Stress physiology and weapon integrity of intertidal mantis shrimp under future ocean conditions

Calcified marine organisms typically experience increased oxidative stress and changes in mineralization in response to ocean acidification and warming conditions. These effects could hinder the potency of animal weapons, such as the mantis shrimp’s raptorial appendage. The mechanical properties of this calcified weapon enable extremely powerful punches to be delivered to prey and aggressors. We examined oxidative stress and exoskeleton structure, mineral content, and mechanical properties of the raptorial appendage and the carapace under long-term ocean acidification and warming conditions. The predatory appendage had significantly higher % Mg under ocean acidification conditions, while oxidative stress levels as well as the % Ca and mechanical properties of the appendage remained unchanged. Thus, mantis shrimp tolerate expanded ranges of pH and temperature without experiencing oxidative stress or functional changes to their weapons. Our findings suggest that these powerful predators will not be hindered under future ocean conditions.


Field pH and temperature data collection
To record pH and temperature in the field, we used a portable pH probe (NBS values subsequently converted to the total seawater scale) in the sea grass beds from which N. bredini individuals were collected at two main collecting sites, Isla Mina and Galeta Marine Laboratory from March 7-11 and March 20-24 (10 days total). We focused on the sea grass specifically because our goal was to capture the range of values experienced by N. bredini, and sea grass photosynthesis and respiration influences the pH environment. Thus, we recorded pH and temperature at high and low tides, and before sunrise and after midday to capture the variation in pH values that is created by sea grass respiration and photosynthesis. We also recorded these parameters at high tide, low tide, and after midday at Isla Mina, but we were unable to document pH and temperature before sunrise because this site was too remote to access at night. On March 24, 2015, two water samples were collected at three time points: before sunrise, at low tide, and after midday (6 samples total) at Galeta Marine Laboratory, and three water samples were collected after midday at Isla Mina. All samples were brought to the Dickson Laboratory at Scripps Institution of Oceanography for analysis of pH, total alkalinity, and density based salinity, as described in the Materials and Methods section of the main manuscript.

Oxidative stress
Bradford assays: To perform the Bradford assays, 200 µL of 1X Bradford dye (Bio-Rad Laboratories, Hercules, CA) and 10 µL of either the sample or bovine serum albumen (BSA) standard were added to a well plate. Samples were measured using an iMark Microplate Absorbance Reader (Bio-Rad Laboratories, Hercules, CA) at 595 nm. Standard curves were generated using BSA at 0.9, 0.7, 0.56, 0.28, 0.14, and 0 µg/µL protein concentrations (all R 2values were > 0.95) to calculate sample concentrations based on the best fit line of the standard curves.
Protein carbonyl analysis: The protein concentrations of the samples were first determined with the Bradford Assay. The samples were then diluted to 10 µL/mL based on protein concentrations. Samples were compared to BSA standards. Standards and samples were plated into a protein binding plate in triplicate and incubated overnight. 100 µL of DNPH solution, 200 µL of blocking solution, 100 µL of anti-DNP antibody, and 100 µL of HRP secondary antibody was added to and removed from the binding plate. Three to five washes with 200 µL of either 1X PBS solution or wash solution were interspersed between each step. 100 µL of substrate solution was added to the plate and 100 µL of stop solution was added after 15 min.
Superoxide dismutase (SOD) and catalase (CAT) analyses: To analyze SOD enzyme abundance, 50 µL of the substrate preparation was combined with 10 µL of the N. bredini tissue homogenate, and then 25 µL of xanthine oxidase preparation. Samples were prepared in triplicate and compared to BSA standards. Prior to reading, the plate was centrifuged at 3,221 g for 3 min and incubated at room temperature for 20 min. CAT abundance was measured with a CAT Activity Kit following similar methods (25 µL of sample, 25 µL of H 2 O 2 , and 25 µL of detection reagent, and 25 µL of substrate). Samples were prepared in triplicate and compared to bovine CAT as standards. Prior to reading, the plate was centrifuged at 3,000 g for 1 min and incubated at room temperature for 15 min. Table S1. Water chemistry of samples from the laboratory and field. Values are mean ± standard deviation of chemical properties of the seawater in the experimental cups over the course of the six-month exposure period and of seawater samples from the two collecting sites, Galeta Marine Lab and Isla Mina, Panama. 155 measurements were taken per experimental cup and the mean pH and temperature values for each cup was then used to calculate the overall means for each treatment. Measurements at Galeta Marine Lab were taken throughout the day and night but only times with the extremes of the measurement range are presented (high tide before sunrise and low tide in the afternoon). For Isla Mina, measurements at low tide in the afternoon are presented. pH, salinity, and total alkalinity (TA) were measured by the Dickson Laboratory and all other parameters were calculated using CO2sys. pH T denotes the total seawater scale. * Indicates significant differences between experimental treatments (α = 0.05). Note that while the pH values of the cups were significantly different between treatments, the mean difference [95% C.I.] between the reduced pH and reduced pH/increased temperature treatments was only 0.02 [0.02, 0.02] and the total alkalinity, pCO 2 , HCO 3 -, and ΩCa were not significantly different. The temperature of the experimental cups was significantly different between treatments, but the mean difference between the ambient and reduced pH treatments was only 0.24°C, which we do not think is biologically relevant.  Figure S1. Mineral maps of Ca and Mg distribution. Merus samples from (a) ambient, (b) reduced pH, and (c) reduced pH/increased temperature treatments are shown. The top row shows the SEM images of the mapped regions. The middle and bottom rows show the maps for Ca and Mg distributions, respectively, that were generated from the EDX analysis. Ca density appears to be uniform across the cuticle and across treatments. Mg density and distribution was higher in the reduced pH treatment compared to the other treatments. Maps for the carapace are not shown because the distribution of the elements in the samples was similar to the merus, although overall, the carapace was less dense than the merus. Scale bars = 20 μm.

Supplementary
Supplementary Figure S2. SDS-Page gel electrophoresis of the experimental samples examined for oxidative stress. The gel image shows no protein degradation or "smear" indicating limited protein degradation. Each protein band (arranged in columns labeled 1-12) is a sample from a different individual that was in either the ambient, reduced pH, or reduced pH/increased temperature treatments. The columns labeled "controls" were animals that had been frozen and stored at -20°C and -80°C to examine whether the sample stored -20°C had noticeably more tissue degradation than the sample stored at -80°C. Dotted reference lines help compare migration of the bands in the gel and identify differences between bands that otherwise look similar (e.g. lanes 1 and 12). The well-defined bands in all samples suggest that the tissues were sufficiently intact to produce reliable results for protein assays (i.e. all tissues stored at -20°C experienced as much detectable tissue degradation as the control stored at -80°C). The intensity of the bands, therefore, does not reflect differences between storage at -20°C or-80°C. Rather, the differences in the levels of band migration in the gel and their respective intensities illustrate individual variation between samples. ……………………………………….
The spectrum was taken at 20 keV. Elemental peaks were consistently detected for C, O, Na, Mg, Cl, and Ca. Unlabeled peaks are Ir, which was used to coat the samples and were not included in the quantitative analysis. In this particular sample, % Ca and % Mg in relation to the other detected elements were 41.25 wt% and 2.69 wt%, respectively.