DOI: 10.5061/dryad.j9kd51c84
title: Ocean warming reduces gastropod survival despite maintenance of feeding and oxygen consumption rates

description: Short-term, sub-lethal response variables are increasingly used to provide
 rapid indications of whole organism responses to future climate
 conditions. Accumulating evidence suggests, however, that these response
 variables may not consistently reflect whole organism responses which
 manifest over longer time scales. Here, we consider the effect of moderate
 warming on longer-term whole organism fitness, as reflected by survival,
 as well as two shorter-term response variables, feeding rate and oxygen
 consumption, for two tropical gastropod species. We found a significant
 reduction in survival under warming, despite no significant effect of
 warming on feeding or oxygen consumption rates. This result demonstrates
 that the maintenance of physiological rates alone is not sufficient to
 sustain organism survival under elevated temperatures; rather an increase
 in physiological process rates is likely required. Consequently, isolated
 short-term physiological processes may not adequately reflect longer-term
 whole organism responses to altered climate. For improved understanding,
 both short- and long-term responses need to be considered.
description: <strong>Experimental design and
 set-up</strong> To determine the effects of
 warming on tropical gastropods, we quantified the response of two species
 (<i>Chlorostoma argyrostoma</i>, <i>Lunella
 coronata</i>) to experimentally manipulate temperatures in a
 laboratory-based tank experiment. We considered adults of these species
 (maximum shell diameter <i>C. argyrostoma</i> 22.2 mm ± 0.4
 mm, <i>L. coronata</i> 24.3 ± 0.5 mm; mean ± SE), which
 typically live to be between 5 and 10 years of age, although they can live
 for 30 years (Walsby 1977, Cooper & Shanks 2011). We exposed
 experimental tanks to current or future temperatures for six weeks from
 July to August 2019. The ‘current’ temperature (25.5 °C) was based on
 long-term measurements of the collection site in the summer experimental
 period (25.4 ± 0.2 °C was the average temperature over the experimental
 months, i.e. July – August, for the last 10 years of data published, i.e.
 2009 – 2018, at the site nearest our collection location, i.e. Eastern
 Buffer station EM2; Hong Kong Environmental Protection Department 2019),
 while the ‘future’ temperature (+ 2.5 °C) simulates ocean temperature
 anticipated between the years 2081-2100 (Stocker <i>et al</i>.
 2013). We used six replicate tanks for each treatment, with replicate
 specimens of each gastropod species in each tank (<i>n</i> = 3
 of each species per tank). <strong>Collection of
 gastropods and holding conditions</strong> We
 collected adult gastropods (<i>C. argyrostoma</i>, <i>L.
 coronata</i>) and algae (<i>Gelidium</i> spp.) in early
 July at Joss House Bay in Tai Miu Wan, Hong Kong (22.27°S, 114.29°E). We
 then brought experimental organisms back to the Simon F.S. Li Marine
 Science Laboratory, Hong Kong, and cleaned all gastropod shells of
 epibiotic organisms. To allow for acclimation, prior to
 the experimental period we kept all gastropods and algae in separate
 holding tanks (19 L seawater; <i>n</i> = 10 individuals per
 tank) filled with natural seawater under current conditions (temperature =
 26.1 °C, salinity = 35, pH = 8.03, 12:12h light:dark regime) for seven
 days. During this period, we provided gastropods algae collected from the
 field to graze upon. <strong>Experimental
 treatments</strong> Following acclimation, we
 transferred individual gastropods into experimental tanks
 (<i>n</i> = 3 gastropods of each species, 6 total individuals
 per tank) where each were held isolated from the others in a cage, with
 the same conditions as in the holding tanks described above. We provided
 each gastropod a piece of algae for food which was replenished weekly
 throughout the experimental period. Half of the tanks we maintained at
 current temperatures (<i>n</i> = 6), while gradually
 increasing temperature of the other half to the future treatment
 (<i>n</i> = 6). The water temperature in the ‘future’
 treatment tanks was warmed at a rate of 0.5 °C per hour to be 2.5 °C
 hotter than the ‘current’ treatment tanks on the day before the
 experimental period. This rate was used to minimise the chance of
 mortality to result from initial heat shock (the first gastropod mortality
 was recorded 9 days after establishment of treatment conditions). Bar
 heaters were used inside of the tanks to establish and maintain
 experimental temperatures. iButtons monitored temperature in each tank,
 where they took recordings every 10 minutes (Thermochron iButton logger,
 Embedded Data Systems). Mean water temperature was significantly different
 between the current and future tanks (mean ± SE, current 25.7 ± &lt;
 0.1, future 28.2 ± &lt;0.1 °C, F<sub>1, 10 </sub>= 98.22,
 <i>p</i> = &lt;0.0001). We maintained water quality by
 removing faecal matter and renewing one-third weekly with fresh marine
 water. <strong>Experimental responses
 </strong> Responses of gastropods to experimental
 treatments were quantified at the end of the experiment in terms of
 survival, algal consumption (feeding), and oxygen consumption.
 We measured survival on the 39<sup>th</sup> and
 40<sup>th</sup> days of the experiments by quantifying the
 number of living gastropods remaining (this was the same as the number
 used in concurrent oxygen measurements). During the experimental period we
 regularly checked the snails and removed any dead individuals which were
 identified by probing the gastropods to prompt a reaction. While we do
 believe the gastropods were physiologically dead, they were certainly dead
 from an ecological viewpoint (that is, unable to attach and retain their
 place on the substrate, move in response to external cues).
 We quantified feeding rates by providing each grazer a piece of
 <i>Gellidium </i>spp. to feed on for 7 days with the feeding
 trial started on the 31<sup>st</sup> day of the experiment and
 concluded on the 38<sup>th</sup>. We quantified the change in
 algal mass (final – initial measurement) by gently patting the algae dry
 and then weighing to the nearest 0.0001 g at the beginning and end of the
 feeding period. The gastropods were provided sufficient algae that they
 were able to feed to satiation (0.1992 ± 0.0082 g; mean ± SE). For each
 tank, we also had control cages in which algal pieces were added without
 grazers; these cages indicated change driven by algal rather than grazer
 responses and were used to correct for any growth or tissue loss not due
 to the herbivores. We calculated consumed algae as: initial mass ×
 correction factor – final mass of the piece of algae. The correction
 factor was: the final mass/initial mass for each control algal unit, which
 we then averaged to provide a single value per tank (following Renaud
 <i>et al</i>. 1990). We measured oxygen
 consumption rates on the 39<sup>th</sup> and
 40<sup>th</sup> days of the experiments (at the same time as
 survival was considered) by transferring each gastropod to an airtight
 chamber (55mL falcon tubes) filled with seawater from the treatment
 conditions, ensuring the absence of any gas bubbles. The chamber was kept
 within the tank to maintain treatment temperature. We also filled chambers
 without gastropods with water; measurements from these were used to
 account for any possible biological activity in the water. Each chamber
 had a pre-calibrated oxygen spot attached to the inner wall (Fibox4 trace,
 PreSens). Using these spots and a fibre optic oxygen sensor, we measured
 oxygen concentration twice in rapid succession every minute for a
 10-minute period. We subtracted the background levels of oxygen
 consumption measured in paired controls from the measured rates of each
 snail before statistical analysis.
 <strong>Statistical analysis</strong>
 We used PERMANOVAs (with Euclidian distance matrices) to examine
 the effects of temperature and species on survival, feeding rates, and
 oxygen consumption. We corrected rates of oxygen consumption and feeding
 for gastropod size effects by dividing the oxygen and algal consumption
 rate measured for each gastropod by the gastropod wet mass. In all
 analyses, we treated temperature and species as orthogonal and fixed, with
 two levels (current and future or <i>C. argyrostoma</i> and
 <i>L. coronata</i>, respectively), and tank used as
 replicates. The PERMANOVAs were done using the PERMANOVA+ routines that
 are an ‘add-in’ to the PRIMER 6 software (Clarke & Gorley 2006,
 Anderson <i>et al</i> 2008). To evaluate the chances of
 committing type II errors, we used dissimilarity-based multivariate
 standard error (MultSE) to assess the effect of increased sample size on
 the precision of our parameter estimates using the SSP package in R
 (Anderson & Santana‐Garcon 2015, R Core Team 2020, Guerra-Castro
 <i>et al</i>. 2021).
description: R Script files walks user through transforming the raw data into
 usable data sets. Analyses are completed in Primer instead of R. There are
 simplified data sets that are prepared for that purpose, however the
 cleaning of the data and transforming raw data into rate data can be found
 in R.