ALBERT

Description: We examined five exoskeletal structures in the California spiny lobster, Panulirus interruptus, to better understand both the extent of exoskeleton variation within a single crustacean species and the relationship between morphology and function. The five structures were the carapace, antenna, rostral horn, mandible, and abdominal segment, each of which provides predator defenses to different degrees, potentially leading to differentiation in structure construction. Here, we characterized and compared the mineralization (wt. % and concentration of Ca and Mg), ultrastructure (cuticle layer thickness), and mechanical properties (hardness and stiffness). Layer thickness (µm) and wt. % mineralization were determined using a scanning electron microscope (SEM) equipped with electron-dispersive x-ray spectroscopy (EDX). Concentration of elements (µmol/mg sample) was measured using inductively-coupled x-ray spectrometry (ICP-MS), while material properties (GPa) were measured using a nanoindenter with a Berkovitch diamond tip.

Description: We examined the response of multiple structures used for predator defense in the California spiny lobster, Panulirus interruptus, to a series of ocean acidification-like conditions. Lobsters were collected by modified commercial traps offshore La Jolla, CA (in the area around 32.8534193, -117.2687516) in October 2016 and held at ambient conditions (pH 7.97, 16.5°C) before exposure to stable or diurnally fluctuating reduced pH conditions established by bubbling CO2 and as measured using best practices (ambient pH/stable, 7.97, 16.5°C; reduced pH/stable 7.67, 16.6°C; reduced pH with low fluctuations, 7.67 ± 0.05, 16.4°C; reduced pH with high fluctuations, 7.67 ± 0.10, 16.4°C). After three months, inductively-coupled x-ray spectrometry (ICP-MS) was performed on cuticle samples at the Scripps Isotope Geochemistry Laboratory (SIGL) for a precise quantification of elements (µmol/mg sample). The carapace spine was air-dried and then trimmed so only the spine remained with no setae, and the abdominal segment was cut as a 1 x 1 cm² from the center of the second abdominal segment and air-dried. Samples were weighed and placed in Teflon vials for digestion with 0.5 ml of concentrated Teflon-distilled (TD) nitric acid (HNO3) on a hotplate at 100°C for 〉24 h. Samples were dried down and diluted by a factor of 4000 with 2% TD HNO3 before being transferred to pre-cleaned centrifuge tubes for analysis. Samples were doped with an indium solution 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. Total procedural blanks represented 〈0.3% of the measurement for Mg and Ca. Raw data were corrected offline for instrument background and drift. Samples were bracketed by internal standards of crab carapace (n=3), which allowed for calculation of absolute values as well as weighted averages of isotopes from natural abundance. The standards yielded external precision of 2% and 3% for Mg and Ca, respectively.

Description: We examined the response of multiple structures used for predator defense in the California spiny lobster, Panulirus interruptus, to a series of ocean acidification-like conditions. Lobsters were collected by modified commercial traps offshore La Jolla, CA (in the area around 32.8534193, -117.2687516) in October 2016 and held at ambient conditions (pH 7.97, 16.5°C) before exposure to stable or diurnally fluctuating reduced pH conditions established by bubbling CO2 and as measured using best practices (ambient pH/stable, 7.97, 16.5°C; reduced pH/stable 7.67, 16.6°C; reduced pH with low fluctuations, 7.67 ± 0.05, 16.4°C; reduced pH with high fluctuations, 7.67 ± 0.10, 16.4°C). After three months, we examined the relative atomic weight composition (weight %/atomic weight of element) of the carapace using a scanning electron microscope (SEM) equipped with electron-dispersive x-ray spectroscopy (EDX). Each cuticle sample was rinsed with deionized water and allowed to air dry. Samples were then freeze-fractured with liquid nitrogen and critical-point dried (AutoSamdri 815 Series A, Tousimis, Rockville, MD, USA) before being mounted on a 90-degree SEM tip and sputter-coated with iridium. Cross-sections of these cuticle samples were examined with ultra-high-resolution scanning electron microscopy under high vacuum (XL30 SFEG with Sirion column and Apreo LoVac, FEI, Hillsboro, OR, USA with Oxford X-MAX 80 EDS detector, Concord, MA, USA) at 10 or 20 kV. One to two samples each of the carapace spine and antenna from individual lobsters were imaged. EDX was measured with with two machines (XL30 SFEG with Sirion column and Apreo LoVac, FEI, Hillsboro, OR, USA with Oxford X-MAX 80 EDS detector, Concord, MA, USA) at 20 kV acceleration voltage. Spectra were taken on the cross-sectional surface of the exocuticle and the endocuticle layers of the carapace spine and antenna base and the core and outer ring of the horn tip.

Description: We examined the response of multiple structures used for predator defense in the California spiny lobster, Panulirus interruptus, to a series of ocean acidification-like conditions. Lobsters were collected by modified commercial traps offshore La Jolla, CA (in the area around 32.8534193, -117.2687516) in October 2016 and held at ambient conditions (pH 7.97, 16.5°C) before exposure to stable or diurnally fluctuating reduced pH conditions established by bubbling CO2 and as measured using best practices (ambient pH/stable, 7.97, 16.5°C; reduced pH/stable 7.67, 16.6°C; reduced pH with low fluctuations, 7.67 ± 0.05, 16.4°C; reduced pH with high fluctuations, 7.67 ± 0.10, 16.4°C). After three months, we examined the atomic weight composition (%) of the carapace using a scanning electron microscope (SEM) equipped with electron-dispersive x-ray spectroscopy (EDX). Each cuticle sample was rinsed with deionized water and allowed to air dry. Samples were then freeze-fractured with liquid nitrogen and critical-point dried (AutoSamdri 815 Series A, Tousimis, Rockville, MD, USA) before being mounted on a 90-degree SEM tip and sputter-coated with iridium. Cross-sections of these cuticle samples were examined with ultra-high-resolution scanning electron microscopy under high vacuum (XL30 SFEG with Sirion column and Apreo LoVac, FEI, Hillsboro, OR, USA with Oxford X-MAX 80 EDS detector, Concord, MA, USA) at 10 or 20 kV. One to two samples each of the carapace spine and antenna from individual lobsters were imaged. EDX was measured with with two machines (XL30 SFEG with Sirion column and Apreo LoVac, FEI, Hillsboro, OR, USA with Oxford X-MAX 80 EDS detector, Concord, MA, USA) at 20 kV acceleration voltage. Spectra were taken on the cross-sectional surface of the exocuticle and the endocuticle layers of the carapace spine and antenna base and the core and outer ring of the horn tip.

Description: We examined the response of multiple structures used for predator defense in the California spiny lobster, Panulirus interruptus, to a series of ocean acidification-like conditions. Lobsters were collected by modified commercial traps offshore La Jolla, CA (in the area around 32.8534193, -117.2687516) in October 2016 and held at ambient conditions (pH 7.97, 16.5°C) before exposure to stable or diurnally fluctuating reduced pH conditions established by bubbling CO2 and as measured using best practices (ambient pH/stable, 7.97, 16.5°C; reduced pH/stable 7.67, 16.6°C; reduced pH with low fluctuations, 7.67 ± 0.05, 16.4°C; reduced pH with high fluctuations, 7.67 ± 0.10, 16.4°C). After three months, we examined cuticle layer thickness (µm) using a scanning electron microscope (SEM) equipped with electron-dispersive x-ray spectroscopy (EDX). Each cuticle sample was rinsed with deionized water and allowed to air dry. Samples were then freeze-fractured with liquid nitrogen and critical-point dried (AutoSamdri 815 Series A, Tousimis, Rockville, MD, USA) before being mounted on a 90-degree SEM tip and sputter-coated with iridium. Cross-sections of these cuticle samples were examined with ultra-high-resolution scanning electron microscopy under high vacuum (XL30 SFEG with Sirion column and Apreo LoVac, FEI, Hillsboro, OR, USA with Oxford X-MAX 80 EDS detector, Concord, MA, USA) at 10 or 20 kV. One to two samples each of the carapace spine and antenna from individual lobsters were imaged and measured for the total cuticle thickness (epicuticle, exocuticle, and endocuticle), as well as thickness of the individual exo- and endocuticle layers.

Description: We examined the response of multiple structures used for predator defense in the California spiny lobster, Panulirus interruptus, to a series of ocean acidification-like conditions. Lobsters were collected by modified commercial traps offshore La Jolla, CA (in the area around 32.8534193, -117.2687516) in October 2016 and held at ambient conditions (pH 7.97, 16.5℃) before exposure to stable or diurnally fluctuating reduced pH conditions established by bubbling CO2 and as measured using best practices (ambient pH/stable, 7.97, 16.5℃; reduced pH/stable 7.67, 16.6℃; reduced pH with low fluctuations, 7.67 ± 0.05, 16.4℃; reduced pH with high fluctuations, 7.67 ± 0.10, 16.4℃). After three months, the carapace spine and rostral horn tip were tested for hardness and stiffness using a nanoindentation materials testing machine (Nano Hardness Tester, Nanovea, Irvine, CA, USA) equipped with a Berkovich tip. Fresh samples (〈12 hours, except for two samples tested within 24 hours) were kept hydrated in seawater until testing. Samples were secured to an aluminum block with cyanoacrylate glue such that the outer surface was facing up. Indentations were performed by applying a load of 40 mN to the outer surface of the sample at loading and unloading rates of 80 mN/min with a 30 sec hold for creep. At least three indents were taken per sample.

Description: Spiny lobsters rely on multiple biomineralized exoskeletal predator defenses that may be sensitive to ocean acidification (OA). Compromised mechanical integrity of these defensive structures may tilt predator-prey outcomes, leading to increased mortality in the lobsters' environment. Here, we tested the effects of OA-like conditions on the mechanical integrity of selected exoskeletal defenses of juvenile California spiny lobster, Panulirus interruptus. Young spiny lobsters reside in kelp forests with dynamic carbonate chemistry due to local metabolism and photosynthesis as well as seasonal upwelling, yielding daily and seasonal fluctuations in pH. Lobsters were exposed to a series of stable and diurnally fluctuating reduced pH conditions for three months (ambient pH/stable, 7.97; reduced pH/stable 7.67; reduced pH with low fluctuations, 7.67 ± 0.05; reduced pH with high fluctuations, 7.67 ± 0.10), after which we examined the intermolt composition (Ca and Mg content), ultrastructure (cuticle and layer thickness), and mechanical properties (hardness and stiffness) of selected exoskeletal predator defenses. Cuticle ultrastructure was consistently robust to pH conditions, while mineralization and mechanical properties were variable. Notably, the carapace was less mineralized under both reduced pH treatments with fluctuations, but with no effect on material properties, and the rostral horn had lower hardness in reduced/high fluctuating conditions without a corresponding difference in mineralization. Antennal flexural stiffness was lower in reduced, stable pH conditions compared to the reduced pH treatment with high fluctuations and not correlated with changes in cuticle structure or mineralization. These results demonstrate a complex relationship between mineralization and mechanical properties of the exoskeleton under changing ocean chemistry, and that fluctuating reduced pH conditions can induce responses not observed under the stable reduced pH conditions often used in OA research. Furthermore, this study shows that some juvenile California spiny lobster exoskeletal defenses are responsive to changes in ocean carbonate chemistry, even during the intermolt period, in ways that can potentially increase susceptibility to predation among this critical life stage.

Description: We examined the response of multiple structures used for predator defense in the California spiny lobster, Panulirus interruptus, to a series of ocean acidification-like conditions. Lobsters were collected by modified commercial traps offshore La Jolla, CA (in the area around 32.8534193, -117.2687516) in October 2016 and held at ambient conditions (pH 7.97, 16.5°C) before exposure to stable or diurnally fluctuating reduced pH conditions established by bubbling CO2 and as measured using best practices (ambient pH/stable, 7.97, 16.5°C; reduced pH/stable 7.67, 16.6°C; reduced pH with low fluctuations, 7.67 ± 0.05, 16.4°C; reduced pH with high fluctuations, 7.67 ± 0.10, 16.4°C). After three months, we examined the composition (wt. % and concentration of Ca and Mg), ultrastructure (cuticle and thickness of exocuticle ), and mechanical properties (hardness and stiffness). Layer thickness (µm) and wt. % mineralization were determined using a scanning electron microscope (SEM) equipped with electron-dispersive x-ray spectroscopy (EDX). Concentration of elements (µmol/mg sample) was measured using inductively-coupled x-ray spectrometry (ICP-MS), while material properties (GPa) were measured using a nanoindenter with a Berkovitch diamond tip.

Description: We examined the response of multiple structures used for predator defense in the California spiny lobster, Panulirus interruptus, to a series of ocean acidification-like conditions. Lobsters were collected by modified commercial traps offshore La Jolla, CA (in the area around 32.8534193, -117.2687516) in October 2016 and held at ambient conditions (pH 7.97, 16.5℃) before exposure to stable or diurnally fluctuating reduced pH conditions established by bubbling CO2 and as measured using best practices (ambient pH/stable, 7.97, 16.5℃; reduced pH/stable 7.67, 16.6℃; reduced pH with low fluctuations, 7.67 ± 0.05, 16.4℃; reduced pH with high fluctuations, 7.67 ± 0.10, 16.4℃). After three months, we examined the composition (wt. % and concentration of Ca and Mg), ultrastructure (cuticle and thickness of exocuticle ), and mechanical properties (hardness and stiffness). Layer thickness (µm) and wt. % mineralization were determined using a scanning electron microscope (SEM) equipped with electron-dispersive x-ray spectroscopy (EDX). Concentration of elements (µmol/mg sample) was measured using inductively-coupled x-ray spectrometry (ICP-MS), while material properties (GPa) were measured using a nanoindenter with a Berkovitch diamond tip. The metadata file describes headers, units, methods, etc in each data file. CSV files are data files for separate measurements and will run with R code stored at GitHub to reproduce analyses and figures.