Environmental pH , O 2 and Capsular Effects on the Geochemical Composition of Statoliths of Embryonic Squid Doryteuthis opalescens

Spawning market squid lay embryo capsules on the seafloor of the continental shelf of the California Current System (CCS), where ocean acidification, deoxygenation and intensified upwelling lower the pH and [O2]. Squid statolith geochemistry has been shown to reflect the squid’s environment (e.g., seawater temperature and elemental concentration). We used real-world environmental levels of pH and [O2] observed on squid-embryo beds to test in the laboratory whether or not squid statolith geochemistry reflects environmental pH and [O2]. We asked whether pH and [O2] levels might affect the incorporation of element ratios (B:Ca, Mg:Ca, Sr:Ca, Ba:Ca, Pb:Ca, U:Ca) into squid embryonic statoliths as (1) individual elements and/or (2) multivariate elemental signatures, and consider future applications as proxies for pH and [O2] exposure. Embryo exposure to high and low pH and [O2] alone and together during development over four weeks only moderately affected elemental concentrations of the statoliths, and uranium was an OPEN ACCESS

Environmental [O 2 ] is decreasing more quickly along the coast of the Southern California Bight than in the offshore pelagic realm [24,25].Ecological theory suggests that organisms will respond with species-specific shifts in size frequency and biogeographic range [8,26].Many of the knowledge gaps regarding population level effects of [O 2 ] and pH/pCO 2 exist because of inadequate tools for assessment, and geochemical proxies have not yet been utilized for squid.
The near-shore squid, Doryteuthis opalescens, is particularly sensitive to environmental change associated with the El Niño Southern Oscillation (ENSO; [27][28][29]), yet basic knowledge of the market squid population dynamics is lacking.This includes assessment of their population connectivity [30], as well as knowledge of critical ecological mechanisms controlling their population size [31].Fishery boom and bust cycles often correlate with environmental change, such as that associated with ENSO [27][28][29].Numerous hypotheses for annual catch fluctuation have been presented, but are difficult to test in the field because of sampling method biases [28,29,32].The developmental, physiological, behavioral and ecological mechanisms that lead to such drastic changes in annual catch have yet to be described, and the magnitude of risk for the population associated with environmental change is unknown.The embryonic life stage may be especially susceptible to low [O 2 ] and pH/pCO 2 exposure during La Niña events, because unlike other stages, embryos are site attached to the seafloor and are completely reliant upon a fixed energy reservoir (i.e., yolk).Without the ability to move, embryos must tolerate exposure to low [O 2 ] and pH/pCO 2 levels using their fixed energy supply.One of the most promising tools available to investigators to understand environmental effects at the population level is through the use of statoliths [30,33,34] that are developed during this time.
Statoliths, made of ~95% CaCO 3 in the aragonite crystal form [35], are used by squid as part of their equilibrium and motion sensory organs (statocysts; [30,32,33,36]).Paired statoliths develop in each market squid embryo during the last two-thirds of the embryogenesis period (Figure 1; [37]), then remain embedded within the statocyst as it grows during each following life stage.After death, the statoliths can sometimes even be preserved in the fossil record [33,38,39].Statolith aragonite crystal grows with a daily banding pattern, and growth is heavily influenced by the environment [33,34,38,40].However, the squid statolith is not in direct contact with the environment, but rather with endolymph fluid within the statocyst [41].
Clear geochemical proxies for exposure to low pH have been established for foraminifera shells [60][61][62], coral skeletons [63] and mollusk shells [64] using either δ 11 boron or uranium:calcium ratios.In addition, δ 18 O has been explored as a proxy for O 2 in statoliths of Illex illecebrosus [35].Here, we explore the element:calcium composition of market squid (Doryteuthis opalescens) statoliths as a potential proxy of pH/pCO 2 and [O 2 ] exposure using levels of pH/pCO 2 and [O 2 ] that reflect the highs and lows observed within embryo beds in southern California.This investigation is the first that we are aware of to test for a proxy of pH exposure using squid statoliths.
Any information about these squid could help to fill large knowledge gaps concerning scenarios of rapid climate change.Low pH/high pCO 2 and low [O 2 ] (hereafter referred to as "low pHOx") can cause species-specific negative effects in isolation or in tandem, and the magnitude of each of these effects is likely to be habitat specific [65,66].We conducted experiments to investigate whether statolith geochemistry can reveal squid exposure to low pHOx, low [O 2 ] only and/or low pH/high pCO 2 only (hereafter, referred to as "low pH") during benthic encapsulated stages.We hypothesize that: (1) conditions associated with upwelled seawater observed in the D. opalescens spawning habitat influence the geochemical composition within embryonic carbonate structures in a manner useful as a proxy and (2) environmental exposure effects can be separated from capsular effects.More specifically, we assessed whether: (A) encapsulated embryos exposed to low pHOx yield distinct individual elemental levels in squid statoliths; (B) multi-elemental signatures can classify statolith exposures independent of individual elemental ratios; and (C) exposure to low pHOx levels yields elemental signatures different from individual effects of low [O 2 ] or low pH.A goal is to form the ability to assess the exposure of early developmental recruits collected from the field in the absence of seawater pH and [O 2 ] measurements.

Materials and Methods
Treatments were held constant over the entire embryogenesis period to allow for chronic exposure.Encapsulated squid embryos were reared under controlled conditions for the majority of embryogenesis.[1] (see System Overview and Experimental Treatments Section).Unlike near-surface waters, seawater in the lower to mid-shelf depths that are regularly utilized by squid [32] experiences reduced environmental variability (i.e., environmental conditions are more stable).In the Southern California Bight, the magnitude of environmental variability (e.g., range of pH and [O 2 ]) decreases with depth.For example, the range of [O 2 ] decreased by 37% and the range of pH by 39% from a 7 to 17-m depth [3].The variability [O 2 ] and pH continues to decrease with depth [1] near areas where squid-embryo capsules were collected (< 10 km).For embryo beds at 80-90 m on the shelf, pH and [O 2 ] conditions can be near constant over month-long periods.A novel laboratory approach using the Multiple Stressor Experimental Aquarium at Scripps (MSEAS; [67]) was used to control pH and [O 2 ] levels.Two experiments were conducted.Each included four tanks: two treatments with two replicate tanks each.For each experiment, capsules were randomized among treatments, aquaria and position within each aquarium.

Collection of Squid Embryos and Seawater Data
For Experiment 1, newly laid squid capsules (encapsulated embryos) were collected by hand from La Jolla Bay, San Diego, USA (32.86°N, 117.27°W), and from capsules laid at Scripps Institution of Oceanography (squid caught from Del Mar, USA; 32.96° N, 117.28°W).For Experiment 2, newly laid capsules were taken from La Jolla Bay, USA (32.87°N, 117.25°W).All capsules were exposed to treatments for 24 days or more, allowing at least one week of exposure to treatment conditions prior to the initiation of statolith development (statoliths are completely grown under controlled conditions).Environmental data were collected continuously from a 30-m depth, 0.5 m above the seafloor, using a site-attached instrument (SeapHOx: described by Frieder et al. [3]) that measured conductivity, temperature, pressure, pH T and [O 2 ] from 23 June 2012 to 4 July 4 2013 (location: 32.86° N, 117.27°W).

System Overview and Experimental Treatments
Embryo capsules of D. opalescens in Experiment 1 were cultured under treatments of constant low (7.55, 90 µmol•kg −1 ) and high levels (7.9, 240 µmol•kg −1 ) of pH T and dissolved oxygen (Table 1, Figure 1).Several step-wise changes in the system level setting were conducted during the course of each experiment to maintain stable environmental conditions [67], and thus, data were not normally distributed.Treatment conditions were distinct for each experiment with respect to pH, Ω aragonite and [O 2 ], but not for temperature and alkalinity (Wilcoxon test; Table 1).Capsules were cultured in 55-L aquaria that had been acid-rinsed in 1 N HCl and then rinsed five times with ultrapure H 2 O (resistivity >18.0 MΩ•cm).Treatments were implemented using MSEAS, a manipulated flow-through aquarium design [67].Seawater was supplied with seawater pumped from a 5 ± 1.5 m depth off of the Scripps Pier.Embryos used for both experiments were collected from the field pre-organogenesis, Stages 11-12 [37,68,69].For Experiment 1, 24 capsules were collected from the field just after being laid and were allowed to acclimate for 3 days at 11 °C.An additional 16 capsules laid in captivity at Scripps Institution of Oceanography were added to the experiment 7 days later.Ten capsules (6 field, 4 aquaria) were randomly assigned and placed into a position in each of four aquaria and were evenly distributed among sources.Twenty capsules were placed into each aquarium.Densities in Experiments 1 (~100 capsules•m −2 ) and 2 (density ~200 capsules•m −2 ) are within the viable range found in the field, where they are reported to range from 1 to 47,720 capsules•m -2 [32,70].Embryos were cultured to embryonic-developmental Stages 28 or 29 (near-hatch paralarvae) [68] to reduce ontogenetic effects [71] and were collected only within the central portion of the capsule in order to reduce capsule-position effects [72] (Figure 2).These stages were indicated by the pigmentation of the ink sac (this occurs earlier in D. opalescens than in D. pealeii), the complete covering of the eyes by the cornea, but not a prominent Hoyle's Organ [37,68,69].Cultures were maintained at constant temperature (11.3 °C ± 0.3 °C, SD), salinity (33.4 ± 0.2) and light levels using 15 W LED lights on a 12:12 h light:dark cycle to reduce these types of environmental effects on statolith development [21,40,72].Salinity, temperature and seawater flow rate were constant among treatments.Statolith development takes place at the start of organogenesis [37] Further, to verify whether or not seawater trace-metal concentration varied among treatments, 100-mL seawater samples were taken weekly using clean-lab protocols to minimize any possibility of contamination [73].Seawater samples were filtered, acidified with 50 µL of 12 N optima HCl and stored in darkness at room temperature (21 °C ± 3 °C) until they were analyzed at Arizona State University for magnesium 24 (Mg), calcium 48 (Ca), strontium 88 (Sr), barium 138 (Ba) and uranium 28 (U).Boron (B) was estimated using discrete salinity values taken daily [74].

Extraction and Mounting of Statoliths for Elemental Analyses
Statolith extraction and mounting procedures followed clean lab protocols [73].Statoliths were removed for analysis when embryos were developed to Stages 28-29 [68].Statoliths were removed by dissection, and then chemical digestion of soft parts (encapsulation and embryo proteins) was carried out by placing embryos on a slide with digesting solution (10 µL of ultrapure water and 5 mL of ultrapure 15% H 2 O 2 buffered with 0.05 N NaOH in ultrapure H 2 O) for 10-20 min, depending on the amount of soft tissue.Digestion solution was removed by pipetting with a clean tip, and statoliths were rinsed three times in ultrapure H 2 O.All remaining H 2 O was removed by evaporation overnight underneath a hood within a Class 100 clean room.One statolith from each embryo was extracted, and 10-20 embryos were dissected from the center position of each capsule (total = 10-20 statoliths from each capsule).Statoliths were mounted on double-sided tape (Scotch TM ), attached to a slide and prepared for laser ablation.The chemical composition of mounting tape was determined by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) [75]; all elemental counts from the tape were at least three orders of magnitude lower than counts found in statoliths.Statolith lengths ranged from 65 to 130 µm.

LA-ICP-MS Instrument Settings and Methods for Elemental Analyses of Statoliths
Statoliths were analyzed by LA-ICP-MS at low resolution for an elemental menu consisting of boron 11  (B), magnesium 24 (Mg), calcium 48 (Ca), manganese 55 (Mn), copper 63 (Cu), zinc 66 (Zn), strontium 88 (Sr), barium 138 (Ba), lead 208 (Pb) and uranium 238 (U).Ablated material from haphazardly selected statoliths from each slide was introduced via a New Wave UP-213 UV laser, frequency-quadrupled to a 213-nm wavelength with a nominal beam width of 40 µm in the spot beam setting, into a Finnigan Element 2 sector field ICP-MS using a micro-flow nebulizer at 20 µL•min −1 .The laser was set to the spot beam mode at 40% power and a frequency of 20 Hz.Consistent plasma conditions were maintained using 1% HNO 3 during analysis of standards, on instrument blanks and on laser-ablated samples [75].Instrument sensitivity was monitored measuring indium (In) and was approximately 1 × 10 6 counts•s −1 for 1 ppb.All slides were assigned a number and then analyzed in a random order.The laser-ablation process completely vaporized each statolith.
To standardize the mass variation of statolith samples, the concentrations of elements in the statoliths are reported as ratios with respect to Ca, the dominant elemental constituent of the statolith [76].Aragonite is a solid, acellular, metabolically inert, crystalline structure [76] that is ~95% CaCO 3 [35] and differs from other calcified structures found in cephalopods that are porous, cellular and metabolically active, such as cuttlebone, which have been shown to become less porous (hypercalcification or increased CaCO 3 density) in response to hypercapnia [47,77].Further, if hypercalcification or hypocalcification is induced by a treatment, we would expect all element:calcium ratios to be significantly increased (hypocalcification) or decreased (hypercalcification). Therefore, we would predict that this method is sensitive to changes in calcium concentration.Element:Ca of the sample was determined by using matrix-matched solution standards of known element:Ca and a mass-bias correction [78].
Detection limits were defined as the intensities (counts per second) of elements present in the instrument blank plus three times the standard deviation [79].The average intensity of each element above the detection limit was as follows (measured as multiples of detection limit): Mg > 1770, Sr > 1,280, Ba > 440, U > 220, Pb and Cu > 50, Zn > 40, Mn > 1.2 and B > 0.8.The percentage of samples where each element was above the detection limit was as follows: Mg 97%, Sr 99%, Ba 100%, U 97%, Pb 98% and B 40%.For the B data, we only included those samples that were above detection limits and eliminated all others from further analysis.

Statistical Analyses of Seawater and Statolith Elemental Composition
Seawater samples were taken weekly from each tank within each treatment.For statolith samples, tanks were considered replicates for statistical testing of the hypotheses.The average number of embryos (i.e., statoliths) analyzed per capsule was 5.4 ± 0.5 (standard error).All data were first examined for variance homogeneity and tested for normality by means of residual analysis prior to using ANOVA models.All seawater elemental data were normal, and statolith elemental data were normal for B:Ca, Sr:Ca and Ba:Ca (no transformations needed).Statolith Mg:Ca, Pb:Ca and U:Ca data were right-skewed, with U:Ca being the most intensely skewed of the data sets.Mg:Ca and Pb:Ca data were square-root transformed, whereas U:Ca data were cube-root transformed; data were normally distributed after transformation.Experiments 1 and 2 were tested separately.Treatment effects were tested using a one-way mixed model ANOVAs nesting the tank (fixed) and capsular (random) factors [80].In Experiment 1, the effects of treatment on the elemental concentration (element:Ca) of statoliths were tested between groups with high pHOx to those of low pHOx.In Experiment 2, the effects for treatment onto the elemental concentration (element:Ca) of statoliths were tested between groups with low [O 2 ] and low pH levels.
Multivariate analysis of similarity (ANOSIM, Euclidean Distance, N = 9999 permutations) was used to test the hypothesis that the treatment influences the multi-elemental composition of the statoliths.Principle component analysis (PCA) was used to investigate elemental signatures among groups using a correlation matrix (i.e., data were not transformed).Statistical analyses were conducted using JMP (Version Pro 11) statistical software (SAS Institute, Cary, NC, USA).

Results and Discussion
Seawater element concentrations were not different among treatments in Experiment 1 or Experiment 2 (Appendix Table A1).Further, chronic exposure to undersaturated seawater (Ω aragonite < 1; Table 1) did not prevent statolith formation.Six elements measured within the statoliths had concentrations detectable at levels sufficient for analyses: B, Mg, Sr, Ba, Pb and U.This is the first report of B being detectable within statoliths of the cephalopod taxon and the first time U has been reported as detectable within statoliths of D. opalescens.Further, several patterns emerged through elemental analyses.

Elemental Variations among Treatment Groups
In Experiment 1, U:Ca was significantly higher (eight times) within the statoliths from the low pHOx treatment (F 1,3 = 16.86,p = 0.0005) than the high pHOx treatment (Figure 3).The only tank effect observed was also for U:Ca in Experiment 1 (F 2,3 = 5.42, p = 0.0130), but this was driven by capsular effects (F 3,13 = 2.65, p = 0.0060) and not by seawater effects (F 1,4 = 0.0302, p = 0.86; Appendix Table A1).Statolith U:Ca averages among capsules in the low pHOx treatment varied over an order of magnitude (capsule values = 0.357, 0.343, 0.248, 0.157, 0.021 µmol/mol).No other individual elements significantly varied between treatments in Experiment 1, and we did not collect any evidence to support a change in calcium concentration indicative of either hypercalcification [47,77] or hypocalcification.Experiment 2 was conducted to reveal whether or not low pH only or low [O 2 ] treatments induced a similar or distinct response.In this experiment, the low [O 2 ] only treatment elicited a distinct response of U:Ca relative to embryos exposed to low pH only (F 1,6 = 5.91, p = 0.0225; Figure 3).The endolymph fluid within the statocyst is highly regulated in squid [41] with similarities to the highly-regulated sacculus found in fish [81,82].Low environmental [O 2 ] may impair the regulation of internal pH by embryonic squid, due to the reduced aerobic metabolic rate.This can lead to insufficient ATP production necessary to fuel active mechanisms for pH regulation and calcification.Interestingly, statoliths from the low-[O 2 ] treatment had a higher Sr:Ca ratio in comparison to the low-pH treatment (F 1,6 = 6.136, p = 0.021; Figure 3).Environmental strontium is critical for statolith formation [59].Strontium has been associated with temperature effects and salinity effects, but this is the first report of a strontium effect associated with [O 2 ] or pH.No other elements exhibited treatment effects.
The capsular effects (integrated effects of outer-embryo structures, capsular and chorion membranes and embryonic processes) were significant for all element:calcium ratios for each experiment (Table 2).The capsular effects are significant for several reasons.First, these findings support the importance of the direct and/or indirect maternal influence on embryonic statolith geochemistry.Evidence for the direct maternal transfer of two essential elements ( 75 Se, 65 Zn) and one non-essential element ( 110 m Ag) has been reported for a cuttlefish [83].Other studies have found evidence indicating a direct role of maternal transfer to embryonic statoliths [73,84].Indirect maternal influence could be caused by a variation in the quality of the capsular and chorion membrane.Although this issue has not been explicitly studied, other investigators have found significant differences of Co uptake among capsules (one capsule of three capsules) of the squid, Loligo vulgaris [49].Second, our findings show that the embryonic-statolith geochemistry is distinct for many elements among capsules, but not distinct for most elements among environmental treatments.These differences may be attributable to differences among capsular units.Each unit may have differences in: (1) elemental uptake in their capsular or chorion membranes [49,50]; (2) utilization of embryonic-epidermal ionocytes [17][18][19]; (3) embryonic metabolism [45,46] and the effect total embryo metabolism per capsule has on the diffusion of environmental [O 2 ] and pCO 2 [20,55,56]; and (4) the rate of active transport of the statocyst membrane [41]; or (5) any combination these factors.Although there are many reports that support environmental "recording" within statolith geochemistry [30,33,34,59], our data suggest that statolith geochemistry records both the environment and capsular effects within each embryo.

Multivariate Analyses
To test whether the elemental composition of statoliths varied among treatments, multivariate analyses were conducted for each experiment using analysis of similarities (ANOSIM; one-way, Euclidean-distance matrix, N = 9999 permutations) and principle component analysis (PCA).The elemental composition between treatments was not distinguished in either Experiments 1 or 2 (ANOSIM: Experiment 1, R = 0.022, p = 0.369; Experiment 2, R = 0.007, p = 0.359).
These multivariate results presented emphasize a limited relationship between statolith elemental chemistry and the environmental pH and [O 2 ].ANOSIM analyses were used to compare statolith chemistry among capsules within treatment (total of four tests; Table 3).Again, the capsules exhibited a significant effect on statolith chemistry within each treatment group, with the exception of the low pHOx treatment (although no effect was detected, this may be caused by a lack of statistical power associated with a small sample size).Elements driving capsular differences were not similar between the experiments (Table 4, Figure 4).These data indicate that any statolith-geochemical record would be an integrated signal between the environmental pH and [O 2 ] and physiological processes within outer embryonic structures.Table 3. Statolith elemental composition differences among capsules within each treatment were tested using a one-way analysis of similarities (Euclidean-distance matrix; elemental menu = B:Ca, Mg:Ca, Sr:Ca, Ba:Ca, U:Ca; N = 9999 permutations for all treatments, except low pHOx (N = 3)).

Statoliths as an Indicator of Environmental Response
The experiments conducted here provide the first evidence that embryonic statolith geochemistry can be affected by environmental [O 2 ] and pH at levels that occur at natal sites.B:Ca and U:Ca were investigated as pH proxies; however, they did not exhibit the direct relationship with environmental pH that has been found with foraminifera shells [60][61][62], coral skeletons [63] and mollusk shells [64].These B:Ca results suggest that, when environmental pH T is low (7.55-7.56)and [O 2 ] is high (241-242 µmol•kg −1 ), squid embryos can regulate the endolymph pH within the statocyst, where the statolith crystal grows.Constant pH was found in a study of the endolymph fluid of squid statocysts [41], and pH has been shown to be highly regulated in the endolymph fluid of saccules of fish (the squid statocyst analog) [81,82].Lower taxonomical groups have a more direct relationship with seawater, with less integration of physiological processes [76].Seawater with low pH T has higher levels of bioavailable U, and in foraminifera, the incorporation of U into biogenic CaCO 3 increases with decreasing [CO 3

2−
] is also regulated within endolymph fluid.These results suggest that both pH and [CO 3 2− ] are highly regulated within the squid embryos and the statocyst, as has been found with other squid [41], and these regulation processes are unaffected by an environmental pH T level of 7.55.One option is that high pCO 2 /low pH in seawater does not significantly affect the blood chemistry of squid embryos [86], due to ion-regulating epithelia regulating internal pH [17][18][19] (Figure 2).However, it is also possible that squid embryos exposed to high pCO 2 /low pH can utilize energy derived from yolk reserves to compensate for putative alterations in their internal pH.Further testing is needed to determine if there is a threshold below which squid are not able to regulate their pH.Moreover, it is essential to know if the D. opalescens embryo is internally acidified during exposure to realistic, high pCO 2 /low pH conditions and the compensatory mechanisms that are involved.
Development of an environmental [O 2 ] proxy is still in its infancy, and more research is needed to test for different mechanisms.However, U:Ca and Sr:Ca were enriched in squid statoliths grown in low [O 2 ] treatments (Figure 3).Environmental strontium is critical in the formation of the statolith [59].Sr:Ca and Ba:Ca are widely reported to have a strong, often negative, relationship with temperature, although for Sr:Ca, the relationship can be more complex [87].U:Ca was recently reported to have a positive relationship with temperature [88].Since all tanks were kept within 1 °C of one another (Figure 1) and did not reach the temperature differentials reported to generate Sr signals for a congener squid species (>2 °C, [48]) and one gastropod (= 4 °C, [73]), our results are not likely related to temperature effects.Curiously, when exposed to low [O 2 ] with low pH (low pHOx), statoliths were not enriched with Sr. Strontium incorporation into squid statoliths may be inversely related to metabolic rate.Low environmental pH/high pCO 2 and high temperatures can cause metabolic depression in squid embryos [45,46] and reduced growth [52].
The only element:calcium measured in this study that might be a useful indicator of low pHOx conditions is U:Ca.We showed that U:Ca is enriched (eight-fold increase) in the statoliths of embryos exposed to low pHOx relative to those exposed to the high pHOx treatment.We propose that this enrichment is driven by low [O 2 ] and exacerbated by the interactive effect of low [O 2 ] and pH (low pHOx).Under low pH stress, low [O 2 ] may impair the regulation of internal pH by embryonic squid, due to the reduced aerobic metabolic rate.This can lead to insufficient ATP production necessary to fuel active mechanisms for pH regulation and calcification.However, this indicator of an environmental response may not be useful as a proxy per se, because it likely tracks a sublethal effect on the embryo.Specifically, the uranium enrichment reflects the loss of pH regulation in the endolymph of the statocyst and may represent a threshold rather than a gradient.
The results presented show that the statolith chemistry records integrated the effects of the environment in concert with physiological processes, here identified as capsular effects (Tables 2 and 4).These data suggest that statolith-chemical composition has a substantial disconnect from the external seawater environment (unlike foraminifera and their shells and some corals and their skeletons).Elemental composition measurements from the capsule jelly, perivitelline fluid [89] and within the endolymph fluid of the statocyst in addition to environmental measurements would help clarify the relationship between the environment and statolith geochemistry.

Conclusions
For the first time that we are aware, we demonstrated that environmental pH and [O 2 ] affect squid statolith geochemistry (uranium:calcium) and that statolith geochemistry is strongly affected by factors associated with the capsule (capsular effects).The only other known study that tested the effects of environmental pH on squid-embryo statolith geochemistry found that only 65 Zn significantly differed from an elemental suite that included 110m Ag, 109 Cd, 57 Co, 203 Hg and 54 Mn [49].Evidence that environmental tracers in squid statoliths can track seawater pH and [O 2 ] is especially useful, because uranium has been shown to be promising for understanding squid life history, migrations and habitat use [88,90,91].
However, we did not find strong evidence that environmental pH and [O 2 ] effects can be resolved for the use of statolith geochemistry as environmental proxies of pH and [O 2 ].We did find strong capsular effects.The mechanism behind the capsular effect on statolith elemental incorporation is presumably due to a process that similarly affects all embryos of the capsule.Mechanisms include maternal transfer [53,73,83,84] and capsular and chorion membrane structural differences [49,50] among capsules.Less likely mechanisms include processes within the embryos [17][18][19]45,46] that are expressed similarly among embryos.These capsular effects are the first evidence (statolith chemistry) of strong physiological differences among the same cohort, and the importance of these differences for the persistence of the D. opalescens population is not known.Future applications might include the use of uranium:calcium as a geochemical marker tracking the initiation and duration of sublethal effects.

Experiment 1 (
November, 2011) compared the statolith geochemical response between embryos exposed to high pH and [O 2 ] (high pHOx) and low pH and [O 2 ] (low pHOx) treatments, and Experiment 2 (March, 2012) compared the statolith geochemical response between embryos exposed to low [O 2 ] and high pH (low [O 2 ]) and low pH and high [O 2 ] (low pH) treatments.Levels were based on field measurements of water conditions made from water depths of 35-88 m water depth, ~6 km off of Del Mar, USA
, and our experiments exposed embryos to treatments one week or more prior to the statolith formation.The low [O 2 ] and pH treatments developed more slowly (5-7 d) compared to the other treatments; thus, samples from the 3 treatments (Experiment 1: low pHOx and Experiment 2: low pH, low [O 2 ]) were gathered at two times, once to match the high pHOx treatment exposure duration and once again 5-7 d later to allow embryos exposed to low [O 2 ] and/or pH to develop to near-hatch Stages 28-29.The levels of [O 2 ], pH, Ω Aragonite and temperature were held constant for each treatment (Figure 1).

Figure 2 .
Figure 2. (a) Two squid-embryo capsules.Capsules are directly exposed to the environment, and each contains between 100 and 300 embryos.Ruler units are in cm; (b) Subsection of the capsule with the capsular membranes removed.Gelatinous material fills the interstitial space between the chorions and the capsular membranes; tick marks at the bottom of the image demarcate mm; (c) Chorion filled with perivitelline fluid and containing a squid embryo; (d) Squid embryo: statocysts are circled in red; (e) Statocysts are filled with endolymph fluid, and each contains a single statolith; (f) Embryonic statolith, made of aragonite.

Figure 3 .
Figure 3. Element:Ca for each treatment analyzed.(A) Experiment 1: Low pHOx results in statoliths with high levels of U:Ca (F 1, 3 = 16.86,p = 0.0005); (B) Experiment 2: Low [O 2 ] results in a higher U:Ca concentration within statoliths in comparison to low pH (F 1, 6 = 5.91, p = 0.0225) and higher Sr:Ca concentrations (F 1, 6 = 6.47, p = 0.0174); (C) Field values are from a single capsule that developed in the field.Treatments were tested using one-way ANOVA.Number within column = Number of capsules analyzed.* Significant.Bar = ±1 standard error from the mean.

Table 2 .
The statolith elemental:calcium composition among capsules within treatments was tested using a one-way mixed model ANOVA (nested within treatment and tank factors).Significance = bold.