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The tolerance of juvenile stone crabs to hypoxia: Size matters
Journal of Experimental Marine Biology and Ecology 523 (2020) 151269
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Short Communication
The tolerance of juvenile stone crabs to hypoxia: Size matters Philip M. Gravinese
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Mote Marine Laboratory, Fisheries Ecology and Enhancement, 1600 Ken Thompson Way, Sarasota, FL 34236, United States
A R T I C LE I N FO
A B S T R A C T
Keywords: Stone crab Hypoxia Juveniles Menippe mercenaria, survivorship Crabs
Over the last fifty years, anthropogenic activities have increased nutrient loading into coastal habitats causing more frequent hypoxic events. Global climate change is expected to increase the frequency and severity of hypoxic conditions as temperatures continue to increase, which will pose physiological and metabolic limits on many coastal species. This study determined the survivorship and tolerance of the commercially important juvenile stone crab (Menippe mercenaria) during short-term hypoxic exposure under laboratory conditions. Small (2.0–3.0 mm carapace width) and large (5.0–18.0 mm carapace width) juveniles were gradually exposed to moderate hypoxic conditions (1.5 mg L−1 oxygen concentration) for 2 h and compared to individuals in normoxic conditions. Smaller juveniles were more sensitive than larger conspecifics and exhibited 80% mortality. Larger juveniles did not exhibit significant mortality (~12% total mortality); however, 35% of the larger crabs displayed immobile by the end of the experiment. These results indicate that juvenile stone crab tolerance to hypoxia is size-dependent and likely changes throughout ontogeny.
1. Introduction Dissolved oxygen concentrations in both the open ocean and in coastal habitats have been declining since the mid-20th century (Breitburg et al., 2018). Over the last fifty years, many hypoxic or low oxygen events in coastal areas have been associated with anthropogenic disturbances (Rabalais et al., 2009; Schmidtko et al., 2017). Increased urbanization and coastal development are diverting high-nutrient runoff, which is resulting in eutrophication and increasing the frequency of moderate to severe oxygen depletion events (Spicer, 2014; Breitburg et al., 2018). Increasing atmospheric CO2 concentrations are also warming the oceans and by 2100, sea surface ocean temperatures are expected to increase by 2–4 °C (IPCC, 2013). This warming trend will exacerbate oxygen depletion in shallow nutrient-enriched coastal systems by further decreasing oxygen solubility and increasing respiration rates of coastal biota (Gilbert et al., 2010; Altieri and Gedan, 2015). Shallow coastal habitats, such as seagrass beds and oyster reefs, serve as critical nursery habitats for the many commercially and ecologically important juvenile crustaceans. These habitats can experience substantial fluxes in dissolved oxygen concentrations driven by respiration and photosynthetic processes, which can result in hypoxic conditions occurring over a few hours or seasonal (several weeks) temporal scales (Johnson and Welsh, 1985; Kenney et al., 1988; Asmus et al., 1994). Hypoxic events can also occur in the aftermath of
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prolonged algae blooms. In some locations, like Florida's gulf coast, the harmful “red tide” alga Karenia brevis can result in large fish kills and the resulting decomposition eventually produces hypoxic or even anoxic events in many shallow nearshore habitats like seagrass beds and oyster reefs (Landsberg et al., 2009). Although red tide blooms can be episodic in Florida, the most recent bloom lasted for > 17 months from 2017 to 2018 and was responsible for large fish kills. Similarly, Florida Bay in Everglades National Park, which is a known nursery ground for juvenile crustaceans (including stone crabs), also suffers from chronic hypoxic events that are often driven by algal blooms (i.e., Synechococcus spp.; Glibert et al., 2009). The onset of hypoxic events caused by algae blooms can be rapid and may result in dissolved oxygen concentrations that are physiologically stressful or lethal for newly settled juvenile crabs, especially if individuals cannot emigrate into normoxic waters (Diaz and Rosenberg, 1995). Previous studies have used commercially important crustaceans as a model to evaluate the impacts of hypoxia. The evidence from those studies suggests that tolerance to hypoxia is likely dependent upon the duration of exposure and an individual's size because changes in oxygen demand, physiology, and sites of gas exchange differ throughout a crab's life-cycle (i.e., larvae vs. post-larvae vs. juvenile vs. adult; Spicer and Eriksson, 2003; Pörtner, 2010; Alter et al., 2015). For example, stage-V larvae of the squat lobster (Pleuroncodes monodon) were able to maintain their oxygen consumption (oxyconform) with decreasing oxygen tensions, while their megalopae stage switched to oxyregulatory
Corresponding author. E-mail address: [email protected].
https://doi.org/10.1016/j.jembe.2019.151269 Received 27 August 2019; Received in revised form 6 November 2019; Accepted 7 November 2019 0022-0981/ © 2019 Elsevier B.V. All rights reserved.
Journal of Experimental Marine Biology and Ecology 523 (2020) 151269
P.M. Gravinese
as individuals ranging between 2.0 and 3.0 mm CW ( μ = 2.5 mm CW ± 0.2 SD; n = 15 per treatment). The range of the small size class represented newly settled juveniles (J1–J2) that were likely within 15 days of metamorphosis (Krimsky and Epifanio, 2010). The larger juvenile size class was defined as individuals ranging between 5.0 and 18.0 mm CW ( μ = 9.0 mm CW ± 3.5 SD; n = 17 per treatment; > J5). Animals were then randomly assigned to either the normoxic (> 5 mg L−1 dissolved oxygen) or the hypoxic treatment (1.5 mg L−1 dissolved oxygen). Normoxic levels used in this experiment were within the range of dissolved oxygen observed over the last twenty years in Cedar Key, Florida (Robbins and Lisle, 2018). Hypoxia in shallow nearshore nursery habitats in Cedar Key is reported to be episodic and ephemeral (Robbins and Lisle, 2018). This experiment therefore exposed all juveniles to their respective treatment conditions for 2 h. Prior to starting experimentation, all juveniles were placed into clean 250 mL glass jars that were sealed with a screw-on lid and maintained at normoxic conditions (dissolved oxygen μ = 6.2 mg L−1 ± 0.44 SD). Each juvenile was maintained independently in its own jar, which served as the unit of replication throughout experimentation. The jar lids had a small hole, which allowed for inserting tubing and air stones that delivered the appropriate gas treatment. The dissolved oxygen was then gradually “ramped down” in the hypoxic treatment to the experimental set point (< 2 mg L−1) over a 30-min period (Fig. 1). The hypoxic treatment was achieved by gently bubbling ultrapure nitrogen gas into each juvenile's chamber. Dissolved oxygen (mg L−1) was recorded at the start (0 min), middle (15 min), and end (30 min/experiment start) of the “ramp down” period in both treatments using a dissolved oxygen probe (HI98193, Hannah Instruments), which was calibrated daily using a two-point calibration (100% saturation and 0% saturation). Dissolved oxygen levels were then recorded every 20 min throughout experimentation. The temperature (29.5 °C ± 0.6) during experimentation was set to approximate the summer Cedar Key seawater temperatures (https:// www.seatemperature.org/north-america/united-states/cedar-key-july. htm). The temperature was maintained by placing the experimental jars into a thermostatically controlled water bath. The water bath was digitally controlled using heaters and temperature probes, which were constantly monitored and maintained by AquaControllers (Apex System, Neptune). Salinity and temperature were also recorded at each sampling interval throughout experimentation using a conductivity meter (Orion Star A322; ThermoScientific).
behavior (i.e., maintain oxygen consumption rates independent of ambient oxygen concentrations; Yannicelli et al., 2013; Leiva et al., 2018). Megalopae of the king crab Paralithodes camtschaticus also switch from being oxyconformers to being oxyregulators after developing into newly settled juveniles (Nakanishi, 1987). Adults of the blue crab Callinectes sapidus are also more tolerant to moderate hypoxia than juvenile conspecifics, which provides additional evidence that the respiratory physiology changes during ontogeny in crustaceans (deFur et al., 1990; Stickle et al., 1989; Das and Stickle, 1993; Alter et al., 2015). Despite these ontogentic changes in the blue crab, however, neither life stage was able to survive short periods of severe hypoxia. Characterizing the tolerance of newly settled juveniles to hypoxic events therefore could provide insights to environmental changes that may impact the future harvest for commercially important crustacean species, an assessment that has yet to be performed for Florida's lucrative stone crab fishery. The Florida stone crab, Menippe mercenaria, is a valuable fishery in the southeastern United States. In Florida alone, stone crab landings support on average a ~US$25–30 million-a-year commercial and an active recreational fishery, however, the annual harvest has declined by ~30% since 1996 (Florida Fish and Wildlife Conservation Commission, 1998–2018; Muller et al., 2011). The stone crab life cycle is similar to other brachyuran crustaceans: the larvae are released in coastal waters and then migrate offshore where they complete larval development before migrating back into nearshore settlement sites (Gravinese, 2007; Gravinese, 2018). Juvenile settlement occurs during late summer and early autumn when elevated temperatures and periodic red tides can persist, causing both short and long term hypoxic events (Krimsky and Epifanio, 2008; Gravinese et al., 2019). Previous work has indicated that larval and sublegal stone crabs are sensitivity to red tides; however, no work has assessed their tolerance to the hypoxic conditions that may follow in the aftermath of a red tide bloom (Gravinese et al., 2018a; Gravinese et al., 2019). The duration of a hypoxic event (few hours to weeks) may therefore serve as density-independent factor that could limit the abundance of new recruits into the fishery. This study was designed to determine the effects of moderate hypoxic exposure on the survivorship and stress response in M. mercenaria juveniles. Understanding the impacts of hypoxia on the tolerance of juveniles will be useful for managers to identify potential bottlenecks that could limit long-term sustainability and management of new recruits into the fishery. 2. Materials and methods 2.1. Study site, collection and maintenance of experimental animals All experiments were conducted from June–August 2017 at Mote Marine Laboratory and Aquarium in Sarasota, Florida, USA. Juvenile stone crabs (N = 64; carapace width < 20 mm) were collected from Cedar Key, Florida, using commercial stone crab traps deployed by Florida Fish and Wildlife Research Institute's Independent Monitoring Program. Juvenile crabs were then transported back to Mote Marine Laboratory in Sarasota, Florida, where they were allowed to acclimate to laboratory conditions for 48 h. During acclimation all juveniles were maintained in independent glass jars (250 mL) and were fed a diet of enriched (Selcon lipid concentrate) 1-day old Artemia (Brine Shrimp Direct). Seawater was changed daily to remove uneaten food and minimize the build-up of metabolic wastes. Animals were maintained on a light:dark cycle (14 h day: 10 h night) that approximated the photoperiod at the time of collection. 2.2. Experimental design
Fig. 1. The mean ( ± SD) dissolved oxygen (mg L−1) concentrations for the normoxic (white circles) and hypoxic (black circles) treatment during the acclimation/ramp down period (0–30 min) vs. the experimental period (0–120 min).
After transport to the laboratory, the carapace width (CW) of each juvenile was measured using a digital caliper. Individuals were then separated into two size classes. The small juvenile size class was defined 2
Journal of Experimental Marine Biology and Ecology 523 (2020) 151269
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2.3. Determining juvenile tolerance The condition of juvenile stone crabs was monitored in each treatment at 20 min intervals for 2 h for both tolerance and survivorship. At each 20 min interval juveniles were scored as being: 1) alive, 2) immobile, or 3) dead. Individuals that were classified as alive were actively walking, immediately responsive to mechanical stimulation (i.e., upon gently prodding the animal attempted to avoid the probe), and aggressively defended against manipulation of their mouth parts. Immobile juveniles were defined as inactive or having limited movement (i.e., lethargic but able to walk), were occasionally responsive to mechanical stimulation, but still defended against manipulation of their mouth parts. Juveniles were classified as dead if they were unresponsive to repeated mechanical stimulation, displayed retracted legs, and allowed manipulation of their mouth parts. 2.4. Statistical analyses The effect of hypoxia on survivorship at each size class (juveniles 2.0–3.0 mm CW and juveniles 5.0–18.0 mm CW) was determined using a failure-time analysis (Cox proportional hazard model) using the survival package in R (Therneau, 2015), with juvenile death serving as the ‘event’, and time since the beginning of the experiment as the ‘time until an event occurs’. The Cox regression coefficients (i.e., hazard ratios) were used to estimate the likelihood an individual juvenile from each size class would experience mortality in the experimental treatment. Comparisons of survivorship among normoxic and hypoxic treatments were made using a Log-rank (LR) test. The stress scores used to characterize each juvenile's tolerance in each treatment were repeated measures and did not meet the assumptions of normality (Shapiro test: P < .001). A Friedman test was therefore used to determine differences in the stress score among treatments. A threshold was then determined for each size class by comparing the minimum amount of time that lapsed before a significant change in individual stress scores was observed using a Dunn's test for multiple pairwise comparisons using the Holm correction using the PMCMR package in R (Pohlert, 2019). All statistical analyses were performed with R software (R Development Core Team, 2016; version 3.6.1).
Fig. 2. Cumulative survivorship of small M. mercenaria juveniles (n = 15 per treatment; top panel) and large M. mercenaria juveniles (n = 17 per treatment; bottom panel) during exposure the hypoxic and normoxic treatment. All animals were exposed to the treatment conditions for 2 h. Shaded regions represent the 95% confidence intervals. The asterisk represents treatments that are significantly different at the α = 0.05 level.
3. Results
used to express the likelihood an individual would die under moderate hypoxic conditions. The hazard ratios indicated that juveniles exposed to moderate hypoxic conditions for 2 h were 8.3 times more likely to die than juveniles in normoxic conditions (95% CL: 2.0–39.2; P = .004). The decrease in juvenile crab survivorship, however, was largely dependent an individual's size class. Stone crabs in the smaller size class (2.0–3.0 mm CW) exhibited a significantly higher mortality in the hypoxic treatment (small juveniles: LR1 = 15.6, P < .0001), while larger juveniles (5.0–18.0 mm CW) did not exhibit a significant decrease in survivorship and were more tolerant to moderate hypoxia (large juveniles: LR1 = 0.40, P = .54). There was an 80% decrease in survivorship of the smaller juveniles when exposed to hypoxic conditions relative to the normoxic treatment (Fig. 2A), whereas larger juveniles only experienced an 11.7% decrease in survivorship relative to the normoxic treatment (Fig. 2B). The hazard ratios indicated that smaller juveniles exposed to moderate hypoxia were 19.7 times more likely to die than conspecifics in the normoxic treatment (95% CL: 2.5–153.2; P = .004). Although larger juveniles did not show a significant decrease in survivorship, the hazard ratios still indicated that larger crabs were 2.1 times more likely to die than conspecifics in the normoxic treatment (95% CL: 0.19–23.5; P = .50).
3.1. Experimental conditions After the 30-min ramp down period, the dissolved oxygen levels in the hypoxic treatment were maintained within a narrow range throughout the 2 h exposure period (hypoxic: 1.5 mg L−1 ± 0.52; Fig. 1). The normoxic treatment was also maintained within a narrow range after the acclimation period (normoxic: 5.9 mg L−1 ± 0.51). Salinity and temperature were also maintained within a narrow range throughout experimentation (Table 1). 3.2. Juvenile survivorship Juvenile stone crab survivorship was significantly reduced in the hypoxic treatment relative to the normoxic treatment (LR1 = 12.2, P = .0004). The Cox regression coefficients (i.e., hazard ratios) were Table 1 The mean ( ± SD) dissolved oxygen (mg L−1), temperature (°C) and salinity after the ramp down period in the hypoxic and normoxic treatments. Treatment
Dissolved oxygen
Salinity
Temperature
Normoxic Hypoxic
5.9 (0.15) 1.5 (0.15)
34.89 (1.0) 34.91 (1.0)
29.4 (0.6) 29.5 (0.7)
3.3. Juvenile tolerance The tolerance of small and large juvenile stone crabs to moderate 3
Journal of Experimental Marine Biology and Ecology 523 (2020) 151269
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Fig. 3. Relative frequency (%) of small (n = 15 per treatment) and large (n = 17 per treatment) juvenile M. mercenaria that were immobile (white bars) and dead (black bars) following exposure to normoxic (A and C) and hypoxic (B and D) conditions for 2 h. The asterisk represents the minimum time elapsed before a significant stress response was observed that was different from the normoxic treatment (control).
moderate hypoxia suggesting that short-term (e.g., 2 h) exposure in near-shore communities could influence recruitment success and increase the susceptibility of juveniles to predation resulting in even further decreases in survival. Smaller juvenile stone crabs were exceptionally sensitive to hypoxia and exhibited significant mortality (40% total mortality; 30.8% relative mortality) within 60 min of exposure, whereas the larger size class only experienced 11.7% mortality during the entire 2 h exposure period. The size-dependent sensitivity of stone crabs may be associated with metabolic rates, which scale allometrically with body mass in crustaceans (Glazier, 2006; Levia et al., 2018). The smaller size class used in this experiment ranged from 2.0–3.0 mm carapace width and likely represents newly metamorphosed juveniles (J1–J2 instar; 2.0 mm CW). Krimsky and Epifanio (2010) estimated this size class to include individuals that were, on average, < 15 days post-settlement. Newly settled juvenile crustaceans are still undergoing substantial developmental changes. The respiratory mechanisms used to physiologically tolerate lower dissolved oxygen conditions may not be fully functional or efficient during these earlier juvenile stages. For example, the structure of the oxygen carrying protein hemocyanin changes throughout crustacean development, which may influence metabolites that have an affinity for oxygen and aid in oxygen transport (Terwilliger and Brown, 1993; Terwilliger, 1998). Changes in hemocyanin throughout ontogeny may therefore partially explain the sensitivity of earlier stage juveniles to hypoxia as they are only a few days removed from the plankton at 2 mm carapace width (Terwilliger and Brown,
hypoxia was dependent upon both size class and exposure time, with smaller juveniles experiencing higher stress scores than conspecifics in normoxic conditions (Χ2 = 66.2, df = 6, P < .0001). Larger juveniles also showed significant stress relative to animals in the normoxic treatment (Χ2 = 38.2, df = 6, P < .0001; Fig. 3). Crabs maintained in the normoxic treatment did not experience a significant increase in stress over the course of the experiment (Fig. 3A and C). The threshold, or minimum time before a significant stress response was observed that was significantly different from the normoxic treatment, for smaller crabs was 60 min (P = .003; Fig. 3B). The threshold response at 60 min in smaller juveniles was representative of elevated stress in 46.2% (relative frequency) of the individuals (Fig. 3B). Although the larger crabs did not exhibit increased mortality, they did display significant sublethal stress, indicating that they were only slightly more tolerant to moderate hypoxia than smaller conspecifics. The exposure threshold in the larger juvenile size class was 100 min (P = .04; Fig. 3D), which was representative of immobility in 33.3% (relative frequency) of the larger juveniles. 4. Discussion The response of juvenile stone crabs to moderate hypoxia was largely driven by differences in an individual's size, with smaller crabs exhibiting significantly reduced survivorship relative to larger juveniles exposed to similar conditions. Although larger juveniles did not experience significant mortality they still displayed sublethal effects to 4
Journal of Experimental Marine Biology and Ecology 523 (2020) 151269
P.M. Gravinese
Furthermore, field studies should aim to correlate the success of new recruits into the fishery with fine-scale environmental changes like hypoxia.
1993; Spicer, 1995; Brown and Terwilliger, 1998). The results of this study suggest that stone crab responses to hypoxia co-vary not only with size but also with exposure time. Smaller juvenile stone crabs also displayed significant immobility after 60 min of exposure (Fig. 3B). The susceptibility of larger juveniles to hypoxia was also indicated by elevated stress and immobility, however, this response took almost two times longer to observe relative to smaller conspecifics (Fig. 3D). The size-specific tolerance of juvenile stone crabs to hypoxia presented here aligns with previous work for other crustaceans, which also demonstrates increasing tolerance with age and throughout ontogeny (Spicer, 1995; Eriksson and Baden, 1997; Leiva et al., 2018). Juvenile stone crabs also appear to be more sensitive to hypoxia than juvenile blue crabs, which can occupy similar post-settlement habitats in Florida. Das and Stickle (1993) found that juvenile blue crabs exposed to anoxic conditions survived for up to three days while exposure to hypoxic conditions (16% saturation - comparable to this experiment) resulted in mortality within six days. Previous work has suggested that this tolerance in blue crabs may provide the species an opportunity to avoid hypoxia by moving into normoxic waters; however, stone crabs do not swim like blue crabs, and animals in this experiment showed no evidence of increased restlessness (Pihl et al., 1991; Bell et al., 2009). In fact, lethargy was the earliest stress response observed in this experiment and always a precursor to mortality after exposure to hypoxic conditions. The high prevalence of immobility observed in both size classes of crabs suggests that hypoxia (and anoxia) may prevent avoidance behavior in juvenile stone crabs, which will likely limit the ability of juvenile crabs to emigrate into normoxic waters. This sublethal effect may be physiologically linked to the lower oxygen-carrying capacity of the hemolymph of juvenile crabs imposing constraints on oxygen transport thus limiting the activity of the crabs under certain conditions; which may explain the rapid onset of immobility observed in this study (Terwilliger and Brown, 1993). Although crabs in this study were only exposed to short-term hypoxia, the sublethal effects may be sufficient to cause significant mortality in some communities, especially if the hypoxic conditions persist, become more extreme, or become anoxic. Furthermore, 2 h of exposure may be more common in some near-shore communities than prolonged events, especially during the summer evenings when temperatures are elevated, after seasonal runoff events, or in the aftermath of a severe red tide bloom. The immobility response reported here is also likely to increase an individual's susceptibility to predation and suggests that emigration from nursery habitats may not be possible for this species. Hypoxic conditions in coastal habitats are increasing in frequency and duration, and models predict that seawater conditions will become increasingly warmer and more acidic toward the end of the century (IPCC, 2013; Breitburg et al., 2018). Ocean acidification and elevated temperature are linked to reduced oxygen saturation levels, which could exacerbate hypoxic conditions in the future (Pörtner et al., 2006; Pörtner, 2008; Melzner et al., 2013; Breitburg et al., 2018). Future increases in temperature will also accelerate metabolism and result in acidosis, which can ultimately impair organisms' oxygen transport systems (Pörtner and Farrell, 2008; Melzner et al., 2013). This is a concern for species, like stone crabs, that are living in subtropical and tropical habitats and close to their thermal limit. Historical trends suggest that seawater temperatures, projected for the 21st century, are warming five times faster than the 0.6 °C warming rate documented in the 20th century (Kerr, 2004). Furthermore, some stone crab habitats (i.e., Florida Keys) have already experienced an increase in seawater temperature over the last century (Kuffner et al., 2015). The crabs used in this study were exposed to moderate hypoxic levels (1.5 mg L−1) and additional decreases in dissolved oxygen driven by elevated temperature could exacerbate the physiological responses reported here, especially since stone crab larvae have been shown to be sensitive to warming (Gravinese et al., 2018b). Additional studies are necessary to determine the impacts of multiple stressors, such as elevated temperature and hypoxia, on juvenile stone crab physiological tolerances.
Declaration of Competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This research was supported by the Steinwachs Family Foundation. I thank R. Gandy and the Florida Fish and Wildlife Research Institute's Independent Monitoring Program who assisted in animal collection and L. Toth for providing both editorial comments and assistance in figure composition. Crabs were collected in compliance with Florida Fish and Wildlife Scientific Activity License #17-1868-F-SR. All data generated or analyzed during this study are included in this published article. References Alter, K., Paschke, K., Gebauer, P., Cumillaf, J.P., Pörtner, H.O., 2015. Differential physiological responses to oxygen availability in early life stages of decapods developing in distinct environments. Mar. Biol. 162 (5), 1111–1124. Altieri, A.H., Gedan, K.B., 2015. Climate change and dead zones. Glob. Chang. Biol. 21, 1395–1406. https://doi.org/10.1111/gcb.12754. pmid: 25385668. Asmus, R.M., Asmus, H., Willie, A., Zubillaga, G.F., Reise, K., 1994. Complementary oxygen and nutrient fluxes in seagrass and mussel banks? In: Dyer, K.R., Orth, R.J. (Eds.), Changes in Fluxes in Estuaries: Implications from Sciences to Management. Olsen and Olsen, Fredensborgg, pp. 227–237. Bell, G.W., Eggleston, D.B., Noga, E.J., 2009. Environmental and physiological controls of blue crab avoidance behavior during exposure to hypoxia. Biol. Bull. 217 (2), 161–172. Breitburg, D., Levin, L.A., Oschlies, A., Grégoire, M., Chavez, F.P., Conley, D.J., Garçon, V., Gilbert, D., Gutiérrez, D., Isensee, K., Jacinto, G.S., 2018. Declining oxygen in the global ocean and coastal waters. Science 359 (6371), eaam7240. Brown, A.C., Terwilliger, N.B., 1998. Ontogeny of hemocyanin function in the Dungeness crab Cancer magister: hemolymph modulation of hemocyanin oxygen-binding. J. Exp. Biol. 201 (6), 819–826. Das, T., Stickle, W.B., 1993. Sensitivity of crabs Callinectes sapidus and C. similis and the gastropod Stramonita haemastoma to hypoxia and anoxia. Mar. Ecol. Prog. Ser. 98, 263–274. Defur, P.L., Mangum, C.P., Reese, J.E., 1990. Respiratory responses of the blue crab Callinectes sapidus to long-term hypoxia. Biol. Bull. 178 (1), 46–54. R Development Core Team, 2016. R: A language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria ISBN 3–900051–07-0. http://www.R-project.org. Diaz, R.J., Rosenberg, R., 1995. Marine benthic hypoxia: a review of its ecological effects and the behavioural responses of benthic macrofauna. Oceanogr. Mar. Biol. Annu. Rev. 33, 245–303. Eriksson, S.P., Baden, S.P., 1997. Behaviour and tolerance to hypoxia in juvenile Norway lobster (Nephrops norvegicus) of different ages. Mar. Biol. 128 (1), 49–54. Florida Fish and Wildlife Conservation Commission, 1998–2018. Commercial Fisheries Landing Summaries. https://public.myfwc.com/FWRI/PFDM/ReportCreator.aspx (Accessed 1 July 2019). Gilbert, D., Rabalais, N.N., Díaz, R.J., Zhang, J., 2010. Evidence for greater oxygen decline rates in the coastal ocean than in the open ocean. Biogeosciences. 7, 2283–2296. https://doi.org/10.5194/bg-7-2283-2010. Glazier, D.S., 2006. The 3/4-power law is not universal: evolution of isometric, ontogenetic metabolic scaling in pelagic animals. Biosci. 56 (4), 325–332. Glibert, P.M., Heil, C.A., Rudnick, D.T., Madden, C.J., Boyer, J.N., Kelly, S., 2009. Florida bay: water quality status and trends, historic and emerging algal bloom problems. Contrib. Mar. Sci. 38, 5–17. Gravinese, P.M., 2007. Behavioral Basis for Depth Regulation in the Larvae of the Florida Stone Crab, Menippe mercenaria. M.S. thesis. Florida Institute of Technology 86 pp. Gravinese, P.M., 2018. Vertical swimming behavior in larvae of the Florida stone crab Menippe mercenaria. J. Plankton Res. 40 (6), 643–654. Gravinese, P.M., Kronstadt, S.M., Clemente, T., Cole, C., Blum, P., Henry, M.S., Pierce, R.H., Lovko, V.J., 2018a. The effects of red tide (Karenia brevis) on reflex impairment and mortality of sublegal Florida stone crabs, Menippe mercenaria. Mar. Environ. Res. 137, 145–148. Gravinese, P.M., Enochs, I.C., Manzello, D.P., van Woesik, R., 2018b. Warming and pCO2 effects on Florida stone crab larvae. Estuar. Coast. Shelf Sci. 204, 193–201. Gravinese, P.M., Saso, E., Lovko, V.J., Blum, P., Cole, C., Pierce, R.H., 2019. Karenia brevis causes high mortality and impaired swimming behavior of Florida stone crab larvae. Harmful Algae 84, 188–194. Intergovernmental Panel on Climate Change, 2013. The Physical Science Basis. Working Group I Contribution to the 5th Assessment Report of the Intergovernmental Panel on
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Journal of Experimental Marine Biology and Ecology journal homepage: www.elsevier.com/locate/jembe
Short Communication
The tolerance of juvenile stone crabs to hypoxia: Size matters Philip M. Gravinese
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Mote Marine Laboratory, Fisheries Ecology and Enhancement, 1600 Ken Thompson Way, Sarasota, FL 34236, United States
A R T I C LE I N FO
A B S T R A C T
Keywords: Stone crab Hypoxia Juveniles Menippe mercenaria, survivorship Crabs
Over the last fifty years, anthropogenic activities have increased nutrient loading into coastal habitats causing more frequent hypoxic events. Global climate change is expected to increase the frequency and severity of hypoxic conditions as temperatures continue to increase, which will pose physiological and metabolic limits on many coastal species. This study determined the survivorship and tolerance of the commercially important juvenile stone crab (Menippe mercenaria) during short-term hypoxic exposure under laboratory conditions. Small (2.0–3.0 mm carapace width) and large (5.0–18.0 mm carapace width) juveniles were gradually exposed to moderate hypoxic conditions (1.5 mg L−1 oxygen concentration) for 2 h and compared to individuals in normoxic conditions. Smaller juveniles were more sensitive than larger conspecifics and exhibited 80% mortality. Larger juveniles did not exhibit significant mortality (~12% total mortality); however, 35% of the larger crabs displayed immobile by the end of the experiment. These results indicate that juvenile stone crab tolerance to hypoxia is size-dependent and likely changes throughout ontogeny.
1. Introduction Dissolved oxygen concentrations in both the open ocean and in coastal habitats have been declining since the mid-20th century (Breitburg et al., 2018). Over the last fifty years, many hypoxic or low oxygen events in coastal areas have been associated with anthropogenic disturbances (Rabalais et al., 2009; Schmidtko et al., 2017). Increased urbanization and coastal development are diverting high-nutrient runoff, which is resulting in eutrophication and increasing the frequency of moderate to severe oxygen depletion events (Spicer, 2014; Breitburg et al., 2018). Increasing atmospheric CO2 concentrations are also warming the oceans and by 2100, sea surface ocean temperatures are expected to increase by 2–4 °C (IPCC, 2013). This warming trend will exacerbate oxygen depletion in shallow nutrient-enriched coastal systems by further decreasing oxygen solubility and increasing respiration rates of coastal biota (Gilbert et al., 2010; Altieri and Gedan, 2015). Shallow coastal habitats, such as seagrass beds and oyster reefs, serve as critical nursery habitats for the many commercially and ecologically important juvenile crustaceans. These habitats can experience substantial fluxes in dissolved oxygen concentrations driven by respiration and photosynthetic processes, which can result in hypoxic conditions occurring over a few hours or seasonal (several weeks) temporal scales (Johnson and Welsh, 1985; Kenney et al., 1988; Asmus et al., 1994). Hypoxic events can also occur in the aftermath of
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prolonged algae blooms. In some locations, like Florida's gulf coast, the harmful “red tide” alga Karenia brevis can result in large fish kills and the resulting decomposition eventually produces hypoxic or even anoxic events in many shallow nearshore habitats like seagrass beds and oyster reefs (Landsberg et al., 2009). Although red tide blooms can be episodic in Florida, the most recent bloom lasted for > 17 months from 2017 to 2018 and was responsible for large fish kills. Similarly, Florida Bay in Everglades National Park, which is a known nursery ground for juvenile crustaceans (including stone crabs), also suffers from chronic hypoxic events that are often driven by algal blooms (i.e., Synechococcus spp.; Glibert et al., 2009). The onset of hypoxic events caused by algae blooms can be rapid and may result in dissolved oxygen concentrations that are physiologically stressful or lethal for newly settled juvenile crabs, especially if individuals cannot emigrate into normoxic waters (Diaz and Rosenberg, 1995). Previous studies have used commercially important crustaceans as a model to evaluate the impacts of hypoxia. The evidence from those studies suggests that tolerance to hypoxia is likely dependent upon the duration of exposure and an individual's size because changes in oxygen demand, physiology, and sites of gas exchange differ throughout a crab's life-cycle (i.e., larvae vs. post-larvae vs. juvenile vs. adult; Spicer and Eriksson, 2003; Pörtner, 2010; Alter et al., 2015). For example, stage-V larvae of the squat lobster (Pleuroncodes monodon) were able to maintain their oxygen consumption (oxyconform) with decreasing oxygen tensions, while their megalopae stage switched to oxyregulatory
Corresponding author. E-mail address: [email protected].
https://doi.org/10.1016/j.jembe.2019.151269 Received 27 August 2019; Received in revised form 6 November 2019; Accepted 7 November 2019 0022-0981/ © 2019 Elsevier B.V. All rights reserved.
Journal of Experimental Marine Biology and Ecology 523 (2020) 151269
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as individuals ranging between 2.0 and 3.0 mm CW ( μ = 2.5 mm CW ± 0.2 SD; n = 15 per treatment). The range of the small size class represented newly settled juveniles (J1–J2) that were likely within 15 days of metamorphosis (Krimsky and Epifanio, 2010). The larger juvenile size class was defined as individuals ranging between 5.0 and 18.0 mm CW ( μ = 9.0 mm CW ± 3.5 SD; n = 17 per treatment; > J5). Animals were then randomly assigned to either the normoxic (> 5 mg L−1 dissolved oxygen) or the hypoxic treatment (1.5 mg L−1 dissolved oxygen). Normoxic levels used in this experiment were within the range of dissolved oxygen observed over the last twenty years in Cedar Key, Florida (Robbins and Lisle, 2018). Hypoxia in shallow nearshore nursery habitats in Cedar Key is reported to be episodic and ephemeral (Robbins and Lisle, 2018). This experiment therefore exposed all juveniles to their respective treatment conditions for 2 h. Prior to starting experimentation, all juveniles were placed into clean 250 mL glass jars that were sealed with a screw-on lid and maintained at normoxic conditions (dissolved oxygen μ = 6.2 mg L−1 ± 0.44 SD). Each juvenile was maintained independently in its own jar, which served as the unit of replication throughout experimentation. The jar lids had a small hole, which allowed for inserting tubing and air stones that delivered the appropriate gas treatment. The dissolved oxygen was then gradually “ramped down” in the hypoxic treatment to the experimental set point (< 2 mg L−1) over a 30-min period (Fig. 1). The hypoxic treatment was achieved by gently bubbling ultrapure nitrogen gas into each juvenile's chamber. Dissolved oxygen (mg L−1) was recorded at the start (0 min), middle (15 min), and end (30 min/experiment start) of the “ramp down” period in both treatments using a dissolved oxygen probe (HI98193, Hannah Instruments), which was calibrated daily using a two-point calibration (100% saturation and 0% saturation). Dissolved oxygen levels were then recorded every 20 min throughout experimentation. The temperature (29.5 °C ± 0.6) during experimentation was set to approximate the summer Cedar Key seawater temperatures (https:// www.seatemperature.org/north-america/united-states/cedar-key-july. htm). The temperature was maintained by placing the experimental jars into a thermostatically controlled water bath. The water bath was digitally controlled using heaters and temperature probes, which were constantly monitored and maintained by AquaControllers (Apex System, Neptune). Salinity and temperature were also recorded at each sampling interval throughout experimentation using a conductivity meter (Orion Star A322; ThermoScientific).
behavior (i.e., maintain oxygen consumption rates independent of ambient oxygen concentrations; Yannicelli et al., 2013; Leiva et al., 2018). Megalopae of the king crab Paralithodes camtschaticus also switch from being oxyconformers to being oxyregulators after developing into newly settled juveniles (Nakanishi, 1987). Adults of the blue crab Callinectes sapidus are also more tolerant to moderate hypoxia than juvenile conspecifics, which provides additional evidence that the respiratory physiology changes during ontogeny in crustaceans (deFur et al., 1990; Stickle et al., 1989; Das and Stickle, 1993; Alter et al., 2015). Despite these ontogentic changes in the blue crab, however, neither life stage was able to survive short periods of severe hypoxia. Characterizing the tolerance of newly settled juveniles to hypoxic events therefore could provide insights to environmental changes that may impact the future harvest for commercially important crustacean species, an assessment that has yet to be performed for Florida's lucrative stone crab fishery. The Florida stone crab, Menippe mercenaria, is a valuable fishery in the southeastern United States. In Florida alone, stone crab landings support on average a ~US$25–30 million-a-year commercial and an active recreational fishery, however, the annual harvest has declined by ~30% since 1996 (Florida Fish and Wildlife Conservation Commission, 1998–2018; Muller et al., 2011). The stone crab life cycle is similar to other brachyuran crustaceans: the larvae are released in coastal waters and then migrate offshore where they complete larval development before migrating back into nearshore settlement sites (Gravinese, 2007; Gravinese, 2018). Juvenile settlement occurs during late summer and early autumn when elevated temperatures and periodic red tides can persist, causing both short and long term hypoxic events (Krimsky and Epifanio, 2008; Gravinese et al., 2019). Previous work has indicated that larval and sublegal stone crabs are sensitivity to red tides; however, no work has assessed their tolerance to the hypoxic conditions that may follow in the aftermath of a red tide bloom (Gravinese et al., 2018a; Gravinese et al., 2019). The duration of a hypoxic event (few hours to weeks) may therefore serve as density-independent factor that could limit the abundance of new recruits into the fishery. This study was designed to determine the effects of moderate hypoxic exposure on the survivorship and stress response in M. mercenaria juveniles. Understanding the impacts of hypoxia on the tolerance of juveniles will be useful for managers to identify potential bottlenecks that could limit long-term sustainability and management of new recruits into the fishery. 2. Materials and methods 2.1. Study site, collection and maintenance of experimental animals All experiments were conducted from June–August 2017 at Mote Marine Laboratory and Aquarium in Sarasota, Florida, USA. Juvenile stone crabs (N = 64; carapace width < 20 mm) were collected from Cedar Key, Florida, using commercial stone crab traps deployed by Florida Fish and Wildlife Research Institute's Independent Monitoring Program. Juvenile crabs were then transported back to Mote Marine Laboratory in Sarasota, Florida, where they were allowed to acclimate to laboratory conditions for 48 h. During acclimation all juveniles were maintained in independent glass jars (250 mL) and were fed a diet of enriched (Selcon lipid concentrate) 1-day old Artemia (Brine Shrimp Direct). Seawater was changed daily to remove uneaten food and minimize the build-up of metabolic wastes. Animals were maintained on a light:dark cycle (14 h day: 10 h night) that approximated the photoperiod at the time of collection. 2.2. Experimental design
Fig. 1. The mean ( ± SD) dissolved oxygen (mg L−1) concentrations for the normoxic (white circles) and hypoxic (black circles) treatment during the acclimation/ramp down period (0–30 min) vs. the experimental period (0–120 min).
After transport to the laboratory, the carapace width (CW) of each juvenile was measured using a digital caliper. Individuals were then separated into two size classes. The small juvenile size class was defined 2
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2.3. Determining juvenile tolerance The condition of juvenile stone crabs was monitored in each treatment at 20 min intervals for 2 h for both tolerance and survivorship. At each 20 min interval juveniles were scored as being: 1) alive, 2) immobile, or 3) dead. Individuals that were classified as alive were actively walking, immediately responsive to mechanical stimulation (i.e., upon gently prodding the animal attempted to avoid the probe), and aggressively defended against manipulation of their mouth parts. Immobile juveniles were defined as inactive or having limited movement (i.e., lethargic but able to walk), were occasionally responsive to mechanical stimulation, but still defended against manipulation of their mouth parts. Juveniles were classified as dead if they were unresponsive to repeated mechanical stimulation, displayed retracted legs, and allowed manipulation of their mouth parts. 2.4. Statistical analyses The effect of hypoxia on survivorship at each size class (juveniles 2.0–3.0 mm CW and juveniles 5.0–18.0 mm CW) was determined using a failure-time analysis (Cox proportional hazard model) using the survival package in R (Therneau, 2015), with juvenile death serving as the ‘event’, and time since the beginning of the experiment as the ‘time until an event occurs’. The Cox regression coefficients (i.e., hazard ratios) were used to estimate the likelihood an individual juvenile from each size class would experience mortality in the experimental treatment. Comparisons of survivorship among normoxic and hypoxic treatments were made using a Log-rank (LR) test. The stress scores used to characterize each juvenile's tolerance in each treatment were repeated measures and did not meet the assumptions of normality (Shapiro test: P < .001). A Friedman test was therefore used to determine differences in the stress score among treatments. A threshold was then determined for each size class by comparing the minimum amount of time that lapsed before a significant change in individual stress scores was observed using a Dunn's test for multiple pairwise comparisons using the Holm correction using the PMCMR package in R (Pohlert, 2019). All statistical analyses were performed with R software (R Development Core Team, 2016; version 3.6.1).
Fig. 2. Cumulative survivorship of small M. mercenaria juveniles (n = 15 per treatment; top panel) and large M. mercenaria juveniles (n = 17 per treatment; bottom panel) during exposure the hypoxic and normoxic treatment. All animals were exposed to the treatment conditions for 2 h. Shaded regions represent the 95% confidence intervals. The asterisk represents treatments that are significantly different at the α = 0.05 level.
3. Results
used to express the likelihood an individual would die under moderate hypoxic conditions. The hazard ratios indicated that juveniles exposed to moderate hypoxic conditions for 2 h were 8.3 times more likely to die than juveniles in normoxic conditions (95% CL: 2.0–39.2; P = .004). The decrease in juvenile crab survivorship, however, was largely dependent an individual's size class. Stone crabs in the smaller size class (2.0–3.0 mm CW) exhibited a significantly higher mortality in the hypoxic treatment (small juveniles: LR1 = 15.6, P < .0001), while larger juveniles (5.0–18.0 mm CW) did not exhibit a significant decrease in survivorship and were more tolerant to moderate hypoxia (large juveniles: LR1 = 0.40, P = .54). There was an 80% decrease in survivorship of the smaller juveniles when exposed to hypoxic conditions relative to the normoxic treatment (Fig. 2A), whereas larger juveniles only experienced an 11.7% decrease in survivorship relative to the normoxic treatment (Fig. 2B). The hazard ratios indicated that smaller juveniles exposed to moderate hypoxia were 19.7 times more likely to die than conspecifics in the normoxic treatment (95% CL: 2.5–153.2; P = .004). Although larger juveniles did not show a significant decrease in survivorship, the hazard ratios still indicated that larger crabs were 2.1 times more likely to die than conspecifics in the normoxic treatment (95% CL: 0.19–23.5; P = .50).
3.1. Experimental conditions After the 30-min ramp down period, the dissolved oxygen levels in the hypoxic treatment were maintained within a narrow range throughout the 2 h exposure period (hypoxic: 1.5 mg L−1 ± 0.52; Fig. 1). The normoxic treatment was also maintained within a narrow range after the acclimation period (normoxic: 5.9 mg L−1 ± 0.51). Salinity and temperature were also maintained within a narrow range throughout experimentation (Table 1). 3.2. Juvenile survivorship Juvenile stone crab survivorship was significantly reduced in the hypoxic treatment relative to the normoxic treatment (LR1 = 12.2, P = .0004). The Cox regression coefficients (i.e., hazard ratios) were Table 1 The mean ( ± SD) dissolved oxygen (mg L−1), temperature (°C) and salinity after the ramp down period in the hypoxic and normoxic treatments. Treatment
Dissolved oxygen
Salinity
Temperature
Normoxic Hypoxic
5.9 (0.15) 1.5 (0.15)
34.89 (1.0) 34.91 (1.0)
29.4 (0.6) 29.5 (0.7)
3.3. Juvenile tolerance The tolerance of small and large juvenile stone crabs to moderate 3
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Fig. 3. Relative frequency (%) of small (n = 15 per treatment) and large (n = 17 per treatment) juvenile M. mercenaria that were immobile (white bars) and dead (black bars) following exposure to normoxic (A and C) and hypoxic (B and D) conditions for 2 h. The asterisk represents the minimum time elapsed before a significant stress response was observed that was different from the normoxic treatment (control).
moderate hypoxia suggesting that short-term (e.g., 2 h) exposure in near-shore communities could influence recruitment success and increase the susceptibility of juveniles to predation resulting in even further decreases in survival. Smaller juvenile stone crabs were exceptionally sensitive to hypoxia and exhibited significant mortality (40% total mortality; 30.8% relative mortality) within 60 min of exposure, whereas the larger size class only experienced 11.7% mortality during the entire 2 h exposure period. The size-dependent sensitivity of stone crabs may be associated with metabolic rates, which scale allometrically with body mass in crustaceans (Glazier, 2006; Levia et al., 2018). The smaller size class used in this experiment ranged from 2.0–3.0 mm carapace width and likely represents newly metamorphosed juveniles (J1–J2 instar; 2.0 mm CW). Krimsky and Epifanio (2010) estimated this size class to include individuals that were, on average, < 15 days post-settlement. Newly settled juvenile crustaceans are still undergoing substantial developmental changes. The respiratory mechanisms used to physiologically tolerate lower dissolved oxygen conditions may not be fully functional or efficient during these earlier juvenile stages. For example, the structure of the oxygen carrying protein hemocyanin changes throughout crustacean development, which may influence metabolites that have an affinity for oxygen and aid in oxygen transport (Terwilliger and Brown, 1993; Terwilliger, 1998). Changes in hemocyanin throughout ontogeny may therefore partially explain the sensitivity of earlier stage juveniles to hypoxia as they are only a few days removed from the plankton at 2 mm carapace width (Terwilliger and Brown,
hypoxia was dependent upon both size class and exposure time, with smaller juveniles experiencing higher stress scores than conspecifics in normoxic conditions (Χ2 = 66.2, df = 6, P < .0001). Larger juveniles also showed significant stress relative to animals in the normoxic treatment (Χ2 = 38.2, df = 6, P < .0001; Fig. 3). Crabs maintained in the normoxic treatment did not experience a significant increase in stress over the course of the experiment (Fig. 3A and C). The threshold, or minimum time before a significant stress response was observed that was significantly different from the normoxic treatment, for smaller crabs was 60 min (P = .003; Fig. 3B). The threshold response at 60 min in smaller juveniles was representative of elevated stress in 46.2% (relative frequency) of the individuals (Fig. 3B). Although the larger crabs did not exhibit increased mortality, they did display significant sublethal stress, indicating that they were only slightly more tolerant to moderate hypoxia than smaller conspecifics. The exposure threshold in the larger juvenile size class was 100 min (P = .04; Fig. 3D), which was representative of immobility in 33.3% (relative frequency) of the larger juveniles. 4. Discussion The response of juvenile stone crabs to moderate hypoxia was largely driven by differences in an individual's size, with smaller crabs exhibiting significantly reduced survivorship relative to larger juveniles exposed to similar conditions. Although larger juveniles did not experience significant mortality they still displayed sublethal effects to 4
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Furthermore, field studies should aim to correlate the success of new recruits into the fishery with fine-scale environmental changes like hypoxia.
1993; Spicer, 1995; Brown and Terwilliger, 1998). The results of this study suggest that stone crab responses to hypoxia co-vary not only with size but also with exposure time. Smaller juvenile stone crabs also displayed significant immobility after 60 min of exposure (Fig. 3B). The susceptibility of larger juveniles to hypoxia was also indicated by elevated stress and immobility, however, this response took almost two times longer to observe relative to smaller conspecifics (Fig. 3D). The size-specific tolerance of juvenile stone crabs to hypoxia presented here aligns with previous work for other crustaceans, which also demonstrates increasing tolerance with age and throughout ontogeny (Spicer, 1995; Eriksson and Baden, 1997; Leiva et al., 2018). Juvenile stone crabs also appear to be more sensitive to hypoxia than juvenile blue crabs, which can occupy similar post-settlement habitats in Florida. Das and Stickle (1993) found that juvenile blue crabs exposed to anoxic conditions survived for up to three days while exposure to hypoxic conditions (16% saturation - comparable to this experiment) resulted in mortality within six days. Previous work has suggested that this tolerance in blue crabs may provide the species an opportunity to avoid hypoxia by moving into normoxic waters; however, stone crabs do not swim like blue crabs, and animals in this experiment showed no evidence of increased restlessness (Pihl et al., 1991; Bell et al., 2009). In fact, lethargy was the earliest stress response observed in this experiment and always a precursor to mortality after exposure to hypoxic conditions. The high prevalence of immobility observed in both size classes of crabs suggests that hypoxia (and anoxia) may prevent avoidance behavior in juvenile stone crabs, which will likely limit the ability of juvenile crabs to emigrate into normoxic waters. This sublethal effect may be physiologically linked to the lower oxygen-carrying capacity of the hemolymph of juvenile crabs imposing constraints on oxygen transport thus limiting the activity of the crabs under certain conditions; which may explain the rapid onset of immobility observed in this study (Terwilliger and Brown, 1993). Although crabs in this study were only exposed to short-term hypoxia, the sublethal effects may be sufficient to cause significant mortality in some communities, especially if the hypoxic conditions persist, become more extreme, or become anoxic. Furthermore, 2 h of exposure may be more common in some near-shore communities than prolonged events, especially during the summer evenings when temperatures are elevated, after seasonal runoff events, or in the aftermath of a severe red tide bloom. The immobility response reported here is also likely to increase an individual's susceptibility to predation and suggests that emigration from nursery habitats may not be possible for this species. Hypoxic conditions in coastal habitats are increasing in frequency and duration, and models predict that seawater conditions will become increasingly warmer and more acidic toward the end of the century (IPCC, 2013; Breitburg et al., 2018). Ocean acidification and elevated temperature are linked to reduced oxygen saturation levels, which could exacerbate hypoxic conditions in the future (Pörtner et al., 2006; Pörtner, 2008; Melzner et al., 2013; Breitburg et al., 2018). Future increases in temperature will also accelerate metabolism and result in acidosis, which can ultimately impair organisms' oxygen transport systems (Pörtner and Farrell, 2008; Melzner et al., 2013). This is a concern for species, like stone crabs, that are living in subtropical and tropical habitats and close to their thermal limit. Historical trends suggest that seawater temperatures, projected for the 21st century, are warming five times faster than the 0.6 °C warming rate documented in the 20th century (Kerr, 2004). Furthermore, some stone crab habitats (i.e., Florida Keys) have already experienced an increase in seawater temperature over the last century (Kuffner et al., 2015). The crabs used in this study were exposed to moderate hypoxic levels (1.5 mg L−1) and additional decreases in dissolved oxygen driven by elevated temperature could exacerbate the physiological responses reported here, especially since stone crab larvae have been shown to be sensitive to warming (Gravinese et al., 2018b). Additional studies are necessary to determine the impacts of multiple stressors, such as elevated temperature and hypoxia, on juvenile stone crab physiological tolerances.
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