Ethics Statement

The present study on planktonic copepods does not include protected or endangered species.

Field work and sampling in the Southern Ocean have been approved by the German Federal Environment Agency (Umweltbundesamt, UBA) as the responsible German national authority according to the national “Act Implementing the Protocol of Environmental Protection to the Antarctic Treaty”.

Sampling and sorting

Copepod sampling, pH measurements and experiments were conducted onboard R/V Polarstern on two separate cruises in early austral autumn (ANT XXVII/3, February to April 2011) and late spring (ANT XXVIII/2, December 2011). Samples were collected at a total of 20 stations south of the Antarctic Polar front (APF), except Station 210 west of South Georgia. The exact positions are shown in Figure 1.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 1. Study area with sampling sites.

Study area with sampling sites (ANT XXVII/3: black dots; ANT XXVIII/2: open circles). Letters indicate additional tows with a Bongo net (B) or a Tucker Trawl (TT).

https://doi.org/10.1371/journal.pone.0077498.g001

Hydrographical data were recorded prior to any zooplankton haul using a Conductivity-Temperature-Depth (CTD) profiler (SBE 911plus). At each station, stratified depth samples were collected with a vertical haul from a maximum depth of 2000 m to the surface using multiple/opening closing nets (Multinet Maxi, mouth opening 0.5 m²; Multinet Midi, mouth opening 0.25 m²; 100 µm mesh size for both). Vertical hauls took a maximum of two hours. Up to nine discrete depth strata chosen according to the stratification of the water column were sampled. A flowmeter was attached to the net and used to calculate the volume of water filtered. Additional tows from a maximum depth of 300 m to the surface were conducted by a Bongo net (mouth opening 0.28 m², mesh size 100 μm) and Tucker Trawl (mouth opening 2.25 m², mesh size 1500 μm) to provide supplementary material for biochemical analyses and experiments. Immediately after capture, all copepods were removed from the cod end of the nets and eventually sorted by species, sex and developmental stage. All copepodite stages of C. acutus, R. gigas, C. propinquus and P. antarctica were kept separately in jars filled with filtered seawater in temperature-controlled refrigerators or in a cooling container at 0°C for a maximum of two hours previous to hemolymph extraction. The remaining zooplankton from each sample was fixed in 4% borax-buffered formaldehyde in seawater solution for later analyses of community structure.

Hemolymph extraction and analysis

Individual copepods were transferred to a Petri dish kept on an ice bed and dried carefully with a fuzz-free tissue to remove all remaining seawater. Hemolymph was extracted manually under a dissecting microscope using borosilicate glass capillaries, which were prepared prior to extraction with an electrode puller providing ultra-fine tips. Each hemolymph sample was diluted in 40 µL of de-ionized water and kept in a deep-freezer at -80°C. The cation composition was analyzed by ion chromatography with a DIONEX ICS 2000 at 40°C using an IonPac CS 16 column with methane sulfonic acid (MSA, 30 mmol L^-1) as an eluent at 0.36 mL min^-1 flow rate. Inorganic ions such as NH₄⁺, Na⁺, Mg²⁺, K⁺, and Ca²⁺ were measured and peaks were identified according to retention times in comparison to a cation standard of known composition (Dionex, Six Cation Standard). Cation concentrations are presented as mmol L^-1.

pH_e-measurements

At least 500 nL of a hemolymph sample were used to measure pH directly onboard using a NanoDrop 3300 fluorometer (Thermo Fischer) and HPTS (8-Hydroxypyrene-1,3,6-trisulfonic acid trisodium salt) as a pH indicator. After sampling a minimum hemolymph volume of 0.5 μL in a pipette (0.1-2 μL), 5% per volume of a HTPS stock solution (50nM HPTS) was added into the pipette. The final HPTS concentration was about 1nM in all measurements. Fluorescence ratios were calculated by dividing the relative fluorescence resulting from 365 nm excitation by the relative fluorescence resulting from excitation at 470 nm (365:470 ratio). pH was calculated using a calibration curve with 50 mM Imidazole (Sigma-Aldrich, Steinheim, Germany) buffered seawater in the pH range from 5.0 to 8.5.

During ANT XXVII/3, pH measurements were conducted at ambient room temperature. Temperature profiles derived from the CTD were used to determine in situ temperatures and results were adjusted according to Ben-Yaakov [28] (temperature coefficient (∆pH/°C ^≈ 0.01)). During ANT XXVIII/2, pH- measurements were carried out in a temperature-controlled laboratory at in situ temperatures. Measurements resulting in units lower than pH 5.5 and above 8.0 should be interpreted with caution, since depending on the characteristics of the fluorescent dye HPTS error increases beyond these pH values.

Statistical analysis

All analyses were performed with R software version 2.14.2. Data were tested for normality with the Shapiro-Wilk-test. According to the distribution, a two-tailed unpaired t-test (confidence interval 95%) or a non-parametric Wilcoxon`s rank-sum test was applied to detect differences between two groups in pH_e and cation concentration. For the comparison of more than one means, either a one-way ANOVA or the non-parametric Kruskal-Vallis test was adopted. To analyze the effect of different pH values on the ammonium content in the hemolymph, a linear regression analyses was performed.

Cation composition

In the hemolymph of the non-diapausing copepods P. antarctica and C. propinquus, a cation composition almost similar to the ionic composition of seawater was measured, although low levels of up to 48 mmol L^-1 NH₄⁺ occurred and Mg²⁺ values were partially reduced to a minimum of 3 mmol L^-1 (Table 1). Highly elevated concentrations of as much as 515 mmol L^-1 NH₄⁺ and greatly reduced levels of down to 15 mmol L^-1 Na⁺ relative to an average seawater concentration of 470 mmol L^-1 were present only in the hemolymph of C. acutus and R. gigas (Table 1, Figure 2). The Na⁺ concentrations in the hemolymph were up to 97% lower than in seawater and explained most of the cation replacement. Additionally, divalent ions such as Mg²⁺ and Ca²⁺ were reduced. In R. gigas, the highest individual ammonium concentration was found in stage CIII between February and April (512 mmol L^-1, Table 1). The lowest individual ammonium concentration of all samples was determined in C. acutus CV (9 mmol L^-1, Table 1) from the surface layer (0 - 50 m) in December. This sample was also characterized by the highest pH value (pH_e 7.8, Table 1) measured for C. acutus CV during the corresponding sampling period. In contrast, the highest individual ammonium concentration within this species occurred in the hemolymph of CV (515 mmol L^-1, Table 1) from the deepest layer (2000 - 1500 m).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 2. pH values and concentration of sodium and ammonium in seawater and hemolymph of Antarctic copepods.

pH values (mean ± standard deviation; A and B, outlying results (more than 1.5 times the interquantile distance away) are represented by open circles) and concentration of sodium and ammonium (mmol L^-1; C and D; error bars reflect standard deviation) in seawater and in the hemolymph of Antarctic copepods from two different sampling periods (Feb. - April: A and C; December: B and D). Nd not determined, CIII – CV copepodite stages 3 - 5, F females. Number of replicates as in Table 1.

https://doi.org/10.1371/journal.pone.0077498.g002

pH of hemolymph

In the hemolymph of the non-diapausing species P. antarctica and C. propinquus, mean pH values of 7.8 ± 0.3 were measured in both seasons (Table 1, Figure 2). Individual measurements were never lower than pH 7.4 in both species, seasons and all developmental stages (Table 1, Figure 2).

Lower pH levels only occurred in the hemolymph of diapausing C. acutus and R. gigas, and differences in the mean hemolymph pH were statistically discernible between diapausing (C. acutus and R. gigas) and non-diapausing (P. antarctica and C. propinquus) species (ANOVA, p<0.0001). In R. gigas, mean hemolymph pH ranged from 5.9 to 6.3 in both seasons (Table 1, Figure 2). Individual measurements did not exceed pH 6.8 from February to April, while individual values higher than pH 7 were detected in December (R. gigas F: 7.2; CIV: 7.3, Table 1).

In C. acutus, mean hemolymph pH varied from 5.7 to 6.2 between February and April and 6.1 to 6.2 in December (Table 1, Figure 2). The overall highest hemolymph pH was measured in a C. acutus female (pH_e 8.1, Table 1) from the surface layer (100 - 0 m) in December. Overall means (without division into developmental stages) between February and April were pH 6.1 ± 0.4 in R. gigas (n = 57) and pH 6.1 ± 0.6 in C. acutus (n = 28). In December the overall mean amounted to pH 6.2 ± 0.6 for both species (R. gigas n = 40, C. acutus n = 115).

The interaction between pH and ammonium concentration in the hemolymph of C. acutus and R. gigas was tested for the expedition in December (Figure 3). The ammonium content in the hemolymph was significantly affected by acidity and increased with lower pH values in both species. The correlation was most pronounced in C. acutus CV (y=-137x+1135, R²=0.41, p<0.01), followed by C. acutus females (y=-104x+929, R²=0.33, p<0.01). In R. gigas, the correlation was less pronounced, but still discernible (all stages combined: y=-123x+1044, R²=0.12, p=0.034) (Figure 3).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 3. Relationship between pH and concentration of ammonium (NH₄⁺).

Relationship between pH and concentration of ammonium (NH₄⁺, mmol L^-1) in the hemolymph of C. acutus (A; copepodite stage CV (a) y=-137x+1135, R²=0.41, p<0.01; females (b) y=-104x+929, R²=0.33, p<0.01) and R. gigas (B; all stages combined, y=-123x+1044, R²=0.12, p=0.034).

https://doi.org/10.1371/journal.pone.0077498.g003

Ammonium-aided buoyancy

The presence of elevated ammonium concentrations within the hemolymph of only copepods known to undergo diapause in winter was confirmed in this study (Table 1; see 12 for review of previously measured values). Simultaneously, Na⁺ and to a lesser extent Mg²⁺ and Ca²⁺ concentrations were reduced in relation to both seawater and the hemolymph of non-diapausing copepods. Variable amounts of K⁺ are not further discussed in this study, since elevated concentrations are most likely the result of injured body tissue in the course of the hemolymph extraction, allowing the leakage of K⁺ from the intracellular into the extracellular space.

Our findings of cation replacement in the hemolymph of diapausing copepods are in good accordance with changes in the ion composition in body fluids from a range of marine organisms known to use low-density fluids for buoyancy regulation [13,15,16,21]. For comparison, concentration of NH₄⁺ in the deep-sea shrimp N. gibbosus was 296 ± 51 mmol L^-1 in the carapace fluid and 217 ± 54 mmol L^-1 in the hemolymph [15].

Compared to the extra cost of swimming or the accumulation of low-density organic compounds such as lipids, the energetic costs involved in the production of ammonium are low, since ammonium is a waste product from the catabolism of proteins and amino acids. Furthermore, in contrast to other buoyancy mechanisms, ammonium-aided buoyancy is independent from high ambient pressures and rapid changes in depth [13] and is therefore well suited for the extensive vertical migrations of copepods.

pH of hemolymph

Ammonia is highly toxic for most organisms and the concentration in the hemolymph of aquatic crustaceans is generally below 0.8 mmol L^-1 [17]. In decapod shrimps for instance, 96-h LC₅₀ values for adult Penaeus paulensis and juvenile Litopenaeus vannamei were, respectively, 2.4 mmol L^-1 [36] and 2.2 mmol L^-1 total ammonia [37]. The protonation of ammonia to form the comparatively less toxic ionized form NH₄⁺ is strongly dependent on the H⁺ concentration, and to a lesser extent upon temperature and salinity of the respective solution. At a pH of 7.8, 0°C and 32-40 psu salinity (pK ^≈ 10.16), 99.6% of total ammonium exists in the ionic form NH₄⁺, whereas 0.4% is present as NH₃ [38]. At ammonium concentrations of ^≈ 500 mmol L^-1 as measured in the hemolymph of diapausing copepods in this study, 0.4% would result in a NH₃ concentration of 2 mmol L^-1, which far exceeds the general hemolymph concentration of ≤ 0.8 mmol L^-1 in aquatic crustaceans [17]. Under acidic conditions of pH ^≈ 6.0, as measured in the hemolymph of diapausing copepods with elevated ammonium concentrations, only 0.007% of total ammonia is present as toxic NH₃ [38], resulting in a concentration of 0.035 mmol L^-1. Since NH₃ is lipid-soluble, uncharged and therefore easily diffusible across phospholipid membranes, it is regarded as the most toxic form in fish [18] and aquatic crustaceans [19].

To avoid ammonia toxicity, ammoniacal deep-sea crustaceans and squids sequester ammonium fluids in specialized vacuoles, chambers or gelatinous layers [14,15,39]. So far, there is no evidence for the existence of such a cellular unit in copepods.

The present study revealed very low pH values only in the hemolymph of diapausing copepods (Table 1, Figure 2). Moreover, the amount of ammonium was correlated to the respective hemolymph pH in both overwintering species (Figure 3). C. acutus and R. gigas had pH values of 6.1 to 6.2 in both seasons. These results are comparable to the pH in the ammonium-rich carapace fluid of N. gibbosus (pH 6.6 ± 0.08) [15] and in the vacuolar fluid of ammoniacal squid (≥ 5.1) [21]. At such low pH levels, virtually all ammonium is present in the non-toxic ionized form, which additionally reduces the loss of ammonia by diffusion [39,40].

Marine planktonic crustaceans are generally sensitive to low pH conditions and exposure to elevated H⁺ concentrations can cause high mortality rates in zooplankton communities [41]. In addition, acidic pH conditions in the intracellular milieu have been shown to be relevant factors depressing metabolic rate in a range of other invertebrates during dormancy or environmental hypercapnia [42–44]. Since metabolic depression in diapausing copepods is essential to save energy during the food-limited winter period, the low pH values found in overwintering copepods might be beneficial for a successful implementation of diapause. Indeed, potential benefits of a low extracellular pH leading to a reduced aerobic energy turnover and thus metabolic depression have already been established for the marine worm Sipunculus nudus during anaerobiosis [45–47].

Inter-individual/intra-specific variability

Differences in both hemolymph pH and cation composition were discernible between diapausing and non-diapausing species, whereas no statistical differences could be determined between seasons, sampling depths or ontogenetic stages. According to the assumption that ammonium-aided buoyancy changes with season and that hemolymph pH controls metabolic depression, ammonium accumulation should be a seasonal phenomenon with maximum levels in overwintering stages at depth and minimum levels in active stages at the surface. Deviations from this prediction in the present data may be explained by the fact that our samples were collected in the transitional periods of autumn and spring where considerable variation in the individual life histories could not be excluded. Future studies should focus on ammonium concentration and hemolymph pH of Antarctic copepods during the active phase in summer and during diapause in winter.

In C. acutus, copepodite stage CV represents the main overwintering cohort. The fact that maximum ammonium concentration in C. acutus was observed in copepodite stage CV from the deepest water layer supports the idea that ammonium-aided buoyancy is most important in diapausing stages at overwintering depth. Nevertheless, adults have to ascend to the surface in spring with reduced lipid deposits [4,6]. Thus, elevated ammonium levels and high buoyancy is beneficial for the return to the surface. Ammonium levels and/or excretion rates may vary throughout the water column from individual to individual in relation to other factors such as maturity level or lipid content.

In R. gigas, intraspecific variation of hemolymph pH was high and most pronounced in stages CIV and CV in spring. Published data show that most likely not all individuals of R. gigas overwinter inactively. Resting stages were found at depth in the marginal ice-zone of the southern Scotia Sea [48], whereas actively feeding and apparently reproducing individuals occurred in the area of the Antarctic Peninsula during winter [49]. Unlike C. acutus, R. gigas has a more flexible, one- or two-year life cycle, meaning that parts of the population can maintain in the surface waters during winter, while the rest descends and enters diapause [3]. In consequence, we possibly caught and measured a “mixture” of individuals with different physiological backgrounds. Nevertheless, our hypothesis was supported by the observation that the highest ammonium content was measured in copepodite stage CIII in autumn (512 mmol L^-1, Table 1), which represents one of the main overwintering stages in R. gigas [50], whereas minimum concentrations were found in fully developed females (50 - 52 mmol L^-1, Table 1), which definitely had passed through diapause, molting and maturation.

In contrast, the correlation between pH and ammonium was stronger in C. acutus CV (Figure 3) and is indicative for a clearly defined one-year life cycle in which successful spawning is closely restricted to the beginning of the productive season [3].

Accumulation of ammonium and the replacement of ions with a higher solute mass (e.g. Na⁺, Mg²⁺ and Ca²⁺) in the hemolymph of Antarctic copepod species known to undergo diapause in winter was confirmed in this study. These findings support the idea of a relation between vertical ontogenetic migration and ammonia aided buoyancy in Antarctic copepods. Moreover, low pH values were measured only in the hemolymph of diapausing copepods with elevated ammonium levels. The ammonium content was statistically correlated to the respective pH of the hemolymph sample and increased with lower pH values. Further research should focus on the effect of low pH for metabolic depression and its possible role as the sought-after trigger for controlling diapause in Antarctic copepods.