- Email: [email protected]
The ontogeny of larval swimming behavior in the crab Hemigrapsus sanguineus: Implications for larval transport
Journal of Experimental Marine Biology and Ecology 462 (2015) 20–28
Contents lists available at ScienceDirect
Journal of Experimental Marine Biology and Ecology journal homepage: www.elsevier.com/locate/jembe
The ontogeny of larval swimming behavior in the crab Hemigrapsus sanguineus: Implications for larval transport Jonathan H. Cohen a,⁎, Cynthia K. Hanson a, Ana I. Dittel a, Douglas C. Miller a, Charles E. Tilburg b a b
College of Earth, Ocean and Environment, School of Marine Science and Policy, University of Delaware, 700 Pilottown Road, Lewes, DE 19958, USA Department of Marine Sciences, University of New England, 11 Hills Beach Road, Biddeford, ME 04005, USA
a r t i c l e
i n f o
Article history: Received 23 June 2014 Received in revised form 9 September 2014 Accepted 3 October 2014 Available online xxxx Keywords: Geotaxis Larval dispersal Phototaxis Vertical migration
a b s t r a c t Many estuarine crab species are exported from this habitat at spawning and are physically transported to coastal waters where they undergo zoeal development. Later, they recruit back to the adult habitat as megalopae. Larval behavior within the context of wind-driven coastal circulation contributes to this process. The present study tested whether the crab Hemigrapsus sanguineus, an invasive species to the mid-Atlantic coast of the United States as well as in Europe, possesses behavioral responses to exogenous cues (gravity and light) and endogenous rhythms that would facilitate this scenario. Throughout larval development, H. sanguineus zoeae were negatively buoyant. In a laboratory test chamber, early stage (Z1) larvae displayed a strong negative geotaxis in darkness which resulted in surface swimming that was not offset by behavioral responses to light exposure. These Z1 stage larvae also possessed an endogenous tidal rhythm in swimming activity that was in phase with ebb tides in the spawning habitat and was independent of light exposure. These behaviors are consistent with export of larvae from estuarine to coastal waters where larval development occurs. Intermediate (Z3) and late (Z5) stage larvae exhibited a predominant negative geotaxis and surface swimming, with some zoeae showing positive geotaxis and/or sinking in the Z5 stage. Visual spectral sensitivity of eyes in Z3 larvae peaked between 450 and 510 nm, with a secondary peak evident at short-wavelengths (~370 nm). In both the Z3 and Z5 stages, larvae descended in response to light exposure, as expected under a negative feedback model of depth regulation common among crab larvae. Photoresponse thresholds of 5.8 × 1012 and 1.2 × 1014 photons m−2 s−1 for Z3 and Z5 stage larvae, respectively, predict both stages to be in the lower water column during the day. After molting to megalopae, H. sanguineus display positive geotaxis and/or sinking in darkness and lack a phototactic response, consistent with settlement in the adult benthic habitat. Thus, larvae of the invasive crab H. sanguineus possess endogenous rhythms and behavioral responses to environmental cues that are similar to those observed in successful native species with a similar coastal larval development pattern. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Planktonic larval stages are critical time points in the life cycles of many benthic marine invertebrates and estuarine-dependent fish species. Costs during larval dispersal are high (Bonte et al., 2012). Apart from direct effects of mortality, which may be higher in planktonic than in later benthic life (Morgan, 1995), surviving larvae must also recruit to favorable habitats. Once recruited, latent effects of planktonic stages (e.g., starvation) persist as the larval phenotype influences the juvenile and adult stages (Marshall and Morgan, 2011; Pechenik, 2006). Therefore it is not surprising that pelagic larvae have developed a suite of behaviors that provide a measure of control over their dispersal patterns which ultimately influence recruitment and population connectivity (Cowen and Sponaugle, 2009; Levin, 2006; Metaxas and Saunders, 2009; Pineda et al., 2007). ⁎ Corresponding author. Tel.: +1 302 645 4298; fax: +1 302 645 4007. E-mail address: [email protected] (J.H. Cohen).
http://dx.doi.org/10.1016/j.jembe.2014.10.003 0022-0981/© 2014 Elsevier B.V. All rights reserved.
Dispersal in brachyuran crabs is of great interest as larval behavior appears particularly well-integrated into physical transport processes. As zooplankton with limited capacity for horizontal movement but extensive capability for movement in the vertical, crab larvae have emerged as a model for studying the role that larval behavior plays in regulating planktonic vertical distribution and, ultimately, recruitment (Forward, 2009; Sulkin, 1984). Conceptually, vertical distribution of crab larvae results from their negative buoyancy which leads to a descent within the water column that is either reinforced or offset by behavioral responses to persistent exogenous environmental stimuli (e.g., gravity, pressure, light) and endogenous swimming activity rhythms (e.g., circatidal, circadian). In this way, a cessation in swimming and subsequent sinking might occur after the animal ascends in the water column and senses low pressure. This sinking could be accentuated by downward swimming away from high surface light levels and/or with Earth's gravitational field. Opposite behavioral responses at depth would oppose the larva's negative buoyancy and result in an ascent within the water column. Depth regulation in this conceptual
J.H. Cohen et al. / Journal of Experimental Marine Biology and Ecology 462 (2015) 20–28
model ultimately underlies successful recruitment of larvae to the adult habitat. Some species maintain depth-regulation behaviors relative to tidal flow that favor estuarine retention throughout their larval development, enabling them to reliably locate and settle in the adult habitat (e.g., Rhithropanopeus harrisii; Forward, 2009). Other species (e.g., Uca spp. and Callinectes sapidus; Sulkin et al., 1980; Welch and Forward, 2001; López-Duarte et al., 2011) respond to similar environmental cues but in different ways that vary over the larval period such that they are exported from estuaries into the coastal ocean only to return and recruit into benthic populations at the end of their larval development (reviewed in Epifanio, 1988; Forward and Tankersley, 2001; Gibson, 2003; Queiroga and Blanton, 2005). The latter scenario of export-and-return is of particular interest for invasive species as the relative proportions of self-recruits to those advected have implications for range expansion (Byers and Pringle, 2006; Delaney et al., 2012; Tilburg et al., 2011). One such invasive species is the Asian shore crab Hemigrapsus sanguineus, which became established in North America near the mouth of Delaware Bay over 25 years ago (Epifanio, 2013; McDermott, 1991). Preliminary work on behavioral responses of H. sanguineus zoeae and megalopae to exogenous environmental cues, including gravity and pressure, has been done by Park et al. (2004). Results of that study generally support an export-and-return model of larval transport (reviewed in Epifanio, 2013) where larvae reside at shallow depths during early zoeal stages by negative geotaxis and perhaps barokinesis, with a progressive loss of the barokinetic response and a switch to positive geotaxis in late-stage zoeae and megalopae. However, several fundamental questions remain regarding H. sanguineus' behavior in relation to larval transport. Specifically, it is unknown whether early stage zoea larvae exhibit endogenous tidal activity rhythms that could facilitate export but also increase their vulnerability to predators (e.g., Morgan and Anastasia, 2008), or if rhythms are absent given that H. sanguineus adult habitat in mid-Atlantic estuaries is near the ocean (Schab et al., 2013). Such a reduction/loss of tidal rhythms in estuarine species spawning near the ocean has been reported for fiddler crabs of the genus Uca and the blue crab C. sapidus (López-Duarte and Tankersley, 2007; López-Duarte et al., 2011). Another important gap in understanding of H. sanguineus larval dispersal concerns responses of larvae and post-larvae to light, which is a major environmental factor in controlling zooplankton vertical distributions (Cohen and Forward, 2009) and, in turn, their horizontal transport (Metaxas and Saunders, 2009; Queiroga and Blanton, 2005). Accordingly, the present study tested endogenous and exogenous behavioral responses of H. sanguineus zoeae and megalopae that could influence larval transport. In addition to confirm sinking rates and geotaxis results of earlier work (Park et al., 2004), we tested for endogenous activity rhythms in newly hatched zoeae, measured visual spectral sensitivity electrophysiologically at an intermediate larval stage, and characterized phototactic responses throughout larval development. Consistent with the export-and-return model of larval transport, we hypothesized that early-stage larvae would have endogenous tidal activity rhythms and behavioral responses to gravity and light that would result in a consistent shallow distribution and export from Delaware Bay, whereas later stages would exhibit behavioral responses to these environmental factors that generate vertical migrations and deeper distributions in the coastal ocean consistent with predator avoidance and a return to coastal habitats at the end of larval development (e.g., Epifanio and Tilburg, 2008; Tilburg et al., 2009). 2. Materials and methods 2.1. Larval rearing Ovigerous female H. sanguineus were collected near Roosevelt Inlet (DE, USA; 38.817° N, 75.200° W) during spring/summer 2012 and 2013. The collection site was described in detail in Schab et al. (2013).
21
Crabs were kept individually in filtered seawater (25 °C, 30 psu) under fluorescent room lighting and the ambient L:D cycle in the lab until zoeae were released. Larvae were reared in groups, separated by brood, through five zoeal stages (Z1–Z5) to the megalopa stage in 30 psu seawater with a 14L:10D photoperiod (4.5 × 1017 photons m−2 s−1, PAR) and temperature of 25 °C within an incubator. Larvae were fed Artemia nauplii daily. Water was changed every other day. At water changes, larvae were monitored for ontogenetic stage (Hwang et al., 1993) and selected for use in behavioral experiments at Z1, Z3, Z5 and megalopa stages. 2.2. Sinking rates Ten individual larvae from each of 5 replicate broods at each ontogenetic stage were anesthetized for 20 min in 0.1 M urethane. This fully immobilized the larvae, which were otherwise alive based on visible heart beats. Following the method of Park et al. (2004), anesthetized larvae were gently pipetted individually onto the surface of an acrylic column (6 × 6 × 45 cm) filled with seawater. The column was prefilled in the experimental room at least 1 h prior to experiments, and the room was temperature controlled at 23 °C to minimize convection within the column. Each larva was allowed to reach terminal velocity by sinking through 15 cm of the column before the time required to travel an additional 10 cm was recorded. Sinking rates were compared among ontogenetic stage by one-way ANOVA with Tukey HSD posthoc testing. 2.3. Visual spectral sensitivity Spectral sensitivity of the eye in Z3 larvae was determined by electroretinogram (ERG) recording using the criterion response method as described in Cohen and Frank (2006). Larvae were immobilized on an acrylic support and placed within a temperature-controlled seawater bath at 18 °C inside a light-tight Faraday cage. Using a micromanipulator under dim white light, the exposed tip of an epoxy-insulated tungsten microelectrode (127 μm shank, A-M Systems) was placed subcorneally in one eye, which was bathed in light from a fused-silica fiber optic (P806, EXFO). A reference electrode was placed in the seawater bath adjacent to the larva. ERGs were recorded from the eye upon 75 ms light flashes from a monochromatic light source (100 W quartz-halogen lamp, CM110 monochromator, Spectral Products) with irradiance controlled by a variable-gradient neutral density filter (Melles Griot). Differential ERGs were amplified 1000× in AC mode (EXT-02-B, NPI Electronic), then digitized and recorded in LabVIEW for analysis of peak-to-peak height of response waveforms. Spectral sensitivity curves were obtained from triplicate Z3 larvae. While these data do not describe spectral sensitivity in H. sanguineus with ontogeny, they provide physiological justification for test wavelengths in the phototaxis experiments. 2.4. Geotaxis and phototaxis Directed swimming of crab larvae to gravity (geotaxis) and light intensity (phototaxis) were measured by digital video recording of swimming activity in a simulated natural underwater angular light distribution modified from Forward et al. (1984). A light-tight enclosure contained an acrylic water bath (40 × 40 × 15 cm) with inside painted flat black and filled with deionized water in which an acrylic cuvette (5 × 5 × 5 cm) containing 50 larvae in seawater was placed at the horizontal center. The stimulus lamp (Oriel 300 W Xe arc lamp) was positioned outside the enclosure. Lamp output was directed through an electromagnetic shutter to control stimulus duration, collimated, filtered to 488 nm by an interference filter (Melles Griot 03FIL002, 10 nm FWHM), and diffused before horizontally entering the lighttight enclosure containing the water bath. Light was filtered to these wavelengths because they are detected by the larval H. sanguineus eye
22
J.H. Cohen et al. / Journal of Experimental Marine Biology and Ecology 462 (2015) 20–28
(see Results Section 3.3) and have been shown to evoke photobehavior in other larval crabs (Forward and Cronin, 1979). Once inside the enclosure, the light beam was reflected off a mirror to pass vertically through a white acrylic diffuser and into the water bath to uniformly illuminate the larvae. Irradiance at the level of the larvae in the cuvette was measured using an optometer and calibrated radiometric probe (models S471 and 247, UDT Instruments). Narrow unpainted sections of the water bath allowed larvae to be backlit with an infrared LED backlight (880 nm, Advanced Illumination), which the larval eye cannot detect (see Results Section 3.3). Larval swimming behavior in the X–Y plane of the cuvette was captured for subsequent motion analysis by a CCD camera (WVBP330, Panasonic) with a 5–50-mm varifocal lens (13VA550, Pelco) and a digital recorder (DN-200, Datavideo). The entire cuvette was in the camera's field of view and its depth of field was sufficient to resolve zoeae at all positions between the front and back walls. For geotaxis/phototaxis experiments, 50 larvae of a given ontogenetic stage (Z1, Z3, Z5, megalopae) from a single brood were dark adapted for 1 h, then transferred under dim red light to the middle of the cuvette, which was then placed in the water bath. Geotaxis was determined from video captured after 15 min in darkness; the number of larvae in equal 5 cm horizontal sections (top, middle, and bottom) of the cuvette was then quantified by simple visual observation of the video. Arana and Sulkin (1993) used a similar method with congeneric H. oregonensis and found that a stable vertical distribution of larvae was established by 5 min. Given the absence of light cues and that hydrostatic pressure cues in the cuvette were far below the response threshold for H. sanguineus (Park et al., 2004), larvae in the top section displayed negative geotaxis, while those in the bottom section either displayed positive geotaxis or a general decrease in locomotor activity and consequent sinking (Sulkin, 1984). Replicate groups of 50 larvae were tested from 5 different broods. A two-way ANOVA with larval stage and column section as factors and Hold–Sidak post-hoc testing was used to characterize larval distribution in darkness resulting from negative geotaxis (present in top section), no/equal geotaxis (even distribution), or positive geotaxis and/or sinking (present in bottom section). Following the 15 min in darkness and video capture for assessment of geotaxis, larvae were then exposed to 5 s flashes of light from 1.2 × 1011 to 1.2 × 1014 photons m− 2 s− 1 with 5 min in darkness between each flash. Preliminary experiments found that this dark recovery interval was sufficient for a consistent phototactic response at intermediate intensities. Video of larval swimming was captured during the 5 s light exposures (photoresponse) and for an equivalent interval in darkness immediately before each light flash (control). The central 3 s segment of each video was analyzed with CellTrak software (Motion Analysis Inc.) for positions of individual larvae that were swimming in the center of the cuvette, thereby avoiding confounding interactions of larvae with top, bottom, and side walls. Larvae that crossed swimming paths during the analysis interval were excluded, resulting in an average of 13 larvae tracked per light level in each replicate. Angular direction of swimming over this interval was calculated for each larva from its initial and final positions. Ascent of larvae was defined as upward movement toward the stimulus light (zenith ± 30°), whereas descent was downward movement away from the stimulus light (nadir ± 30°). Ascending larvae could either be undergoing positive phototaxis or negative geotaxis, whereas descending larvae could either be undergoing negative phototaxis or positive geotaxis; comparison of photoresponse results with geotaxis results was used to differentiate among these orientation cues. The lowest light intensity required at each ontogenetic stage to evoke a significant change in swimming direction during a light stimulus relative to paired controls of swimming in darkness (i.e., the photoresponse threshold) was determined by one-way repeated measures ANOVAs with Dunnett's post-hoc testing versus the control, with separate tests run at each stage for ascending and descending photoresponses.
2.5. Endogenous activity rhythms Only Z1 larvae were used for endogenous rhythm experiments because previous studies with other species have found tidal rhythms, if present, to be strong in laboratory-hatched first-stage zoeae (e.g., López-Duarte and Tankersley, 2007; Zeng and Naylor, 1996; Queiroga and Blanton, 2005), while rearing of larvae in the laboratory away from tidal entrainment cues could lead to the loss of rhythms in later stages. Z1 larvae that hatched within 24 h of ovigerous female collection were fed Artemia nauplii for 1 day before being placed in covered acrylic columns (5.5 × 3.5 × 30 cm) filled with 30 psu seawater. Columns were located within a light-tight walk-in environmental chamber held at 18 °C and constant darkness (D:D). Each column was backlit with far-red light which H. sanguineus zoeae cannot detect (N740 nm, see Results Section 3.3) and imaged by a CCD camera (TKC9200U, JVC; YV10x5B-2 lens, Fujinon). Video was recorded simultaneously from all cameras by a multi-channel digital recorder (ECORR264-9x1, Everfocus). Larvae were observed in darkness for 96 h without feeding to not confound endogenous swimming activity with feeding activity. Ten replicate columns, each with 50 larvae from one of 8 different broods, were tested during the summer of 2012 and 2013. Video was analyzed for the proportion of zoeae in the top half (upper 10 cm) of the columns at 0.5 h-intervals throughout the recording period; given their negative buoyancy (see Results Section 3.1) the presence of larvae in the upper half of the column was taken to indicate active swimming. To confirm that any tidal rhythms present in Z1 stage larvae persist during the light phase, 4 additional groups of 50 larvae from separate broods were tested as described above, but under constant (L:L) blue-filtered fluorescent lighting (9.3 × 1016 photons m− 2 s− 1, transparent blue sleeve, Creative Lightings). The activity time series for each column was analyzed for significant periodicity as described in Cohen and Forward (2005) by autocorrelation analysis, with free-running period estimates determined by maximum entropy spectral analysis, then cross-correlated with tidal height and L:D cycles at the collection site of the ovigerous female crabs during the time of the experiment. Tidal height was a time series of values at 0.5 h intervals for water height above mean lower low water at the Lewes, DE tidal station 3.5 km from the collection site (Station ID 8557380, NOAA Tides and Currents), and the L:D cycle was a time series of values at 0.5 h intervals for day (sunrise to sunset; value = 10) and night (sunset to sunrise; value = 0). Circular statistics (Rayleigh tests and Watson–Williams F-tests, Oriana v. 4.02) were used to analyze significant peak lags from cross-correlations to identify the phase relationship between activity and tidal/light time series and to compare the effect of D:D and L:L treatments. 3. Results 3.1. Sinking rates Anesthetized H. sanguineus larvae sank with increasing rates over ontogeny (one-way ANOVA, p b 0.001) (Fig. 1). Sinking rates differed among the first (Z1) through fifth (Z5) zoeal stage (pairwise Tukey– Kramer HSD post hoc tests, p b 0.05), but not between Z5 stage larvae and megalopae. 3.2. Geotaxis H. sanguineus larvae added to the middle of the cuvette were not uniformly distributed among the top, middle, and bottom sections after 15 min in darkness (two-way ANOVA, p b 0.001 for interaction of larval stage and cuvette section). Z1 stage larvae displayed strong negative geotaxis, with more larvae present in the top section of the cuvette than in either the middle or bottom sections (Holm–Sidak tests, p b 0.05) (Fig. 2). At the Z3 stage, negative geotaxis in darkness was also observed as larvae were most abundant in the top section of the
J.H. Cohen et al. / Journal of Experimental Marine Biology and Ecology 462 (2015) 20–28
23
Fig. 1. Sinking rates of H. sanguineus zoeae and megalopae. Mean (±SE, n = 50) sinking rates are plotted for each ontogenetic stage tested. Individuals were anesthetized in urethane prior to measurement. Letters indicate significant differences in sinking rate among ontogenetic stages (1-way ANOVA with Tukey HSD post-hoc testing, α = 0.05).
cuvette at the same proportion as in the Z1 stage; while few Z3 stage larvae were present in the other sections, more were in the bottom section than the middle (Holm–Sidak tests, p b 0.05) (Fig. 2). Z5 stage larvae were again negatively geotactic with more larvae in the top section after 15 min in darkness, but a greater proportion of larvae were present in the bottom section than in either of the earlier zoeal stages (Holm–Sidak tests, p b 0.05) (Fig. 2). In contrast to zoeal larvae, megalopae displayed either strong positive geotaxis in darkness or reduced locomotor activity and subsequent descent, as they were primarily present in the bottom section of the cuvette (Holm–Sidak tests, p b 0.05) (Fig. 2). 3.3. Spectral sensitivity and phototaxis Spectral sensitivity of eyes in H. sanguineus Z3 stage larvae determined electrophysiologically peaked between 450 and 510 nm, with a secondary peak evident at short-wavelengths (~ 370 nm) (Fig. 3). When behavioral responses to 488-nm filtered light were tested in a simulated natural angular light field, Z1 stage larvae did not exhibit a photoresponse as they failed to show either an ascent or a descent response above dark control levels at any light intensity (one-way RM ANOVAs, p = 0.786, 0.655, respectively) (Fig. 4). In contrast, Z3 larvae descended upon stimulation by light, with a photoresponse threshold of 5.8 × 1012 photons m−2 s−1 apparent as an increase in the percentage descending at this light level, and a concomitant decrease in the percentage ascending among all responding individuals (one-way RM ANOVAs, p b 0.001; Dunnett's tests, p b 0.05) (Fig. 4). A similar increase in the descent response with light exposure was observed in larvae at the Z5 stage, with a photoresponse threshold of 1.2 × 1014 photons m−2 s−1 for descending larvae (one-way RM ANOVA, p b 0.001, Dunnett's test, p b 0.05) but no significant change in the percentage ascending at any light level versus control swimming in darkness (one-way RM ANOVA, p = 0.001; Dunnett's test, p N 0.05) (Fig. 4). As noted above, megalopae were positioned near the bottom of the cuvette in darkness, and no light intensity tested evoked a change in either ascent or descent responses relative to swimming in darkness (one-way RM ANOVAs p = 0.843, 0.308, respectively) (Fig. 4). 3.4. Endogenous activity rhythms Z1 stage larvae displayed rhythmic patterns of swimming in all replicate laboratory columns under both constant dark (D:D) conditions and constant light (L:L) conditions (Fig. 5) as indicated by autocorrelation
Fig. 2. Geotaxis of H. sanguineus zoeae and megalopae. Mean (±SE, n = 5) proportion of 50 individuals in the top, middle, and bottom sections of a cuvette (5 × 5 × 5 cm) after 15 min in darkness. Each ontogenetic stage is plotted in a separate panel. Letters indicate significant differences in proportion of larvae among column depths (2-way ANOVA with Holm–Sidak post-hoc testing, α = 0.05).
exceeding the white-noise confidence interval. Despite differences in tidal times at the collection site of ovigerous female crabs during D:D experiments (i.e., subjective tidal times), alignment of the activity time series by tidal time (Fig. 5A) showed increased swimming activity at the time of ebb tides, with peaks occurring during both the subjective light and dark phases. A similar pattern was present for L:L experiments
24
J.H. Cohen et al. / Journal of Experimental Marine Biology and Ecology 462 (2015) 20–28
Fig. 3. Visual spectral sensitivity of H. sanguineus Z3 stage larvae. Electroretinogram (ERG) responses to 75 ms flashes from a monochromatic stimulus light at 350–650 nm were used to determine the irradiance required to generate a 40 μV criterion response. Plotted (circles, dotted line) are the inverse irradiances at the criterion, normalized and averaged (±SE) for the 3 replicates. Also plotted (solid line) is the spectral emission of the 488-nm filtered lamp measured in the natural angular light field used for phototaxis experiments.
where tidal times were the same for all replicates, and peaks in larval activity occurred during both the day and night (Fig. 5B). MESA period estimates were tidal (~ 12 h) in 6 of 10 D:D replicates and in all L:L replicates (Table 1). Z1 stage larvae tested in D:D conditions and those tested in L:L conditions showed mean lags in swimming activity of 5.4 and 4.0 h, respectively, relative to the time of ambient high tide (Rayleigh tests, p = 0.0024, p = 0.009). Thus, peaks in larval swimming activity occurred on ebb tides (Fig. 6A). Experimental light condition did not affect the timing of swimming activity as the distributions of lags relative to tides for D:D and L:L columns were similar (Watson–Williams F-test, p = 0.178); the mean lag in swimming activity for all columns was 4.8 h after the time of high tide (Rayleigh test, p = 0.021) (Fig. 6A). In contrast, lags in swimming activity calculated relative to the ambient L:D cycle differed between D:D and L:L columns (Watson–Williams Ftest, p b 0.001) (Fig. 6B). All D:D columns displayed activity during the first half of the subjective day phase (mean = 11:14; Rayleigh test, p b 0.001), coinciding with ebb tide at the collection site of ovigerous crabs during the time when most (8 of 10) of these experiments were conducted. Swimming activity in L:L columns occurred in either the latter half of the subjective day phase or just after the time of sunset (mean = 18:30; Rayleigh test, p = 0.05) (Fig. 6B), again coinciding with ebb tide when these experiments were conducted. 4. Discussion H. sanguineus is typical of many crab species in that larvae are released in estuarine waters near the adult habitat, are advected from the estuary to complete larval development in the coastal ocean, and are then transported back to the adult habitat by wind-driven coastal circulation (Epifanio, 1988; Epifanio and Garvine, 2001; Epifanio and Tilburg, 2008). In coastal waters off Delaware Bay, downshelf larvae can be transported upshelf by upwelling (northeastward) winds that result in northward surface currents and then into Delaware Bay by downwelling (southwestward) winds that result in barotropic (i.e., entire water column) onshore currents (Epifanio and Garvine, 2001; Tilburg et al., 2005). Larval vertical distribution factors heavily in the success of such transport processes (reviewed in Queiroga and Blanton, 2005). A conceptual model for how depth regulation is achieved through swimming behavior has been proposed for crab larvae, which are negatively buoyant and sink when swimming ceases (reviewed in Sulkin, 1984). This descent can be reinforced or countered by swimming activity in response to persistent
exogenous environmental variables in the water column including gravity, pressure, and light, as well as endogenous activity rhythms entrained to the diel cycle and/or tides. Thus, larval vertical distribution and the behaviors that control it have increasingly been recognized as important factors in facilitating successful export of larvae to the coastal ocean, and for their return from these offshore development areas to estuaries, where megalopae ultimately settle and colonize adult habitats (Forward and Tankersley, 2001; Gibson, 2003; Queiroga and Blanton, 2005). In the case of H. sanguineus in Delaware Bay, an estuarine plume extends southward from the Bay mouth and along the inner continental shelf (Sanders and Garvine, 2001). In surface plankton sampling encompassing both night and day periods, Epifanio et al. (2013) found Z1 stage larvae to be retained in the estuarine plume, while advanced stage larvae (Z2–Z5) and megalopae were concentrated in a convergent frontal boundary on the plume's offshore edge. Association with this frontal water would result in upshelf transport of later stages back to Delaware Bay from upwelling winds (Tilburg et al., 2005), while larval development completed in the estuarine plume would result in advection farther south and away from source populations in Delaware Bay (Epifanio, 1988; Epifanio et al., 2013). Could larval behavior enhance transport from the estuary early in development and facilitate return of late-stage larvae to suitable adult habitat while avoiding predators? In the present study, we found behavioral responses to endogenous and exogenous cues in H. sanguineus larvae that would expedite entrainment in the estuarine plume early in larval development, and a change in responses to exogenous cues that would aid in shoreward transport at later ontogenetic stages. Z1 stage larvae in H. sanguineus displayed a strong negative geotaxis, observed previously by Park et al. (2004), which maintained negatively buoyant larvae at the water surface and outweighed any response to light during short-term laboratory experiments. Additionally, Z1 larvae displayed an endogenous rhythmic pattern in swimming activity that peaked during ebb tides irrespective of the subjective L:D cycle, and was not suppressed by constant light exposure. Taken together, these laboratory findings predict that Z1 stage larvae in the field would be in the upper water column but would have even shallower depth distributions during ebb tides. Similar tidal rhythms in swimming that lag ambient high tides by 2.5–4 h have been suggested to underlie ebb-tide transport in newly hatched Z1 stage larvae of estuarine fiddler crabs in semi-diurnal tidal regimes, with this rhythm being weaker or absent in coastal species (López-Duarte and Tankersley, 2009; López-Duarte et al., 2011; Morgan and Anastasia, 2008). Among the few species in which both endogenous tidal rhythms and photobehavior have been studied, the fiddler crab Uca pugilator lacked distinct photobehavior at the Z1 stage but possessed an endogenous activity rhythm in phase with ebb tides, as was observed in H. sanguineus larvae at this stage. In contrast, endogenous tidal activity rhythms were absent in the blue crab C. sapidus that are released farther offshore, while photobehaviors were present (López-Duarte and Tankersley, 2007). These and other studies (e.g., Forward and Cohen, 2004) show that endogenous tidal rhythms used for selective tidal stream transport generally persist during both light and dark phases of the diel cycle, despite increased risk of visual predation during daytime residence in surface waters. Larvae are released from H. sanguineus females on nocturnal ebb tides throughout the lunar cycle, which may help reduce predation risk (Park et al., 2005; Saigusa and Kawagoye, 1997), but the collective suite of an endogenous tidal rhythm and exogenously driven behaviors with respect to gravity and light would keep them in estuarine surface waters (where velocities are greatest) when this water is moving offshore. With a Z1 larval duration of ~4–5 days (Epifanio et al., 1998), this behavior would also aid in continued advection from the estuary to the coastal ocean as larvae are entrained in the Delaware Coastal Current (Sanders and Garvine, 2001).
J.H. Cohen et al. / Journal of Experimental Marine Biology and Ecology 462 (2015) 20–28
25
Fig. 4. Phototaxis of H. sanguineus zoeae and megalopae. Panels are for experiments with Z1 (A, B), Z3 (C, D), Z5 (E, F), and megalopae (G, H). Mean (±SE, n = 5) proportion of actively swimming zoeae or megalopae ascending (zenith ± 30°; left panels) and descending (nadir ± 30°; right panels) during the middle 3 s period of a 5 s light flash are plotted (open circles) over a range of irradiance stimuli. Control swimming activity measured in darkness over an equivalent time interval immediately before each light flash did not differ among light levels (1-way ANOVAs for each ontogenetic stage and direction of swimming, p N 0.05), so mean (± SE, n = 8) control responses are plotted (solid circles) for each panel. Photoresponse thresholds (see Section 2.4) are marked with an asterisk (*), and stages with no significant difference (nsd) among stimuli and the control are indicated.
At the other end of larval development, H. sanguineus megalopae were similar to Z1 stage larvae in that both were non-responsive to light stimuli. However, megalopae displayed a strong but opposite response to gravity than was observed in the Z1 stage; megalopae show positive geotaxis and/or sinking in darkness resulting in individuals residing on the bottom of the test chamber irrespective of light intensity. Such behavior is common among brachyuran megalopae (Queiroga and Blanton, 2005) and confirms earlier work by Park et al. (2004). When coupled with the observed lack of a light effect on megalopal swimming, positive geotaxis and/or sinking responses would allow megalopae to avoid predators but still be transported
toward and up the estuary during downwelling wind events that result in barotropic onshore flow (Epifanio and Garvine, 2001; Epifanio and Tilburg, 2008). Beyond transport, benthic residence at this life stage is critical for settlement of H. sanguineus megalopae in the estuary and molting to the first crab stage, processes accelerated by a variety of biotic and abiotic cues associated with estuarine substrates (reviewed in Epifanio et al., 2013). Behavioral contributions to the timing of larval release, subsequent transport into coastal waters, and ultimately settlement back in estuaries are relatively well understood processes in decapod larvae (e.g., Epifanio, 1988; Forward and Tankersley, 2001; Forward et al.,
26
J.H. Cohen et al. / Journal of Experimental Marine Biology and Ecology 462 (2015) 20–28
Fig. 5. Activity rhythms of H. sanguineus Z1 stage larvae. Proportion of 50 larvae present in the upper half of laboratory columns in D:D conditions (mean ± SE, n = 10) (A) and L:L conditions (mean ± SE, n = 4) (B) for the first 48 h of each time series. Replicate groups of larvae were tested at different tidal times; therefore, data have been aligned according to tidal time (inset panels, average tidal height above mean lower low water). The average time of subjective night during experiments is indicated by grey shading.
Table 1 Period estimates for endogenous activity rhythm experiments. Values are for each experimental column (Activity), along with the ambient tidal height (Tides) and light:dark cycle (L:D cycle) at the collection site of ovigerous female crabs during the time of the experiment. Period estimate (h) Light treatment D:D
L:L
Activity 26.4 13.2 13.7 14.4 11.9 17.4 23.0 12.4 12.6 25.7 12.2 13.6 13.4 14.1
Tides 12.1 12.3 12.3 12.6 12.6 12.4 12.4 12.1 12.1 12.3 11.8 11.8 11.8 11.8
L:D cycle 22.6 22.5 24.6 23.0 23.0 23.0 23.0 23.0 23.0 23.0 22.9 22.9 22.9 22.9
2001; Gibson, 2003). In contrast, behavior during larval development in coastal waters and its influence on larval survival during this period and eventual shoreward transport are far less clear (Gallego et al., 2012; McConaugha, 1992). As larvae develop and grow larger, they become more vulnerable to visual predators unless they possess structural or behavioral strategies for crypsis (Christy, 2011; Johnsen, 2014; Morgan and Christy, 1997), particularly as optical properties of the water column shift to favor visual predation farther from the estuary (e.g., Forward et al., 1988). Consistent with this increase in larval vulnerability, behavior of H. sanguineus larvae in response to gravity and light changes as expected if larvae undergo the predator-avoidance behavioral strategy of diel vertical migration (DVM; Cohen and Forward, 2009). In contrast to earlier stage Z1 larvae, Z3 larvae of H. sanguineus descend when they detect light. The observed photoresponse is a negative phototaxis given that Z3 larvae retain a strong negative geotactic response favoring upward swimming in darkness. This behavioral pattern represents a negative feedback model for depth regulation that is common in crab larvae (Sulkin, 1984; Forward, 2009). Larvae in brighter shallower waters descend by either passive sinking or negative
J.H. Cohen et al. / Journal of Experimental Marine Biology and Ecology 462 (2015) 20–28
27
Fig. 6. Phase relationships between H. sanguineus Z1 stage larval activity rhythms and environmental variables. Lags of peak swimming activity determined from cross-correlation analysis of each replicate column against subjective tidal (A) and L:D (B) cycles are plotted. In panel A, the durations of ebbing and flooding tides are each 6.2 h, and the arrow indicates the mean vector for all data (Rayleigh test, p = 0.021, mean vector length = 0.667). In panel B the average times of sunrise (SR) and sunset (SS) for all replicates are indicated; the solid arrow indicates the mean vector for D:D data (Rayleigh test, p b 0.001, mean vector length = 0.909), the dashed arrow indicates the mean vector for L:L data (Rayleigh test, p = 0.05, mean vector length = 0.830).
phototaxis until they are in apparent darkness when light levels fall below their lower visual threshold, at which point they ascend due to negative geotaxis in darkness. Thus, larvae will oscillate in depth about their lower visual threshold during the day, and then ascend in the water column via this mechanism along with additional light-mediated cues at sunset (e.g., Forward, 1985) as they undergo a nocturnal DVM pattern (Cohen and Forward, 2009). For Z3 stage larvae of H. sanguineus undergoing development in the Delaware Coastal Current, this behavior would enable them to travel northward by upwelling wind-driven surface currents (Epifanio, 1988; Tilburg et al., 2009) during night, while also minimizing predation risk during the day. Consistent with this, Epifanio et al. (2013) found later stage H. sanguineus larvae to be more abundant in the offshore frontal region of the Delaware Coastal Current than in the main stream of the current, suggesting northward transport. Higher resolution field sampling would help to determine the extent to which the observed larval behaviors contribute to the cross-shelf distribution of larvae at intermediate stages. Additionally, the presence of tidal rhythms in intermediate stage larvae is unknown for H. sanguineus or other crabs with larval development in the coastal ocean (Queiroga and Blanton, 2005), but should be investigated. In the Z5 zoeal stage, H. sanguineus larvae displayed bimodal behaviors. Some responded to gravity and light cues according to the negative feedback model as described for Z3 larvae, while others responded more like H. sanguineus megalopae with strong positive geotaxis and/or sinking and limited photoresponses. However, those Z5 larvae that behaved according to the model were less sensitive to light than were Z3 larvae. These thresholds determined in the present study should not be viewed as definitive because they depend on the stimulus resolution used in behavioral experiments, which was purposefully kept at half-log steps to limit light exposure of dark-adapted larvae. Nevertheless, the relative difference observed between photoresponse thresholds for Z3 stage larvae (5.8 × 1012 photons m− 2 s− 1) and Z5 stage larvae (1.2 × 1014 photons m − 2 s − 1) would result in later-stage larvae residing ~ 10 m shallower during the daytime than earlier-stage larvae given optical properties of the Delaware Coastal Current (e.g., Biermann, 2009). A shallower daytime vertical distribution of Z5 stage H. sanguineus larvae, the depth of which is set by the lower visual threshold (Sulkin, 1984), would lead to even greater transport northward toward source populations by upwelling wind-driven surface currents than
for deeper-dwelling Z3 stage larvae. Other Z5 larvae adopting even deeper depths driven by positive geotaxis and minimal response to light would be subjected to cross-shelf transport by the barotropic onshore flow from downwelling events as proposed above for H. sanguineus megalopae (Epifanio and Garvine, 2001; Epifanio and Tilburg, 2008). The observed changes in behavior of H. sanguineus throughout its larval development are consistent with transport out of the estuary, followed by growth in the coastal ocean, and finally return to the adult coastal/estuarine habitat. Schab et al. (2013) reported a recent decline in adult H. sanguineus populations in rocky habitats near the mouth of Delaware Bay that previously favored this invasive species over the native mud crab Panopeus herbstii. Low supply of H. sanguineus larvae to local populations as compared to other crab species (e.g., Epifanio et al., 2013; Steppe and Epifanio, 2006), as well as high variability in recruitment due to impacts of episodic wind events (e.g., Jones and Epifanio, 1995), was suggested to explain this decline. Larval H. sanguineus possess endogenous rhythms and behavioral responses to environmental cues that are similar to those observed in successful native species with similar coastal larval development patterns (e.g., López-Duarte and Tankersley, 2007; Sulkin, 1975; Sulkin et al., 1980). Accordingly, our results suggest that H. sanguineus larval supply to the adult habitat involves behavior coupled with favorable hydrodynamic conditions, which support the interpretation of Schab et al. (2013) that declines in H. sanguineus populations may have resulted from periods of poor recruitment. Further study of larval supply and recruitment patterns in H. sanguineus from mid-Atlantic populations, coupled with numerical modeling (e.g., Delaney et al., 2012; Tilburg et al., 2006), would be beneficial for understanding population dynamics of this invasive crab species.
Acknowledgements The study was supported by a grant (RHCE6 DESG) from the Delaware Sea Grant College Program. We thank Emily Olson, Meghan Owings, and Heather Cronin for the help with experiments; Corey Schab, Corie Charpentier, Olivia Graham, Scott Miller, and Terrell Carter for assistance in larval culturing; and Charles Epifanio for valuable comments on the article. [RH]
28
J.H. Cohen et al. / Journal of Experimental Marine Biology and Ecology 462 (2015) 20–28
References Arana, M., Sulkin, S., 1993. Behavioral basis of depth regulation in the first zoeal stage of the Pacific shore crab, Hemigrapsus oregonensis (Brachyura: Grapsidae). Pac. Sci. 47, 256–262. Biermann, J.L., 2009. The distribution of Callinectes sapidus megalopae at the mouths of Chesapeake and Delaware Bays: implications for larval ingressM.S. thesis University of Maryland, College Park. Bonte, D., Van Dyck, H., Bullock, J.M., Coulon, A., Delgado, M., Gibbs, M., Lehouck, V., Matthysen, E., Mustin, K., Saastamoinen, M., Schtickzelle, N., Stevens, V.M., Vandewoestijne, S., Baguette, M., Barton, K., Benton, T.G., Chaput-Bardy, A., Clobert, J., Dytham, C., Hovestadt, T., Meier, C.M., Palmer, S.C.F., Turlure, C., Travis, J.M.J., 2012. Costs of dispersal. Biol. Rev. 87, 290–312. Byers, J.E., Pringle, J.M., 2006. Going against the flow: retention, range limits and invasions in advective environments. Mar. Ecol. Prog. Ser. 313, 27–41. Christy, J.H., 2011. Timing of hatching and release of larvae by brachyuran crabs: patterns, adaptive significance and control. Integr. Comp. Biol. 51, 62–72. Cohen, J.H., Forward Jr., R.B., 2005. Diel vertical migration in the marine copepod Calanopia americana. II. The proximate role of exogenous light cues and endogenous rhythms. Mar. Biol. 147, 399–410. Cohen, J.H., Forward Jr., R.B., 2009. Zooplankton diel vertical migration — a review of proximate control. Oceanogr. Mar. Biol. Annu. Rev. 47, 77–110. Cohen, J.H., Frank, T.M., 2006. Visual physiology of the Antarctic amphipod Abyssorchomene plebs. Biol. Bull. 211, 140–148. Cowen, R.K., Sponaugle, S., 2009. Larval dispersal and marine population connectivity. Annu. Rev. Mar. Sci. 1, 443–466. Delaney, D.G., Edwards, P.K., Leung, B., 2012. Predicting regional spread of non-native species using oceanographic models: validation and identification of gaps. Mar. Biol. 159, 269–282. Epifanio, C.E., 1988. Transport of invertebrate larvae between estuaries and the continental shelf. Trans. Am. Fish. Soc. Symp. Ser. 3, 104–114. Epifanio, C.E., 2013. Invasion biology of the Asian shore crab Hemigrapsus sanguineus: a review. J. Exp. Mar. Biol. Ecol. 441, 33–49. 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. Epifanio, C.E., Tilburg, C.E., 2008. Transport of blue crab larvae in the Middle Atlantic Bight: a wet and windy journey. J. Mar. Res. 66, 723–749. Epifanio, C.E., Dittel, A.I., Park, S., Schwalm, S., Fouts, A., 1998. Early life history of Hemigrapsus sanguineus, a non-indigenous crab in the Middle Atlantic Bight (USA). Mar. Ecol. Prog. Ser. 170, 231–238. Epifanio, C.E., Tilburg, C.E., Dittel, A.I., 2013. Abundance of invasive and native crab larvae in the mouth of Delaware Bay: Hemigrapsus sanguineus and Uca pugnax. J. Shellfish Res. 32, 543–550. Forward Jr., R.B., 1985. Behavioral responses of larvae of the crab Rhithropanopeus harrisii (Brachyura: Xanthidae) during diel vertical migration. Mar. Biol. 90, 9–18. Forward Jr., R.B., 2009. Larval biology of the crab Rhithropanopeus harrisii (Gould): a synthesis. Biol. Bull. 216, 243–256. Forward Jr., R.B., Cohen, J.H., 2004. Factors affecting the circatidal rhythm in vertical swimming of ovigerous blue crabs, Callinectes sapidus, involved in the spawning migration. J. Exp. Mar. Biol. Ecol. 299, 255–266. Forward Jr., R.B., Cronin, T.W., 1979. Spectral sensitivity of larvae from intertidal crustaceans. J. Comp. Phys. 133, 311–315. Forward Jr., R.B., Tankersley, R.A., 2001. Selective tidal stream transport of marine animals. Oceanogr. Mar. Biol. Annu. Rev. 39, 305–353. Forward Jr., R.B., Cronin, T.W., Stearns, D.E., 1984. Control of diel vertical migration: photoresponses of a larval crustacean. Limnol. Oceanogr. 29, 146–154. Forward Jr., R.B., Cronin, T.W., Douglass, J.K., 1988. The visual pigments of crabs II. Environmental adaptations. J. Comp. Physiol. A 162, 479–490. Forward Jr., R.B., Tankersley, R.A., Rittschof, D., 2001. Cues for metamorphosis of brachyuran crabs: an overview. Am. Zool. 41, 1108–1122. Gallego, A., North, E.W., Houde, E.D., 2012. Understanding and quantifying mortality in pelagic, early life stages of marine organisms — old challenges and new perspectives. J. Mar. Sys. 93, 1–3. Gibson, R.N., 2003. Go with the flow: tidal migration in marine animals. Hydrobiologia 503, 153–161. Hwang, S.G., Lee, C., Kim, C.H., 1993. Complete larval development of Hemigrapsus sanguineus (Decapoda, Brachyura, Grapsidae) reared in the laboratory. Kor. J. Syst. Zool. 9, 69–86. Johnsen, S., 2014. Hide and seek in the open sea: pelagic camouflage and visual countermeasures. Annu. Rev. Mar. Sci. 6, 369–392. Jones, M.B., Epifanio, C.E., 1995. Settlement of brachyuran megalopae in Delaware Bay: an analysis of time series data. Mar. Ecol. Prog. Ser. 125, 67–76. Levin, L.A., 2006. Recent progress in understanding larval dispersal: new directions and digressions. Integr. Comp. Biol. 46, 282–297.
López-Duarte, P.C., Tankersley, R.A., 2007. Circatidal swimming behavior of brachyuran crab zoea larvae: implications for ebb-tide transport. Mar. Biol. 151, 2037–2051. López-Duarte, P.C., Tankersley, R.A., 2009. Developmental shift in the selective tidalstream transport behavior of larvae of the fiddler crab Uca minax (LeConte). J. Exp. Mar. Biol. Ecol. 368, 169–180. López-Duarte, P.C., Christy, J.H., Tankersley, R.A., 2011. A behavioral mechanism for dispersal in fiddler crab larvae (genus Uca) varies with adult habitat, not phylogeny. Limnol. Oceanogr. 56, 1879–1892. Marshall, D.J., Morgan, S.G., 2011. Ecological and evolutionary consequences of linked lifehistory stages in the sea. Curr. Biol. 21, R718–R725. McConaugha, J.R., 1992. Decapod larvae: dispersal, mortality, and ecology. A working hypothesis. Am. Zool. 32, 512–523. McDermott, J., 1991. A breeding population of the western Pacific crab Hemigrapsus sanguineus (Crustacea: Decapoda: Grapsidae) established on the Atlantic coast of North America. Biol. Bull. 181, 195–198. Metaxas, A., Saunders, M., 2009. Quantifying the “bio-” components in biophysical models of larval transport in marine benthic invertebrates: advances and pitfalls. Bio. Bull. 216, 257–272. Morgan, S.G., 1995. Life and death in the plankton: larval mortality and adaptation. In: McEdward, L. (Ed.), Ecology of Marine Invertebrate Larvae. CRC Press, Boca Raton, pp. 279–321. Morgan, S.G., Anastasia, J.R., 2008. Behavioral tradeoff in estuarine larvae favors seaward migration over minimizing visibility to predators. Proc. Natl. Acad. Sci. USA 105, 222–227. Morgan, S.G., Christy, J.H., 1997. Planktivorous fishes as selective agents for reproductive synchrony. J. Exp. Mar. Biol. Ecol. 209, 89–101. Park, S., Epifanio, C.E., Grey, E.K., 2004. Behavior of larval Hemigrapsus sanguineus (de Haan) in response to gravity and pressure. J. Exp. Mar. Biol. Ecol. 307, 197–206. Park, S., Epifanio, C.E., Iglay, R.B., 2005. Patterns of larval release by the Asian shore crab Hemigrapsus sanguineus (De Haan): periodicity at diel and tidal frequencies. J. Shellfish Res. 24, 591–595. Pechenik, J.A., 2006. Larval experience and latent effects — metamorphosis is not a new beginning. Integr. Comp. Biol. 46, 323–333. Pineda, J., Hare, J.A., Sponaugle, S., 2007. Larval transport and dispersal in the coastal ocean and consequences for population connectivity. Oceanogr. 20, 22–39. Queiroga, H., Blanton, J., 2005. Interactions between behaviour and physical forcing in the control of horizontal transport of decapod crustacean larvae. Adv. Mar. Biol. 47, 109–214. Saigusa, M., Kawagoye, O., 1997. Circatidal rhythm of an intertidal crab, Hemigrapsus sanguineus: synchrony with unequal tide height and involvement of a lightresponse mechanism. Mar. Biol. 129, 87–96. Sanders, T.M., Garvine, R.W., 2001. Fresh water delivery to the continental shelf and subsequent mixing: an observational study. J. Geophys. Res. 106, 27087–27101. Schab, C.M., Park, S., Waidner, L.A., Epifanio, C.E., 2013. Return of the native: historical comparison of invasive and indigenous populations near the mouth of Delaware Bay. J. Shellfish Res. 32, 1–8. Steppe, C.N., Epifanio, C.E., 2006. Synoptic distribution of crab larvae near the mouth of Delaware Bay: influence of nearshore hydrographic regimes. Estuar. Coast. Shelf Sci. 70, 645–662. Sulkin, S.D., 1975. The influence of light in the depth regulation of crab larvae. Biol. Bull. 148, 333–343. Sulkin, S.D., 1984. Behavioral basis of depth regulation in the larvae of brachyuran crabs. Mar. Ecol. Prog. Ser. 15, 181–205. Sulkin, S.D., van Heukelem, W., Kelly, P., van Heukelem, L., 1980. The behavioral basis of larval recruitment in the crab Callinectes sapidus Rathbun: a laboratory investigation of ontogenetic changes in geotaxis and barokinesis. Biol. Bull. 159, 402–417. Tilburg, C.E., Reager, J.T., Whitney, M.M., 2005. The physics of blue crab larval recruitment in Delaware Bay: a model study. J. Mar. Res. 63, 471–495. Tilburg, C.E., Houser, L.T., Steppe, C.N., Garvine, R.W., Epifanio, C.E., 2006. Effects of coastal transport on larval patches: models and observations. Estuar. Coast. Shelf Sci. 67, 145–160. Tilburg, C.E., Dittel, A.I., Epifanio, C.E., 2009. High concentrations of blue crab (Callinectes sapidus) larvae along the offshore edge of a coastal current: effects of convergent circulation. Fish. Oceanogr. 18, 135–146. Tilburg, C.E., Dittel, A.I., Miller, D.C., Epifanio, C.E., 2011. Transport and retention of the mitten crab (Eriocheir sinensis) in a Mid-Atlantic estuary: predictions from a larval transport model. J. Mar. Res. 69, 137–165. Welch, J.M., Forward Jr., R.B., 2001. Flood tide transport of blue crab, Callinectes sapidus, postlarvae: behavioral responses to salinity and turbulence. Mar. Biol. 139, 911–918. Zeng, C., Naylor, E., 1996. Synchronization of endogenous tidal vertical migration rhythms in laboratory-hatched larvae of the crab Carcinus maenas. J. Exp. Mar. Biol. Ecol. 198, 269–289.
Contents lists available at ScienceDirect
Journal of Experimental Marine Biology and Ecology journal homepage: www.elsevier.com/locate/jembe
The ontogeny of larval swimming behavior in the crab Hemigrapsus sanguineus: Implications for larval transport Jonathan H. Cohen a,⁎, Cynthia K. Hanson a, Ana I. Dittel a, Douglas C. Miller a, Charles E. Tilburg b a b
College of Earth, Ocean and Environment, School of Marine Science and Policy, University of Delaware, 700 Pilottown Road, Lewes, DE 19958, USA Department of Marine Sciences, University of New England, 11 Hills Beach Road, Biddeford, ME 04005, USA
a r t i c l e
i n f o
Article history: Received 23 June 2014 Received in revised form 9 September 2014 Accepted 3 October 2014 Available online xxxx Keywords: Geotaxis Larval dispersal Phototaxis Vertical migration
a b s t r a c t Many estuarine crab species are exported from this habitat at spawning and are physically transported to coastal waters where they undergo zoeal development. Later, they recruit back to the adult habitat as megalopae. Larval behavior within the context of wind-driven coastal circulation contributes to this process. The present study tested whether the crab Hemigrapsus sanguineus, an invasive species to the mid-Atlantic coast of the United States as well as in Europe, possesses behavioral responses to exogenous cues (gravity and light) and endogenous rhythms that would facilitate this scenario. Throughout larval development, H. sanguineus zoeae were negatively buoyant. In a laboratory test chamber, early stage (Z1) larvae displayed a strong negative geotaxis in darkness which resulted in surface swimming that was not offset by behavioral responses to light exposure. These Z1 stage larvae also possessed an endogenous tidal rhythm in swimming activity that was in phase with ebb tides in the spawning habitat and was independent of light exposure. These behaviors are consistent with export of larvae from estuarine to coastal waters where larval development occurs. Intermediate (Z3) and late (Z5) stage larvae exhibited a predominant negative geotaxis and surface swimming, with some zoeae showing positive geotaxis and/or sinking in the Z5 stage. Visual spectral sensitivity of eyes in Z3 larvae peaked between 450 and 510 nm, with a secondary peak evident at short-wavelengths (~370 nm). In both the Z3 and Z5 stages, larvae descended in response to light exposure, as expected under a negative feedback model of depth regulation common among crab larvae. Photoresponse thresholds of 5.8 × 1012 and 1.2 × 1014 photons m−2 s−1 for Z3 and Z5 stage larvae, respectively, predict both stages to be in the lower water column during the day. After molting to megalopae, H. sanguineus display positive geotaxis and/or sinking in darkness and lack a phototactic response, consistent with settlement in the adult benthic habitat. Thus, larvae of the invasive crab H. sanguineus possess endogenous rhythms and behavioral responses to environmental cues that are similar to those observed in successful native species with a similar coastal larval development pattern. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Planktonic larval stages are critical time points in the life cycles of many benthic marine invertebrates and estuarine-dependent fish species. Costs during larval dispersal are high (Bonte et al., 2012). Apart from direct effects of mortality, which may be higher in planktonic than in later benthic life (Morgan, 1995), surviving larvae must also recruit to favorable habitats. Once recruited, latent effects of planktonic stages (e.g., starvation) persist as the larval phenotype influences the juvenile and adult stages (Marshall and Morgan, 2011; Pechenik, 2006). Therefore it is not surprising that pelagic larvae have developed a suite of behaviors that provide a measure of control over their dispersal patterns which ultimately influence recruitment and population connectivity (Cowen and Sponaugle, 2009; Levin, 2006; Metaxas and Saunders, 2009; Pineda et al., 2007). ⁎ Corresponding author. Tel.: +1 302 645 4298; fax: +1 302 645 4007. E-mail address: [email protected] (J.H. Cohen).
http://dx.doi.org/10.1016/j.jembe.2014.10.003 0022-0981/© 2014 Elsevier B.V. All rights reserved.
Dispersal in brachyuran crabs is of great interest as larval behavior appears particularly well-integrated into physical transport processes. As zooplankton with limited capacity for horizontal movement but extensive capability for movement in the vertical, crab larvae have emerged as a model for studying the role that larval behavior plays in regulating planktonic vertical distribution and, ultimately, recruitment (Forward, 2009; Sulkin, 1984). Conceptually, vertical distribution of crab larvae results from their negative buoyancy which leads to a descent within the water column that is either reinforced or offset by behavioral responses to persistent exogenous environmental stimuli (e.g., gravity, pressure, light) and endogenous swimming activity rhythms (e.g., circatidal, circadian). In this way, a cessation in swimming and subsequent sinking might occur after the animal ascends in the water column and senses low pressure. This sinking could be accentuated by downward swimming away from high surface light levels and/or with Earth's gravitational field. Opposite behavioral responses at depth would oppose the larva's negative buoyancy and result in an ascent within the water column. Depth regulation in this conceptual
J.H. Cohen et al. / Journal of Experimental Marine Biology and Ecology 462 (2015) 20–28
model ultimately underlies successful recruitment of larvae to the adult habitat. Some species maintain depth-regulation behaviors relative to tidal flow that favor estuarine retention throughout their larval development, enabling them to reliably locate and settle in the adult habitat (e.g., Rhithropanopeus harrisii; Forward, 2009). Other species (e.g., Uca spp. and Callinectes sapidus; Sulkin et al., 1980; Welch and Forward, 2001; López-Duarte et al., 2011) respond to similar environmental cues but in different ways that vary over the larval period such that they are exported from estuaries into the coastal ocean only to return and recruit into benthic populations at the end of their larval development (reviewed in Epifanio, 1988; Forward and Tankersley, 2001; Gibson, 2003; Queiroga and Blanton, 2005). The latter scenario of export-and-return is of particular interest for invasive species as the relative proportions of self-recruits to those advected have implications for range expansion (Byers and Pringle, 2006; Delaney et al., 2012; Tilburg et al., 2011). One such invasive species is the Asian shore crab Hemigrapsus sanguineus, which became established in North America near the mouth of Delaware Bay over 25 years ago (Epifanio, 2013; McDermott, 1991). Preliminary work on behavioral responses of H. sanguineus zoeae and megalopae to exogenous environmental cues, including gravity and pressure, has been done by Park et al. (2004). Results of that study generally support an export-and-return model of larval transport (reviewed in Epifanio, 2013) where larvae reside at shallow depths during early zoeal stages by negative geotaxis and perhaps barokinesis, with a progressive loss of the barokinetic response and a switch to positive geotaxis in late-stage zoeae and megalopae. However, several fundamental questions remain regarding H. sanguineus' behavior in relation to larval transport. Specifically, it is unknown whether early stage zoea larvae exhibit endogenous tidal activity rhythms that could facilitate export but also increase their vulnerability to predators (e.g., Morgan and Anastasia, 2008), or if rhythms are absent given that H. sanguineus adult habitat in mid-Atlantic estuaries is near the ocean (Schab et al., 2013). Such a reduction/loss of tidal rhythms in estuarine species spawning near the ocean has been reported for fiddler crabs of the genus Uca and the blue crab C. sapidus (López-Duarte and Tankersley, 2007; López-Duarte et al., 2011). Another important gap in understanding of H. sanguineus larval dispersal concerns responses of larvae and post-larvae to light, which is a major environmental factor in controlling zooplankton vertical distributions (Cohen and Forward, 2009) and, in turn, their horizontal transport (Metaxas and Saunders, 2009; Queiroga and Blanton, 2005). Accordingly, the present study tested endogenous and exogenous behavioral responses of H. sanguineus zoeae and megalopae that could influence larval transport. In addition to confirm sinking rates and geotaxis results of earlier work (Park et al., 2004), we tested for endogenous activity rhythms in newly hatched zoeae, measured visual spectral sensitivity electrophysiologically at an intermediate larval stage, and characterized phototactic responses throughout larval development. Consistent with the export-and-return model of larval transport, we hypothesized that early-stage larvae would have endogenous tidal activity rhythms and behavioral responses to gravity and light that would result in a consistent shallow distribution and export from Delaware Bay, whereas later stages would exhibit behavioral responses to these environmental factors that generate vertical migrations and deeper distributions in the coastal ocean consistent with predator avoidance and a return to coastal habitats at the end of larval development (e.g., Epifanio and Tilburg, 2008; Tilburg et al., 2009). 2. Materials and methods 2.1. Larval rearing Ovigerous female H. sanguineus were collected near Roosevelt Inlet (DE, USA; 38.817° N, 75.200° W) during spring/summer 2012 and 2013. The collection site was described in detail in Schab et al. (2013).
21
Crabs were kept individually in filtered seawater (25 °C, 30 psu) under fluorescent room lighting and the ambient L:D cycle in the lab until zoeae were released. Larvae were reared in groups, separated by brood, through five zoeal stages (Z1–Z5) to the megalopa stage in 30 psu seawater with a 14L:10D photoperiod (4.5 × 1017 photons m−2 s−1, PAR) and temperature of 25 °C within an incubator. Larvae were fed Artemia nauplii daily. Water was changed every other day. At water changes, larvae were monitored for ontogenetic stage (Hwang et al., 1993) and selected for use in behavioral experiments at Z1, Z3, Z5 and megalopa stages. 2.2. Sinking rates Ten individual larvae from each of 5 replicate broods at each ontogenetic stage were anesthetized for 20 min in 0.1 M urethane. This fully immobilized the larvae, which were otherwise alive based on visible heart beats. Following the method of Park et al. (2004), anesthetized larvae were gently pipetted individually onto the surface of an acrylic column (6 × 6 × 45 cm) filled with seawater. The column was prefilled in the experimental room at least 1 h prior to experiments, and the room was temperature controlled at 23 °C to minimize convection within the column. Each larva was allowed to reach terminal velocity by sinking through 15 cm of the column before the time required to travel an additional 10 cm was recorded. Sinking rates were compared among ontogenetic stage by one-way ANOVA with Tukey HSD posthoc testing. 2.3. Visual spectral sensitivity Spectral sensitivity of the eye in Z3 larvae was determined by electroretinogram (ERG) recording using the criterion response method as described in Cohen and Frank (2006). Larvae were immobilized on an acrylic support and placed within a temperature-controlled seawater bath at 18 °C inside a light-tight Faraday cage. Using a micromanipulator under dim white light, the exposed tip of an epoxy-insulated tungsten microelectrode (127 μm shank, A-M Systems) was placed subcorneally in one eye, which was bathed in light from a fused-silica fiber optic (P806, EXFO). A reference electrode was placed in the seawater bath adjacent to the larva. ERGs were recorded from the eye upon 75 ms light flashes from a monochromatic light source (100 W quartz-halogen lamp, CM110 monochromator, Spectral Products) with irradiance controlled by a variable-gradient neutral density filter (Melles Griot). Differential ERGs were amplified 1000× in AC mode (EXT-02-B, NPI Electronic), then digitized and recorded in LabVIEW for analysis of peak-to-peak height of response waveforms. Spectral sensitivity curves were obtained from triplicate Z3 larvae. While these data do not describe spectral sensitivity in H. sanguineus with ontogeny, they provide physiological justification for test wavelengths in the phototaxis experiments. 2.4. Geotaxis and phototaxis Directed swimming of crab larvae to gravity (geotaxis) and light intensity (phototaxis) were measured by digital video recording of swimming activity in a simulated natural underwater angular light distribution modified from Forward et al. (1984). A light-tight enclosure contained an acrylic water bath (40 × 40 × 15 cm) with inside painted flat black and filled with deionized water in which an acrylic cuvette (5 × 5 × 5 cm) containing 50 larvae in seawater was placed at the horizontal center. The stimulus lamp (Oriel 300 W Xe arc lamp) was positioned outside the enclosure. Lamp output was directed through an electromagnetic shutter to control stimulus duration, collimated, filtered to 488 nm by an interference filter (Melles Griot 03FIL002, 10 nm FWHM), and diffused before horizontally entering the lighttight enclosure containing the water bath. Light was filtered to these wavelengths because they are detected by the larval H. sanguineus eye
22
J.H. Cohen et al. / Journal of Experimental Marine Biology and Ecology 462 (2015) 20–28
(see Results Section 3.3) and have been shown to evoke photobehavior in other larval crabs (Forward and Cronin, 1979). Once inside the enclosure, the light beam was reflected off a mirror to pass vertically through a white acrylic diffuser and into the water bath to uniformly illuminate the larvae. Irradiance at the level of the larvae in the cuvette was measured using an optometer and calibrated radiometric probe (models S471 and 247, UDT Instruments). Narrow unpainted sections of the water bath allowed larvae to be backlit with an infrared LED backlight (880 nm, Advanced Illumination), which the larval eye cannot detect (see Results Section 3.3). Larval swimming behavior in the X–Y plane of the cuvette was captured for subsequent motion analysis by a CCD camera (WVBP330, Panasonic) with a 5–50-mm varifocal lens (13VA550, Pelco) and a digital recorder (DN-200, Datavideo). The entire cuvette was in the camera's field of view and its depth of field was sufficient to resolve zoeae at all positions between the front and back walls. For geotaxis/phototaxis experiments, 50 larvae of a given ontogenetic stage (Z1, Z3, Z5, megalopae) from a single brood were dark adapted for 1 h, then transferred under dim red light to the middle of the cuvette, which was then placed in the water bath. Geotaxis was determined from video captured after 15 min in darkness; the number of larvae in equal 5 cm horizontal sections (top, middle, and bottom) of the cuvette was then quantified by simple visual observation of the video. Arana and Sulkin (1993) used a similar method with congeneric H. oregonensis and found that a stable vertical distribution of larvae was established by 5 min. Given the absence of light cues and that hydrostatic pressure cues in the cuvette were far below the response threshold for H. sanguineus (Park et al., 2004), larvae in the top section displayed negative geotaxis, while those in the bottom section either displayed positive geotaxis or a general decrease in locomotor activity and consequent sinking (Sulkin, 1984). Replicate groups of 50 larvae were tested from 5 different broods. A two-way ANOVA with larval stage and column section as factors and Hold–Sidak post-hoc testing was used to characterize larval distribution in darkness resulting from negative geotaxis (present in top section), no/equal geotaxis (even distribution), or positive geotaxis and/or sinking (present in bottom section). Following the 15 min in darkness and video capture for assessment of geotaxis, larvae were then exposed to 5 s flashes of light from 1.2 × 1011 to 1.2 × 1014 photons m− 2 s− 1 with 5 min in darkness between each flash. Preliminary experiments found that this dark recovery interval was sufficient for a consistent phototactic response at intermediate intensities. Video of larval swimming was captured during the 5 s light exposures (photoresponse) and for an equivalent interval in darkness immediately before each light flash (control). The central 3 s segment of each video was analyzed with CellTrak software (Motion Analysis Inc.) for positions of individual larvae that were swimming in the center of the cuvette, thereby avoiding confounding interactions of larvae with top, bottom, and side walls. Larvae that crossed swimming paths during the analysis interval were excluded, resulting in an average of 13 larvae tracked per light level in each replicate. Angular direction of swimming over this interval was calculated for each larva from its initial and final positions. Ascent of larvae was defined as upward movement toward the stimulus light (zenith ± 30°), whereas descent was downward movement away from the stimulus light (nadir ± 30°). Ascending larvae could either be undergoing positive phototaxis or negative geotaxis, whereas descending larvae could either be undergoing negative phototaxis or positive geotaxis; comparison of photoresponse results with geotaxis results was used to differentiate among these orientation cues. The lowest light intensity required at each ontogenetic stage to evoke a significant change in swimming direction during a light stimulus relative to paired controls of swimming in darkness (i.e., the photoresponse threshold) was determined by one-way repeated measures ANOVAs with Dunnett's post-hoc testing versus the control, with separate tests run at each stage for ascending and descending photoresponses.
2.5. Endogenous activity rhythms Only Z1 larvae were used for endogenous rhythm experiments because previous studies with other species have found tidal rhythms, if present, to be strong in laboratory-hatched first-stage zoeae (e.g., López-Duarte and Tankersley, 2007; Zeng and Naylor, 1996; Queiroga and Blanton, 2005), while rearing of larvae in the laboratory away from tidal entrainment cues could lead to the loss of rhythms in later stages. Z1 larvae that hatched within 24 h of ovigerous female collection were fed Artemia nauplii for 1 day before being placed in covered acrylic columns (5.5 × 3.5 × 30 cm) filled with 30 psu seawater. Columns were located within a light-tight walk-in environmental chamber held at 18 °C and constant darkness (D:D). Each column was backlit with far-red light which H. sanguineus zoeae cannot detect (N740 nm, see Results Section 3.3) and imaged by a CCD camera (TKC9200U, JVC; YV10x5B-2 lens, Fujinon). Video was recorded simultaneously from all cameras by a multi-channel digital recorder (ECORR264-9x1, Everfocus). Larvae were observed in darkness for 96 h without feeding to not confound endogenous swimming activity with feeding activity. Ten replicate columns, each with 50 larvae from one of 8 different broods, were tested during the summer of 2012 and 2013. Video was analyzed for the proportion of zoeae in the top half (upper 10 cm) of the columns at 0.5 h-intervals throughout the recording period; given their negative buoyancy (see Results Section 3.1) the presence of larvae in the upper half of the column was taken to indicate active swimming. To confirm that any tidal rhythms present in Z1 stage larvae persist during the light phase, 4 additional groups of 50 larvae from separate broods were tested as described above, but under constant (L:L) blue-filtered fluorescent lighting (9.3 × 1016 photons m− 2 s− 1, transparent blue sleeve, Creative Lightings). The activity time series for each column was analyzed for significant periodicity as described in Cohen and Forward (2005) by autocorrelation analysis, with free-running period estimates determined by maximum entropy spectral analysis, then cross-correlated with tidal height and L:D cycles at the collection site of the ovigerous female crabs during the time of the experiment. Tidal height was a time series of values at 0.5 h intervals for water height above mean lower low water at the Lewes, DE tidal station 3.5 km from the collection site (Station ID 8557380, NOAA Tides and Currents), and the L:D cycle was a time series of values at 0.5 h intervals for day (sunrise to sunset; value = 10) and night (sunset to sunrise; value = 0). Circular statistics (Rayleigh tests and Watson–Williams F-tests, Oriana v. 4.02) were used to analyze significant peak lags from cross-correlations to identify the phase relationship between activity and tidal/light time series and to compare the effect of D:D and L:L treatments. 3. Results 3.1. Sinking rates Anesthetized H. sanguineus larvae sank with increasing rates over ontogeny (one-way ANOVA, p b 0.001) (Fig. 1). Sinking rates differed among the first (Z1) through fifth (Z5) zoeal stage (pairwise Tukey– Kramer HSD post hoc tests, p b 0.05), but not between Z5 stage larvae and megalopae. 3.2. Geotaxis H. sanguineus larvae added to the middle of the cuvette were not uniformly distributed among the top, middle, and bottom sections after 15 min in darkness (two-way ANOVA, p b 0.001 for interaction of larval stage and cuvette section). Z1 stage larvae displayed strong negative geotaxis, with more larvae present in the top section of the cuvette than in either the middle or bottom sections (Holm–Sidak tests, p b 0.05) (Fig. 2). At the Z3 stage, negative geotaxis in darkness was also observed as larvae were most abundant in the top section of the
J.H. Cohen et al. / Journal of Experimental Marine Biology and Ecology 462 (2015) 20–28
23
Fig. 1. Sinking rates of H. sanguineus zoeae and megalopae. Mean (±SE, n = 50) sinking rates are plotted for each ontogenetic stage tested. Individuals were anesthetized in urethane prior to measurement. Letters indicate significant differences in sinking rate among ontogenetic stages (1-way ANOVA with Tukey HSD post-hoc testing, α = 0.05).
cuvette at the same proportion as in the Z1 stage; while few Z3 stage larvae were present in the other sections, more were in the bottom section than the middle (Holm–Sidak tests, p b 0.05) (Fig. 2). Z5 stage larvae were again negatively geotactic with more larvae in the top section after 15 min in darkness, but a greater proportion of larvae were present in the bottom section than in either of the earlier zoeal stages (Holm–Sidak tests, p b 0.05) (Fig. 2). In contrast to zoeal larvae, megalopae displayed either strong positive geotaxis in darkness or reduced locomotor activity and subsequent descent, as they were primarily present in the bottom section of the cuvette (Holm–Sidak tests, p b 0.05) (Fig. 2). 3.3. Spectral sensitivity and phototaxis Spectral sensitivity of eyes in H. sanguineus Z3 stage larvae determined electrophysiologically peaked between 450 and 510 nm, with a secondary peak evident at short-wavelengths (~ 370 nm) (Fig. 3). When behavioral responses to 488-nm filtered light were tested in a simulated natural angular light field, Z1 stage larvae did not exhibit a photoresponse as they failed to show either an ascent or a descent response above dark control levels at any light intensity (one-way RM ANOVAs, p = 0.786, 0.655, respectively) (Fig. 4). In contrast, Z3 larvae descended upon stimulation by light, with a photoresponse threshold of 5.8 × 1012 photons m−2 s−1 apparent as an increase in the percentage descending at this light level, and a concomitant decrease in the percentage ascending among all responding individuals (one-way RM ANOVAs, p b 0.001; Dunnett's tests, p b 0.05) (Fig. 4). A similar increase in the descent response with light exposure was observed in larvae at the Z5 stage, with a photoresponse threshold of 1.2 × 1014 photons m−2 s−1 for descending larvae (one-way RM ANOVA, p b 0.001, Dunnett's test, p b 0.05) but no significant change in the percentage ascending at any light level versus control swimming in darkness (one-way RM ANOVA, p = 0.001; Dunnett's test, p N 0.05) (Fig. 4). As noted above, megalopae were positioned near the bottom of the cuvette in darkness, and no light intensity tested evoked a change in either ascent or descent responses relative to swimming in darkness (one-way RM ANOVAs p = 0.843, 0.308, respectively) (Fig. 4). 3.4. Endogenous activity rhythms Z1 stage larvae displayed rhythmic patterns of swimming in all replicate laboratory columns under both constant dark (D:D) conditions and constant light (L:L) conditions (Fig. 5) as indicated by autocorrelation
Fig. 2. Geotaxis of H. sanguineus zoeae and megalopae. Mean (±SE, n = 5) proportion of 50 individuals in the top, middle, and bottom sections of a cuvette (5 × 5 × 5 cm) after 15 min in darkness. Each ontogenetic stage is plotted in a separate panel. Letters indicate significant differences in proportion of larvae among column depths (2-way ANOVA with Holm–Sidak post-hoc testing, α = 0.05).
exceeding the white-noise confidence interval. Despite differences in tidal times at the collection site of ovigerous female crabs during D:D experiments (i.e., subjective tidal times), alignment of the activity time series by tidal time (Fig. 5A) showed increased swimming activity at the time of ebb tides, with peaks occurring during both the subjective light and dark phases. A similar pattern was present for L:L experiments
24
J.H. Cohen et al. / Journal of Experimental Marine Biology and Ecology 462 (2015) 20–28
Fig. 3. Visual spectral sensitivity of H. sanguineus Z3 stage larvae. Electroretinogram (ERG) responses to 75 ms flashes from a monochromatic stimulus light at 350–650 nm were used to determine the irradiance required to generate a 40 μV criterion response. Plotted (circles, dotted line) are the inverse irradiances at the criterion, normalized and averaged (±SE) for the 3 replicates. Also plotted (solid line) is the spectral emission of the 488-nm filtered lamp measured in the natural angular light field used for phototaxis experiments.
where tidal times were the same for all replicates, and peaks in larval activity occurred during both the day and night (Fig. 5B). MESA period estimates were tidal (~ 12 h) in 6 of 10 D:D replicates and in all L:L replicates (Table 1). Z1 stage larvae tested in D:D conditions and those tested in L:L conditions showed mean lags in swimming activity of 5.4 and 4.0 h, respectively, relative to the time of ambient high tide (Rayleigh tests, p = 0.0024, p = 0.009). Thus, peaks in larval swimming activity occurred on ebb tides (Fig. 6A). Experimental light condition did not affect the timing of swimming activity as the distributions of lags relative to tides for D:D and L:L columns were similar (Watson–Williams F-test, p = 0.178); the mean lag in swimming activity for all columns was 4.8 h after the time of high tide (Rayleigh test, p = 0.021) (Fig. 6A). In contrast, lags in swimming activity calculated relative to the ambient L:D cycle differed between D:D and L:L columns (Watson–Williams Ftest, p b 0.001) (Fig. 6B). All D:D columns displayed activity during the first half of the subjective day phase (mean = 11:14; Rayleigh test, p b 0.001), coinciding with ebb tide at the collection site of ovigerous crabs during the time when most (8 of 10) of these experiments were conducted. Swimming activity in L:L columns occurred in either the latter half of the subjective day phase or just after the time of sunset (mean = 18:30; Rayleigh test, p = 0.05) (Fig. 6B), again coinciding with ebb tide when these experiments were conducted. 4. Discussion H. sanguineus is typical of many crab species in that larvae are released in estuarine waters near the adult habitat, are advected from the estuary to complete larval development in the coastal ocean, and are then transported back to the adult habitat by wind-driven coastal circulation (Epifanio, 1988; Epifanio and Garvine, 2001; Epifanio and Tilburg, 2008). In coastal waters off Delaware Bay, downshelf larvae can be transported upshelf by upwelling (northeastward) winds that result in northward surface currents and then into Delaware Bay by downwelling (southwestward) winds that result in barotropic (i.e., entire water column) onshore currents (Epifanio and Garvine, 2001; Tilburg et al., 2005). Larval vertical distribution factors heavily in the success of such transport processes (reviewed in Queiroga and Blanton, 2005). A conceptual model for how depth regulation is achieved through swimming behavior has been proposed for crab larvae, which are negatively buoyant and sink when swimming ceases (reviewed in Sulkin, 1984). This descent can be reinforced or countered by swimming activity in response to persistent
exogenous environmental variables in the water column including gravity, pressure, and light, as well as endogenous activity rhythms entrained to the diel cycle and/or tides. Thus, larval vertical distribution and the behaviors that control it have increasingly been recognized as important factors in facilitating successful export of larvae to the coastal ocean, and for their return from these offshore development areas to estuaries, where megalopae ultimately settle and colonize adult habitats (Forward and Tankersley, 2001; Gibson, 2003; Queiroga and Blanton, 2005). In the case of H. sanguineus in Delaware Bay, an estuarine plume extends southward from the Bay mouth and along the inner continental shelf (Sanders and Garvine, 2001). In surface plankton sampling encompassing both night and day periods, Epifanio et al. (2013) found Z1 stage larvae to be retained in the estuarine plume, while advanced stage larvae (Z2–Z5) and megalopae were concentrated in a convergent frontal boundary on the plume's offshore edge. Association with this frontal water would result in upshelf transport of later stages back to Delaware Bay from upwelling winds (Tilburg et al., 2005), while larval development completed in the estuarine plume would result in advection farther south and away from source populations in Delaware Bay (Epifanio, 1988; Epifanio et al., 2013). Could larval behavior enhance transport from the estuary early in development and facilitate return of late-stage larvae to suitable adult habitat while avoiding predators? In the present study, we found behavioral responses to endogenous and exogenous cues in H. sanguineus larvae that would expedite entrainment in the estuarine plume early in larval development, and a change in responses to exogenous cues that would aid in shoreward transport at later ontogenetic stages. Z1 stage larvae in H. sanguineus displayed a strong negative geotaxis, observed previously by Park et al. (2004), which maintained negatively buoyant larvae at the water surface and outweighed any response to light during short-term laboratory experiments. Additionally, Z1 larvae displayed an endogenous rhythmic pattern in swimming activity that peaked during ebb tides irrespective of the subjective L:D cycle, and was not suppressed by constant light exposure. Taken together, these laboratory findings predict that Z1 stage larvae in the field would be in the upper water column but would have even shallower depth distributions during ebb tides. Similar tidal rhythms in swimming that lag ambient high tides by 2.5–4 h have been suggested to underlie ebb-tide transport in newly hatched Z1 stage larvae of estuarine fiddler crabs in semi-diurnal tidal regimes, with this rhythm being weaker or absent in coastal species (López-Duarte and Tankersley, 2009; López-Duarte et al., 2011; Morgan and Anastasia, 2008). Among the few species in which both endogenous tidal rhythms and photobehavior have been studied, the fiddler crab Uca pugilator lacked distinct photobehavior at the Z1 stage but possessed an endogenous activity rhythm in phase with ebb tides, as was observed in H. sanguineus larvae at this stage. In contrast, endogenous tidal activity rhythms were absent in the blue crab C. sapidus that are released farther offshore, while photobehaviors were present (López-Duarte and Tankersley, 2007). These and other studies (e.g., Forward and Cohen, 2004) show that endogenous tidal rhythms used for selective tidal stream transport generally persist during both light and dark phases of the diel cycle, despite increased risk of visual predation during daytime residence in surface waters. Larvae are released from H. sanguineus females on nocturnal ebb tides throughout the lunar cycle, which may help reduce predation risk (Park et al., 2005; Saigusa and Kawagoye, 1997), but the collective suite of an endogenous tidal rhythm and exogenously driven behaviors with respect to gravity and light would keep them in estuarine surface waters (where velocities are greatest) when this water is moving offshore. With a Z1 larval duration of ~4–5 days (Epifanio et al., 1998), this behavior would also aid in continued advection from the estuary to the coastal ocean as larvae are entrained in the Delaware Coastal Current (Sanders and Garvine, 2001).
J.H. Cohen et al. / Journal of Experimental Marine Biology and Ecology 462 (2015) 20–28
25
Fig. 4. Phototaxis of H. sanguineus zoeae and megalopae. Panels are for experiments with Z1 (A, B), Z3 (C, D), Z5 (E, F), and megalopae (G, H). Mean (±SE, n = 5) proportion of actively swimming zoeae or megalopae ascending (zenith ± 30°; left panels) and descending (nadir ± 30°; right panels) during the middle 3 s period of a 5 s light flash are plotted (open circles) over a range of irradiance stimuli. Control swimming activity measured in darkness over an equivalent time interval immediately before each light flash did not differ among light levels (1-way ANOVAs for each ontogenetic stage and direction of swimming, p N 0.05), so mean (± SE, n = 8) control responses are plotted (solid circles) for each panel. Photoresponse thresholds (see Section 2.4) are marked with an asterisk (*), and stages with no significant difference (nsd) among stimuli and the control are indicated.
At the other end of larval development, H. sanguineus megalopae were similar to Z1 stage larvae in that both were non-responsive to light stimuli. However, megalopae displayed a strong but opposite response to gravity than was observed in the Z1 stage; megalopae show positive geotaxis and/or sinking in darkness resulting in individuals residing on the bottom of the test chamber irrespective of light intensity. Such behavior is common among brachyuran megalopae (Queiroga and Blanton, 2005) and confirms earlier work by Park et al. (2004). When coupled with the observed lack of a light effect on megalopal swimming, positive geotaxis and/or sinking responses would allow megalopae to avoid predators but still be transported
toward and up the estuary during downwelling wind events that result in barotropic onshore flow (Epifanio and Garvine, 2001; Epifanio and Tilburg, 2008). Beyond transport, benthic residence at this life stage is critical for settlement of H. sanguineus megalopae in the estuary and molting to the first crab stage, processes accelerated by a variety of biotic and abiotic cues associated with estuarine substrates (reviewed in Epifanio et al., 2013). Behavioral contributions to the timing of larval release, subsequent transport into coastal waters, and ultimately settlement back in estuaries are relatively well understood processes in decapod larvae (e.g., Epifanio, 1988; Forward and Tankersley, 2001; Forward et al.,
26
J.H. Cohen et al. / Journal of Experimental Marine Biology and Ecology 462 (2015) 20–28
Fig. 5. Activity rhythms of H. sanguineus Z1 stage larvae. Proportion of 50 larvae present in the upper half of laboratory columns in D:D conditions (mean ± SE, n = 10) (A) and L:L conditions (mean ± SE, n = 4) (B) for the first 48 h of each time series. Replicate groups of larvae were tested at different tidal times; therefore, data have been aligned according to tidal time (inset panels, average tidal height above mean lower low water). The average time of subjective night during experiments is indicated by grey shading.
Table 1 Period estimates for endogenous activity rhythm experiments. Values are for each experimental column (Activity), along with the ambient tidal height (Tides) and light:dark cycle (L:D cycle) at the collection site of ovigerous female crabs during the time of the experiment. Period estimate (h) Light treatment D:D
L:L
Activity 26.4 13.2 13.7 14.4 11.9 17.4 23.0 12.4 12.6 25.7 12.2 13.6 13.4 14.1
Tides 12.1 12.3 12.3 12.6 12.6 12.4 12.4 12.1 12.1 12.3 11.8 11.8 11.8 11.8
L:D cycle 22.6 22.5 24.6 23.0 23.0 23.0 23.0 23.0 23.0 23.0 22.9 22.9 22.9 22.9
2001; Gibson, 2003). In contrast, behavior during larval development in coastal waters and its influence on larval survival during this period and eventual shoreward transport are far less clear (Gallego et al., 2012; McConaugha, 1992). As larvae develop and grow larger, they become more vulnerable to visual predators unless they possess structural or behavioral strategies for crypsis (Christy, 2011; Johnsen, 2014; Morgan and Christy, 1997), particularly as optical properties of the water column shift to favor visual predation farther from the estuary (e.g., Forward et al., 1988). Consistent with this increase in larval vulnerability, behavior of H. sanguineus larvae in response to gravity and light changes as expected if larvae undergo the predator-avoidance behavioral strategy of diel vertical migration (DVM; Cohen and Forward, 2009). In contrast to earlier stage Z1 larvae, Z3 larvae of H. sanguineus descend when they detect light. The observed photoresponse is a negative phototaxis given that Z3 larvae retain a strong negative geotactic response favoring upward swimming in darkness. This behavioral pattern represents a negative feedback model for depth regulation that is common in crab larvae (Sulkin, 1984; Forward, 2009). Larvae in brighter shallower waters descend by either passive sinking or negative
J.H. Cohen et al. / Journal of Experimental Marine Biology and Ecology 462 (2015) 20–28
27
Fig. 6. Phase relationships between H. sanguineus Z1 stage larval activity rhythms and environmental variables. Lags of peak swimming activity determined from cross-correlation analysis of each replicate column against subjective tidal (A) and L:D (B) cycles are plotted. In panel A, the durations of ebbing and flooding tides are each 6.2 h, and the arrow indicates the mean vector for all data (Rayleigh test, p = 0.021, mean vector length = 0.667). In panel B the average times of sunrise (SR) and sunset (SS) for all replicates are indicated; the solid arrow indicates the mean vector for D:D data (Rayleigh test, p b 0.001, mean vector length = 0.909), the dashed arrow indicates the mean vector for L:L data (Rayleigh test, p = 0.05, mean vector length = 0.830).
phototaxis until they are in apparent darkness when light levels fall below their lower visual threshold, at which point they ascend due to negative geotaxis in darkness. Thus, larvae will oscillate in depth about their lower visual threshold during the day, and then ascend in the water column via this mechanism along with additional light-mediated cues at sunset (e.g., Forward, 1985) as they undergo a nocturnal DVM pattern (Cohen and Forward, 2009). For Z3 stage larvae of H. sanguineus undergoing development in the Delaware Coastal Current, this behavior would enable them to travel northward by upwelling wind-driven surface currents (Epifanio, 1988; Tilburg et al., 2009) during night, while also minimizing predation risk during the day. Consistent with this, Epifanio et al. (2013) found later stage H. sanguineus larvae to be more abundant in the offshore frontal region of the Delaware Coastal Current than in the main stream of the current, suggesting northward transport. Higher resolution field sampling would help to determine the extent to which the observed larval behaviors contribute to the cross-shelf distribution of larvae at intermediate stages. Additionally, the presence of tidal rhythms in intermediate stage larvae is unknown for H. sanguineus or other crabs with larval development in the coastal ocean (Queiroga and Blanton, 2005), but should be investigated. In the Z5 zoeal stage, H. sanguineus larvae displayed bimodal behaviors. Some responded to gravity and light cues according to the negative feedback model as described for Z3 larvae, while others responded more like H. sanguineus megalopae with strong positive geotaxis and/or sinking and limited photoresponses. However, those Z5 larvae that behaved according to the model were less sensitive to light than were Z3 larvae. These thresholds determined in the present study should not be viewed as definitive because they depend on the stimulus resolution used in behavioral experiments, which was purposefully kept at half-log steps to limit light exposure of dark-adapted larvae. Nevertheless, the relative difference observed between photoresponse thresholds for Z3 stage larvae (5.8 × 1012 photons m− 2 s− 1) and Z5 stage larvae (1.2 × 1014 photons m − 2 s − 1) would result in later-stage larvae residing ~ 10 m shallower during the daytime than earlier-stage larvae given optical properties of the Delaware Coastal Current (e.g., Biermann, 2009). A shallower daytime vertical distribution of Z5 stage H. sanguineus larvae, the depth of which is set by the lower visual threshold (Sulkin, 1984), would lead to even greater transport northward toward source populations by upwelling wind-driven surface currents than
for deeper-dwelling Z3 stage larvae. Other Z5 larvae adopting even deeper depths driven by positive geotaxis and minimal response to light would be subjected to cross-shelf transport by the barotropic onshore flow from downwelling events as proposed above for H. sanguineus megalopae (Epifanio and Garvine, 2001; Epifanio and Tilburg, 2008). The observed changes in behavior of H. sanguineus throughout its larval development are consistent with transport out of the estuary, followed by growth in the coastal ocean, and finally return to the adult coastal/estuarine habitat. Schab et al. (2013) reported a recent decline in adult H. sanguineus populations in rocky habitats near the mouth of Delaware Bay that previously favored this invasive species over the native mud crab Panopeus herbstii. Low supply of H. sanguineus larvae to local populations as compared to other crab species (e.g., Epifanio et al., 2013; Steppe and Epifanio, 2006), as well as high variability in recruitment due to impacts of episodic wind events (e.g., Jones and Epifanio, 1995), was suggested to explain this decline. Larval H. sanguineus possess endogenous rhythms and behavioral responses to environmental cues that are similar to those observed in successful native species with similar coastal larval development patterns (e.g., López-Duarte and Tankersley, 2007; Sulkin, 1975; Sulkin et al., 1980). Accordingly, our results suggest that H. sanguineus larval supply to the adult habitat involves behavior coupled with favorable hydrodynamic conditions, which support the interpretation of Schab et al. (2013) that declines in H. sanguineus populations may have resulted from periods of poor recruitment. Further study of larval supply and recruitment patterns in H. sanguineus from mid-Atlantic populations, coupled with numerical modeling (e.g., Delaney et al., 2012; Tilburg et al., 2006), would be beneficial for understanding population dynamics of this invasive crab species.
Acknowledgements The study was supported by a grant (RHCE6 DESG) from the Delaware Sea Grant College Program. We thank Emily Olson, Meghan Owings, and Heather Cronin for the help with experiments; Corey Schab, Corie Charpentier, Olivia Graham, Scott Miller, and Terrell Carter for assistance in larval culturing; and Charles Epifanio for valuable comments on the article. [RH]
28
J.H. Cohen et al. / Journal of Experimental Marine Biology and Ecology 462 (2015) 20–28
References Arana, M., Sulkin, S., 1993. Behavioral basis of depth regulation in the first zoeal stage of the Pacific shore crab, Hemigrapsus oregonensis (Brachyura: Grapsidae). Pac. Sci. 47, 256–262. Biermann, J.L., 2009. The distribution of Callinectes sapidus megalopae at the mouths of Chesapeake and Delaware Bays: implications for larval ingressM.S. thesis University of Maryland, College Park. Bonte, D., Van Dyck, H., Bullock, J.M., Coulon, A., Delgado, M., Gibbs, M., Lehouck, V., Matthysen, E., Mustin, K., Saastamoinen, M., Schtickzelle, N., Stevens, V.M., Vandewoestijne, S., Baguette, M., Barton, K., Benton, T.G., Chaput-Bardy, A., Clobert, J., Dytham, C., Hovestadt, T., Meier, C.M., Palmer, S.C.F., Turlure, C., Travis, J.M.J., 2012. Costs of dispersal. Biol. Rev. 87, 290–312. Byers, J.E., Pringle, J.M., 2006. Going against the flow: retention, range limits and invasions in advective environments. Mar. Ecol. Prog. Ser. 313, 27–41. Christy, J.H., 2011. Timing of hatching and release of larvae by brachyuran crabs: patterns, adaptive significance and control. Integr. Comp. Biol. 51, 62–72. Cohen, J.H., Forward Jr., R.B., 2005. Diel vertical migration in the marine copepod Calanopia americana. II. The proximate role of exogenous light cues and endogenous rhythms. Mar. Biol. 147, 399–410. Cohen, J.H., Forward Jr., R.B., 2009. Zooplankton diel vertical migration — a review of proximate control. Oceanogr. Mar. Biol. Annu. Rev. 47, 77–110. Cohen, J.H., Frank, T.M., 2006. Visual physiology of the Antarctic amphipod Abyssorchomene plebs. Biol. Bull. 211, 140–148. Cowen, R.K., Sponaugle, S., 2009. Larval dispersal and marine population connectivity. Annu. Rev. Mar. Sci. 1, 443–466. Delaney, D.G., Edwards, P.K., Leung, B., 2012. Predicting regional spread of non-native species using oceanographic models: validation and identification of gaps. Mar. Biol. 159, 269–282. Epifanio, C.E., 1988. Transport of invertebrate larvae between estuaries and the continental shelf. Trans. Am. Fish. Soc. Symp. Ser. 3, 104–114. Epifanio, C.E., 2013. Invasion biology of the Asian shore crab Hemigrapsus sanguineus: a review. J. Exp. Mar. Biol. Ecol. 441, 33–49. 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. Epifanio, C.E., Tilburg, C.E., 2008. Transport of blue crab larvae in the Middle Atlantic Bight: a wet and windy journey. J. Mar. Res. 66, 723–749. Epifanio, C.E., Dittel, A.I., Park, S., Schwalm, S., Fouts, A., 1998. Early life history of Hemigrapsus sanguineus, a non-indigenous crab in the Middle Atlantic Bight (USA). Mar. Ecol. Prog. Ser. 170, 231–238. Epifanio, C.E., Tilburg, C.E., Dittel, A.I., 2013. Abundance of invasive and native crab larvae in the mouth of Delaware Bay: Hemigrapsus sanguineus and Uca pugnax. J. Shellfish Res. 32, 543–550. Forward Jr., R.B., 1985. Behavioral responses of larvae of the crab Rhithropanopeus harrisii (Brachyura: Xanthidae) during diel vertical migration. Mar. Biol. 90, 9–18. Forward Jr., R.B., 2009. Larval biology of the crab Rhithropanopeus harrisii (Gould): a synthesis. Biol. Bull. 216, 243–256. Forward Jr., R.B., Cohen, J.H., 2004. Factors affecting the circatidal rhythm in vertical swimming of ovigerous blue crabs, Callinectes sapidus, involved in the spawning migration. J. Exp. Mar. Biol. Ecol. 299, 255–266. Forward Jr., R.B., Cronin, T.W., 1979. Spectral sensitivity of larvae from intertidal crustaceans. J. Comp. Phys. 133, 311–315. Forward Jr., R.B., Tankersley, R.A., 2001. Selective tidal stream transport of marine animals. Oceanogr. Mar. Biol. Annu. Rev. 39, 305–353. Forward Jr., R.B., Cronin, T.W., Stearns, D.E., 1984. Control of diel vertical migration: photoresponses of a larval crustacean. Limnol. Oceanogr. 29, 146–154. Forward Jr., R.B., Cronin, T.W., Douglass, J.K., 1988. The visual pigments of crabs II. Environmental adaptations. J. Comp. Physiol. A 162, 479–490. Forward Jr., R.B., Tankersley, R.A., Rittschof, D., 2001. Cues for metamorphosis of brachyuran crabs: an overview. Am. Zool. 41, 1108–1122. Gallego, A., North, E.W., Houde, E.D., 2012. Understanding and quantifying mortality in pelagic, early life stages of marine organisms — old challenges and new perspectives. J. Mar. Sys. 93, 1–3. Gibson, R.N., 2003. Go with the flow: tidal migration in marine animals. Hydrobiologia 503, 153–161. Hwang, S.G., Lee, C., Kim, C.H., 1993. Complete larval development of Hemigrapsus sanguineus (Decapoda, Brachyura, Grapsidae) reared in the laboratory. Kor. J. Syst. Zool. 9, 69–86. Johnsen, S., 2014. Hide and seek in the open sea: pelagic camouflage and visual countermeasures. Annu. Rev. Mar. Sci. 6, 369–392. Jones, M.B., Epifanio, C.E., 1995. Settlement of brachyuran megalopae in Delaware Bay: an analysis of time series data. Mar. Ecol. Prog. Ser. 125, 67–76. Levin, L.A., 2006. Recent progress in understanding larval dispersal: new directions and digressions. Integr. Comp. Biol. 46, 282–297.
López-Duarte, P.C., Tankersley, R.A., 2007. Circatidal swimming behavior of brachyuran crab zoea larvae: implications for ebb-tide transport. Mar. Biol. 151, 2037–2051. López-Duarte, P.C., Tankersley, R.A., 2009. Developmental shift in the selective tidalstream transport behavior of larvae of the fiddler crab Uca minax (LeConte). J. Exp. Mar. Biol. Ecol. 368, 169–180. López-Duarte, P.C., Christy, J.H., Tankersley, R.A., 2011. A behavioral mechanism for dispersal in fiddler crab larvae (genus Uca) varies with adult habitat, not phylogeny. Limnol. Oceanogr. 56, 1879–1892. Marshall, D.J., Morgan, S.G., 2011. Ecological and evolutionary consequences of linked lifehistory stages in the sea. Curr. Biol. 21, R718–R725. McConaugha, J.R., 1992. Decapod larvae: dispersal, mortality, and ecology. A working hypothesis. Am. Zool. 32, 512–523. McDermott, J., 1991. A breeding population of the western Pacific crab Hemigrapsus sanguineus (Crustacea: Decapoda: Grapsidae) established on the Atlantic coast of North America. Biol. Bull. 181, 195–198. Metaxas, A., Saunders, M., 2009. Quantifying the “bio-” components in biophysical models of larval transport in marine benthic invertebrates: advances and pitfalls. Bio. Bull. 216, 257–272. Morgan, S.G., 1995. Life and death in the plankton: larval mortality and adaptation. In: McEdward, L. (Ed.), Ecology of Marine Invertebrate Larvae. CRC Press, Boca Raton, pp. 279–321. Morgan, S.G., Anastasia, J.R., 2008. Behavioral tradeoff in estuarine larvae favors seaward migration over minimizing visibility to predators. Proc. Natl. Acad. Sci. USA 105, 222–227. Morgan, S.G., Christy, J.H., 1997. Planktivorous fishes as selective agents for reproductive synchrony. J. Exp. Mar. Biol. Ecol. 209, 89–101. Park, S., Epifanio, C.E., Grey, E.K., 2004. Behavior of larval Hemigrapsus sanguineus (de Haan) in response to gravity and pressure. J. Exp. Mar. Biol. Ecol. 307, 197–206. Park, S., Epifanio, C.E., Iglay, R.B., 2005. Patterns of larval release by the Asian shore crab Hemigrapsus sanguineus (De Haan): periodicity at diel and tidal frequencies. J. Shellfish Res. 24, 591–595. Pechenik, J.A., 2006. Larval experience and latent effects — metamorphosis is not a new beginning. Integr. Comp. Biol. 46, 323–333. Pineda, J., Hare, J.A., Sponaugle, S., 2007. Larval transport and dispersal in the coastal ocean and consequences for population connectivity. Oceanogr. 20, 22–39. Queiroga, H., Blanton, J., 2005. Interactions between behaviour and physical forcing in the control of horizontal transport of decapod crustacean larvae. Adv. Mar. Biol. 47, 109–214. Saigusa, M., Kawagoye, O., 1997. Circatidal rhythm of an intertidal crab, Hemigrapsus sanguineus: synchrony with unequal tide height and involvement of a lightresponse mechanism. Mar. Biol. 129, 87–96. Sanders, T.M., Garvine, R.W., 2001. Fresh water delivery to the continental shelf and subsequent mixing: an observational study. J. Geophys. Res. 106, 27087–27101. Schab, C.M., Park, S., Waidner, L.A., Epifanio, C.E., 2013. Return of the native: historical comparison of invasive and indigenous populations near the mouth of Delaware Bay. J. Shellfish Res. 32, 1–8. Steppe, C.N., Epifanio, C.E., 2006. Synoptic distribution of crab larvae near the mouth of Delaware Bay: influence of nearshore hydrographic regimes. Estuar. Coast. Shelf Sci. 70, 645–662. Sulkin, S.D., 1975. The influence of light in the depth regulation of crab larvae. Biol. Bull. 148, 333–343. Sulkin, S.D., 1984. Behavioral basis of depth regulation in the larvae of brachyuran crabs. Mar. Ecol. Prog. Ser. 15, 181–205. Sulkin, S.D., van Heukelem, W., Kelly, P., van Heukelem, L., 1980. The behavioral basis of larval recruitment in the crab Callinectes sapidus Rathbun: a laboratory investigation of ontogenetic changes in geotaxis and barokinesis. Biol. Bull. 159, 402–417. Tilburg, C.E., Reager, J.T., Whitney, M.M., 2005. The physics of blue crab larval recruitment in Delaware Bay: a model study. J. Mar. Res. 63, 471–495. Tilburg, C.E., Houser, L.T., Steppe, C.N., Garvine, R.W., Epifanio, C.E., 2006. Effects of coastal transport on larval patches: models and observations. Estuar. Coast. Shelf Sci. 67, 145–160. Tilburg, C.E., Dittel, A.I., Epifanio, C.E., 2009. High concentrations of blue crab (Callinectes sapidus) larvae along the offshore edge of a coastal current: effects of convergent circulation. Fish. Oceanogr. 18, 135–146. Tilburg, C.E., Dittel, A.I., Miller, D.C., Epifanio, C.E., 2011. Transport and retention of the mitten crab (Eriocheir sinensis) in a Mid-Atlantic estuary: predictions from a larval transport model. J. Mar. Res. 69, 137–165. Welch, J.M., Forward Jr., R.B., 2001. Flood tide transport of blue crab, Callinectes sapidus, postlarvae: behavioral responses to salinity and turbulence. Mar. Biol. 139, 911–918. Zeng, C., Naylor, E., 1996. Synchronization of endogenous tidal vertical migration rhythms in laboratory-hatched larvae of the crab Carcinus maenas. J. Exp. Mar. Biol. Ecol. 198, 269–289.