Increased sensitivity of subantarctic marine invertebrates to metals under a changing climate

Study location and test species
Subantarctic Macquarie Island lies in the Southern Ocean, just north of the Antarctic Convergence at 54 degrees 30' S, 158 degrees 57' E. Its climate is driven by oceanic processes, resulting in highly stable daily and inter-seasonal air and sea temperatures (Pendlebury and Barnes-Keoghan, 2007). Temperatures in intertidal rock pools (0.5 to 2 m deep) were logged with Thermochron ibuttons over two consecutive summers and averaged 6.5 (plus or minus 0.5) degrees C. The island is relatively pristine and in many areas there has been no past exposure to contamination. To confirm sites used for invertebrate collections were free from metal contamination, seawater samples were taken and analysed by inductively coupled plasma optical emission spectrometry (ICP-OES; Varian 720-ES; S1)

The four invertebrate species used in this study were drawn from a range of taxa and ecological niches (Figure 1). The isopod Limnoria stephenseni was collected from floating fronds of the kelp Macrosystis pyrifera, which occurs several hundred meters offshore. The copepod Harpacticus sp. and bivalve Gaimardia trapesina were collected from algal species in the high energy shallow, subtidal zone. Finally, the flatworm Obrimoposthia ohlini was collected from the undersides of boulders throughout the intertidal zone. We hypothesised L. stephenseni would be particularly sensitive to changes in salinity and temperature due to its distribution in the deeper and relatively stable subtidal areas, while O. ohlini would be less sensitive due to its distribution high in the intertidal zone and exposure to naturally variable conditions. We reasoned that the remaining two species, G. trapesina, and Harpacticus sp. were intermediate in the conditions to which they are naturally exposed and hence would likely be intermediate in their response.

Test procedure
The combined effect of salinity, temperature and copper on biota was determined using a multi-factorial design. A range of copper concentrations were tested with each combination of temperatures and salinities, so that there were up to 9 copper toxicity tests simultaneously conducted per species (Table 1). Experiments on L. stephenseni and Harpacticus sp. were done on Macquarie Island within 2 to 3 days of collection, during which they were acclimated to laboratory conditions. While, G. trapesina and O. ohlini were transported by ship to Australia in a recirculating aquarium system and maintained in a recirculating aquarium at the Australian Antarctic Division in Hobart, both at 6 degreesC. These two taxa were used in experiments within 3 months of their collection. A limited number of G. trapesina and O. ohlini were available, resulting in fewer combinations of stressors tested.

Controls for the temperature and salinity treatments were set at ambient levels of 35 plus or minus 0.1 ppt and 5.5 to 6 degreesC for all species. The lowered control temperature for the bivalve reflected the cooler seasonal temperatures at time of testing and lower position within the intertidal. Previous tests conducted under these ambient conditions provided information on the ranges of relevant copper concentrations, appropriate test durations, and water change regimes for each taxon (Holan et al., 2017, Holan et al., 2016b). From these previous studies, we determined that a test duration of 14 d was sometimes required with 7 d often being the best outcome for most species due to high control survival and sufficient response across concentrations. The bivalve G. trapesina was an exception to this due to unfavourable water quality after 3 days in previous work (Holan et al., 2016). For the other three species, this longer duration for acute tests, compared to tests with tropical and temperature species (24 to 96 h) was consistent with previous Antarctic studies that have required longer durations in order to elicit an acute response in biota (King and Riddle, 2001, Marcus Zamora et al., 2015, Sfiligoj et al., 2015). Experimental variables (volume of water, density of test organisms, copper concentrations, temperatures and salinities) differed for each experiment due to differences between each species (Table 1). The temperature increases that were tested (2 to 4 degreesC) reflected the increased sea and air temperatures predicted for the region tested by 2100 (Collins et al., 2013).

Treatments were prepared 24 h prior to the addition of animals. Seawater was filtered to 0.45 microns and water quality was measured using a TPS 90-FL multimeter at the start and end of tests. Dissolved oxygen was greater than 80% saturation and pH was 8.1 to 8.3 at the start of tests. All experimental vials and glassware were washed with 10% nitric acid and rinsed with MilliQ water three times before use. Salinity of test solutions was prepared by dilution through the addition of MilliQ water. Copper treatments using the filtered seawater at altered salinities were prepared using 500mg/L CuSO4 (Analytical grade, Univar) in MilliQ water stock solution. Samples of test solutions for metal analysis by ICP-OES were taken at the start and end of tests (on days 0 and 14). Details of ICP-OES procedures are described in the Supplemental material (S4). Samples were taken using a 0.45 µm syringe filter that had been acid and Milli-Q rinsed. Samples were then acidified with 1% diluted ultra-pure nitric acid (65% Merck Suprapur). Measured concentrations at the start of tests were within 96% of nominal concentrations. In order to determine approximate exposure concentrations for each treatment, we averaged the 0 d and 14 d measured concentrations (Table 1). Tests were conducted in temperature controlled cabinets at a light intensity of 2360 lux. At the Macquarie Island station a light-dark regime of 16:8 h was used to mimic summer conditions. In the laboratories in Kingston, Australia, a 12:12 h regime was used to simulate Autum light conditions (as appropriate for the time of testing). Test individuals were slowly acclimated to treatment temperatures over 1 to 2 h before being added to treatments. Temperatures were monitored using Thermochron ibutton data loggers within the cabinets for the duration of the tests.

Determination of mortality of individuals differed for each taxon. Mortality was recorded for Gaimardia trapesina when shells were open due to dysfunctional adductor muscles; for Obrimoposthia ohlini when individuals were inactive and covered in mucous; for Limnoria stephenseni when individuals were inactive after gentle stimulation with a stream of water from a pipette; and for Harpacticus sp. when urosomes were perpendicular to prosomes (as used in other studies with copepods; see Kwok and Leung, 2005). All dead individuals were removed from test vials.

研究地点与受试物种
亚南极麦夸里岛（Subantarctic Macquarie Island）位于南大洋，地处南极辐合带以北，坐标为南纬54°30′、东经158°57′。该岛气候受海洋过程调控，日均与季际气温及海温均极为稳定（Pendlebury和Barnes-Keoghan，2007）。研究人员采用Thermochron iButton温度记录仪对潮间带岩石池（水深0.5~2 m）的水温进行了连续两个夏季的监测，测得平均水温为6.5±0.5 ℃。该岛整体相对原始，多数区域未受污染。为确认无脊椎动物采集点未受金属污染，研究人员采集了海水样本，并采用电感耦合等离子体光发射光谱法（inductively coupled plasma optical emission spectrometry，ICP-OES；Varian 720-ES；S1）进行了分析。

本研究选用的4种无脊椎动物分属不同分类群与生态位（图1）。等足类（isopod）斯蒂芬森蛀木水虱（Limnoria stephenseni）采自离岸数百米处的巨藻（Macrosystis pyrifera）漂浮藻体；桡足类（copepod）猛水蚤属未定种（Harpacticus sp.）与双壳类（bivalve）盖姆尼亚蛤（Gaimardia trapesina）采自高能量浅海潮下带的藻类生境；最后，扁形涡虫（flatworm）奥氏涡虫（Obrimoposthia ohlini）采自整个潮间带区域的石块底面。研究团队提出如下假说：斯蒂芬森蛀木水虱（L. stephenseni）因栖息于较深且相对稳定的潮下带生境，对盐度与温度变化尤为敏感；而奥氏涡虫（O. ohlini）因分布于潮间带高位区域，长期暴露于自然波动的环境中，敏感性相对较低。其余两个物种——盖姆尼亚蛤（G. trapesina）与猛水蚤属未定种（Harpacticus sp.）——所栖息的环境波动程度介于两者之间，因此其对胁迫的响应也大概率处于中间水平。

实验流程
本研究采用多因素实验设计，探究盐度、温度与铜离子对生物的联合胁迫效应。针对温度与盐度的每一种组合，设置了一系列铜离子浓度梯度，因此每个受试物种最多可同时开展9组铜毒性实验（表1）。斯蒂芬森蛀木水虱与猛水蚤属未定种的实验于麦夸里岛开展，采集后2~3天内即可启动实验，实验前需将生物驯化至实验室环境。而盖姆尼亚蛤与奥氏涡虫则通过循环水族箱系统海运至澳大利亚，于霍巴特的澳大利亚南极分部的循环水族箱中暂养，水温维持在6 ℃。这两个类群均在采集后3个月内开展实验。由于盖姆尼亚蛤与奥氏涡虫的样本量有限，因此测试的胁迫因子组合数相对较少。

所有受试物种的温度与盐度对照均设置为环境水平：盐度35±0.1 ppt，温度5.5~6 ℃。双壳类的对照温度设置较低，这契合了采样时的季节低温环境，以及其栖息于潮间带较低位置的生境特征。此前在该环境条件下开展的预实验，已明确了各分类群适用的铜离子浓度范围、适宜实验时长与换水方案（Holan等，2017；Holan等，2016b）。基于上述前期研究，团队确定：多数物种的实验时长以7天为最优，此时对照组存活率高且各浓度组均有足够的响应；但部分物种需采用14天的实验周期。盖姆尼亚蛤属于例外，前期研究显示其在实验3天后水质会出现恶化（Holan等，2016）。相较于热带及温带物种的急性实验时长（24~96 h），其余三个物种的较长实验周期与此前南极相关研究的结论一致——这些研究均表明，需延长实验时长才能在生物中诱导出明确的急性胁迫响应（King和Riddle，2001；Marcus Zamora等，2015；Sfiligoj等，2015）。由于各物种的生物学特性存在差异，每个实验的变量参数（水体体积、受试生物密度、铜离子浓度、温度与盐度）均有所不同（表1）。本次测试的温度梯度（升高2~4 ℃）契合了该区域至2100年的海温与气温上升预测（Collins等，2013）。

实验处理液需在接种生物前24小时配制。海水经0.45 μm过滤，实验前后均采用TPS 90-FL型多参数水质仪测定水质。实验初始时，水体溶解氧饱和度≥80%，pH值介于8.1~8.3之间。所有实验用瓶与玻璃器皿均先用10%硝酸浸泡洗涤，再用Milli-Q超纯水冲洗三次后方可使用。实验溶液的盐度通过添加Milli-Q超纯水进行稀释配制。针对不同盐度的过滤海水，采用500 mg/L的硫酸铜（CuSO4，分析纯，Univar品牌）Milli-Q超纯水储备液配制铜离子处理液。分别在实验初始（第0天）与结束（第14天）时采集实验溶液样本，用于电感耦合等离子体光发射光谱法（ICP-OES）金属含量分析。ICP-OES的具体操作流程详见补充材料（S4）。样本采集采用经酸液与Milli-Q超纯水冲洗过的0.45 μm针式过滤器。采集后，样本需用1%稀释的超纯硝酸（65% Merck Suprapur）进行酸化处理。实验初始时测得的铜离子浓度与理论标称浓度的偏差不超过4%（即实测值为标称值的96%以上）。为确定每个处理组的实际暴露浓度，研究人员取第0天与第14天的实测浓度平均值作为最终暴露浓度（表1）。实验均在控温培养箱中开展，光照强度设置为2360 lux。在麦夸里岛站点，实验采用16:8 h的光暗周期以模拟夏季环境；而在澳大利亚金斯顿的实验室中，则采用12:12 h的光暗周期以匹配实验开展时的秋季光照条件。受试个体需在1~2小时内逐步驯化至处理温度，之后再接入实验体系。实验全程采用培养箱内的Thermochron iButton数据记录仪监测温度变化。

不同分类群的个体死亡率判定标准各不相同。盖姆尼亚蛤的死亡率以闭壳肌功能失常导致的壳张开为判定标准；奥氏涡虫以个体失去活动能力且体表覆盖黏液为判定标准；斯蒂芬森蛀木水虱以经滴管水流轻柔刺激后仍无活动反应为判定标准；猛水蚤属未定种则以腹部体节（urosomes）与头胸部体节（prosomes）呈垂直状态为判定标准（该方法已应用于桡足类相关研究；详见Kwok和Leung，2005）。所有死亡个体均需从实验瓶中移除。