DOI: 10.6078/d1g994
title: Nanostring data from female and male crabs (Petrolisthes cinctipes) according to intertidal zone height and sampling date

description: Thermal extremes alter population processes, which can result in part from
 temperature-induced movement at different spatial and temporal scales.
 Thermal thresholds for animal movement likely change based on underlying
 thermal physiology and life-history stage, a topic that requires greater
 study. The intertidal porcelain crab Petrolisthes cinctipes currently
 experiences temperatures that can reach near-lethal levels in the
 high-intertidal zone at low tide. However, the thermal thresholds that
 trigger migration to cooler microhabitats, and the extent to which crabs
 move in response to temperature, remain unknown. Moreover, the influence
 of reproductive status on these thresholds is rarely investigated. We
 integrated demographic, molecular, behavioral, and physiological
 measurements to determine if behavioral thermal limits varied due to
 reproductive state. Demographic data showed a trend for gravid, egg
 bearing, crabs to appear more often under rocks in the cooler intertidal
 zone where crab density is highest. In situ expression of 31 genes related
 to stress, metabolism, and growth in the field differed significantly
 based on intertidal elevation, with mid-intertidal crabs expressing the
 gene for the reproductive yolk protein vitellogenin (vg) earlier in the
 season. Furthermore, we experimentally demonstrate that VG protein levels
 in female hemolymph increases with density. We tested for temperatures
 that elicit movement and found that gravid females engage in heat
 avoidance behavior at lower temperatures (i.e., have a lower voluntary
 thermal maximum, VTmax) than non-gravid females. VTmax was positively
 correlated with the temperature of peak firing rate for distal afferent
 nerve fibers in the walking leg, a physiological relationship that could
 correspond to the mechanistic underpinning for temperature dependent
 movement. The vulnerability of marine organisms to global change is
 predicated by their ability to utilize and integrate physiological and
 behavioral strategies in response to temperature to maximize survival and
 reproduction. Interactions between fine-scale temperature variation and
 reproductive biology can have important consequences for the ecology of
 species, and is likely to influence how populations respond to ongoing
 climate change.
description: Total RNA
 was extracted from the bodies of the crabs. The walking legs and claws of
 the frozen crabs were removed, and the remaining bodies were ground to a
 powder in liquid nitrogen with a mortar and pestle. RNA was extracted from
 the powder using guanidine isothiocyanate extraction with Tri Reagent
 (Molecular Research Center, USA) according to the manufacturer's
 protocol. Tri Reagent was added to each 50 mg powder sample along with
 nuclease-free stainless-steel beads and shaken in a TissueLyser (Qiagen)
 at 30 Hz for 10 minutes. Phase separation was performed with BCP
 (Molecular Research Center, USA), and isopropanol and a high salt buffer
 were used to precipitate the RNA. The RNA was washed twice with 75%
 ethanol and dissolved in 50 μL of RNase free water. RNA quality was
 assessed with A260/280 ratios and 1% agarose gel electrophoresis with
 ethidium bromide staining. RNA was stored at -80°C. The measurement of
 target and housekeeping gene expression was conducted by the University of
 California, San Francisco Center for Advanced Technology using the
 Nanostring platform. Nanostring generates read counts for each gene from
 each sample without cDNA synthesis or PCR amplification. 25 μL of RNA per
 sample at a concentration of 100 ng/μL was provided to generate expression
 data of 31 target genes and eight housekeeping genes. Target genes were
 chosen based on their association with important biological processes
 including stress physiology (e.g., <em>heat-shock proteins, V-type
 proton ATPases</em>, genes associated with ubiquitination),
 metabolism (e.g., <em>cytochromes, acyl-coa synthetase</em>),
 growth (e.g., <em>cuticular proteins</em>), and reproduction
 (e.g., <em>vitellogenin, vitelline egg coat protein</em>).
 Gene expression was standardized relative to the expression of internal
 positive controls (for target and housekeeping genes) and housekeeping
 genes (for target genes only) for each individual. For the positive
 controls, we generated a positive control correction factor for each
 individual by dividing the geometric mean of positive controls for the
 individual by the geometric mean of positive controls across all
 individuals. The counts of all genes for that individual were then
 multiplied by this factor. Target genes for an individual were
 standardized to housekeeping genes in a similar manner, whereby target
 gene expression was multiplied by a correction factor taken as the
 geometric mean of housekeeping gene expression for that individual divided
 by the geometric mean of housekeeping gene expression across all
 individuals.