No compromise between metabolism and behavior of decorator crabs in reduced pH conditions

Many marine calcifiers experience metabolic costs when exposed to experimental ocean acidification conditions, potentially limiting the energy available to support regulatory processes and behaviors. Decorator crabs expend energy on decoration camouflage and may face acute trade-offs under environmental stress. We hypothesized that under reduced pH conditions, decorator crabs will be energy limited and allocate energy towards growth and calcification at the expense of decoration behavior. Decorator crabs, Pelia tumida, were exposed to ambient (8.01) and reduced (7.74) pH conditions for five weeks. Half of the animals in each treatment were given sponge to decorate with. Animals were analyzed for changes in body mass, exoskeleton mineral content (Ca and Mg), organic content (a proxy for metabolism), and decoration behavior (sponge mass and percent cover). Overall, decorator crabs showed no signs of energy limitation under reduced pH conditions. Exoskeleton mineral content, body mass, and organic content of crabs remained the same across pH and decoration treatments, with no effect of reduced pH on decoration behavior. Despite being a relatively inactive, osmoconforming species, Pelia tumida is able to maintain multiple regulatory processes and behavior when exposed to environmental pH stress, which underscores the complexity of responses within Crustacea to ocean acidification conditions.

Water chemistry. Daily readings of pH and temperature were taken from each header tank and experimental cup using a portable probe (HQ40d, probe PHC201, accuracy 0.01 pH, 0.01 °C temperature, Hach, Loveland, Colorado, United States). All pH probes were calibrated weekly using NBS buffer solutions. Water samples were also taken from each header tank and a subset of experimental cups at the beginning (1 random cup from each pH treatment) and end (2 random cups from each pH treatment) of the experiment in accordance with standard operating procedures 44 . Water samples were submitted to the Dickson laboratory at SIO for analysis of pH SWS , density-based salinity, and total alkalinity (TA) at 25 °C (Table 1). Carbonate and aragonite saturation states, concentrations of carbonate and bicarbonate, and pCO 2 were calculated using CO 2 Sys 2.01 (Table 1). For calculations, dissociation constants of K 1 and K 2 were from Mehrbach 45 , refit by Dickson and Millero 46 . The HSO 4 constant was from Dickson 47 , the [B} T value was from Uppstrom 48 , and the seawater pH scale was used. Water samples were used to calculate the average difference between the Hach pH probe readings and spectrophotometric pH values per sampling set. The mean pH offset was then used to correct daily measures of pH from the Hach probe (35 total per cup), which were then averaged for all experimental cups in each treatment (Table 1).
Decoration behavior. Immediately prior to the start of the experiment, crabs were kept in water to minimize stress and carefully cleaned of all decorations under a dissecting microscope using tweezers and a probe. Caution was taken to minimize damage to setae. Animals were then patted dry using Kim wipes, measured for carapace width and length using digital calipers, and weighed on a balance (RADWAG PS 3500/C/2, RADWAG, Radom, Poland). This procedure did not induce noticeable stress in P. tumida, which is characterized by overturning and difficulty with righting (personal observation).
Within each experimental treatment (ambient and reduced pH), 12 crabs were allowed to decorate (decorated crabs) while the other 12 crabs were not allowed to decorate (undecorated crabs). Decorations were provided each week only to the decorated crabs in each treatment. Pelia tumida primarily uses sponge for decoration, but it also covers with red algae, bryozoans, and hydroids (pers. observ.). As most animals were thoroughly covered in sponge at the time of collection, this was the decoration chosen for the experiment. Two sponge species, Halichondria panacea and Haliclona permollis, were collected from the Scripps Pier flume and held in a separate flow-through tank. Preliminary observation revealed that crabs showed no preference between the two sponge species. New sponge cut to 3 cm × 3 cm × 1 cm pieces were given to each decorated crab once per week so that they could decorate ad libitum for 24 hours, after which time the sponge was removed. Decorator crabs typically decorate immediately once a decoration source is provided, so this time was considered sufficient for crabs to decorate 49,50 .
Weekly photographs were taken of the dorsal carapace of each crab using an Iphone 6 camera to track decoration throughout the duration of the experiment. To minimize disturbance to the animals, crabs were kept in seawater while being transferred to a small container for imaging. A small grid was included in the image for calibration. Both carapace area and total area of sponge (including sponge extending beyond the carapace) were traced and calculated using ImageJ software (Fig. 1). Percent cover was calculated as total area of sponge divided by carapace area. Though some decoration is placed on the legs, it was minimal compared to the decoration on the carapace; thus only carapace decoration was evaluated. To equalize handling stress among treatments, undecorated crabs were sham-handled each time that the decorated crabs were photographed, using the same protocol, www.nature.com/scientificreports www.nature.com/scientificreports/ but with no photograph taken. At the end of the experiment, final decoration percent cover was quantified by photographing the dorsal carapace of each crab using a HD digital camera (Leica DFC290, Buffalo Grove, 206 Illinois, United States) attached to a stereomicroscope (Leica M165 C, Buffalo Grove, Illinois, United States). From these images, percent sponge cover was measured and calculated using the same method as described for the weekly measurements.
Immediately after imaging, decorations were carefully removed from each crab, dried in a fume hood for 6 days, and then weighed using a digital microbalance (Sartorius 1602 MP 6, Data Weighing Systems, Inc., Elk Grove, Illinois, United States). Decoration behavior was characterized as both the mass of sponge carried and the percent cover of sponge.
Once cleaned, crabs were measured and weighed by the procedure described above. The crabs were then euthanized by being placed in a −20 °C freezer for 30 minutes before being dissected for analyses of exoskeleton morphology and organic content. None of the crab dissections revealed evidence of apolysis, the earliest sign of premolt, so all crabs used for analysis were considered to be in the intermolt stage.
Exoskeleton calcification. Cleaned crab carapaces were bisected so that one half could be analyzed for structure and elemental content using scanning electron microscopy (SEM) and the other half of the carapace analyzed for elemental composition using inductively coupled plasma mass spectroscopy (ICP-MS).
For SEM, carapace samples were fractured at a consistent location in the mesobranchial region using forceps, and then dried in a critical point drier (AutoSamdri 815 Series A, Tousimis, Rockville, Maryland, United States), secured to a double 90° SEM mount revealing the cross-section, and sputter coated with iridium. Cross-sections of carapace samples were then examined with an ultra-high resolution SEM equipped with EDX (XL30 SFEG with Sirion column, Field Emission Incorporated, Hillsboro, Oregon, United States). SEM imaging was done at 10 kV acceleration voltage. An overview image of the whole cuticle cross-section along with magnified images of the epicuticle, exocuticle and endocuticle layers were taken for each animal. Total cuticle thickness comprised all three visible layers and was averaged from 5 measurements taken from each whole cuticle image.
Mineral composition of the carapace cuticle was examined using EDX by magnifying each image so that the whole cuticle filled the screen. Spectra were collected at a 20 kV acceleration voltage and a minimum of 5,000 counts per second. A semi-quantitative analysis of all elements in the cuticle cross-section was conducted on all samples. Ca and Mg, which are key elements in cuticle mineralization, along with C, O, Na, Cl, Al, P, and S were found consistently in all samples, with some samples also containing small amounts of Si and K. We focused specifically on the amount of Ca and Mg in each sample, which was calculated as the weight percent (wt.%) relative to all detected elements, excluding the iridium coating.
For elemental trace analysis using ICP-MS, cleaned carapace samples were weighed and placed in Teflon vials for digestion with 0.5 mL of concentrated Teflon-distilled (TD) nitric acid (HNO 3 ) on a hotplate at 120 °C for >24 h. Samples were dried and diluted by a factor of 4000 with 2% TD HNO 3 before being transferred to pre-cleaned centrifuge tubes for analysis. Samples were doped with an indium solution at this time to monitor instrumental drift. Measurements were done using a ThermoScientific iCAPq c ICP-MS (Thermo Fisher Scientific GmbH, Bremen, Germany) in standard mode. Masses of Mg and Ca were sequentially measured for 30 ratios, resulting in internal precision of <2% (2 s.d.). Elements were corrected for total mole fraction (ambient pH/decorated n = 10, ambient pH/undecorated n = 8, reduced pH/decorated n = 10, reduced pH/undecorated n = 9). Raw data were corrected off line for instrument background and drift. Samples were bracketed by internal standards of crab carapace (n = 2), which allowed for calculations of absolute values. The standards yielded external precision of better than 1% for Mg and Ca (2 s.d.). organic content. The metabolic state of an organism can be inferred through its overall organic content 32,51,52 . Here, we refer to organic content as the overall mass of proteins, lipids and carbohydrates. A crab with www.nature.com/scientificreports www.nature.com/scientificreports/ sufficient energy resources presumably has relatively higher organic content, and thus higher lipid stores and protein, compared to animals with higher energetic costs, which require the burning of this organic content to supply energy. Therefore, we designate animals with lower organic content as having higher energetic costs and higher metabolism 32 . Organic content is thus considered a proxy for metabolic activity.
After removal of the carapace, all internal organs and soft tissues, including the hepatopancreas (the primary storage organ), were carefully removed using a probe and forceps to excise all tissue from the cephalothorax. Due to the small size and improbability of separating each organ, all internal tissues were massed together for analysis. To prevent degradation of tissue, samples were placed in a sealed petri dish and stored in a −80 °C freezer for up to 50 days until analysis was performed.
Organic content of each crab was attained through thermogravimetric analysis (TGA) of the tissue sample using a SDT Q600 (TA Instruments, New Castle, Delaware, United States). Samples were heated in a 9 μL alumina crucible, from room temperature to 800 °C, at a rate of 10 °C/min in air. Previous studies using TGA on biological samples aided in interpretation and temperature designations of our TGA curves [53][54][55] . Within our sample curves, water was observed to burn off at approximately 200 °C, proteins, lipids and carbohydrates burned from 200 °C to 450 °C, and carbonaceous materials burned from 450 °C to the end of the procedure (Fig. 2). The temperature of 450 °C was chosen as the end point of the organic content burn off because it was consistent with temperatures in previous studies on microalgae (476 °C 54 ), and it was invariable throughout all samples. Organic content was estimated by measuring the loss of tissue mass between 180 °C and 420 °C. Percent water content was also calculated because studies have shown that changes in water content are inversely related to changes in organic content, so we would therefore expect an increase in water content as metabolism increases and organic content decreases 56 . The use of TGA ensures that no water or carbonaceous materials impacted the determination of organic content. We chose to measure tissue pyrolysis instead of respirometry as a proxy for metabolism primarily because of complications with using live decoration that also consumes oxygen, as noted by Berke and Woodin 32 . Caloric intake was not used due to difficulty in collecting and measuring the debris of unconsumed food. statistical analyses. All data were tested for normality using a Shapiro-Wilk test and for homogeneity of variance using Levene's test. Decoration, change in mass, mineralization, and organic content were compared across treatments using two-way ANCOVAs with initial crab mass as a covariate. Sex was included as a factor because both males and females were used in the experiment. Mineralization data (wt.% Ca, wt.% Mg, Ca, Mg) were log transformed prior to analysis. Statistical analyses were performed in R Version 3.2.3. All data are reported as mean ± standard deviation.

Results
Water chemistry. Experimental pH was stable throughout the experiment for both ambient (8.01 ± 0.03) and reduced (7.74 ± 0.02) pH treatments. Temperature also remained consistent and the same among treatments throughout the experiment (ambient pH: 21.8 ± 1.2 °C; reduced pH: 21.8 ± 1.2 °C) (Mann-Whitney U test, U = 577632.0, N1 = 1084, N2 = 1102, P = 0.18), except for the last three days over which the temperature steadily decreased to 17.4 °C in both treatments. survival and mass. Similar levels of mortality occurred among treatments, with 4 deaths in ambient pH/ undecorated (3 female, 1 male), 2 in ambient pH/decorated (both female), 3 in reduced pH/undecorated (all male), and 3 in the reduced pH/decorated (all male). These deaths occurred randomly over the duration of the experiment and could not be attributed to any specific factor. We do not expect that fatality was due to handling www.nature.com/scientificreports www.nature.com/scientificreports/ during decoration removal because of the time course of deaths and because the procedure had no effect on crabs during preliminary work. It is possible that limited diet breadth or feeding frequency may have caused inadvertent stress, which is supported by the loss in mass across treatments, as described below. The experiment was ended after five weeks to prevent further mortality. During this time, only 2 animals molted (one from each of the undecorated treatments) and were excluded from analyses. This left sample sizes of N = 9 ambient pH/ decorated, N = 8 ambient pH/undecorated, N = 9 reduced pH/decorated, and N = 8 reduced pH/undecorated for all analyses. Growth associated with molting was not assessed in this experiment, but intermolt changes in mass were. Most crabs experienced a loss in mass (ambient pH/decorated: −7.54 ± 5.54%; ambient pH/undecorated: −0.77 ± 11.80%; reduced pH/decorated: −2.87 ± 17.46%; reduced pH/undecorated: −7.27 ± 6.53%) (Fig. 3). The percent change in mass was highly variable among individuals, but did not differ among treatments (two-way ANCOVA: initial crab mass = covariate, F (3,24) = 1.19, P = 0.34), and there was no effect of initial crab body mass (F = 2.58, P = 0.12), but female crabs lost more mass (F = 4.70, P = 0.04). The variance in percent change in mass did not differ among treatments (Levene's test, F = 2.13, P = 0.12).

Discussion
The decorator crab Pelia tumida shows tolerance to pH levels that mimic forecasted near-term changes in ocean chemistry (decrease 0.3-0.4 in pH by 2100 43 ). Such experimental conditions are sufficient to elicit a variety of detectable responses in other crustacean species, including changes in survival, metabolism, and calcification 1,6,57 , but not for any of the variables measured in this study on P. tumida. While vulnerabilities may manifest in other biological aspects, P. tumida appears to be resilient to moderate pH reductions, at least over the time-scale of this experiment. Length of exposure to reduced pH conditions is an important factor because some species take as long as a year to reveal measurable effects 58 , while others do so in as little as 3 weeks 11 . The natural environmental variability within this species' range may factor heavily in their resilience, as coastal waters off California are thought to be naturally stressful because of changes in temperature and calcite saturation states that result from upwelling events 59 . The experimental pH used in this study on P. tumida was well below what they experience in the subtidal habitat they were collected from, which ranges from 7.9 to 8.13 (based on data logged every 15 min for 16 consecutive months; data from Kram, S. L., Takeshita, Y., Dickson, A., Martz, T. & Smith, J. E. Scripps Ocean Acidification Real-time [SOAR] Dataset, Scripps Institution of Oceanography). Yet, inhabiting a variable pH environment does not necessarily confer resilience. Forecasted changes in ocean pH may overextend the physiological tolerance limits of near-shore animals, resulting in detectable responses. This is evidenced in metabolic and thermal tolerance changes of intertidal porcelain crabs 20,60 and changes in growth and calcification of intertidal barnacles 13,61 . Pelia tumida likely has a broad physiological tolerance range similar to that of mantis shrimp, which experience no stress at pH values far below what they encounter in nature 15 . This is surprising given that P. tumida is a relatively inactive, slow-moving, osmoconforming species, which are traits thought to reduce the capacity of animals to respond to changes in external pH 1,17,18 .
Calcification is a critical metric in evaluating the response of crustaceans to ocean acidification conditions, and our conclusion that this process is unaffected in P. tumida is limited to intermolt maintenance processes. Examples of increased calcification in crustaceans responding to reduced pH conditions occurs during the molting process, because calcification peaks immediately following ecdysis, when new exoskeleton is formed and hardened through cross-linking and deposition of calcium carbonate. Too few crabs molted in this experiment to evaluate molt-related calcification. Albeit small compared to post-molt calcification, there is a flux of calcium across the epithelium and a net uptake of calcium in the exoskeleton that takes place during the intermolt stage [62][63][64] . This was recently observed in intermolt mantis shrimp, where calcium content of the cuticle continued to increase up to 6 months following molting, indicating long-term accretion 15 . More significantly, we did not detect any evidence that dissolution of the exoskeleton occurred, as might be expected in osmoconformers if their hemolymph pH decreases. Neither Ca nor Mg quantities of the exoskeleton differed under reduced pH conditions, indicating that these elements remained in the exoskeleton rather than accruing in the hemolymph as a consequence of dissolution. There appears to be no metabolic effects related to the calcification process in P. tumida. While the calcification regulatory processes were unaffected by reduced pH conditions over the course of 5 weeks, it remains a possibility that effects may emerge over a longer exposure time.
Furthermore, like most crustacean ocean acidification studies that examine calcification, we focused only on one region of the exoskeleton. Due to the small size of P. tumida, we only sampled the carapace, but it is possible that other regions of the crab exoskeleton are more sensitive to reduced pH conditions, such as the chelae or setae. In mantis shrimp, for example, changes in Mg content occurred in the merus of the raptorial appendage, but not the carapace, under reduced pH 15 . This region-specific response to reduced pH was also observed in the velvet crab, Necora puber, which had increased Mg in the chelae, but not the carapace 14 . This variable response within an individual is not surprising given that the calcification process itself is malleable, enabling localized control of mineral content for regions that support specialized functions. Inherent variation in calcification within an individual animal requires that localized measurements of mineral content from multiple exoskeleton regions be considered if accurate descriptions of the crustacean response to environmental stressors are to be made.
Maintaining homeostasis of physiological processes under environmental stress requires additional energy for some organisms, materializing as increased metabolism and reduced growth rates. This energetic trade-off has been observed in a variety of marine calcifiers whose strong acid-base regulation maintains calcification under reduced pH conditions, but at metabolic expense 6,10,13 . For instance, when exposed to moderate increases in pCO 2 [991 µatm 65 and 490 µatm, 1,100 µatm, 2,400 µatm 66 ], another decorating majid crab, Hyas araneus, sustained net calcification, but experienced increased metabolism and stress. We predicted a similar response in the decorator crab P. tumida, but it showed no indication of metabolic costs, as measured by organic content, under reduced pH conditions. The current study did not, however, take into account the complexity of a species' metabolic response to environmental pH. When H. araneus was exposed to a high pCO 2 level of 1,960 µatm, metabolic rate decreased rather than increased 65 , revealing a disparity of outcomes specific to carbon chemistry parameters. Reduced metabolic rates under increased pCO 2 conditions have been observed in other crustaceans as well, including the velvet swimming crab, Necora puber 14 , and the prawn, Metapenaeus joyneri 26 . Metabolic depression is thus another (2019) 9:6262 | https://doi.org/10.1038/s41598-019-42696-8 www.nature.com/scientificreports www.nature.com/scientificreports/ potential outcome for animals exposed to environmental stress, but it is typically associated with reduced growth rates 18,67,68 . Pelia tumida showed no signs of elevated or depressed metabolism, based on organic content, suggesting that the pH conditions used in this experiment either did not impose a sufficient stress to affect metabolism or that the crab's energy budget already accounts for physiological energetics under stress 22 .
Surprisingly, we did not detect an energetic cost of decorating for P. tumida, in contradiction to expectation and previous studies on decoration behavior energetics in decorator crabs and sea urchins 29,32 . The cost of decoration behavior has only been quantified in one species of decorator crab, and is likely inconstant given the diversity of species and the interconnection of habitat, body size, ontogeny and sexual dimorphism with the evolution of decorating in this group. The energetics required for decorating are presumably unique to the individual species. Decorator crab species that do not undergo an ontogenetic shift in decoration behavior, such as P. tumida, tend to be small (5-40 mm carapace width 32 ), presenting the possibility that decorating may not be as costly for smaller crabs. Yet, Oregonia gracilis demonstrates a cost of decoration for both juveniles (3-6 mm) and subadults (11-18 mm) 32 , refuting the idea of a size refuge from decoration costs. Species may minimize decoration costs by using different types and amounts of decoration material or by reducing activity and being slow-moving 69 . Indeed, small decorator crabs facilitate blending in with their background by remaining still and inactive during daylight hours 28 .
Our main hypothesis was that under energetically stressful conditions, decoration behavior would be reduced to allocate energy to other important physiological processes, as was observed in the sea urchin Strongylocentrotus droebachiensis 29 . However, P. tumida did not change its decoration behavior (percent cover and total mass of sponge) when maintained under reduced pH conditions. This outcome is sensible because there were no indications of energy limitation related to pH conditions; both change in crab mass (wet weight) and organic content were the same across treatments. Reduced pH alone does not appear to induce sufficient sub-lethal stress to affect metabolism or behavior in P. tumida. However, if ocean acidification is considered in concert with other climate change stressors, such as increasing temperature, declining oxygen, or changes in salinity, the interactive effects may induce measureable stress in P. tumida.
Typically studies of energetic trade-offs carefully control and monitor energy intake 70,71 , which is why studies on decoration behavior energetics often use starvation conditions 32 . We chose not to introduce starvation stress in this experiment in an attempt to explore realistic responses to reduced pH conditions. The natural energy intake of Pelia tumida is unknown, but they inhabit areas with abundant algae and invertebrates and are likely not food-limited in nature. In this study, crabs were fed to satiation three times per week, but either this amount was not adequate or the singular diet did not provide complete nutrition because almost all crabs experienced a small loss in mass by the end of the experiment. The consistency suggests that it is more than measurement error in wet weights. In spite of crabs being food limited, pH conditions had no effect on metabolism, calcification, and decoration behavior. It is also germane that while we fed crabs consistent, measured amounts of food throughout the experiment, quantifying unconsumed food was not feasible. We therefore have no precise measure of the amount of food consumed per individual. If decorated animals in the reduced pH treatment had higher energetic costs, but satiated this with increased feeding, it was not detectable. Additionally, some species of decorator crabs are known to use their decorations as a food cache 72 . Percent sponge cover in individuals was not stable over the course of the experiment, indicating that some sponge may have fallen off, deteriorated, or possibly been consumed. Thus, some crabs may have consumed more food than others, providing them with higher caloric intake or different food quality. The variation in decoration material over time was the same for crabs in ambient and reduced pH treatments, thereby dispelling the possibility that decoration food cache was used to supplement energy in response to pH stress. Overall, metabolic costs may not have been detected because the decorator crabs were not energy limited in this experiment, an important factor for some species in coping with ocean acidification conditions 73 .

Conclusions
Crustaceans exhibit responses to experimental ocean acidification conditions that vary among species, life histories, habitats, and experimental conditions, making it opportune, yet challenging, to decipher the critical aspects driving tolerance in this diverse group of animals. The current study on P. tumida revealed no changes at the organismal level, in terms of physiology (organic content; lipids, carbohydrates and proteins), morphology (Ca and Mg content), or behavior (decoration percent cover and mass), under reduced pH conditions. This species of decorator crab is able to maintain multiple biological processes while responding to decreases in external pH, which contrasts with other studies focusing on energy limitation and behavioral aspects in response to reduced pH and emphasizes the need for further crustacean research to include more species and a wider view of possible ecological responses.

Data Availability
The datasets generated and analyzed during the current study are available from the corresponding author on request.