DOI: 10.5285/221909cf-eac3-4a64-b1d0-3c1cdccdfe3c
title: In situ growth and physiological data from two Antarctic anemone species, Isotealia antarctica and Urticinopsis antarctica at Rothera Research Station (2020-2023)

description: To further our understanding of Antarctic predator growth and seasonal physiology, field growth rates were measured for two soft-bodied Antarctic anemone benthic predators, Isotealia antarctica and Urticinopsis antarctica, using in situ sampling of anemones on uniquely marked tiles from Rothera Research Station from 2020-2023. Ex situ measurements of oxygen consumption and seven-day faecal output were obtained from recently collected specimens in aquaria and compared between summer and winter. Winter physiological data for Antarctic species are rare, and we tested the hypothesis that generalist feeders or predators continue to feed during the winter. There is a dearth of basic life history and physiological data from Southern Ocean species, particularly from benthic sessile predators. This is an important data gap because species inhabiting the Southern Ocean live in a more temperature stable but seasonally varying environment than temperate and tropical counterparts. 

This work was supported by core funding from the NERC, UKRI, UK to the British Antarctic Survey.
description: Barcoding: A ~10 mm snip of a single tentacle was collected from each anemone in the trial group (n = 28), which was preserved in ethanol for barcoding (574bp fragment of the 18s gene). DNA was extracted from tentacles using the DNeasy extraction kit (Qiagen) according to manufacturer's instructions. A 574bp fragment of the 18S rDNA gene was amplified using NSF4 (CTGGTTGATYCTGCCAGT) and NSR581 (ATTACCGCGGCTGCTGGC) primers using MyTaq PCR ready mix (Meridian Bioscience) and a PCR annealing temperature of 60°C. PCR products were sequenced using the NSR581 primer by Source Bioscience, with verification using NSF4, if required (Supplemental file S2). Initial species identities were confirmed by blastn sequence similarity searching against the NCBI nucleotide database. Assignment of different individual animals to each species was then performed using ClustalX.

Buoyant weight: Buoyant weight was measured in the 45 experimental animals at time zero and at subsequent sampling points after 1 and 2 years on individuals recollected after these times. They were weighed after being gently removed from their tiles and cleaned of debris. Animals were placed on a scale suspended in ambient sea water and weights were recorded (± 0.001 g) using a Sartorius LA3200D balance.

Respirometry: Respiration measurements were conducted in appropriately sized respiration chambers, chosen after preliminary trials aimed to identify chamber sizes and trial durations needed for approximately 20% reduction of oxygen in respirometers. Prior to respiration measurements, animals were placed within the chambers and left for 24 hours to habituate to the experimental conditions; chambers were left open to allow continual water exchange with the flow through aquarium, with a 1 mm mesh preventing the anemone escape. This gave anemones time to settle within their respirometer and reduce metabolic rates to routine levels. Prior to measuring oxygen consumption, chamber water was exchanged, all air bubbles were removed, and the lids sealed. In addition, three control chambers, without anemones, were sealed to account for any changes in background oxygen levels. Oxygen concentrations were then recorded within each chamber at 'time 0' (3 repeat measurements per chamber at each sampling interval) using a Fibox-3 oxygen system (PreSens GmBH with SP-PSt3-NAUspots and OxyView PST3-V5.31 b software). Measurements were collected at the start and at the end of a six-hour period, when oxygen saturation did not fall below 70% in any chamber. Prior to taking samples, chambers were gently inverted three times to mix the contents and ensure that three stable readings were recorded. Temperature was recorded from the aquarium tank that the respiration chambers were placed in. At the end of the respirometry measurements, the volume of each anemone was measured by displacement of the volume of sea water. This volume was then subtracted from the volume of the respirometer to calculate the final volume of water within the chamber from which oxygen was extracted. The Fibox-3 system uses oxygen spots inside chambers. During each calibration, the amplitude from each spot was checked to ensure it read over 10,000, a measure of the integrity of oxygen sensitive foil. Measurements were standardised by calibrating each chamber spot 24 hours post measurement. The Mean Sea Level (QFF) atmospheric pressure was recorded before measurements commenced. Spots in chambers were calibrated with 100% saturated oxygen, which had been vigorously aerated for 30 minutes and then left to stand for 10 minutes. They were then calibrated for 0% oxygen using seawater with added sodium dithionite (2.5.%) at the ambient experimental temperatures.

Faecal collection: Immediately after collection, anemones were placed in individual, standardised floating containers of 5632cm3, in the Bonner laboratory flow through aquarium. Faeces were pipetted from each container every 24 hours for seven days. Samples were labelled according to individual anemone ID and the day of faecal collection. Container water was refreshed after each faecal collection to maintain water quality. Faecal samples (n = 282) were first rinsed in MiliQ water and placed in pre-weighed foil boats. Samples were dried to a constant weight at 60°C and then ignited at 460°C for a 12-hour dwelling cycle in a muffle furnace. Ash free dry mass (AFDM), an estimate of organic content, was calculated by subtracting the ash mass from the dried mass.
description: Respirometry data was collected using Fibox-3 oxygen system, PreSens GmBH with SP-PSt3-NAUspots and OxyView PST3-V5.31 b software.
Anemones were weighted on a Sartoritus LA320D balance
Statistics were completed using R Studio version 4.3.1
description: Respirometry data relies on the calibration of PreSens GmBH with SP-PSt3-NAUspots after each respirometry event. These spots are calibrated with 0 % oxygen water and 100 % saturated oxygen water. 
The Sartoritus LA320D balances are calibrated every year and are rotated.

Losses of data_growth metrics_supplementalS4 (57KB), contain NA's because author was unable to obtain metrics for each individual anemone for ash free weights because anemone contain high water content and take a long time to dry. Furnace space was limited at Rothera.