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
