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Freezing in the Antarctic limpet, Nacella concinna
Cryobiology 61 (2010) 128–132
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Freezing in the Antarctic limpet, Nacella concinna q T.C. Hawes a,*, M.R. Worland b, J.S. Bale a a b
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK British Antarctic Survey, High Cross, Madingley Road, Cambridge CB3 OET, UK
a r t i c l e
i n f o
Article history: Received 6 October 2009 Accepted 15 June 2010 Available online 19 June 2010 Keywords: Freezing Mucus Osmolality Mollusc Intertidal Antarctic
a b s t r a c t The process of organismal freezing in the Antarctic limpet, Nacella concinna, is complicated by molluscan biology. Internal ice formation is, in particular, mediated by two factors: (a) the provision of an inoculative target for ice formation in the exposed mucus-secreting foot; and (b) osmoconformity to the marine environment. With regard to the first, direct observations of the independent freezing of pedal mucus support the hypothesis that internal ice formation is delayed by the mucal film. As to the second, ice nucleation parametrics of organismal tissue (head, midgut, gonad, foot) and mucus in both inter- and subtidal populations were characterized by high melting points (range = 4.61 to 6.29 °C), with only c.50% of a given sample osmotically active. At this stage it would be premature to ascribe a cryo-adaptive function to the mucus as the protective effects are more readily attributed to the physical properties of the secretion (i.e. viscosity) and their corresponding effects on the rate of heat transfer. As it is difficult to thermally distinguish between the freezing of mucus and the rest of the animal, the question as to whether it is tolerant of internal as well as external ice formation remains problematic, although it may be well suited to the osmotic stresses of organismal freezing. Ó 2010 Elsevier Inc. All rights reserved.
Introduction The Antarctic limpet, Nacella concinna Strebel 1908, is one of the most common marine macro-invertebrates of Maritime Antarctica, with a distribution that ranges from South Georgia down the length of the Antarctic Peninsula [5]. It has a wide depth range – from rocky shores to >110 m [23,24,30] and two types are recognized: intertidal and subtidal [3,7,30]. Its conspicuousness and relative abundance have made it a convenient model organism for Antarctic marine studies. Numerous aspects of its biology have been examined including: reproduction [22]; mucus production [20]; osmoconformity [5,6]; stenothermy [21] freshwater tolerance [5]; protein metabolism [8]; and heat shock protein expression [2]. However, although it has been, and continues to, provide a model for the examination of the stresses characteristic of Antarctic marine and intertidal environments, its cryobiology has received relatively little attention. In N. concinna, physiological freezing phenomena are mediated, in particular, by two components of molluscan biology: (a) steno-
q Statement of funding: The research described in this paper was funded by a CGS grant (7/26) from the NERC Antarctic funding initiative, UK. * Corresponding author. Present address: Department of Zoology, University of Otago, P.O. Box 56, Dunedin, New Zealand. E-mail address: [email protected] (T.C. Hawes).
0011-2240/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2010.06.006
haline osmoconformity, which facilitates physiological synchronization with the marine environment [5,6]; and (b) a muscular, mucus-secreting foot, which enables both free movement on, and ‘cemented’ attachment to, the habitat substrate [20]. These two characters acquire an added significance in the context of sub-zero temperatures: the former predisposes them to tolerate the osmotic stresses of freezing; the latter provides an inoculative target for the transmission of the freezing front. This paper examines the role of both in the cryobiology of N. concinna. Although the mechanical challenges of ice (i.e. scouring from brash ice) have received consideration at population scales [25,27], relatively little is known about N. concinna’s relations with ice at physiological scales beyond its lower thermal limits [4]. Hargens and Shabica [9] experimentally siphoned mucus off from test animals and showed that controls that were allowed to retain their mucus were able to survive lower temperatures. However, although cited frequently as a fascinating ‘oddity’ in the cryobiological literature, the role of mucus in cryoprotection has not been revisited. Recently, Waller et al. [29] noted a gap between whole body crystallization temperatures (Tcs) and lower lethal temperatures (LLTs) that they attributed to partial freeze tolerance. Here, in tandem with organismal ice nucleation parametrics and measurements of LLTs, we return to Hargens and Shabica’s [9] original theory and re-examine N. concinna’s responses to freezing in the context of mucus production.
T.C. Hawes et al. / Cryobiology 61 (2010) 128–132
Materials and methods Collection and treatment of animals Fieldwork was carried out between December 2007 and February 2008 at the British Antarctic Survey’s research station at Rothera Point, Adelaide Island, on the west coast of the Antarctic Peninsula (67°340 S, 66°080 W). Subtidal specimens were collected by SCUBA (Rothera Dive Team) and maintained in an aquarium at 0–1 °C. Intertidal specimens were collected by wading along the shoreline near the station during low tide and maintained in buckets with c.5 l seawater (to provide a shallow ‘intertidal’ environment) from the marine aquaria, changed daily, and kept in a climate controlled incubator at 0–1 °C. Animals were acclimated for 1 week before use.
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lengths of 150 were measured: mean = 24.3 mm ± 0.04). Mucus production was determined gravimetrically using a Mettler-Toledo microbalance (UMXW d = 0.1 lg) (Mettler-Toledo Ltd., UK). After the end of the exposure, the limpets were removed from the sample tubes for survival determination after 48 h. The 50 ml seawater in which each individual had been sitting (now combined with mucus secreted during the experiment) was collected. The seawater was evaporated in a furnace (Carbolite RWF11/13, Carbolite, UK) at 120 °C and the ash collected after 24 h. The salinity of seawater used was 35 (3.5%). Given that the salt constant is constant for all samples, subtracting 3.5% from the mass of the ash provided a measure of the salt-free mass of the mucus produced by each individual. Differences in mucus production were compared using general linear modeling (GLM). Ice nucleation parametrics
Lower lethal temperatures (LLTs) Intertidal limpets were cooled in batches of 10 from their acclimation temperature at 0.1 °C min1 to different exposure temperatures (6, 7, 8, 10, 11 °C), held there for 1 h, and then rewarmed to their acclimation temperature at the same rate. Individuals were placed in 50 ml plastic tubes filled with seawater and suspended in a refrigerated circulator (Thermo Haake Phoenix P2 Circulator; Thermo Haake International, Germany). For each treatment 20–40 animals were tested. At the end of the experiment limpets were placed in individual 50 ml plastic beakers filled with seawater, turned upside and kept in a climate controlled incubator at 0–1 °C. Survival was determined as the proportion of animals self-righting after 48 h [21]. Estimated percentiles for limpet lower lethal temperatures were calculated by Probit analysis.
Intertidal and subtidal samples were dissected and ice nucleation parametrics determined for head, midgut, gonad, and foot tissue. Similar measurements were made for mucus, samples of which were collected by pipette from limpets blotted dry of seawater, held upside down and then allowed to secrete freely. Parametrics were calculated from Tc and melting points (MPs) determined by differential scanning calorimetry (DSC) (Mettler-Toledo DSC 820 thermal analysis system; Mettler-Toledo Ltd., Leicester, UK), and gravimetric measurement of water content [13]. Results The mean Tc of intertidal limpets was 6.60 ± 0.27 °C (n = 24). Fig. 1 shows the survival (a) and Probit estimated survival
Temperature of crystallization measurements (Tc) Whole-animal Tc measurements were determined following the protocol described by Waller et al. [29] using copper-constantan thermocouples held against dry limpets in 50 ml plastic tubes, submerged in a refrigerating circulator (see above), then slowly cooled at 0.1 °C min1. Exotherms were recorded using an 8 channel datalogger (Pico Technology Ltd., UK) [12]. Casual observations of freezing (see Results) were subsequently made with an individual limpet placed in a small aluminum open-top container (10 4 cm) and thermocouples held against the exposed limpet’s ventral surface and the sides of the container. The open-top container was placed on a metal cold plate through which cooled alcohol was circulated from a refrigerating circulator (see above) and the temperature of the container surface slowly lowered (0.1 °C min1) to sub-zero temperatures. After a freezing event was detected, the limpet was gently prised off the side of the container, turned upside down and examined. These observations prompted a repeat of the original Tc protocol, with a revised element: this time animals were removed immediately after the detection of an exotherm and examined for evidence of tissue freezing. Mucus production in relation to low temperature exposure To examine the relationship between survival of low temperature exposure and mucus production in both intertidal and subtidal samples, a separate experiment was undertaken in which limpets were exposed to a range of low temperature treatments: 6 °C for 1, 6, and 12 h; and 6 °C for 6 h with 6 h pre-acclimation at 1 °C. Exposure protocols followed those described above to determine LLT except for the duration of exposure. To control for body size differences in mucus production [20], only ‘medium’ sized limpets (c.23–27 mm shell length) were used in the experiment. (Shell
Fig. 1. (a) Percentage survival (measured as self-righting response after 48 h) of intertidal Nacella concinna exposed to sub-zero temperatures; (b) parametric cumulative failure plot of survival in limpets exposed to sub-zero temperatures as determined by Probit analysis and with LT10, LT50, and LT90 indicated (survival determined as righting response 48 h after exposure) (centre line = mean; outer lines = upper and lower 95% confidence limits).
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probabilities (b) of limpets exposed to sub-zero temperatures. The LT50 of limpets was 6.88 ± 0.42 °C (Fig. 1b). Given that c.50% of the sample survived below their Tc, there would normally be some confidence in attributing some degree of freeze tolerance to the limpets [29]. However, direct observations revealed that the exotherms used as signatures of whole-animal Tcs were, in fact, produced by the freezing of the mucal film of the animals, not the animals themselves. Casual observations of a limpet frozen on a cold plate and turned over, revealed that while the mucus was frozen solid, the pedal foot of the organism was unfrozen and readily resumed activity upon warming (Fig. 2). These casual observations were corroborated by repeating the protocol used to determine Tc (6.93 ± 0.30) on a further eight animals. Upon removal after the initiation of the Tc exotherm, the same observations were made: the mucus had frozen, but the pedal foot remained visibly unfrozen, resuming activity almost immediately. Subsequent examination of exotherms from Tc measurements gave no clear indication of subsequent separate freezing events for the animals themselves, making it impossible to determine at what later point the animals themselves had frozen. Comparison of pedal mucus production in both intertidal and subtidal specimens exposed to a range of low temperature treatments, found no significant differences between survivors and non-survivors, but significant differences between treatments (df = 3; F = 5.71; p = 0.001) and populations (df = 1; F = 11.65; p = 0.001) (Table 1). Table 2 compares the ice nucleation parametrics of organismal components of inter- and subtidal populations with mucus. There were no significant differences in osmotically active portions, but there were significant differences
Fig. 2. Limpet turned over after freezing on a cold plate; the pedal foot (pf) is unfrozen but the mucus (m) is clearly seen as a frozen strand adhered to the wall of the container on which it was originally attached.
between tissue/fluids for both Tcs (df = 4; F = 4.42; p = 0.003) and MPs (df = 4; F = 18.94; p < 0.001) (see Table 3 for posthoc comparisons).
Discussion Freeze tolerance is well known in limpets [1,17,19,26]. However, although, in all likelihood, N. concinna is freeze tolerant to some degree, observations of the independent freezing of the mucal film, render the definitive cryotyping [sensu 10,11] of this species problematic. Hopefully, further attempts to distinguish between the freezing of mucus and the organism using thermal diagnostics may be more fruitful with the hindsight provided by the results described here; alternatively, histological methods [15] offer another avenue. Regardless of such complications, the role of mucus in the limpets’ cryobiology is, of course, fascinating, in itself, and provides the first corroboration of Hargens and Shabica’s [9] findings. Adopting a conservative interpretation of the observations, it would, however be premature to ascribe a cryo-adaptive role to the mucus. It is certainly cryoprotective, but this protection, is for the moment, most sensibly attributed to the physical properties of mucus as a substance, i.e., viscosity. The more viscous a substance, the more the thermal lag it induces – a phenomenon that can be readily observed by attempting to freeze drops of 1 M glycerol at sub-zero temperatures. Hargens and Shabica [9] demonstrate this for limpet mucus in their study: comparing the delayed rate of equilibrium freezing in mucus with the faster rate of seawater. Measures of mucus production in this study fit comfortably within the ranges of daily synthesis previously reported for N. concinna (0.49–1.47 mg dry mass d1) [20]. Although there were significant differences between treatments and populations, there were no significant differences between survivors and non-survivors. Differences between treatments are readily attributed to the different exposure durations. Differences between populations – generally subtidal limpets produced more mucus than intertidal limpets – are not readily explained without more information about the differences between the populations. Indeed, the differences are unexpected in that, intuitively, greater rates of production would be expected in intertidal samples, given that they are routinely exposed to greater wave disturbance (requiring clamping) and that terrestrial locomotion presumably requires considerable lubrication. Both populations will, of course, use mucus to facilitate both clamping attachment and lubricated locomotion. In fact, these dual functions of mucus may provide an explanation for the lack of sig-
Table 1 Comparative mucus production in inter- and subtidal limpet populations that survived and did not survive low temperature exposure treatments (mean error = SE ± 1). Population
Low temperature exposure treatment
n
Treatment # Intertidal
Subtidal
Mucus production (mg) Survivors (S)
n
Non-survivors (NS)
Post hoc significant differences (Tukey’s) Between treatments
Between populations
6 °C 1 h
1A
9
0.84 ± 0.17
19
0.85 ± 0.04
vs. 4B (p = 0.002)
(S) vs. 1B (p = 0.021) (NS) vs. 1B (p = 0.014)
6 °C 6 h
2A
9
0.75 ± 0.03
21
0.81 ± 0.05
6 °C 12 h 6 °C 6 h + 6 h pre-acclimation
3A 4A
4 7
0.92 ± 0.02 0.92 ± 0.03
14 12
0.85 ± 0.04 0.97 ± 0.05
vs. 3B (p = 0.029) vs. 4B (p < 0.001) vs. 4B (p = 0.042)
6 °C 1 h
1B
9
0.97 ± 0.03
10
0.86 ± 0.04
6 °C 6 h 6 °C 12 h 6 °C 6 h + 6 h pre-acclimation
2B 3B 4B
13 7 19
0.92 ± 0.06 0.98 ± 0.05 1.09 ± 0.05
25 12 21
0.91 ± 0.05 0.98 ± 0.03 1.02 ± 0.04
vs. 1A(S) (p = 0.021) vs. 1A(NS) (p = 0.014) vs. vs. vs. vs.
2A 1A 2A 3A
(p = 0.029) (p = 0.002) (p < 0.001) (p = 0.042)
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T.C. Hawes et al. / Cryobiology 61 (2010) 128–132 Table 2 Ice nucleation parametrics of Nacella concinna tissue and fluid samples (n = 10 for each sample) (mean error = SE ± 1). Population
Tissue/ fluid
Dry mass (mg)
Percentage water (%)
Tc (°C)
MP (°C)
Osmolality (Osmols)
Osmotically active water (%)
Osmotically inactive water (%)
Ratio OI: OA
Intertidal
Head Midgut Foot Gonad Mucus
9.69 ± 1.67 19.65 ± 1.87 0.54 ± 3.71 7.14 ± 2.08 0.39 ± 0.13
81.46 ± 2.00 71.16 ± 1.12 79.96 ± 2.16 73.00 ± 2.55 95.44 ± 1.37
11.03 ± 0.72 9.64 ± 0.33 11.68 ± 0.97 11.01 ± 0.66 14.83 ± 0.93
6.17 ± 0.24 5.48 ± 0.11 5.46 ± 0.25 5.19 ± 0.13 4.61 ± 0.15
3.32 ± 0.13 2.95 ± 0.06 2.93 ± 0.13 2.79 ± 0.27 2.48 ± 0.08
46.65 ± 0.826 48.03 ± 1.85 49.51 ± 4.54 46.50 ± 2.69 50.79 ± 11.08
53.35 ± 0.86 51.97 ± 1.85 50.49 ± 4.54 53.50 ± 5.87 49.21 ± 11.08
1:0.87 1:0.92 1:0.98 1:0.87 1:1.03
Subtidal
Head Midgut Foot Gonad Mucus
8.43 ± 1.44 24.57 ± 3.75 3.68 ± 0.68 4.31 ± 1.56 0.96 ± 0.05
81.2 ± 1.42 69.79 ± 2.16 79.13 ± 1.36 69.53 ± 6.33 96.05 ± 0.55
11.95 ± 0.71 11.42 ± 0.94 12.79 ± 0.82 13.55 ± 1.45 13.22 ± 0.87
6.29 ± 0.16 5.6 ± 0.22 5.89 ± 0.26 4.99 ± 0.17 4.85 ± 0.11
3.38 ± 0.08 3.01 ± 0.12 3.16 ± 0.14 2.68 ± 0.09 2.61 ± 0.06
50.13 ± 4.54 45.95 ± 2.37 47.37 ± 4.68 48.35 ± 2.00 47.79 ± 2.31
49.87 ± 4.54 54.05 ± 2.37 52.63 ± 4.68 51.65 ± 2.00 52.21 ± 2.31
1:1 1:0.85 1:0.9 1:0.93 1:0.92
Table 3 Posthoc (Tukey’s) significant differences between Tcs and MPs in inter- and subtidal populations (I = intertidal; S = subtidal). Parametric
Factor
Interaction
Difference of means
P
Tc
Tissue/fluid
Head vs. mucus Midgut vs. mucus Midgut (I) vs. mucus (I)
2.536 3.49 5.185
0.038 0.001 0.003
Head vs. midgut Head vs. foot Head vs. gonad Head vs. mucus Midgut vs. mucus Foot vs. gonad Foot vs. mucus Head (I) vs. gonad (I) Head (I) vs. mucus (I) Head(I) vs. gonad (S) Head (I) vs. mucus (S) Head (I) vs. mucus (I) Gonad (I) vs. head (S) Mucus (I) vs. head (S) Mucus (I) vs. midgut (S) Mucus (I) vs. foot (S) Head (S) vs. gonad (S) Head (S) vs. mucus (S) Foot (S) vs. gonad (S) Foot (S) vs. mucus (S)
0.690 0.560 1.141 1.504 0.814 0.581 0.186 0.977 1.560 1.181 1.325 0.872 1.100 1.683 0.991 1.275 1.304 1.448 0.896 1.040
0.004 0.034 <0.001 <0.001 <0.001 0.025 <0.001 0.017 <0.001 0.002 <0.001 0.040 0.003 <0.001 0.010 <0.001 <0.001 <0.001 0.040 <0.001
Phenotype MP
Tissue/ fluid
Phenotype
nificant differences between survivors and non-survivors. The amount of mucus produced may be less important than the type produced. Smith et al. [28] found significant differences in the composition of adhesive (for attachment) and non-adhesive (for movement) mucus in the limpet Lottia limatula, with both protein and carbohydrate content approximately two times greater in the former. Given that adhesive mucus enables a cementing of the animal to surfaces, it would presumably be the more viscous of the two and thus more capable of delaying the ice front. Casual observations of mucus frozen independently and photographed (Fig. 2) were of an animal that had been clamped down. Likewise, although no measurements were taken (these differences were not appreciated during the experiment), many of the test animals exposed to sub-zero temperatures were firmly attached to sample tubes and required considerable effort to be removed. Indeed it is worth noting that previous investigations found that attachment accounted for 80% of mucus production over a 24 h period [20]. Although the effect of temperature on composition (i.e. the secretion of a cryo-adaptive mucus at sub-zero temperatures) cannot be ruled out, the attachment hypothesis described above would seem the most appropriate for future scrutiny. Another explanation, is that mucus simply confers protection as long as it is present (note the results of removal [9]) and that if all animals are producing mucus
(with a difference of only c.<25%), differences in survivorship must be attributed to other components of fitness. Ice nucleation parametric of tissue and fluids from N. concinna reveal the extent to which salinity mediates the freezing process. The connections between salinity tolerance and freezing have long been recognized in intertidal animals [14,15]. Indeed, in intertidal invertebrates, acclimation to higher salinity levels has been shown to increase freeze tolerance, although there is a threshold at which salt accumulation becomes toxic and detrimental to fitness [16,18]. Although our results are organismally compartmentalized, the lack of significant difference between tissue and fluids means that we can comfortably say that only c.50% of body water is osmotically active in N. concinna. This is considerably less than that found in other investigations [1], although we note that the calorimetric methods may not be directly comparable. High internal salt concentrations suggest that N. concinna would be well suited to survive freezing through: (a) tolerance of the osmotic stresses of freezing and (b) reduction of the total amount of ice formed in its tissues. Limpets offer an intriguing model for examining the ways in which organismal cryobiology is complicated by molluscan biology. The results described here highlight the mediating role played by mucus secretions and physiological osmoconformity in the
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freezing process. The roles of both warrant further investigation and elucidation. Acknowledgments Research was carried out while TCH was an Honorary Research Associate at the University of Birmingham. Fieldwork was funded by a CGS Grant (7/26) from the NERC Antarctic Funding Initiative. Thanks to the Rothera Dive Team and Dr. Simon Morley for subtidal collections. References [1] A. Ansart, P. Vernon, Cold hardiness in molluscs, Acta. Oecol. 24 (2003) 95–102. [2] M.S. Clark, P. Geissler, C. Waller, K.P.P. Fraser, D.K.A. Barnes, L.S. Peck, Low heat shock thresholds in wild Antarctic inter-tidal limpets (Nacella concinna), Cell Stress Chaperon. 13 (2008) 51–58. [3] J. Davenport, Tenacity of the Antarctic limpet Nacella concinna, J. Mollus. Stud. 54 (1988) 355–356. [4] J. Davenport, Comparisons of the biology of the intertidal sub-Antarctic limpets Nacella concinna and Kerguelenella lateralis, J. Mollus. Stud. 63 (1997) 39–48. [5] J. Davenport, Meltwater effects on intertidal Antarctic limpets, Nacella concinna, J. Mar. Biol. Assoc. UK 81 (2001) 643–649. [6] J. Davenport, H. McAlister, Environmental conditions and physiological tolerances of intertidal fauna in relation to shore zonation at Husvik, South Georgia, J. Mar. Biol. Assoc. UK 76 (1996) 985–1002. [7] C.M. De Aranzamendi, R. Sahade, M. Tatián, M.B. Chiaperro, Genetic differentiation between morphotypes in the Antarctic limpet Nacella concinna as revealed by inter-simple sequence repeat markers, Mar. Biol. (2008) 875–885. [8] K.P.P. Fraser, A. Clarke, L.S. Peck, Low-temperature protein metabolism: seasonal changes in protein synthesis and RNA dynamics in the Antarctic limpet Nacella concinna Strebel 1908, J. Exp. Biol. 205 (2002) 3077–3086. [9] A.R. Hargens, S.V. Shabica, Protection against lethal freezing temperatures by mucus in an Antarctic limpet, Cryobiology 10 (1973) 331–337. [10] T.C. Hawes, A postscript on cryotypes, J. Exp. Biol. 211 (2008) 3518. [11] T.C. Hawes, J.S. Bale, Plasticity in arthropod cryotypes, J. Exp. Biol. 210 (2007) 2585–2592. [12] T.C. Hawes, C.E. Couldridge, J.S. Bale, M.R. Worland, P. Convey, Habitat temperature and the temporal scaling of cold hardening in the High Arctic collembolan, Hypogastrura tullbergi (Schäffer), Ecol. Entomol. 31 (2006) 450– 459.
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Cryobiology journal homepage: www.elsevier.com/locate/ycryo
Freezing in the Antarctic limpet, Nacella concinna q T.C. Hawes a,*, M.R. Worland b, J.S. Bale a a b
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK British Antarctic Survey, High Cross, Madingley Road, Cambridge CB3 OET, UK
a r t i c l e
i n f o
Article history: Received 6 October 2009 Accepted 15 June 2010 Available online 19 June 2010 Keywords: Freezing Mucus Osmolality Mollusc Intertidal Antarctic
a b s t r a c t The process of organismal freezing in the Antarctic limpet, Nacella concinna, is complicated by molluscan biology. Internal ice formation is, in particular, mediated by two factors: (a) the provision of an inoculative target for ice formation in the exposed mucus-secreting foot; and (b) osmoconformity to the marine environment. With regard to the first, direct observations of the independent freezing of pedal mucus support the hypothesis that internal ice formation is delayed by the mucal film. As to the second, ice nucleation parametrics of organismal tissue (head, midgut, gonad, foot) and mucus in both inter- and subtidal populations were characterized by high melting points (range = 4.61 to 6.29 °C), with only c.50% of a given sample osmotically active. At this stage it would be premature to ascribe a cryo-adaptive function to the mucus as the protective effects are more readily attributed to the physical properties of the secretion (i.e. viscosity) and their corresponding effects on the rate of heat transfer. As it is difficult to thermally distinguish between the freezing of mucus and the rest of the animal, the question as to whether it is tolerant of internal as well as external ice formation remains problematic, although it may be well suited to the osmotic stresses of organismal freezing. Ó 2010 Elsevier Inc. All rights reserved.
Introduction The Antarctic limpet, Nacella concinna Strebel 1908, is one of the most common marine macro-invertebrates of Maritime Antarctica, with a distribution that ranges from South Georgia down the length of the Antarctic Peninsula [5]. It has a wide depth range – from rocky shores to >110 m [23,24,30] and two types are recognized: intertidal and subtidal [3,7,30]. Its conspicuousness and relative abundance have made it a convenient model organism for Antarctic marine studies. Numerous aspects of its biology have been examined including: reproduction [22]; mucus production [20]; osmoconformity [5,6]; stenothermy [21] freshwater tolerance [5]; protein metabolism [8]; and heat shock protein expression [2]. However, although it has been, and continues to, provide a model for the examination of the stresses characteristic of Antarctic marine and intertidal environments, its cryobiology has received relatively little attention. In N. concinna, physiological freezing phenomena are mediated, in particular, by two components of molluscan biology: (a) steno-
q Statement of funding: The research described in this paper was funded by a CGS grant (7/26) from the NERC Antarctic funding initiative, UK. * Corresponding author. Present address: Department of Zoology, University of Otago, P.O. Box 56, Dunedin, New Zealand. E-mail address: [email protected] (T.C. Hawes).
0011-2240/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2010.06.006
haline osmoconformity, which facilitates physiological synchronization with the marine environment [5,6]; and (b) a muscular, mucus-secreting foot, which enables both free movement on, and ‘cemented’ attachment to, the habitat substrate [20]. These two characters acquire an added significance in the context of sub-zero temperatures: the former predisposes them to tolerate the osmotic stresses of freezing; the latter provides an inoculative target for the transmission of the freezing front. This paper examines the role of both in the cryobiology of N. concinna. Although the mechanical challenges of ice (i.e. scouring from brash ice) have received consideration at population scales [25,27], relatively little is known about N. concinna’s relations with ice at physiological scales beyond its lower thermal limits [4]. Hargens and Shabica [9] experimentally siphoned mucus off from test animals and showed that controls that were allowed to retain their mucus were able to survive lower temperatures. However, although cited frequently as a fascinating ‘oddity’ in the cryobiological literature, the role of mucus in cryoprotection has not been revisited. Recently, Waller et al. [29] noted a gap between whole body crystallization temperatures (Tcs) and lower lethal temperatures (LLTs) that they attributed to partial freeze tolerance. Here, in tandem with organismal ice nucleation parametrics and measurements of LLTs, we return to Hargens and Shabica’s [9] original theory and re-examine N. concinna’s responses to freezing in the context of mucus production.
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Materials and methods Collection and treatment of animals Fieldwork was carried out between December 2007 and February 2008 at the British Antarctic Survey’s research station at Rothera Point, Adelaide Island, on the west coast of the Antarctic Peninsula (67°340 S, 66°080 W). Subtidal specimens were collected by SCUBA (Rothera Dive Team) and maintained in an aquarium at 0–1 °C. Intertidal specimens were collected by wading along the shoreline near the station during low tide and maintained in buckets with c.5 l seawater (to provide a shallow ‘intertidal’ environment) from the marine aquaria, changed daily, and kept in a climate controlled incubator at 0–1 °C. Animals were acclimated for 1 week before use.
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lengths of 150 were measured: mean = 24.3 mm ± 0.04). Mucus production was determined gravimetrically using a Mettler-Toledo microbalance (UMXW d = 0.1 lg) (Mettler-Toledo Ltd., UK). After the end of the exposure, the limpets were removed from the sample tubes for survival determination after 48 h. The 50 ml seawater in which each individual had been sitting (now combined with mucus secreted during the experiment) was collected. The seawater was evaporated in a furnace (Carbolite RWF11/13, Carbolite, UK) at 120 °C and the ash collected after 24 h. The salinity of seawater used was 35 (3.5%). Given that the salt constant is constant for all samples, subtracting 3.5% from the mass of the ash provided a measure of the salt-free mass of the mucus produced by each individual. Differences in mucus production were compared using general linear modeling (GLM). Ice nucleation parametrics
Lower lethal temperatures (LLTs) Intertidal limpets were cooled in batches of 10 from their acclimation temperature at 0.1 °C min1 to different exposure temperatures (6, 7, 8, 10, 11 °C), held there for 1 h, and then rewarmed to their acclimation temperature at the same rate. Individuals were placed in 50 ml plastic tubes filled with seawater and suspended in a refrigerated circulator (Thermo Haake Phoenix P2 Circulator; Thermo Haake International, Germany). For each treatment 20–40 animals were tested. At the end of the experiment limpets were placed in individual 50 ml plastic beakers filled with seawater, turned upside and kept in a climate controlled incubator at 0–1 °C. Survival was determined as the proportion of animals self-righting after 48 h [21]. Estimated percentiles for limpet lower lethal temperatures were calculated by Probit analysis.
Intertidal and subtidal samples were dissected and ice nucleation parametrics determined for head, midgut, gonad, and foot tissue. Similar measurements were made for mucus, samples of which were collected by pipette from limpets blotted dry of seawater, held upside down and then allowed to secrete freely. Parametrics were calculated from Tc and melting points (MPs) determined by differential scanning calorimetry (DSC) (Mettler-Toledo DSC 820 thermal analysis system; Mettler-Toledo Ltd., Leicester, UK), and gravimetric measurement of water content [13]. Results The mean Tc of intertidal limpets was 6.60 ± 0.27 °C (n = 24). Fig. 1 shows the survival (a) and Probit estimated survival
Temperature of crystallization measurements (Tc) Whole-animal Tc measurements were determined following the protocol described by Waller et al. [29] using copper-constantan thermocouples held against dry limpets in 50 ml plastic tubes, submerged in a refrigerating circulator (see above), then slowly cooled at 0.1 °C min1. Exotherms were recorded using an 8 channel datalogger (Pico Technology Ltd., UK) [12]. Casual observations of freezing (see Results) were subsequently made with an individual limpet placed in a small aluminum open-top container (10 4 cm) and thermocouples held against the exposed limpet’s ventral surface and the sides of the container. The open-top container was placed on a metal cold plate through which cooled alcohol was circulated from a refrigerating circulator (see above) and the temperature of the container surface slowly lowered (0.1 °C min1) to sub-zero temperatures. After a freezing event was detected, the limpet was gently prised off the side of the container, turned upside down and examined. These observations prompted a repeat of the original Tc protocol, with a revised element: this time animals were removed immediately after the detection of an exotherm and examined for evidence of tissue freezing. Mucus production in relation to low temperature exposure To examine the relationship between survival of low temperature exposure and mucus production in both intertidal and subtidal samples, a separate experiment was undertaken in which limpets were exposed to a range of low temperature treatments: 6 °C for 1, 6, and 12 h; and 6 °C for 6 h with 6 h pre-acclimation at 1 °C. Exposure protocols followed those described above to determine LLT except for the duration of exposure. To control for body size differences in mucus production [20], only ‘medium’ sized limpets (c.23–27 mm shell length) were used in the experiment. (Shell
Fig. 1. (a) Percentage survival (measured as self-righting response after 48 h) of intertidal Nacella concinna exposed to sub-zero temperatures; (b) parametric cumulative failure plot of survival in limpets exposed to sub-zero temperatures as determined by Probit analysis and with LT10, LT50, and LT90 indicated (survival determined as righting response 48 h after exposure) (centre line = mean; outer lines = upper and lower 95% confidence limits).
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probabilities (b) of limpets exposed to sub-zero temperatures. The LT50 of limpets was 6.88 ± 0.42 °C (Fig. 1b). Given that c.50% of the sample survived below their Tc, there would normally be some confidence in attributing some degree of freeze tolerance to the limpets [29]. However, direct observations revealed that the exotherms used as signatures of whole-animal Tcs were, in fact, produced by the freezing of the mucal film of the animals, not the animals themselves. Casual observations of a limpet frozen on a cold plate and turned over, revealed that while the mucus was frozen solid, the pedal foot of the organism was unfrozen and readily resumed activity upon warming (Fig. 2). These casual observations were corroborated by repeating the protocol used to determine Tc (6.93 ± 0.30) on a further eight animals. Upon removal after the initiation of the Tc exotherm, the same observations were made: the mucus had frozen, but the pedal foot remained visibly unfrozen, resuming activity almost immediately. Subsequent examination of exotherms from Tc measurements gave no clear indication of subsequent separate freezing events for the animals themselves, making it impossible to determine at what later point the animals themselves had frozen. Comparison of pedal mucus production in both intertidal and subtidal specimens exposed to a range of low temperature treatments, found no significant differences between survivors and non-survivors, but significant differences between treatments (df = 3; F = 5.71; p = 0.001) and populations (df = 1; F = 11.65; p = 0.001) (Table 1). Table 2 compares the ice nucleation parametrics of organismal components of inter- and subtidal populations with mucus. There were no significant differences in osmotically active portions, but there were significant differences
Fig. 2. Limpet turned over after freezing on a cold plate; the pedal foot (pf) is unfrozen but the mucus (m) is clearly seen as a frozen strand adhered to the wall of the container on which it was originally attached.
between tissue/fluids for both Tcs (df = 4; F = 4.42; p = 0.003) and MPs (df = 4; F = 18.94; p < 0.001) (see Table 3 for posthoc comparisons).
Discussion Freeze tolerance is well known in limpets [1,17,19,26]. However, although, in all likelihood, N. concinna is freeze tolerant to some degree, observations of the independent freezing of the mucal film, render the definitive cryotyping [sensu 10,11] of this species problematic. Hopefully, further attempts to distinguish between the freezing of mucus and the organism using thermal diagnostics may be more fruitful with the hindsight provided by the results described here; alternatively, histological methods [15] offer another avenue. Regardless of such complications, the role of mucus in the limpets’ cryobiology is, of course, fascinating, in itself, and provides the first corroboration of Hargens and Shabica’s [9] findings. Adopting a conservative interpretation of the observations, it would, however be premature to ascribe a cryo-adaptive role to the mucus. It is certainly cryoprotective, but this protection, is for the moment, most sensibly attributed to the physical properties of mucus as a substance, i.e., viscosity. The more viscous a substance, the more the thermal lag it induces – a phenomenon that can be readily observed by attempting to freeze drops of 1 M glycerol at sub-zero temperatures. Hargens and Shabica [9] demonstrate this for limpet mucus in their study: comparing the delayed rate of equilibrium freezing in mucus with the faster rate of seawater. Measures of mucus production in this study fit comfortably within the ranges of daily synthesis previously reported for N. concinna (0.49–1.47 mg dry mass d1) [20]. Although there were significant differences between treatments and populations, there were no significant differences between survivors and non-survivors. Differences between treatments are readily attributed to the different exposure durations. Differences between populations – generally subtidal limpets produced more mucus than intertidal limpets – are not readily explained without more information about the differences between the populations. Indeed, the differences are unexpected in that, intuitively, greater rates of production would be expected in intertidal samples, given that they are routinely exposed to greater wave disturbance (requiring clamping) and that terrestrial locomotion presumably requires considerable lubrication. Both populations will, of course, use mucus to facilitate both clamping attachment and lubricated locomotion. In fact, these dual functions of mucus may provide an explanation for the lack of sig-
Table 1 Comparative mucus production in inter- and subtidal limpet populations that survived and did not survive low temperature exposure treatments (mean error = SE ± 1). Population
Low temperature exposure treatment
n
Treatment # Intertidal
Subtidal
Mucus production (mg) Survivors (S)
n
Non-survivors (NS)
Post hoc significant differences (Tukey’s) Between treatments
Between populations
6 °C 1 h
1A
9
0.84 ± 0.17
19
0.85 ± 0.04
vs. 4B (p = 0.002)
(S) vs. 1B (p = 0.021) (NS) vs. 1B (p = 0.014)
6 °C 6 h
2A
9
0.75 ± 0.03
21
0.81 ± 0.05
6 °C 12 h 6 °C 6 h + 6 h pre-acclimation
3A 4A
4 7
0.92 ± 0.02 0.92 ± 0.03
14 12
0.85 ± 0.04 0.97 ± 0.05
vs. 3B (p = 0.029) vs. 4B (p < 0.001) vs. 4B (p = 0.042)
6 °C 1 h
1B
9
0.97 ± 0.03
10
0.86 ± 0.04
6 °C 6 h 6 °C 12 h 6 °C 6 h + 6 h pre-acclimation
2B 3B 4B
13 7 19
0.92 ± 0.06 0.98 ± 0.05 1.09 ± 0.05
25 12 21
0.91 ± 0.05 0.98 ± 0.03 1.02 ± 0.04
vs. 1A(S) (p = 0.021) vs. 1A(NS) (p = 0.014) vs. vs. vs. vs.
2A 1A 2A 3A
(p = 0.029) (p = 0.002) (p < 0.001) (p = 0.042)
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T.C. Hawes et al. / Cryobiology 61 (2010) 128–132 Table 2 Ice nucleation parametrics of Nacella concinna tissue and fluid samples (n = 10 for each sample) (mean error = SE ± 1). Population
Tissue/ fluid
Dry mass (mg)
Percentage water (%)
Tc (°C)
MP (°C)
Osmolality (Osmols)
Osmotically active water (%)
Osmotically inactive water (%)
Ratio OI: OA
Intertidal
Head Midgut Foot Gonad Mucus
9.69 ± 1.67 19.65 ± 1.87 0.54 ± 3.71 7.14 ± 2.08 0.39 ± 0.13
81.46 ± 2.00 71.16 ± 1.12 79.96 ± 2.16 73.00 ± 2.55 95.44 ± 1.37
11.03 ± 0.72 9.64 ± 0.33 11.68 ± 0.97 11.01 ± 0.66 14.83 ± 0.93
6.17 ± 0.24 5.48 ± 0.11 5.46 ± 0.25 5.19 ± 0.13 4.61 ± 0.15
3.32 ± 0.13 2.95 ± 0.06 2.93 ± 0.13 2.79 ± 0.27 2.48 ± 0.08
46.65 ± 0.826 48.03 ± 1.85 49.51 ± 4.54 46.50 ± 2.69 50.79 ± 11.08
53.35 ± 0.86 51.97 ± 1.85 50.49 ± 4.54 53.50 ± 5.87 49.21 ± 11.08
1:0.87 1:0.92 1:0.98 1:0.87 1:1.03
Subtidal
Head Midgut Foot Gonad Mucus
8.43 ± 1.44 24.57 ± 3.75 3.68 ± 0.68 4.31 ± 1.56 0.96 ± 0.05
81.2 ± 1.42 69.79 ± 2.16 79.13 ± 1.36 69.53 ± 6.33 96.05 ± 0.55
11.95 ± 0.71 11.42 ± 0.94 12.79 ± 0.82 13.55 ± 1.45 13.22 ± 0.87
6.29 ± 0.16 5.6 ± 0.22 5.89 ± 0.26 4.99 ± 0.17 4.85 ± 0.11
3.38 ± 0.08 3.01 ± 0.12 3.16 ± 0.14 2.68 ± 0.09 2.61 ± 0.06
50.13 ± 4.54 45.95 ± 2.37 47.37 ± 4.68 48.35 ± 2.00 47.79 ± 2.31
49.87 ± 4.54 54.05 ± 2.37 52.63 ± 4.68 51.65 ± 2.00 52.21 ± 2.31
1:1 1:0.85 1:0.9 1:0.93 1:0.92
Table 3 Posthoc (Tukey’s) significant differences between Tcs and MPs in inter- and subtidal populations (I = intertidal; S = subtidal). Parametric
Factor
Interaction
Difference of means
P
Tc
Tissue/fluid
Head vs. mucus Midgut vs. mucus Midgut (I) vs. mucus (I)
2.536 3.49 5.185
0.038 0.001 0.003
Head vs. midgut Head vs. foot Head vs. gonad Head vs. mucus Midgut vs. mucus Foot vs. gonad Foot vs. mucus Head (I) vs. gonad (I) Head (I) vs. mucus (I) Head(I) vs. gonad (S) Head (I) vs. mucus (S) Head (I) vs. mucus (I) Gonad (I) vs. head (S) Mucus (I) vs. head (S) Mucus (I) vs. midgut (S) Mucus (I) vs. foot (S) Head (S) vs. gonad (S) Head (S) vs. mucus (S) Foot (S) vs. gonad (S) Foot (S) vs. mucus (S)
0.690 0.560 1.141 1.504 0.814 0.581 0.186 0.977 1.560 1.181 1.325 0.872 1.100 1.683 0.991 1.275 1.304 1.448 0.896 1.040
0.004 0.034 <0.001 <0.001 <0.001 0.025 <0.001 0.017 <0.001 0.002 <0.001 0.040 0.003 <0.001 0.010 <0.001 <0.001 <0.001 0.040 <0.001
Phenotype MP
Tissue/ fluid
Phenotype
nificant differences between survivors and non-survivors. The amount of mucus produced may be less important than the type produced. Smith et al. [28] found significant differences in the composition of adhesive (for attachment) and non-adhesive (for movement) mucus in the limpet Lottia limatula, with both protein and carbohydrate content approximately two times greater in the former. Given that adhesive mucus enables a cementing of the animal to surfaces, it would presumably be the more viscous of the two and thus more capable of delaying the ice front. Casual observations of mucus frozen independently and photographed (Fig. 2) were of an animal that had been clamped down. Likewise, although no measurements were taken (these differences were not appreciated during the experiment), many of the test animals exposed to sub-zero temperatures were firmly attached to sample tubes and required considerable effort to be removed. Indeed it is worth noting that previous investigations found that attachment accounted for 80% of mucus production over a 24 h period [20]. Although the effect of temperature on composition (i.e. the secretion of a cryo-adaptive mucus at sub-zero temperatures) cannot be ruled out, the attachment hypothesis described above would seem the most appropriate for future scrutiny. Another explanation, is that mucus simply confers protection as long as it is present (note the results of removal [9]) and that if all animals are producing mucus
(with a difference of only c.<25%), differences in survivorship must be attributed to other components of fitness. Ice nucleation parametric of tissue and fluids from N. concinna reveal the extent to which salinity mediates the freezing process. The connections between salinity tolerance and freezing have long been recognized in intertidal animals [14,15]. Indeed, in intertidal invertebrates, acclimation to higher salinity levels has been shown to increase freeze tolerance, although there is a threshold at which salt accumulation becomes toxic and detrimental to fitness [16,18]. Although our results are organismally compartmentalized, the lack of significant difference between tissue and fluids means that we can comfortably say that only c.50% of body water is osmotically active in N. concinna. This is considerably less than that found in other investigations [1], although we note that the calorimetric methods may not be directly comparable. High internal salt concentrations suggest that N. concinna would be well suited to survive freezing through: (a) tolerance of the osmotic stresses of freezing and (b) reduction of the total amount of ice formed in its tissues. Limpets offer an intriguing model for examining the ways in which organismal cryobiology is complicated by molluscan biology. The results described here highlight the mediating role played by mucus secretions and physiological osmoconformity in the
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