Conceived and designed the experiments: SD MB. Performed the experiments: HSB NS SD. Analyzed the data: HSB NS AD. Contributed reagents/materials/analysis tools: SD. Wrote the paper: HSB NS AD MB.
The authors have declared that no competing interests exist.
As the oceans simultaneously warm, acidify and increase in
We examined the interactive effects of near-future ocean warming and increased acidification/
This study of the effects of ocean warming and CO2 driven acidification on development and calcification of marine invertebrate larvae reared in experimental conditions from the outset of development (fertilization) shows the positive and negative effects of these stressors. In simultaneous exposure to stressors the dwarfing effects of acidification were dominant. Reduction in size of sea urchin larvae in a high
As the oceans warm and absorb increasing amounts of CO2, marine biota are faced with a suite of stressors causing major change to marine ecosystems
Temperature, pH,
Despite the well known controlling influence of temperature on development and the thermal thresholds exhibited by embryos, investigation of the impacts of climate change on marine life histories has largely focussed on ocean acidification as the sole stressor
We investigated the interactive effect of ocean warming and acidification on the larvae of
Our experiments were placed in a climate and regionally relevant setting for the SE Australia climate change hot spot (warming: +3–6°C; acidification: −0.3–0.5 pH units)
The range of morphology of
A-B. Control pH 8.15, largest larvae were from +3°C (27°C) treatments. PO, post oral arms; BR, body rod. C-D. pH 7.8. E-F. pH 7.6. With increased acidity/
Percentage of normal
Source | df | MS | TK | ||
Temp |
2 | 3.595 | 35.3 | 0.0029 | (24, 27) >30 |
pH |
2 | 0.230 | 8.0 | 0.0402 | (8.15, 7.8) (7.8, 7.6), 8.15 >7.6 |
temp × pH | 4 | 0.126 | 2.2 | 0.1598 | |
Female |
2 | 1.348 | 30.6 | <0.0001 | |
Temp × female | 4 | 0.101 | 2.3 | 0.0691 | |
pH × female | 4 | 0.028 | 0.7 | 0.6243 | |
temp × pH × female | 8 | 0.057 | 1.3 | 0.2595 | |
Residual | 54 | 0.044 | |||
Total | 80 |
*Significant,
The mean difference in PO arm length (larval asymmetry) differed among the 24°C and 27°C treatments (
Mean arm asymmetry in
Source | df | MS | ||
pH | 2 | 0.4 | 0.6 | 0.545 |
Temp |
1 | 0.9 | 17.1 | 0.001 |
pH × temp | 2 | 0.02 | 0.4 | 0.687 |
Residual | 12 | 0.05 | ||
Total | 17 |
*Significant,
Five-day echinoplutei had well developed PO arms and these were the longest skeletal element (
A. Mean post oral (PO) arm length and B. total length of calcite rods (TLC) of
Parameter | Source | df | MS | TK | ||
PO | pH |
2 | 2105 | 28 | <0.0001 | 8.15>(7.8, 7.6) |
Temp |
1 | 3621 | 48 | <0.0001 | ||
pH × temp | 2 | 183 | 2.4 | 0.1297 | ||
Residual | 12 | 75 | ||||
Total | 17 | |||||
TLC | pH |
2 | 11136 | 27 | <0.0001 | 8.15>(7.8, 7.6) |
Temp |
1 | 12448 | 30 | 0.0001 | ||
pH × temp | 2 | 624 | 1.5 | 0.2623 | ||
Residual | 12 | 416 | ||||
Total | 17 |
*Significant,
In this first study of the effects of simultaneous exposure to warming and CO2 driven acidification on calcification in marine invertebrate larvae reared in experimental conditions from fertilization, we show the positive and negative effects of these stressors. Larval growth in
Temperature is considered to be the primary environmental factor controlling the physiology, phenology, planktonic larval duration and biogeography of marine invertebrates
Developmental thermotolerance varies greatly between echinoids with a +4°C warming above ambient approaching the thermal limit of many species
A +3°C warming enhanced larval growth of
Increased ocean acidity, hypercapnia and decreased carbonate mineral saturation are inextricably linked and are all likely to exert negative effects on larvae. This may be through direct pH effects on metabolic systems such as those involved with calcite precipitation (eg. carbonic anhydrase) and cellular protection (eg. heat shock proteins) and direct hypercapnic suppression of metabolism
Calcification in sea urchin larvae occurs internally, under a different chemical environment than surrounding seawater through an amorphous phase of CaCO3 that would dissolve if exposed to low pH seawater
The effects of ocean acidification on marine calcifiers vary among phyla, species, life history stages and latitudes/habitats. With regard to the pelagic life stage, some larvae show deleterious effects of near future
Despite the pervasive effect of ocean warming on development, this factor is rarely considered in studies of climate change impacts. Larval performance may differ in experiments when temperature is brought into the mix of factors assessed. For regions with significant warming such as SE Australia, temperature is the most immediate and contemporary climate change stressor. Many progeny will not reach the calcified larval stage in a warm ocean, regardless of pH/
While our results clearly showed the effects of climate change stressors on larval development, egg source also exerted a significant influence. We did not set out to test maternal effects, but it is important to be cognisant of this factor in considering the larval responses. Maternal provisioning influences larval tolerance and ecological outcomes for invertebrate larvae
With respect to the benthic life phase of marine calcifiers, numerous studies investigate the response of juveniles or adults sourced from field collections or aquaculture translocated from present day to acidified conditions
We focussed on the pelagic life phase because this is the crucial dispersal stage and is considered to be most vulnerable to environmental perturbations
For each egg source ca. 2000 eggs (∼20 eggs ml−1) were placed in rearing containers (100 ml), three for each temperature-pH treatment (see below), in flow-through experimental FSW (flow rate ca. 0.13 ml sec−1, 300–400 turnovers day−1) for 20 minutes prior to the introduction of sperm. The containers had a window cut from each side as an overflow and a 45 µm mesh set back from the overflow to retain eggs. The number of sperm required to achieve a sperm to egg ratio of ca. 1000:1, was determined through haemocytometer counts. The sperm was briefly activated (1–2 sec) in experimental FSW prior to addition to containers holding eggs. The flow-through system was turned off (5 min) during fertilisation and was then turned back on to remove excess sperm. This fertilisation procedure was repeated in separate experiments with the eggs of the three females.
The embryos were reared in experimental conditions to the 5 day echinopluteus stage and were not fed to avoid the potentially confounding influence of algal introduction.
The embryos were reared in experimental flow-through FSW in three temperature (control = 24°C, +3°C, +6°C) and three pH (control = 8.15, −0.3, −0.5 pH units) levels in all combinations with three containers of embryos per treatment. Experimental pH was adjusted using an automatic CO2 injection system. Two pH controllers (Tunze), set at pH 7.6 and pH 7.8, were attached to two header tanks (60 L). The controllers, pH probes, solenoid valves and gas cylinders were connected in series and injected pure CO2 gas into the header when required, where it was dissolved using a vortex mixing device (Red Sea). The header tanks were continuously bubbled with air to aid mixing and to maintain dissolved oxygen (DO) >90%. A constant volume was maintained in the headers using a float valve. A control header was bubbled with air only. This water was fed into sub-header tanks (20 L) where it was warmed to the required temperature, +3°C (27°C) and +6°C (30°C), using aquarium heaters or unmanipulated for the ambient control. Seawater was delivered to rearing containers using irrigation drip valves. Temperature, pHNBS, DO and salinity at the level of the experimental containers with developing embryos and larvae were measured daily with a WTW multiprobe. Filtered ambient in flow water was not manipulated and had a mean temperature 23.51°C (SE = 0.04, n = 10, Range 23.3–23.8°C) and mean pH 8.13 (SE = 0.004, n = 10, Range pH 8.08–8.16). The experimental water conditions measured at the level of the rearing containers remained stable (+3°C: Mean 26.13°C, SE = 0.14, Range 25.6–26.6°C; +6°C: Mean 30.35°C, SE = 0.25, Range 29.1–31.9°C) (pH −0.3 units: Mean 7.8, SE = 0.006, Range pH 7.76–7.87; pH −0.5 units: Mean 7.61, SE = 0.004, Range pH 7.58–7.65). Total alkalinity (TA
T | 24°C | 27°C | 30°C | ||||||
pH | 8.15 | 7.8 | 7.6 | 8.15 | 7.8 | 7.6 | 8.15 | 7.8 | 7.6 |
448 (3) | 1142 (8) | 1886 (13) | 455 (3) | 1169 (8) | 1938 (14) | 460 (3) | 1196 (9) | 1990 (14) | |
5.1 (0.00) | 2.6 (0.02) | 1.7 (0.02) | 5.5 (0.05) | 2.8 (0.03) | 1.9 (0.20) | 5.9 (0.05) | 3.1 (0.03) | 2.0 (0.02) | |
3.4 (0.03) | 1.7 (0.02) | 1.1 (0.01) | 3.7 (0.03) | 1.9 (0.02) | 1.2 (0.01) | 4.0 (0.04) | 2.1 (0.02) | 1.4 (0.01) |
Mean (SEM)
Specimens from each rearing container (
Larval growth was documented in an image analysis study of photographs of larvae reared at 24°C and 27°C. The 30°C treatments were excluded due to high mortality and insufficient larvae to measure. Haphazardly selected plutei positioned flat to the plane of focus were photographed using a digital camera mounted on a compound microscope. For each female 35 larvae (taken across the three rearing containers) from each treatment were measured using Image J (NIH, USA). Thus a total of 630 larvae were used (3 females ×35 larvae ×3 pH×2 temperatures). For each larva the length of the two post oral (PO) arms body rods (BR) were measured. The mean length of the two PO arms was determined and the difference in their length was calculated as a measure of arm asymmetry. Total length of calcite rods (TLC), determined as the sum of all skeletal elements was used as a proxy for biocalcification.
For the percentage of normal development data where larvae from three separate rearing containers were scored per female a three factor ANOVA with pH and temperature as fixed orthogonal factors and egg source as a random factor was used. Percentage data were arcsine transformed prior to analysis. Homogeneity of variance was checked using Cochran’s test. For the data on difference in PO arm lengths (arm asymmetry), PO length and TLC where a single mean data point derived from 35 larvae sourced from across 3 rearing containers was determined, a two factor ANOVA with pH and temperature as fixed factors was used. The raw data on arm asymmetry was heterogeneous and was ln(
The reviewers are thanked for helpful comments.