Journal of Experimental Marine Biology and Ecology 326 (2005) 144 – 156 www.elsevier.com/locate/jembe
Detecting growth under environmental extremes: Spatial and temporal patterns in nucleic acid ratios in two Antarctic bivalves J. Norkko a,b,*,1, A. Norkko a,1, S.F. Thrush a, V.J. Cummings c b
a National Institute of Water and Atmospheric Research, P.O. Box 11-115, Hamilton, New Zealand School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand c National Institute of Water and Atmospheric Research, P.O. Box 14-901, Wellington, New Zealand
Received 2 May 2005; received in revised form 19 May 2005; accepted 24 May 2005
Abstract Growth of Antarctic benthic organisms is very slow due to temperature and food availability, and subtle differences in growth rate may be difficult to detect. Nucleic acid ratios (RNA/DNA, RNA/protein or total RNA concentration) are measures of protein synthesis potential and may be used to assess short-term growth rate in a range of marine organisms. We quantified nucleic acid ratios in the scallop Adamussium colbecki and the clam Laternula elliptica at five locations in the Ross Sea, Antarctica. We were able to detect species-specific, habitat-specific, and seasonal differences in nucleic acid ratios and related these to associated differences in primary productivity. By using nucleic acid ratios, future studies could relatively easily obtain a measure of growth rate from a multitude of locations with contrasting habitat characteristics, food availability and temperature regimes around the Antarctic continent. This would yield a unique understanding of spatial and temporal patterns in bivalve growth in this extreme environment. D 2005 Elsevier B.V. All rights reserved. Keywords: Adamussium colbecki; Food availability; Laternula elliptica; Nucleic acid ratios; Primary productivity; RNA
1. Introduction Due to very low and nearly constant water temperatures, the basal metabolism of the Antarctic ben-
* Corresponding author. Tel.: +358 50 4363546; fax: +358 9 61394494. E-mail address:
[email protected] (J. Norkko). 1 Present address: Finnish Institute of Marine Research, PB 33, 00931 Helsinki, Finland. 0022-0981/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2005.05.014
thic fauna is low (Peck, 2002). However, while average annual growth rates of the benthic fauna can be extremely slow (Arntz et al., 1994), they may increase rapidly when food is abundant (Clarke, 1988). Thus the short peak in primary production in summer providing a pulsed influx of organic material to benthic communities is of great importance, although its significance may vary depending on location (Dayton and Oliver, 1977). The coastal Ross Sea spans over six latitudinal degrees and there are strong environmental gradients
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in this area, with variable sea-ice conditions, light conditions and productivity (Dayton and Oliver, 1977; Dayton, 1990; Faranda et al., 2000), making it an ideal natural setting for testing predictions about responses of benthic fauna to fluctuations in food availability. While solar radiation changes predictably along the latitudinal gradient, variable ice and snow cover, oceanographic circulation patterns, coastline topography, and the location of polynyas affect the coastal ecosystems on a regional scale, resulting in markedly different patterns in primary productivity. Food for the benthic communities can be derived from (a) the annual phytoplankton bloom in summer (either in the open ocean or in near-shore moats), (b) ice algae, (c) benthic microalgae, (d) macroalgae, or (e) advected organic material that originated elsewhere (Arntz et al., 1994; Gili et al., 2001). The relative importance of each food source is likely to vary between locations (Dayton et al., 1986; Norkko et al., in preparation). By integrating information on the relationships between the physiology of benthic species and environmental variables from several sites along strong gradients in light conditions, circulation patterns and variable primary productivity, the ability to predict how and why ecological patterns may change over different spatial and temporal scales is improved. As growth in Antarctic bivalves is slow compared to many temperate and subtropical species (Brey and Clarke, 1993), traditional measurements of short-term growth can be difficult to obtain. Nucleic acid ratios (RNA/DNA, RNA/protein or total RNA concentration; a measure of protein synthesis potential) may be used as a sensitive, indirect measure of instantaneous short-term growth rate in a range of marine organisms (Houlihan, 1991; Buckley et al., 1999; Dahlhoff, 2004). Moreover, nucleic acids ratios appear most sensitive to growth-facilitating factors (e.g. increased food availability, Norkko et al., in press), which suggests that they should be useful for quantifying short-term growth rates in the Antarctic context, where the seasonality in food availability is a major factor structuring benthic communities (Arntz et al., 1994). The nucleic acid approach has recently been trialled in field-based studies in Antarctica. In pelagic species, high RNA/DNA ratios have been found in Antarctic krill in connection with high phytoplankton
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concentrations (Cullen et al., 2003; Shin et al., 2003). Similarly, RNA/DNA ratios were higher in fish outside the ice edge compared to fish in an ice-covered zone, which corresponded to large differences in primary production (Geiger et al., 2000). Conversely, RNA/DNA ratios in five species of copepod were analysed at the same sites around the ice edge, and no differences were found between zones (Geiger et al., 2001). In coastal benthic species, higher nucleic acid ratios were found in summer in the holothurian Heterocucumis steineni (Fraser et al., 2004) and in the limpet Nacella concinna (Fraser et al., 2002). Nevertheless, we are not aware of any published studies measuring nucleic acid ratios in field-collected Antarctic bivalves in an attempt to describe spatial and temporal patterns in instantaneous growth rates. Although the physiology of Antarctic bivalves is receiving considerable attention, there is a paucity of comparisons between near-shore locations over larger spatial scales. Typically, investigations into bivalve physiology have been conducted on bivalves collected from only few locations and have not been explicitly linked to geographic differences in environmental factors influencing growth (e.g. Berkman, 1990; Ahn and Shim, 1998; Brockington, 2001; Chiantore et al., 2003). Geographical differences in bivalve physiology and population dynamics have been predicted. For example, the scallop Adamussium colbecki exhibits large differences in depth distribution at different sites along the Victoria Land Coast in the Ross Sea, which probably is related to site-specific differences in ice cover (Chiantore et al., 2001). Further, Brey and Hain (1992) compared growth rings of the bivalve Lissarca notorcadensis from different areas of the Weddell Sea (north of 638S and south of 708S), finding slower growth rates further south, and suggested differences in growth and reproduction to be related to food availability. Again, comparisons across strong environmental gradients may provide valuable information on physiological responses in relation to environmental forcing. The aim of this study was to investigate spatial and temporal differences in nucleic acid ratios in Antarctic bivalves in relation to food availability and other environmental factors. Nucleic acid ratios were quantified in two important suspension-feeding bivalves, A. colbecki (Smith 1902) and Laternula elliptica (King and Broderip), in locations with contrasting
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habitat characteristics, spanning a 330-km latitudinal gradient in the coastal western Ross Sea. In addition, nucleic acid ratios from bivalves collected in spring were compared with bivalves collected in late summer, after the annual peak in primary production, to investigate the effect of this peaked influx of food on short-term growth rates. This sampling allowed investigation of nucleic acid ratios in terms of (a) differences between species, (b) small- and large-scale spatial variability along the latitudinal gradient, (c) the influence of different environmental variables, and (d) the effect of summer peaks in primary production. Bivalve nucleic acid ratios were predicted to be species-specific and closely related to spatial and temporal differences in food quantity and quality.
2. Methods 2.1. Study species Both the epibenthic scallop A. colbecki (hereafter Adamussium) and the deep-burrowing clam L. elliptica (hereafter Laternula) have circumpolar distributions and are key species in Antarctic coastal ecosystems (Dell, 1972). Due to differences in natural history characteristics between the two species, especially the timing and mode of reproduction, distinct patterns in nucleic acid ratios are predicted. Adamussium is a suspension feeder, which feeds on phytoplankton, resuspended benthic diatoms, foraminifera and detritus (Stockton, 1984; Berkman, 1990; Ansell et al., 1998, Chiantore et al., 1998). It has a maximum observed shell height of 12 cm (Berkman, 1990). Small specimens (b 5 cm in shell height) grow at a rate of 10 mm per year, while larger specimens grow b 1 mm per year (Ralph and Maxwell, 1977; Stockton, 1984; Chiantore et al., 2003; Heilmayer et al., 2003). Recapture by chance of two individuals 8 and 12 years after marking revealed very slow growth in adult Adamussium at New Harbour (0.3–0.5 mm per year) and a potential centuryscale lifespan (Berkman et al., 2004). In McMurdo Sound (778S) and at Adelaide Island (678S) Adamussium spawns in early spring, producing unprotected planktotrophic larvae (Berkman et al., 1991; Tyler et al., 2003). However, Chiantore et al. (2001, 2002) observed mature females in late summer in Terra
Nova Bay (748S), indicating that either there is some geographic variability in the timing of spawning, or the individuals have mature oocytes in preparation for the next spring. Laternula may burrow up to 50 cm into muddy or gravelly sediment (Ralph and Maxwell, 1977; Zamorano et al., 1986). It feeds on phytoplankton and resuspended benthic diatoms, and has a large muscular siphon, which is important for energy storage (Ahn et al., 2003). The shell of Laternula may be N 10 cm long and reaches this size in 12–13 years (Ralph and Maxwell, 1977; Urban and Mercuri, 1998). In contrast to Adamussium, Laternula has lecithotrophic larvae protected by an egg capsule (Berkman et al., 1991). However, like Adamussium some latitudinal variability in the timing of spawning seems apparent. Bosch and Pearse (1988) observed mass spawning beginning in March in McMurdo Sound (778S), while peak spawning occurs in January–February at King George Island (628S, Urban and Mercuri, 1998; Ahn et al., 2003). 2.2. Study area and field sampling Bivalves were collected by SCUBA diving in spring 2002 from approximately 20 m depth at four locations in McMurdo Sound: Dunlop Island, Spike Cape, New Harbour and Cape Evans (Fig. 1; Table 1). Additional bivalve samples were obtained from New Harbour in summer 2003 to enable a temporal comparison with the samples collected in spring. In addition, summer samples were obtained from Terra Nova Bay further north along the Victoria Land coast (Fig. 1). The locations have different habitat characteristics, water circulation patterns, sea-ice conditions and primary production, all of which are expected to influence bivalve short-term growth rates. At Dunlop Island (77814.2VS, 163828.0VE, sampled 22–25 October) and Spike Cape (77818.0VS, 163834.0VE, sampled 30 October–2 November) the habitat is a mix of cobble, gravel and sandy sediment. The sea-ice at both locations breaks out most years and was approximately 2.5 m thick during this investigation. Explorers Cove in New Harbour (77834.5VS, 163831.7VE, sampled 5–8 November and 25 January) is a sandy embayment, where the sea-ice only breaks out every 5 to 10 years. During this investigation the ice was approximately 3.2 m
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A)
Ross Ice shelf Terra Nova Bay McMurdo Sound
Antarctica
B)
Ross Sea
40 km
Dunlop Is. Spike Cape
New Harbour
McMurdo Sound
Ross Island
Cape Evans Scott Base Ross Ice Shelf
Fig. 1. (A) Location of McMurdo Sound and Terra Nova Bay on the Antarctic continent and (B) map of McMurdo Sound with the study locations marked with black dots.
thick and the last known break-up was in 1999. Due to the thick and persistent ice cover, and predominant northward water currents consisting of plankton poor water from underneath the Ross Ice Shelf, New Harbour does not experience the strong phytoplankton bloom characteristic of the eastern side of McMurdo Sound (Dayton and Oliver, 1977). At Cape Evans (77838.1VS, 166824.9VE, sampled 12– 14 November) the habitat is dominated by boulders and cobbles, interspersed with gravel and sand. The sea-ice at Cape Evans breaks out most years and was approximately 2.5 m thick during this investigation. Terra Nova Bay (74841.9VS, 164807.5VE) is situated 280 km north of Dunlop Island. Unlike the western side of McMurdo Sound, where the water temperature is constantly between 1.7 and 1.9 8C, the temperature at Terra Nova Bay varies between + 1 8C in summer and 1.9 8C in winter. The sea-ice breaks
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out most years, although the timing may vary (Mangoni et al., 2004). In addition, the productive Terra Nova Bay polynya remains ice free throughout most of the winter (Arrigo et al., 1998). Within the Terra Nova Bay area, sampling was conducted at Tethys Bay (24–27 January) and Road Cove (16 February). In Tethys Bay the habitat is a mix of boulders, rocks and sandy sediment. In Road Cove the habitat is sandy and totally dominated by Adamussium. To maximise information on possible co-variables influencing their growth, the bivalves were collected in close proximity to 20 m transects, which were concurrently sampled for assessment of structural and functional benthic biodiversity, and sediment characteristics (Cummings et al., in preparation; Norkko et al., in preparation). Benthic chlorophyll a was analysed as a measure of food availability and was extracted from freeze-dried sediments by boiling in 90% ethanol. The extract was measured spectrophotometrically and an acidification step was included to separate degradation products (phaeophytin) from chlorophyll a (Sartory, 1982). Stable isotope signatures of the benthic fauna, including Adamussium and Laternula, and potential food sources were also obtained as part of the biodiversity survey, providing
Table 1 Number of Adamussium colbecki and Laternula elliptica sampled for nucleic acid analysis at different locations in McMurdo Sound (M) and Terra Nova Bay (T) Time
Location
Site Depth Adamussium Laternula (m)
Spring
Dunlop Island (M)
1 2 3 1 2 3 1 2 3 1 1 1
Spike Cape (M) New Harbour (M)
Cape Evans (M) Summer New Harbour (M) Road Cove (T) – Tethys Bay (T) 1 2 3 Site depth is also given.
19 21 15.5 18.5 20 15 24.5 20 17 12 18 24.5
15 5 5 15 7 5 15 15 15 – – 16
12 – 8 7 7 3 – – – 8 4 –
40 21 20 24
15 2 – 3
– 5 5 5
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a better understanding of the trophic structure of the benthic community and the relative importance of different food sources in different locations (Norkko et al., in preparation). At Dunlop Island, Spike Cape, New Harbour and Tethys Bay three sites were sampled. The sites were separated by approximately 50 m, except at New Harbour where they were separated by 300–600 m. Up to 16 adult Adamussium and/or Laternula were collected per site (Table 1). The most extensive sampling was done at New Harbour, where 15 Adamussium were sampled from three different sites with differing light levels, distance from shore and benthic primary production. Due to very low densities, no Laternula were obtained from New Harbour. At Cape Evans Laternula were sampled from 12 and 18 m depth at one site only. The two depths were chosen based on apparent differences in benthic primary production and proximity to ice algae. Adamussium were not found at Cape Evans. From Tethys Bay a total of only five Adamussium were able to be collected. Instead, additional Adamussium were collected by dredging from 40 m depth in a nearby area at Road Cove. 2.3. Processing of bivalve samples Individual Adamussium and/or Laternula were brought to the surface, measured (shell height for Adamussium and shell length for Laternula) and immediately dissected. Adductor muscle from Adamussium and siphon muscle from Laternula was sampled for analysis of RNA, DNA and protein. Tissue samples were snap-frozen in liquid nitrogen. The samples were transported to New Zealand in liquid nitrogen, stored at 70 8C and freeze dried prior to analysis. The tissue was pulverised in a glass mortar and subsampled for RNA, DNA and protein quantification. Total RNA and DNA were extracted separately using TRI Reagentk and DNAzolk, respectively (Molecular Research Centre, Inc. # TR118/200 and DN127/200; Chomczynski, 1993; Chomczynski et al., 1997), and quantified spectrophotometrically. For the RNA and DNA analyses 12–15 and 6–8 mg tissue was used, respectively, and an additional ethanol wash was added to the manufacturer’s protocol to ensure samples of satisfactory purity. At 260 nm one absor-
bance unit corresponds to 40 Ag mL 1 pure RNA or 50 Ag mL 1 pure double-stranded DNA, and the results were expressed as Ag RNA or DNA mg 1 tissue dry weight. Total protein was estimated from 8 to 10 mg pulverised tissue, which was incubated in 0.5 mol/L NaOH at 37 8C overnight and assayed using the bicinchoninic acid method with bovine serum albumin as standard (Smith et al., 1985, Sigma # BCA-1). The results were expressed as percent protein of tissue dry weight. 2.4. Data analysis To determine whether shell size and benthic chlorophyll a varied between locations or over time, t-test or ANOVA followed by Tukey’s multiple comparison tests was used. Differences between locations in Adamussium RNA were analysed similarly. Due to a negative correlation between Laternula RNA and shell length, Laternula RNA was analysed using ANCOVA, with shell length as covariate. At Cape Evans differences in Laternula RNA between the two depths were analysed using t-test. For comparisons between locations, data from all three sites within a location were used. For Cape Evans, the average values of the two depths were used. Within New Harbour Adamussium RNA at the three sites was also compared, as distances and differences in habitat characteristics between sites, and the number of bivalves sampled per site were sufficiently large at this location. Prior to analysis data was examined for homogeneity of variances ( F test) and normality (Kolmogorov–Smirnov). P b 0.05 was used as the general significance level.
3. Results 3.1. Environmental variables Water temperature was 1.9 8C at all locations in McMurdo Sound in spring and water clarity was high, with no indication that the phytoplankton bloom had started. At Terra Nova Bay in summer, water temperature was around 0 8C and visibility was only 1–10 m, indicating a bloom. Only benthic chlorophyll a could be measured at each location
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and was used as a proxy for primary productivity, although it does not necessarily reflect pelagic primary production, but will incorporate sedimented pelagic material. New Harbour and Cape Evans had the lowest and the highest benthic chlorophyll a, respectively (Fig. 2A). The lower benthic chlorophyll a at Tethys Bay compared with Cape Evans, despite higher temperature in Tethys Bay, could be due to the plankton bloom shading the microphytobenthos, while the bloom had not yet sedimented to the seafloor. The ratio of chlorophyll a to phaeophytin provides information on food quality. All locations had more degraded than healthy microphytobenthos (chlorophyll a/phaeophytin ratio b 1, Fig. 2B) and the lowest ratio was recorded at Tethys Bay, possibly also due to shading from the plankton bloom.
Fig. 2. (A) Benthic chlorophyll a concentration and (B) chlorophyll a: phaeophytin ratio at the sampling locations (mean F S.D., n = 15, except at CE, where n = 9). DI = Dunlop Island, SC = Spike Cape, NH = New Harbour, CE = Cape Evans, TB = Tethys Bay.
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3.2. Nucleic acid ratios RNA concentrations and RNA/protein ratios were highly correlated for both study species (Adamussium: r = 0.876, P b 0.0001; Laternula: r = 0.967, P b 0.001) and only RNA data is presented in detail here. In general, RNA, protein and DNA contents were markedly higher in Laternula than in Adamussium (Tables 2 and 3, Fig. 3). DNA exhibited inconsistent patterns and high variability in both species (Tables 2 and 3), which prevented its use for standardising RNA content using RNA/DNA ratios. Thus DNA and RNA/ DNA ratios were not analysed further. 3.3. Adamussium colbecki A narrow size range of Adamussium was sampled (shell height 6.6–10.0 cm). On a location-by-location basis, no strong or consistent correlation was found between RNA and size, with the relationship varying from positive to negative. Thus size was not included as a co-factor in the analysis of Adamussium RNA. However, when all individuals were included, there was a general weak trend towards a negative correlation between RNA and shell height (r = 0.441, P b 0.0001). Within McMurdo Sound, significant differences in Adamussium RNA were found between locations in spring ( F 2,94 = 16.206, P b 0.0001), with significantly higher RNA at Spike Cape and Dunlop Island compared with New Harbour ( P b 0.05, Fig. 3A). Smallerscale variability in Adamussium RNA was also detected within New Harbour ( F 2,42 = 5.484, P = 0.0077), with RNA at Sites 2 and 3 higher than at Site 1 ( P b 0.05, Fig. 4A). Sites 2 and 3 are shallower and closer to the near-shore moat, where high primary production has been observed in summer (Stockton, 1984). This coincided with higher benthic chlorophyll a concentrations and higher chlorophyll a : phaeophytin ratios at Sites 2 and 3 (Fig. 4B,C). This gradient in primary productivity was further supported by diatoms living on the Adamussium shells, as diatom coverage also increased towards Site 3 (C. Cerrano, unpublished data). In addition, Adamussium RNA at Site 1 in New Harbour was 15% higher in summer than in spring (4.5 vs. 3.9 Ag mg 1, t 29 = 4.86, P b 0.0001). This coincided with a 170% increase in benthic chlorophyll a between spring and summer at this site (S. Bowser,
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Table 2 Adamussium colbecki: Shell height, and adductor muscle RNA, protein and DNA content at different locations in McMurdo Sound (M) and Terra Nova Bay (T) (mean F S.D., see Table 1 for n) Time
Location
Site/Depth
Shell height (cm)
RNA (Ag mg
Spring
Dunlop Island (M)
1 2 3 1 2 3 1 2 3 1 40 m 1, 2, 3
8.6 F 0.6 8.8 F 0.6 8.3 F 0.4 8.5 F 0.4 7.6 F 1.7 8.8 F 0.6 8.7 F 0.3 9.2 F 0.6 8.3 F 0.6 8.1 F 0.4 8.4 F 0.5 7.4 F 0.5 8.5 F 0.6
4.6 F 0.5 4.4 F 0.7 4.4 F 0.5 4.8 F 0.4 9.4 F 1.4 4.7 F 0.3 3.9 F 0.3 4.2 F 0.5 4.4 F 0.5 4.5 F 0.5 5.9 F 0.5 7.4 F 1.3 4.7 F 0.9
Spike Cape (M)
New Harbour (M)
Summer
New Harbour (M) Road Cove (T) Tethys Bay (T) Average of all times and locations
1
)
Protein (%)
DNA (Ag mg
25 F 2 25 F 3 28 F 3 23 F 2 30 F 2 25 F 2 25 F 2 24 F 2 25 F 2 27 F 2 26 F 1 28 F 2 25 F 2
0.6 F 0.3 0.6 F 0.3 0.7 F 0.2 0.9 F 0.4 4.2 F 1.6 1.3 F 0.4 0.4 F 0.1 0.5 F 0.2 0.4 F 0.2 0.7 F 0.2 1.8 F 0.3 2.0 F 0.5 0.8 F 0.6
1
)
As only a total of five Adamussium were found at the three sites at Tethys Bay, the location mean is reported. Adamussium were not found at Cape Evans.
personal communication, http://www.bowserlab.org/ datastore/datastorelaunch.htm). On a larger spatial scale, significant differences in Adamussium RNA were found in summer between McMurdo Sound (New Harbour, Site 1) and Terra Nova Bay (Tethys Bay and Road Cove; F 2,33 = 42.057, P b 0.0001, Fig. 3A). Adamussium at the Terra Nova Bay locations had significantly higher RNA than Adamussium collected at the same time in New Harbour ( P b 0.01). Within Terra Nova Bay, Adamussium at the deeper site in Road Cove (40 m) had lower RNA ( P b 0.01) than at the shallower site in
Tethys Bay (20 m), where the highest Adamussium RNA values overall were recorded. 3.4. Laternula elliptica For Laternula the overall range in shell length was 3.2–11.8 cm, although 70% of the individuals were 7–9 cm. The effect of Laternula size on RNA was assessed using individuals of a wider size range collected at Dunlop Island (3.2–9.2 cm). This revealed a negative correlation between RNA and shell length (r = 0.741, P = 0.0002). As the size range for
Table 3 Laternula elliptica: Shell length, and siphon muscle RNA, protein and DNA content at different locations in McMurdo Sound (M) and Terra Nova Bay (T) (mean F S.D., see Table 1 for n) Time
Location
Site/Depth
Shell length (cm)
RNA (Ag mg
Spring
Dunlop Island (M)
1 2 3 1 2 3 1 (12 m) 1 (18 m) 1 2 3
7.9 F 0.7 – 4.9 F 1.5 8.1 F 0.8 6.6 F 0.8 8.4 F 1.2 7.3 F 1.6 8.0 F 0.7 7.9 F 1.1 10.1 F1.3 8.8 F 0.6 7.6 F 1.7
9.8 F 0.7 – 11.2 F 1.2 9.4 F 1.3 10.2 F 1.1 10.4 F 1.8 9.0 F 0.4 7.7 F 0.7 8.1 F1.2 8.7 F 0.8 7.9 F 1.7 9.4 F 1.4
Spike Cape (M)
Cape Evans (M) Summer
Tethys Bay (T)
Average of all times and locations
Laternula were not sampled at New Harbour or Road Cove.
1
)
Protein (%) 29 F 2 – 30 F 2 28 F 2 28 F 2 27 F 1 30 F 1 33 F 1 33 F 2 31 F1 32 F 2 30 F 2
DNA (Ag mg 5.2 F 0.6 – 5.2 F 1.0 4.5 F 1.7 5.9 F 1.9 5.9 F 0.6 3.2 F 0.5 2.7 F 0.8 2.7 F 1.0 3.2 F 0.3 2.3 F 0.5 4.2 F 1.6
1
)
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Fig. 3. RNA concentration in (A) adductor muscle of Adamussium colbecki and (B) siphon muscle of Laternula elliptica at the sampling locations in spring and summer (mean F S.D., see Table 1 for n). NH = New Harbour, DI = Dunlop Island, SC = Spike Cape, CE = Cape Evans, TB = Tethys Bay, RC = Road Cove.
Laternula was greater than for Adamussium, differences in RNA between locations were analysed with ANCOVA, including shell length as a covariate. Significant differences in Laternula RNA were found between locations (Table 4; Fig. 3B), with higher RNA at Spike Cape and Dunlop Island compared with Cape Evans ( P b 0.01). As Laternula were sampled in spring in McMurdo Sound and in summer in Terra Nova Bay (Tethys Bay), direct comparisons between the two areas should be treated cautiously. However, Laternula RNA in Terra Nova Bay in summer was similar to Cape Evans in spring, i.e. relatively low. Laternula RNA was higher at 12 m depth than at 18 m depth at Cape Evans (t 10 = 4.163, P = 0.0019). There was no difference in shell length between these two depths (t 10 = 0.731, P = 0.4816) and no differ-
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Fig. 4. (A) RNA concentration in adductor muscle of Adamussium colbecki, (B) benthic chlorophyll a concentration and (C) chlorophyll a: phaeophytin ratio in spring at the three sites in New Harbour, McMurdo Sound (mean F S.D., n = 15 in A, and 5 in B and C). Sites 2 and 3 are shallower and closer to the productive near-shore moat.
ence in benthic chlorophyll a (t 7 = 0.511, P = 0.6253), although at shallower depths the bivalves were in closer proximity to ice algae. Table 4 Laternula elliptica: Results of ANCOVA testing differences in RNA between locations (Dunlop Island, Spike Cape, Cape Evans and Tethys Bay) with shell length as covariate Factor
SS
df
F
P
Location Shell Location Shell Residual
10.33 6.90 5.08 59.55
3 1 3 55
3.18 6.37 1.56
0.0310 0.0145 0.2086
Significant results are indicated in bold.
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4. Discussion In the Antarctic, growth of benthic organisms is generally very slow and assumed to be related to strong gradients in food availability. This study investigated temporal and spatial patterns in nucleic acid ratios in two Antarctic bivalves, Adamussium colbecki and Laternula elliptica. Even though relatively slowgrowing adults of both species were sampled, nucleic acid ratios differed between the two species, with contrasting relationships between RNA and primary productivity, and with markedly higher RNA in Laternula. RNA also differed with location and season. Thus, despite slow bivalve growth, we were able to detect differences in growth rate indirectly by using RNA as a measure of short-term growth rate. 4.1. Importance of food availability Significant differences in Adamussium RNA were found between locations within McMurdo Sound and even bigger differences were found between McMurdo Sound and Terra Nova Bay. In addition, smaller-scale variability was found within New Harbour in relation to patterns of benthic primary production. All patterns in Adamussium RNA could be related to food availability, with higher RNA in locations with higher primary production and higher RNA in late summer after the peak in primary production. The lower RNA of Adamussium at New Harbour compared to Dunlop Island and Spike Cape in spring is consistent with the theory that a thick, persistent ice cover and a relatively long distance to open water results in lower productivity (Dayton et al., 1986). The higher Adamussium RNA in summer compared with spring at New Harbour further supports the idea of significantly increasing growth rates after peaks in primary production (Clarke, 1988). In this case, the influx of food is likely to originate from the highly productive near-shore moat that forms each summer in New Harbour and supports high Adamussium densities (Stockton, 1984). As water temperature is near constant in New Harbour (between 1.92 and 1.72 8C, S. Bowser, http://www.bowserlab.org/datastore/ datastorelaunch.htm), the elevated RNA in summer is likely a result of increased food availability rather than seasonal temperature variation. The higher Adamussium RNA in Terra Nova Bay probably reflects
the combination of slightly warmer water temperatures, higher productivity and longer growth season of Terra Nova Bay compared to McMurdo Sound which, in combination, result in higher benthic secondary productivity (Faranda et al., 2000; Chiantore et al., 2001). The patterns in RNA also correspond with the slower Adamussium shell growth recorded at New Harbour compared with Terra Nova Bay (Chiantore et al., 2003; Berkman et al., 2004). Adamussium and Laternula have very different modes of living (epifaunal vs. deep burrowing, respectively), but nevertheless utilise similar food sources, as indicated by relatively similar stable isotope signatures of gill tissue in both species within a location (Norkko et al., in preparation). However, stable isotope signatures change between locations, indicating that bivalves may utilise different food sources at different locations. For example, at Terra Nova Bay both Adamussium and Laternula feed mainly on phytoplankton and sediment (microphytobenthos and detritus), while at New Harbour ice algae and sediment are the most important food sources for both species (Norkko et al., in preparation). Given these similar stable isotope signatures, it is interesting that the opposite RNA pattern was exhibited by Laternula, with lower RNA at Cape Evans compared to Dunlop Island and Spike Cape. Cape Evans is considered a highly productive location in McMurdo Sound (Dayton and Oliver, 1977; Dayton et al., 1986) and had the highest sediment chlorophyll a values during the present study, but this is not reflected in the Laternula RNA. The higher Laternula RNA at the shallower depth at Cape Evans cannot be explained by benthic chlorophyll a, as this was similar at both depths. Possibly benthic chlorophyll a is a better indicator of food availability for Adamussium, as they live on the sediment surface and may themselves affect resuspension, whereas the link to Laternula is not as strong. The proximity to ice algae could potentially favour bivalves at shallower depths. The dissimilar pattern in RNA between the two species may be due to differences in feeding behaviour, mobility or reproductive strategies, but this requires further study. For example, species with planktotrophic larvae such as Adamussium may spawn to coincide with the plankton bloom, while species with lecithotrophic larvae such as Laternula may survive on egg yolk reserves during winter and are thus less depen-
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dent on food supply (Pearse et al., 1991). The reproductive effort may influence nucleic acid ratios in tissues other than gonads and, for example, Paon and Kenchington (1995) found a negative correlation between RNA/DNA ratios of adductor muscle and gonad over the reproductive cycle in the scallop Placopecten magellanicus. Thus the different reproductive strategies may have implications for the timing of energy allocation, potentially explaining the contrasting patterns in nucleic acid ratios observed in Adamussium and Laternula. Antarctic suspension feeders may not be completely food limited, as they are able to utilise a whole range of food sources, including resuspended detritus, which remains in the system for long periods due to slow breakdown (Gili et al., 2001). Nevertheless, as food availability appears to be the main factor driving changes in growth in Antarctic benthos, any changes in primary production will have profound influences on benthic communities. As suspension feeders, both Adamussium and Laternula play an important part in this benthic–pelagic coupling and enhance the flux of terrigenous mineral particles and organic material from the phytoplankton bloom to the benthic community (Ahn, 1993; Chiantore et al., 1998). Thus, changes in their health or abundance may have cascading effects on the surrounding communities. As sea-ice conditions are driving primary production (Dayton et al., 1986; Grebmeier and Barry, 1991; Seibel and Dierssen, 2003; Arrigo and van Dijken, 2004; Mangoni et al., 2004), effects of any changes in climate should be most prominent on benthic communities along the coast. 4.2. Metabolism Antarctic benthic species generally exhibit low metabolic rates (Peck, 2002), which increase only when food availability increases (Clarke, 1988). Lower winter metabolic rates have been found in both Laternula and Adamussium (Brockington, 2001; Heilmayer and Brey, 2003). The siphons of Laternula may remain withdrawn below the sediment surface for up to 4 months in winter, during which time the bivalves do not feed (Brockington, 2001). Thus, significantly lower Laternula RNA would be expected in winter, but this remains to be tested. Adamussium and Laternula are the only large
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bivalves in Antarctica and Ahn et al. (2003) suggest that they are successfully adapted to the very seasonal food availability because of the capacity of their muscle tissues to store large amounts of protein in preparation for prolonged starvation. The somewhat higher protein content of Laternula compared to Adamussium may be an adaptation enabling Laternula to completely cease feeding during winter months. Despite slow growth rates, coastal areas around Antarctica support benthic communities with high diversity and high biomass (Brey and Clarke, 1993). Antarctic bivalves may loose less tissue in winter than temperate bivalves, due to the low metabolic cost of living and low protein turnover at low temperatures (Clarke, 1998; Brockington, 2001; Ahn et al., 2003). However, if the temperature rises, mitochondrial maintenance costs rapidly exceed oxygen supply mechanisms in stenothermal Antarctic species, which sets the upper critical temperature for each species (Po¨rtner et al., 1999; Peck et al., 2002). Cold adaptation or temperature compensation should only be assessed at the molecular level as, for example, growth and oxygen consumption are the summed result of many processes, some of which may be completely, partially or not at all temperature compensated (Clarke, 1991; Peck, 2002). For example, Heilmayer and Brey (2003) found no evidence of cold adaptation of whole-body metabolic rates or summer growth rates in Adamussium, while in vitro protein synthesis capacities of Adamussium and the European scallop Aequipecten opercularis were similar at 0 8C and 25 8C, respectively, with higher RNA and RNA/ protein ratios in Adamussium (Storch et al., 2003). Species living at low temperatures may have elevated RNA concentrations either to counteract the thermally induced reduction in RNA translational efficiency (Fraser et al., 2002) or as a result of slow RNA turnover (Storch et al., 2003). However, New Zealand scallops Pecten novaezelandiae appear to have RNA concentrations markedly similar to Adamussium in McMurdo Sound (approximately 3.5–4.5 Ag mg 1, J. Norkko unpublished data), but higher protein content (approximately 30%), resulting in lower RNA/ protein ratios compared with Adamussium. The inherent high capacity for protein synthesis in Antarctic bivalves may be an adaptation allowing rapid upregulation of protein synthesis during peaks in primary production (Storch et al., 2003).
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5. Conclusions Antarctic benthos exhibit very slow growth due to temperature, light regime and food availability. Nevertheless, we found season and location-related differences in short-term growth rates in two key bivalve species. Nucleic acid ratios provide a snapshot of bivalve growth rate at the time of sampling and, by using nucleic acid ratios, future studies could relatively easily obtain a measure of growth rate from a multitude of locations with contrasting habitat characteristics, food availability and temperature regimes around the Antarctic continent. This would yield a unique understanding of spatial and temporal patterns in bivalve growth in this extreme environment. This may then be integrated with information on how the structure and function of Antarctic benthic communities are related to sitespecific physical variables and patterns in primary productivity, which will improve our ability to predict how key species in these ecosystems may respond to environmental changes.
Acknowledgements Thank you for invaluable help with sample collection; Owen Anderson, Neil Andrew, Rod Budd, Carlo Cerrano, Greig Funnell, Marta Guidetti, Peter Marriott and Steve Mercer. Mille grazie, Programma Nazionale Di Recherche´ in Antartide and the support of Riccardo Cattaneo-Vietti, Mariachiara Chinatore and Michela Castellano for facilitating work at Terra Nova Bay and Massimo Patania and Igor Zamaro for assistance with diving. Thanks also to Sam Bowser (United States Antarctic Program) for hosting us at New Harbour, collecting scallops in late summer in New Harbour, and sharing his chlorophyll and temperature data. This research was supported by a Kelly Tarlton’s Antarctic Encounter and Underwater World Antarctic Research Scholarship to JN, with logistic support provided by Antarctica New Zealand and PNRA. Further support was received from the New Zealand Ministry of Fisheries (ZBD2002/01), and a National Institute of Water and Atmospheric Research (New Zealand) PhD scholarship and a stipend from Svenska Kulturfonden (Carl Cedercreutz Fund, Finland) to JN.
Rufus Wells provided advice on methods for analysing nucleic acid ratios and Mike Thorndyke at Kristineberg Marine Research Station, Sweden, generously provided desk space during manuscript preparation. [SS]
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