Exoskeletal predator defenses of juvenile California spiny lobsters (Panulirus interruptus) are affected by fluctuating ocean acidification-like conditions

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

Integrity of crustacean predator defenses under ocean acidification and warming conditions

Crustaceans are a diverse group of species, but all rely on an exoskeleton that is shed and formed anew throughout their lifetime. Exoskeletons perform a wide range of functions, sometimes acting as armor, a means to produce sound, a tool to crush hard prey, or even a window to facilitate transparency. The exoskeleton and its functions, however, are likely vulnerable to ocean acidification and ocean warming, which may alter its composition and the energy allocated towards its production. I investigated the effects of these future ocean conditions on two southern Californian crustaceans, the California spiny lobster Panulirus interruptus and the grass shrimp Hippolyte californiensis, which rely on their exoskeleton for different predator defenses. P. interruptus is an iconic feature of southern California’s kelp forest ecosystem but also a potential prey item for many of its large predators. Spiny lobsters use their antennae, mandibles, carapace, and horns to avoid predation. Each of these structures is specialized for a certain defense, displaying differences in composition, structure, and material properties that allow the antennae, for example, to remain flexible to avoid breaking when pushing predators away while imbuing hardness in crushing structures like the mandible (Chapter 1). Juvenile lobsters exposed to ocean acidification-like conditions largely maintained their predator defenses, displaying some differences in the composition across the exoskeleton but no strong effects to defense functionality, including the non-exoskeletal defenses of detecting chemical cues and the tail-flip escape response (Chapter 3). Additionally, larval P. interruptus, exposed to both ocean acidification and warming conditions, grew slightly smaller in reduced pH but maintained their transparency in both conditions (Chapter 2). In contrast, H. californiensis resides in eelgrass meadows where a primary defense strategy is cryptic colouration, accomplished via a transparent exoskeleton with underlying pigment. When exposed to both ocean acidification and ocean warming-like conditions, shrimp maintained their transparency and did not respond negatively to either condition (Chapter 4). Together, this work on both species demonstrates that a diversity of predator defenses in temperate crustaceans, included those afforded by the exoskeleton, appear to be relatively resilient to both future ocean acidification and ocean warming conditions

Composition, ultrastructure, and material properties of California spiny lobster (Panulirus interruptus) exoskeleton

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

Carapace material properties of juvenile California spiny lobsters (Panulirus interruptus) in response to ocean acidification

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

Cuticle relative atomic weight composition of juvenile California spiny lobsters (Panulirus interruptus) in response to ocean acidification

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

Carapace properties of juvenile California spiny lobsters (Panulirus interruptus) in response to ocean acidification - files to run with R code

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

Seawater carbonate chemistry and carapace material properties, cuticle atomic weight composition, elemental concentrations and thickness of juvenile California spiny lobsters (Panulirus interruptus)

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

Exoskeletal predator defenses of juvenile California spiny lobsters (Panulirus interruptus) are affected by fluctuating ocean acidification

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

Cuticle thickness of juvenile California spiny lobsters (Panulirus interruptus) in response to ocean acidification

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

Cuticle atomic weight composition of juvenile California spiny lobsters (Panulirus interruptus) in response to ocean acidification

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