittschof et al. (1990) proposed that the peptide pheromones are generated by proteolytic digestion of the egg membrane by enzymes released by the embryos. Ovigerous blue crabs aggregate in high salinity areas near the mouths of estuaries. In preparation for larval release, crabs with mature embryos undergo a spawning migration to coastal areas where the larvae are released and then transported offshore for development. During the spawning migration, ovigerous crabs undergo ebb-tide transport, in which they swim in the water column during ebb tide at night and are probably on the bottom at other times (Tankersley et al., 1998). The cues that initiate ebb-tide transport and the underlying behavior are unknown. Since pheromones from the eggs induce female crabs to move into position for larval release and undergo vigorous abdominal pumping that breaks open the eggs (Forward and Lohmann, 1983; De Vries et al., 1991), it is attractive to speculate that the same or similar substances released from the eggs, specifically those containing latestage embryos, may initiate the spawning migration of blue crabs.
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Acknowledgements This material is based in part on research supported by the National Science Foundation Grants No. OCE-9819355/9901146/0096205 and No. OCE-0095092/0094930. We thank M.A. Sigala and M.G. Wieber for their help with collecting crabs. [SS]
References Churchill, E.P., 1921. Life history of the blue crab. Bull. U. S. Bur. Fish. 1917 – 1918 36, 95 – 128. Davis, C.C., 1968. Mechanisms of hatching in aquatic invertebrate eggs. Oceanogr. Mar. Biol. Annu. Rev. 6, 325 – 376. Davis, C.C., 1981. Mechanisms of hatching in aquatic invertebrate eggs: II. Oceanogr. Mar. Biol. Annu. Rev. 19, 95 – 123. DeCoursey, P., 1979. Egg hatching rhythms in three species of fiddler crabs. In: Naylor, E., Hartnoll, R.G. (Eds.), Cyclic Phenomena in Marine Plants and Animals. Proceedings of the 13th European Marine Biological Symposium. Pergamon, Oxford, pp. 399 – 406. DeCoursey, P., 1983. Biological timing. In: Bliss, D.E., Vernberg, F.J., Vernberg, W.B. (Eds.), The Biology of the Crustacea. Behavior and Ecology, vol. 7. Academic Press, New York, pp. 107 – 162. De Vries, M.C., Forward Jr., R.B., 1989. Rhythms in larval release of the sublittoral crab Neopanope sayi and the supralittoral crab Sesarma cinereum. Mar. Biol. 100, 241 – 248. De Vries, M.C., Forward Jr., R.B., 1991. Mechanisms of crustacean egg hatching: evidence for enzyme release by crab embryos. Mar. Biol. 110, 281 – 291. De Vries, M.C., Epifanio, C.E., Dittel, A.I., 1983. Lunar rhythms in the egg hatching of the subtidal crustacean: Callinectes arcuatus Ordway (Decapoda: Brachyura). Estuar. Coast. Shelf Sci. 17, 717 – 724. De Vries, M.C., Rittschof, D., Forward Jr., R.B. 1991. Chemical mediation of larval release behaviors in the crab Neopanope sayi. Biol. Bull. 180, 1 – 11. Epifanio, C.E., 1995. Transport of blue crab (Callinectes sapidus) larvae in the waters off the mid-Atlantic states. Bull. Mar. Sci. 57, 713 – 725. Epifanio, C.E., Garvine, R.W., 2001. Larval transport on the Atlantic Continental Shelf of North America: a review. Estuar. Coast. Shelf Sci. 52, 51 – 77. Forward Jr., R.B., 1987. Larval release rhythms of decapod crustaceans: an overview. Bull. Mar. Sci. 41, 165 – 176. Forward Jr., R.B., Cronin, T.W., 1979. Spectral sensitivity of larvae from intertidal crustaceans. J. Comp. Physiol. 133, 311 – 315. Forward Jr., R.B., Lohmann, K.J. 1983. Control of egg hatching in the crab Rhithropanopeus harrisii (Gould). Biol. Bull. 165, 154 – 166. Forward Jr., R.B., Lohmann, K., Cronin, T.W., 1982. Rhythms in larval release by an estuarine crab (Rhithroopanopeus harrisii). Biol. Bull. 163, 287 – 300. Forward Jr., R.B., Rittschof, D., De Vries, M.C., 1987. Peptide pheromones synchronize crustacean egg hatching and larval release. Chem. Senses 12, 491 – 498. Millikin, M.R., Williams, A.B., 1984. Synopsis of biological data on the blue crab, Callinectes sapidus Rathbun. FAO Fisheries Synopsis 138: NOAA Tech. Rep. NMFS 1., 43 pp. Morgan, S.G., 1987. Adaptive significance of hatching rhythms and dispersal patterns of estuarine crab larvae: avoidance of physiological stress of larval export? J. Exp. Mar. Biol. Ecol. 113, 71 – 78. Morgan, S.G., 1994. Influence of tidal regime on the timing of larval release: implications for recruitment success. In: Dyer, K.R., Orth, R.J. (Eds.), Changes in Fluxes in Estuaries: Implications from Science to Management. Olsen & Olsen, Denmark, pp. 323 – 328. Morgan, S.G., 1995a. The timing of larval release. In: McEdward, L. (Ed.), Ecology of Marine Invertebrate Larvae. CRC Press, Boca Raton, FL, pp. 157 – 192. Morgan, S.G., 1995b. Life and death in the plankton: larval mortality and adaptation. In: McEdward, L. (Ed.), Ecology of Marine Invertebrate Larvae. CRC Press, Boca Raton, FL, pp. 279 – 321.
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Morgan, S.G., Christy, J.H., 1995. Adaptive significance of the timing of larval release by crabs. Am. Nat. 145, 457 – 479. Rittschof, D., 1993. Body odors and neutral-basic peptide mimics: a review of responses by marine organisms. Am. Zool. 33, 487 – 493. Rittschof, D., Forward Jr., R.B., Mott, D.D., 1985. Larval release in the crab Rhithropanopeus harrisii (Gould): chemical cues from hatching eggs. Chem. Senses 10, 567 – 577. Rittschof, D., Forward Jr., R.B., Simons, D.A., Reddy, P.A., Erickson, B.W., 1989. Peptide analogs of the mud crab pumping pheromone. Chem. Senses 14, 137 – 148. Rittschof, R., Forward Jr., R.B., Erickson, B.W., 1990. Larval release in brachyuran crustaceans: functional similarity of the peptide receptor and the catalytic site of trypsin. J. Chem. Ecol. 16, 1359 – 1370. Saigusa, M., 1981. Adaptive significance of a semilunar rhythm in the terrestrial crab Sesarma. Biol. Bull. 160, 311 – 321. Saigusa, M., 1982. Larval release rhythms coinciding with solar day and tidal cycles in the terrestrial crab Sesarma—harmony with semilunar timing and its adaptive significance. Biol. Bull. 162, 371 – 386. Saigusa, M., 1992. Control of hatching in an estuarine terrestrial crab: I. Hatching of embryos detached from the female and emergence of mature larvae. Biol. Bull. 183, 401 – 408. Saigusa, M., 1993. Control of hatching in an estuarine terrestrial crab: 2. Exchange of a cluster of embryos between 2 females. Biol. Bull. 184, 186 – 202. Saigusa, M., 1996. Two kinds of active factor in crab hatch water: ovigerous-hair stripping substance (OHSS) and a proteinase. Biol. Bull. 191, 234 – 240. Saigusa, M., Hidaka, T., 1978. Semilunar rhythm in a zoea-release activity of the land crab Sesarma. Oecologia (Berl.) 37, 163 – 176. Tankersley, R.A., Wieber, M.G., Sigala, M.A., Kachurak, K.A., 1998. Migratory behavior of ovigerous blue crabs Callinectes sapidus: evidence for selective tidal-stream transport. Biol. Bull. 195, 168 – 173. Zar, J.H., 1996. Biostatistical Analysis, 3rd edn. Prentice-Hall, New Jersey.
Larval release behaviors in the blue crab Callinectes sapidus: role of chemical cues R.A. Tankersley a,*, T.M. Bullock b, R.B. Forward Jr. c, D. Rittschof c b
a Department of Biological Sciences, Florida Institute of Technology, Melbourne, FL 32901, USA Department of Biological Sciences, University of Maryland Baltimore County, Baltimore, MD 21250, USA c Duke University Nicholas School of Earth and Environmental Sciences, 135 Duke Marine Lab Rd., Beaufort, NC 28516, USA
Received 24 August 2001; received in revised form 20 January 2002; accepted 16 March 2002
Abstract Egg hatching by brachyuran crabs is often precisely timed relative to environmental cycles and may be controlled by the female, the developing embryos, or both. The current conceptual model for larval release in subtidal brachyuran crabs is that the exact time of release is controlled by the developing embryos. At the time of hatching, the eggs release pheromones that induce stereotypic larval release behaviors in ovigerous females consisting of rapid abdominal pumping. This behavior breaks open the eggs and results in synchronized hatching. To test this model, we examined the role of pheromone substances released by developing and hatching eggs in initiating this pumping behavior in ovigerous blue crabs Callinectes sapidus. Pumping behavior was used as a bioassay to determine if pumping activity changes with the developmental state of the eggs and to test the response of ovigerous crabs to (1) substances released by hatching eggs (hatch water), (2) substances present in homogenized eggs containing early- and late-stage embryos (homogenized egg water), and (3) substances released by developing eggs containing early- and late-stage embryos (egg conditioned water). Pumping activity associated with egg maintenance increased with embryo development. Pumping activity increased with increasing concentration of hatch water and the threshold concentrations for females possessing early- and late-stage eggs were similar. Water containing homogenized eggs also evoked larval release behaviors and response thresholds were the same for females exposed to early- and late-stage egg treatments. Egg conditioned water prepared from eggs containing late-stage embryos was more potent than water prepared from eggs with earlystage embryos. Collectively, these results support the model that larval release in C. sapidus is controlled by pheromones released from hatching eggs and indicate that (1) the responsiveness of ovigerous C. sapidus to the pheromones is relatively independent of the stage of embryo development, (2) homogenates of both early- and late-stage eggs contain similar pheromone
*
Corresponding author. Tel.: +1-321-674-8195; fax: +1-321-674-7238. E-mail address: [email protected] (R.A. Tankersley).
0022-0981/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 0 9 8 1 ( 0 2 ) 0 0 1 3 5 - 1
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concentrations, (3) pheromones are released during development and at the time of hatching, and (4) the concentration of pheromones released from developing eggs increases as the embryos mature. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Callinectes sapidus; Blue crab; Larval release; Chemical cues; Pheromone; Larval hatching
1. Introduction Larval release in brachyuran crabs frequently corresponds with lunar, diel, or tidal cycles (reviewed by DeCoursey, 1983; Forward, 1987). The timing of larval release is critical to the reproductive success of the female crab since it insures that larvae are released at times and locations which minimize the threat of predation and adverse environmental factors and maximize the chances of larvae reaching appropriate nursery grounds (reviewed by Morgan, 1995a,b). In crabs that respond to one or more of these environmental cycles, release usually occurs near the time of nocturnal high tide and during new or full moon (e.g., Saigusa and Hidaka, 1978; Saigusa, 1981, 1982; Forward et al., 1982; De Vries et al., 1983; Morgan, 1987, 1994; De Vries and Forward, 1989; Morgan and Christy, 1995). For most crabs, larval release is a brief event, lasting only a few minutes. Eggs hatch while the female vigorously pumps her abdomen in a stereotypic larval release behavior in which she rises on her walking legs, probes her egg mass with the tips of her legs, and rapidly flexes her abdomen (Saigusa and Hidaka, 1978; DeCoursey, 1979; Forward et al., 1982; De Vries and Forward, 1991). During embryo development, this fanning behavior helps to ventilate the eggs, whereas during larval release, it acts to synchronize hatching by mechanically stimulating and rupturing the egg membrane (Davis, 1968, 1981). For most brachyuran crabs, larval release is under endogenous control. However, the actual timing of hatching may be controlled by the female, the developing embryos, or both (reviewed by Forward, 1987). Although in the terrestrial crab Sesarma haematocheir the female initiates the hatching process and influences the hatching synchrony of the embryos (Saigusa, 1992, 1993), in the subtidal xanthid crabs Rhithropanopeus harrisii and Neopanope sayi, egg hatching is mediated by chemical communication between the larvae and the female (Forward et al., 1982, 1987; Forward and Lohmann, 1983; Rittschof et al., 1985, 1989; De Vries et al., 1991). Near the time of hatching, pheromones associated with the developing embryos induce ovigerous females to exhibit stereotypic larval release behaviors. In R. harrisii, the pheromones responsible for triggering this pumping response have been examined and consist of a heterogeneous group of small peptides ( < 500 Da) each containing arginine (Rittschof et al., 1985, 1989; Forward et al., 1987; Rittschof, 1993). Based on these results, Forward et al. (1987) and De Vries et al. (1991) developed a conceptual model for the chemical control of hatching time in subtidal brachyuran crabs. According to the model, embryos are incubated by the female for a period of time before hatching. During this period, an unknown interaction between the embryos and the female results in synchronized development of the eggs. At the time of egg hatching, peptide
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pheromones are released from the eggs that stimulate abdominal pumping. As eggs hatch, the concentration of the pheromones increases, thereby stimulating the female to initiate more vigorous pumping. This pumping activity of the female physically disrupts the eggs, resulting in synchronized release of larvae. The goal of the present study was to investigate the generality of the conceptual model for the control of hatching time in subtidal brachyuran crabs. Specifically, we examined whether the eggs of the blue crab Callinectes sapidus contain substances that stimulate ovigerous females to initiate the pumping response associated with larval release. After mating in low salinity regions of estuaries, newly inseminated female blue crabs migrate seaward to the inlets (Churchill, 1921; Millikin and Williams, 1984; Tankersley et al., 1998). Once near the mouth of the estuary, newly fertilized eggs are extruded and attached to the pleopods of the female’s abdomen forming a large egg mass or ‘‘sponge’’. The attached eggs are carried by the female for c 2 weeks and as embryonic development proceeds the color of the egg mass changes sequentially from a yellow/orange color to dark brown/black (Churchill, 1921; Millikin and Williams, 1984). Larvae are released synchronously near the time of morning high tide (Tankersley, unpublished data) and are transported in ebb currents to shelf waters where they undergo development (reviewed by Epifanio, 1995; Epifanio and Garvine, 2001). We used the stereotypic larval release behavior of ovigerous crabs (abdomen pumping) as a biological assay to determine if female blue crabs respond to (1) pheromone substances released by hatching eggs, (2) substances present in egg homogenates containing early- and late-stage embryos, and (3) pheromones released by developing eggs prior to hatching. Our results suggest that the hatching model developed for R. harrisii (Forward and Lohmann, 1983) and N. sayi (De Vries et al., 1991) applies to C. sapidus.
2. Materials and methods 2.1. Collection and maintenance of ovigerous crabs Ovigerous blue crabs, C. sapidus, were collected using dip nets and commercial crab traps during June –August 1998 from the Newport River Estuary near the Duke University Marine Laboratory, Beaufort, NC, USA (34j43VN; 76j40VW). Those collected with dip nets were captured near the surface during ebb tides at night as they migrated toward Beaufort Inlet, NC, using ebb-tidal transport (Tankersley et al., 1998). Crabs were maintained in the laboratory in plastic aquaria (30 18 10 cm) containing c 3 l of seawater (32 – 34 psu; 24– 26 jC) that was filtered to remove particles that were > 5 Am. The water in the aquaria was changed daily and the crabs were fed ribbed mussels (Geukensia demissa) every other day. A photoperiod that approximated the natural light/ dark cycle at the time of collection was employed. Following collection, ovigerous females were classified according to the developmental stage of their egg masses (De Vries et al., 1983) by removing small groups of eggs (100 – 200) from the sponge and examining them under dissecting microscope. For most assays, egg masses were grouped into two categories based upon yolk content and embryo eye
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development. Early-stage eggs were typically yellow/orange in color, contained embryos that lacked eyespots, and were >6 days from hatching (Stages 1 –5 of De Vries et al., 1983). Late-stage eggs were darker in color (brown/black), contained little yolk ( < 25%), possessed embryos with developing eyespots, and were V 6 days from hatching (Stages 6– 9 of De Vries et al., 1983). 2.2. Pumping activity To determine if the abdominal pumping activity of female C. sapidus changed with the developmental state of the eggs, the pumping rate of 124 crabs carrying eggs of different developmental stages was determined by placing each crab in a circular glass aquarium (23 cm diameter 14 cm) containing 2 l of filtered ( < 5 Am) seawater and recording the number of pumps in a 5-min period. All crabs were tested within 3 h of collection and each crab was tested only once. Observations were made under dim red light ( c 650 nm). Since crabs are very insensitive to this wavelength (Forward and Cronin, 1979), this procedure helped to minimize disturbance and potential artifacts caused by the presence of the observer. Crabs were separated into four groups based upon egg stage (i.e., Stages 1– 2, 3 –4, 5 – 6, and 7 –9; De Vries et al., 1983) and the pumping frequencies of crabs were compared using a Kruskal – Wallis test and nonparametric multiple comparisons based on rank sums (Zar, 1996). The proportion of crabs that exhibited some degree pumping activity during the 5-min observation period was also compared using v2-contingency analysis (Zar, 1996). 2.3. Biological assays We used an abdominal-pumping assay similar to the one described by Forward and Lohmann (1983) and De Vries et al. (1991) to determine if pheromones present in developing eggs of female C. sapidus and released at the time of hatching stimulate larval release behaviors. The assay consisted of placing an ovigerous crab in a circular glass aquarium (23 cm diameter 14 cm) containing 2 l of filtered ( < 0.45 Am) seawater (34 psu) and recording the number of abdomen pumps in a 2.5-min period. The crab was then transferred to a second bowl containing 2 l of a test solution and the count was repeated. Crabs were considered to respond ‘‘positively’’ if their pumping rate increased upon exposure to the test solution and were scored as ‘‘unresponsive’’ if pumping activity declined or stayed the same. The percentage of crabs that responded positively served as a relative measure of the biological activity of the test solution. Although preliminary experiments indicated that the responsiveness of female crabs to chemical stimulation was independent of the time of day, this two-bowl protocol controlled for possible variability (i.e., tidal or diel) in pumping behavior during the testing period since it was based on relative rather than absolute levels of activity (Forward et al., 1987; De Vries et al., 1991). For each test solution, 20– 38 ovigerous crabs were tested in a graded series of seven concentrations. The first set of bowls in the series contained filter seawater (i.e., concentration = 0) and served as a control for the potential effects of the assay procedure on crab pumping activity. To minimize the chances of adaptation, testing in the remaining six concentrations proceeded from lowest to highest and at least 30 min elapsed before a
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crab was retested in the next concentration in the series (Forward et al., 1987). Between trials, crabs were moved from test solutions to a container of clean filtered seawater. Preliminary experiments indicated that the responsiveness of the crabs remained consistent using this procedure. Crabs were tested only once in each concentration and test solutions were changed after 10 crabs to minimize changes in water composition. Responses of crabs to test solutions (i.e., % responding) were compared to control solutions (i.e., filtered seawater) using a v2-test for multiple proportions and an associated Dunnett-type multiple comparison test (Zar, 1996). 2.4. Preparation of test solutions 2.4.1. Larval-hatch water To determine if substances released at the time of hatching stimulate larval release behaviors in ovigerous C. sapidus, we exposed crabs with intact egg masses to water in which C. sapidus had released larvae and monitored the abdominal-pumping activity using the assay described above. Larval-hatch water was prepared by placing ovigerous crabs in clean holding tanks containing 2 l of 0.45 Am filtered seawater approximately 2 –3 h before the predicted time of larval release (i.e., morning high tides; Tankersley, unpublished data). Immediately following egg hatching, the female crab was removed from the container. The titer of the pheromones in hatch water was estimated by counting the number of larvae in subsamples of hatch water and the concentration was expressed in larvae ml 1. The water was then filtered through a 100-Am filter to remove the larvae, egg membranes, and any unhatched embryos. Eggs were assumed to contain equal concentrations of the pheromones. Hatch water was then diluted with clean 0.45 Am filtered seawater to produce the appropriate target concentrations. We compared the minimum concentration that induced a pumping response (threshold concentration) in crabs possessing early- (N = 25) and late-stage eggs (N = 30) to determine whether the sensitivity of ovigerous females to chemical cues in hatch water varies with the developmental state of the female’s embryos. 2.4.2. Homogenized-egg water To determine if the substances that stimulate pumping behavior are present in eggs prior to larval release and whether their effectiveness or concentration varies with the developmental stage of the embryos, we measured the pumping response of female crabs with intact egg masses to water containing crushed early- and late-stage eggs. Sponges were carefully removed from the abdomen of an ovigerous female, homogenized, and diluted in 0.45 Am filtered seawater to obtain the appropriate test concentration. The concentration of eggs in the stock solution was estimated by weighing and counting the number of eggs in subsamples removed from the sponge prior to homogenization. Thus, concentrations are expressed in eggs ml 1. As in the hatch-water assays, we compared the sensitivities of females with early- and late-stage eggs to test waters containing homogenized late-stage eggs to determine whether sensitivity changed with embryo development. Since threshold concentrations were similar for the two groups of females (see Results), the responsiveness of ovigerous crabs to homogenized early-stage eggs was only determined for crabs possessing late-stage embryos.
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2.4.3. Egg-conditioned water To determine if pheromones responsible for stimulating larval release behaviors in crabs are released from developing eggs prior to hatching, we examined the pumping response of ovigerous females with late-stage eggs to water conditioned with eggs containing developing embryos. Egg-conditioned water was prepared by gently removing an egg mass from an ovigerous crab and placing it in a flask containing 1.5 l of 0.45 Am filtered seawater. The excised egg mass was incubated under gentle aeration for 18 h before it was removed and the water was checked for the presence of newly hatched zoeae larvae or broken eggs. Egg-conditioned water that contained larvae or broken eggs was discarded since it was assumed to contain substances from broken eggs or pheromones that were released during hatching. Egg counts, based on subsamples of the egg mass, were used to estimate the concentration of chemical substances present in conditioned water. Aliquots of the stock solution were diluted with 0.45 Am filtered seawater to produce the desired test concentrations (eggs ml 1). To determine if the concentration of stimulatory substances leaching from the eggs varied with the developmental state of the embryos, we compared the pumping response of ovigerous crabs possessing late-stage embryos to water condition with either early- or late-stage eggs.
3. Results 3.1. Pumping activity Background pumping activity of ovigerous C. sapidus varied with the developmental stage of the embryos (H = 18.06, df = 3, P < 0.001), yet pumping rates were similar for crabs possessing eggs V Stage 6 and only increased significantly in crabs with late-stage embryos (Stages 7– 9) (Fig. 1A; Nonparametric multiple contrasts). The percentage of crabs that pumped during the 5-min observation period differed significantly with embryo age (v2 = 18.65, df = 3, P < 0.001). Pumping was relatively rare (10%) in females with very young embryos (Stages 1– 2) and increased sharply to c 40 –60% in crabs with embryos z Stage 3 (Fig. 1B). The percentage of crabs that pumped did not differ significantly among females with later-stage embryos (Stages z 3; Fig. 1B; Tukey multiple comparison test). 3.2. Response to test solutions 3.2.1. Larval-hatch water Pumping activity of ovigerous females with both early- and late-stage embryos increased significantly when exposed to increasing concentrations of hatch water (Fig. 2; early-stage females: v2 = 13.15, df = 6, P < 0.05; N = 25; late-stage females: v2 = 26.09, df = 6, P < 0.001; N = 30), indicating that pheromones stimulating abdominal pumping are released from the eggs at the time of hatching. The responsiveness of female crabs did not vary significantly with embryo development. The lowest concentrations that induced responses that were significantly different from the controls (i.e., a threshold concen-
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Fig. 1. Frequency of spontaneous pumping activity in ovigerous C. sapidus (X¯ F S.E.) (A) and the percentage of crabs that displayed some pumping activity during a 5-min observation period (B) as a function of the developmental stage of the eggs. Egg stage was determined using the classification scheme of De Vries et al. (1983). Values with similar letters are not significantly different at P < 0.05. Numbers in parenthesis in panel B represent the sample size (N) for each group.
tration) were 40 and 30 larvae ml 1 for early- and late-stage females, respectively (Fig. 2, Dunnett test, P < 0.05). Although the responsiveness of females with early- and late-stage eggs was similar at 30 larvae ml 1 (Fig. 2), differences in sample sizes between the two groups (N = 25 vs. 30) enabled this value to be statistically different ( P < 0.05) from controls for females with late-stage eggs but not for those with early-stage eggs. Consequently, these differences in thresholds were the result of slight differences in the
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Fig. 2. Mean percentage of ovigerous C. sapidus with egg masses containing early- (Stages 1 – 5; solid line) and late-stage (Stages 6 – 9; dashed line) embryos that responded to different concentrations of hatch water. For each group of crabs, the lowest concentration at which the pumping response was significantly different from the controls is indicated by an asterisk.
statistical power of the analyses and were not considered to be biologically relevant. At concentrations above 30 larvae ml 1, the responsiveness of both groups of crabs plateaued, yet the percentages of crabs with early-stage embryos that responded were consistently lower than those with late-stage embryos. This difference is probably a consequence of differences in the background pumping activity of the two groups of crabs (Fig. 1) rather than an indication that females with mature eggs are more sensitive or responsive to chemicals present in hatch water. 3.2.2. Homogenized-egg water Increased concentrations of homogenized late-stage egg water resulted in a significant increase in the responsiveness of females with both early- (v2 = 19.21, df = 6, P < 0.01; N = 28) and late-stage embryos (v2 = 49.44, df = 6, P < 0.001; N = 38) (Fig. 3). Females with late-stage eggs had a slightly lower response threshold than those with early-stage eggs (20 vs. 30 eggs ml 1; Dunnett test, P < 0.05), yet the overall pattern of the dose – response curves and the maximum percent responding to concentrations z 60 eggs ml 1 were similar. To determine if the concentration of the pheromone varied with the developmental stage of the embryos, we repeated the assay with water containing homogenized earlystage eggs. Since the responsiveness of females to substances in late-stage egg water (Fig. 3) only varied slightly with the developmental stage of the embryos in the egg mass, we only examined the responsiveness of females with late-stage eggs (N = 27). As with late-
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Fig. 3. Percentage of ovigerous C. sapidus with egg masses containing early- (Stages 1 – 5; solid line) and latestage (Stages 6 – 9; dashed line) embryos that responded to water containing homogenized eggs containing latestage embryos. For each group of crabs, the lowest concentration at which the pumping response was significantly different from the controls is indicated by an asterisk.
Fig. 4. Percentage of ovigerous C. sapidus with egg masses containing late-stage (Stages 6 – 9) embryos that responded to water containing homogenized eggs containing early-stage embryos. The lowest concentration at which the pumping response was significantly different from the controls is indicated by an asterisk.
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stage egg water, the percent pumping response increased sharply with increasing concentrations of early-stage egg water (v2 = 18.20, df = 6, P < 0.01; N = 27, Fig. 4) and plateaued between 40% and 50% at concentrations z 20 eggs ml 1. The concentration necessary to elicit a significant response was equal for crabs exposed to early- and late-stage egg water (20 eggs ml 1; Figs. 3 and 4), indicating that the concentration of substances in eggs does not vary significantly with embryo development. 3.2.3. Egg-conditioned water Ovigerous C. sapidus with late embryos responded significantly to water conditioned with both early- (v2 = 16.49, df = 6, P < 0.05; N = 20) and late-stage eggs (v2 = 28.58, df = 6, P < 0.001; N = 20) (Fig. 5), indicating that pheromones are released by developing eggs prior to hatching. For both types of water, pumping response increased significantly with increasing concentration, but the concentration necessary to elicit a significant response was lower for late-stage water (50 eggs ml 1) than for early-stage water (400 eggs ml 1) (Dunnett test, P < 0.05). At concentrations z 400 egg ml 1, the responsiveness of crabs leveled off but at a consistently higher level for crabs exposed to late-stage water (65%) than for crabs placed in water conditioned with early-stage eggs (45 –50%). Since both solutions were tested using late-stage females, differences in the dose – response curves suggest that the concentration of pheromones released from eggs prior to hatching increases as the embryos mature or as eggs age.
Fig. 5. Percentage of ovigerous C. sapidus with egg masses containing late-stage (Stages 6 – 9) embryos that responded to water that had been conditioned for 18 h. with eggs containing early- (Stages 1 – 5; solid line) or late-stage (Stages 6 – 9; dashed line) embryos. For each group of crabs, the lowest concentration at which the pumping response was significantly different from the controls is indicated by an asterisk.
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4. Discussion Substances associated with hatching and developing eggs of C. sapidus induced sterotypic larval release behaviors. Many of the responses of C. sapidus in the present study are consistent with the model for larval release for the subtidal crabs R. harrisii (Forward and Lohmann, 1983) and N. sayi (De Vries et al., 1991). In these species, the developing embryos control the exact time of hatching, while the female controls the synchrony. When the eggs become ready to hatch, several eggs hatch spontaneously, liberating pheromones into the water that stimulate the female to undergo stereotypic larval release behavior consisting of rapid abdomen pumping. This action breaks open additional eggs, releasing more of the pheromone, which induces addition pumping and egg hatching. This positive-feedback system results in the synchronized release of larvae over a short period of time (seconds to minutes). Demonstrating that this model applies to C. sapidus depends upon establishing that the embryos release pheromones at the time of hatching that induce the stereotypic abdominal pumping behavior associated with larval release. Abdomen pumping not only occurs during larval release but is observed throughout embryonic development. The level of spontaneous pumping increased with embryo age in C. sapidus (Fig. 1A), which is consistent with results for the crab N. sayi (De Vries et al., 1991), but contrary to those for R. harrisii (Forward and Lohmann, 1983). The increase in pumping activity may serve to enhance the synchronized development of the eggs, especially as hatching approaches. Alternatively, De Vries et al. (1991) provided a physiological explanation for this behavior, suggesting that crabs also pump their abdomens to increase water transport around the eggs, thus supplying oxygen to or removing waste from the developing embryos. Sponges of C. sapidus contain up to 2 million eggs (Millikin and Williams, 1984) and are considerably larger than those of N. sayi (2000 – 4000 eggs; Forward, unpublished data) and R. harrisii (1000 eggs; Forward, unpublished data). Thus, the observed relationship between embryonic development and spontaneous pumping behavior may be a consequence of the combined effects of the slow diffusion rate of water through the egg mass and the greater oxygen demand and waste production of more developed embryos (De Vries et al., 1991). If the embryos release pheromones that induce the female to undergo larval release behavior, then the water in which eggs hatch should contain these pheromones. Clearly, increasing concentrations of hatch water induced increased abdomen pumping by C. sapidus (Fig. 2). The lowest concentration to induce a significant increase in pumping was similar for blue crabs with young and mature embryos, but the level of responsiveness was consistently lower for females with early-stage eggs. Studies of R. harrisii (Forward and Lohmann, 1983) and N. sayi (De Vries et al., 1991) also found that the pumping response increased as hatch water concentration increased and that crabs with young embryos were less responsive than those with mature embryos. Since abdomen pumping is not evoked in non-ovigerous crabs (e.g., Forward and Lohmann, 1983), female blue crabs probably become sensitive to the pheromone at the time of egg deposition. Homogenized early- and late-stage embryos were equally effective at inducing the pumping response (Figs. 3 and 4), which indicates that the concentration of pheromones in the eggs is constant throughout embryonic development. This result conflicts with that for
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R. harrisii (Forward and Lohmann, 1983), which had a greater pumping response upon exposure to homogenized late-stage egg water than to early-stage egg water. Forward and Lohmann (1983) concluded that the concentration of the active chemical in the eggs increased as the embryos matured, suggesting that the pheromone may be produced and stored by the embryo during development. Although the concentration of pheromones present in C. sapidus eggs did not change with egg stage, the amount leaching from the eggs varied significantly with embryo development (Fig. 5). Eggs with both early- and late-stage embryos released the pheromones but release increased significantly as the embryos matured, which suggests that the egg membrane becomes progressively more permeable to the substances as development progresses. This result conflicts with the study of N. sayi. De Vries et al. (1991) demonstrated that the pheromones involved in stimulating pumping are not released from developing eggs because substances associate with ovigerous N. sayi females did not produce a significant pumping response in the absence of hatching. Yet, embryos of several brachyuran crabs, including N. sayi, Uca pugilator, S. cinereum, and S. haematocheir, are known to release proteolytic enzymes near the time of hatching which aid larval release by digesting the layers of the egg case (De Vries and Forward, 1991; Saigusa, 1996). Similar enzymes secreted during development may alter the permeability of the egg membranes to pheromones responsible for inducing abdomen pumping. Collectively, the results of the present study support the model for subtidal brachyuran crabs that pheromones from hatching eggs induce larval release behavior in ovigerous females that results in the synchronized release of larvae. However, the chemical identity of the pheromones is unknown for C. sapidus. Since De Vries et al. (1991) demonstrated that hatching water from different species induced the pumping response in N. sayi, the identity of the chemical cue may be similar among subtidal crabs species. The most extensive chemical identification studies have been done with R. harrisii. The initial study found that the pheromones were a group of small peptides with arginine composing approximately 50% of the amino acids (Rittschof et al., 1985). Further studies demonstrated that they were composed of short peptides with a neutral amino acid at the amino-terminus and arginine at the carboxyl-terminus (Forward et al., 1987; Rittschof et al., 1989). Rittschof et al. (1990) proposed that the peptide pheromones are generated by proteolytic digestion of the egg membrane by enzymes released by the embryos. Ovigerous blue crabs aggregate in high salinity areas near the mouths of estuaries. In preparation for larval release, crabs with mature embryos undergo a spawning migration to coastal areas where the larvae are released and then transported offshore for development. During the spawning migration, ovigerous crabs undergo ebb-tide transport, in which they swim in the water column during ebb tide at night and are probably on the bottom at other times (Tankersley et al., 1998). The cues that initiate ebb-tide transport and the underlying behavior are unknown. Since pheromones from the eggs induce female crabs to move into position for larval release and undergo vigorous abdominal pumping that breaks open the eggs (Forward and Lohmann, 1983; De Vries et al., 1991), it is attractive to speculate that the same or similar substances released from the eggs, specifically those containing latestage embryos, may initiate the spawning migration of blue crabs.
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Acknowledgements This material is based in part on research supported by the National Science Foundation Grants No. OCE-9819355/9901146/0096205 and No. OCE-0095092/0094930. We thank M.A. Sigala and M.G. Wieber for their help with collecting crabs. [SS]
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