Energy Balance and Cold Adaptation in the Octopus Pareledone Charcoti

Journal of Experimental Marine Biology and Ecology L 245 (2000) 197±214 www.elsevier.nl/locate/jembe

Energy balance and cold adaptation in the octopus Pareledone charcoti

H.I. Dalya,b,* , L.S. Peck a aBritish Antarctic Survey, High Cross, Madingley Road, Cambridge CB30ET, UK bDepartment of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, Scotland, UK Received 18 May 1999; received in revised form 23 July 1999; accepted 25 October 1999

Abstract

A complete energy balance equation is calculated for the Antarctic octopus Pareledone charcoti at 08C. Energy used in respiration, growth, and excretion of nitrogenous and faecal waste, was recorded along with the total consumption of energy through food, for three specimens of P. charcoti (live weights: 73, 51 and 29 g). Growth rates were very slow for cephalopods, with a mean daily increase in body weight of only 0.11%. Assimilation ef®ciencies were high, between 95.4 and 97.0%, which is consistent with previous work on octopods. The respiration rate in P. 21 charcoti was low, with a mean of 2.45 mg O2 h for a standard animal of 150 g wet mass at 08C. 21 In the North Sea octopus Eledone cirrhosa, respiration rates of 9.79 mg O2 h at 11.58C and 4.47 21 mg O2 h at 4.58C for a standard animal of 150 g wet mass were recorded. Respiration rates between P. charcoti and E. cirrhosa were compared using a combined Q10 value between P. charcoti at 08C and E. cirrhosa at 4.58C. This suggests that P. charcoti are respiring at a level predicted by E. cirrhosa rates at 4.5 and 11.58C extrapolated to 08C along the curve Q10 5 3, with no evidence of metabolic compensation for low temperature.  2000 Elsevier Science B.V. All rights reserved.

Keywords: Energy balance; Cold adaptation; Antarctic; Octopus; Pareledone charcoti

1. Introduction

Analyses of energy utilisation in cephalopods are rare. A complete energy balance equation has been calculated for only two species of tropical octopus, Octopus cyanea and Octopus maya (Van Heukelem, 1976), with a partial energy budget constructed for

*Corresponding author. Tel.: 144-1224-273-796; fax: 144-1224-272-396. E-mail address: [email protected] (H.I. Daly)

0022-0981/00/$ ± see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0022-0981(99)00161-6 198 H.I. Daly, L.S. Peck / J. Exp. Mar. Biol. Ecol. 245 (2000) 197 ±214 the temperate Octopus vulgaris (O'Dor and Wells, 1987). Recently, energetics in cephalopods from temperate and tropical waters has been reviewed by Wells and Clarke (1996). Energy balance in cold water species has received little attention and the recent successful maintenance of live Antarctic Pareledone charcoti in the BAS aquarium in Cambridge has provided an opportunity for a detailed study of energy balance in a polar species. The ¯ow of energy through an animal is normally represented by the energy balance equation: C 5 P 1 G 1 R 1U 1 F where C is the total consumption of energy in the food, P and G are the values of somatic and gonadal growth, respectively, R is the energy used in respiration, U is the amount utilised in nitrogenous waste excretion and F is the energetic loss through faeces. The component R in the equation above can be divided into several portions which describe the utilisation of energy in more detail (Clarke, 1993; Wells and Clarke,

1996). Each portion of the respiratory costs involved in somatic growth (RS ), reproductive investment (RGBA), basal metabolism (R ) or activity (R ) may vary independently with factors such as food availability or maturity. A substantial portion, termed the speci®c dynamic action (SDA), the increase in metabolic rate associated with feeding, may comprise the energetic cost of somatic (RSG) and gonadal (R ) growth (Clarke, 1993; Peck, 1998). These separate components of respiration highlight the potential for balancing the ®nite energy resource between each component, for maximum ef®ciency. In cephalopods, partitioning of resources probably differs between species, with the RA component comprising a greater proportion of R in squid, due to their active swimming life-style, than in the more sedentary octopuses. As well as constructing an energy budget for P. charcoti, the present study compares the respiration rates with a related species of the Eledoninae which occurs in temperate regions, Eledone cirrhosa. Also known as the lesser octopus, E. cirrhosa, is found around the British coastline and in the North Sea. The seasonal temperature range in the inshore North Sea is approximately 4±128C (McIntyre, 1958), and specimens have been maintained in the marine aquarium at the University of Aberdeen Zoology Department for a number of months at temperatures of 14±158C (Boyle, 1981). This study compares the respiration rates of P. charcoti at Antarctic temperatures of around 08C, to those of E. cirrhosa acclimated from 11.58C down to 4.58C. Extrapolation of E. cirrhosa respiration rates to 08C will allow a direct comparison between the species. One of the most widely used indices to assess the effect of temperature change on metabolism is the Q10 coef®cient (Schmidt-Nielsen, 1991), which is essentially a measure of the temperature sensitivity of an organism. The Q10 value is calculated using the equation (Schmidt-Nielsen, 1991):

R 10/(t 212t ) Q 5 ]2 10 SD R1 where R12and R are the respiration rates at temperatures t 12and t in8C, respectively. In general, an unstressed animal in its normal temperature range should have a Q10 of around 2 (Cossins and Bowler, 1987), as it is observed across a wide range of organisms H.I. Daly, L.S. Peck / J. Exp. Mar. Biol. Ecol. 245 (2000) 197 ±214 199 that metabolic reactions are slowed by approximately half when ambient temperature falls by 108C. A Q10 of less than 2 denotes insensitivity or acclimation to temperature change and a Q10 of more than 2 denotes increased sensitivity, with an expected range of 1±3 (Calow, 1981). Q10 calculations for E. cirrhosa acclimated to two temperatures and a combined Q10 including both E. cirrhosa and P. charcoti has allowed the assessment and comparison of the effects of temperature.

2. Materials and methods

During the austral summers of 1993 and 1994, inshore trawls at depths of 50±150 m at Signy Island in the South Orkney Islands (608 439 S, 458 389 W) caught a number of specimens of P. charcoti. The specimens were maintained in the aquarium at the BAS station on Signy Island prior to being transferred by ship to the UK. The annual temperature range at this site varies from around 21.88C in winter to 10.38Cin summer (Clarke et al., 1988; Clarke and Leakey, 1996). In May 1993, a single male octopus (specimen 1) initially weighing 64 g live weight, was returned to the UK. It began feeding shortly after arrival and appeared healthy throughout the 18 months that it was maintained in the aquarium. The specimen fed irregularly on frozen/thawed krill Euphausia superba, live amphipods Bovallia gigantea and live mussels Mytilus edulis, and increased in weight to 74 g before dying in November 1994 after releasing a single spermatophore. In May 1994, a further 13 P. charcoti were returned, from which two males (specimens 2 and 3) initially weighing 47 and 27 g wet weight, successfully established in the aquarium. They fed regularly on live isopods, Antarcturus sp., and mussels M. edulis, and increased in body weight until they both died in November 1994 weighing 51 and 30 g, respectively. The three healthy specimens showed apparently normal behaviour. They were all held in the cold aquarium system in Cambridge which is cooled to 060.58C (Ward and Peck, 1997), and were maintained in a light regime of 12-h dimmed light and 12-h darkness. For the duration of the trial all the P. charcoti specimens were fed on M. edulis. All food was weighed before addition to each holding tank, and remains removed and weighed after feeding. Frequently, mussels had to be opened using a scalpel before the octopuses would feed. On their arrival in the aquarium live Antarctic isopods, Antarcturus sp., were successfully used to feed the octopuses. Unfortunately, only a small stock of live isopods were available for use as food. The frozen/thawed krill, E. superba, were used as food for specimen 1 on its arrival, and this was only initially successful when individual defrosted krill were placed under the web on a length of wire and moved gently until the octopus closed its arms and web around it. After feeding successfully for a few weeks during the trial all the specimens rejected any further food offered. Samples of the type of food consumed were dried to constant weight to obtain a conversion factor for wet to dry weights. All three specimens were weighed at intervals of 7±14 days throughout the trial to monitor total growth. Specimens were removed from the tanks with nets and weighed in a plastic beaker. The mass of any water released from the mantle cavity of the octopus was obtained after returning the individual to the aquarium and subtracted from total 200 H.I. Daly, L.S. Peck / J. Exp. Mar. Biol. Ecol. 245 (2000) 197 ±214 mass. The specimens were not anaesthetised, and the period of handling during weighing was kept brief to minimise stress to the animal. A 1015-ml glass respirometer chamber was used for trials with the 73 g specimen and a cylindrical 237 ml plastic respirometer was used for the two smaller specimens (Fig. 1). Both chambers were supplied with seawater from a raised header tank and had controllable ¯ow-rates and ports ®lled with silicon sealant for taking samples (Fig. 1).

To determine the oxygen consumption rates (VO2 ), each specimen was placed in the respirometer and it was sealed, making sure that there were no air bubbles trapped in the system. A gentle ¯ow of oxygenated water was passed through the respirometer, at a rate of approximately 600 ml min21 , for 1 h, while observing the behaviour of the specimen and recording the activity and time required for the animal to settle. After this period, the ¯ow was stopped at taps A and B (Fig. 1), to seal the system. The oxygen concentration was determined from 25 ml samples, taken using a gas-tight syringe through a port ®lled with silicon sealant at the top of the chamber (Fig. 1). The sample taken was suf®ciently small to make replacement of the volume removed unnecessary.

Fig. 1. The glass (A) and plastic (B) respirometers used in oxygen consumption and nitrogen excretion trials. Oxygenated water from a header tank ¯owed in tap (a) and out at tap (b), and was collected to monitor ¯ow-rate. H.I. Daly, L.S. Peck / J. Exp. Mar. Biol. Ecol. 245 (2000) 197 ±214 201

To ensure complete mixing of the water, the chamber was gently inverted three times before sampling. Very limited mantle ventilatory movements were observed, and the inversion movement did not appear to unduly disturb the animals. Samples were taken every 10±30 min and oxygen content obtained via injection into a couloximeter (Hersch, 1973; Peck and Uglow, 1990). During trials the respiration chambers were screened from the room to minimise visual disturbance. Oxygen levels in the respirometer were not allowed to fall below 100 mmHg to minimise the possibility of stress caused by low oxygen tension. Trials where air bubbles were observed in the chamber, or when the specimen became highly active were abandoned, so that only resting metabolism with minimum activity was recorded. Nitrogen excretion trials were carried out alternating with the respiration trials over the same time period and using the same respiration chambers. Oxygenated water was passed through the chamber with the specimen inside at approximately 600 ml min21 for 1 h before the chamber was sealed. Repeat samples of 20 ml were taken at the beginning and end of an hour-long trial, with the chamber being inverted three times before sampling to ensure complete mixing. Ammonia concentration was measured using the phenolhypochlorite method described by Liddicoat et al. (1975), as modi®ed by Catalano (1987), and scaled to a sample volume of 10 ml (Clarke et al., 1994). Calibration was achieved by spiking seawater samples with a range of concentrations of ammonium sulphate solution.

VO2 and nitrogen excretion rates were obtained from comparisons with parallel controls (no animal in chamber). All visible faeces were removed from the holding tanks daily and wet weight recorded. Although the faeces were delicate and were easily broken up on contact, care was taken during collection, and an estimated 80% was successfully recovered. As only small amounts of faeces were produced the error introduced into the energy budget during collection was small. Faeces were oven dried on ®lter paper and the dry weight recorded. Healthy specimens of E. cirrhosa were obtained by the Department of Zoology, University of Aberdeen from local commercial trawl ®shermen. In October 1994 several E. cirrhosa specimens were transferred by rail from Aberdeen to the cold marine aquarium at BAS, Cambridge. The smallest specimens available were used to allow a more direct comparison of respiration rates, as they were closest in size to the P. charcoti used earlier. Smaller specimens also experienced less stress when being placed in the respirometer chambers. Although there was mortality of some E. cirrhosa during transportation, two male specimens, with wet weights of 300 and 323 g, were successfully established in the aquarium, were active and appeared healthy. The specimens were held at 11.58C on arrival, which was the approximate sea temperature at the time of capture, and not disturbed for 48 h to allow them to settle and acclimate. A large glass respiration chamber, similar in shape to A (Fig. 1), but of 2701-ml volume, with taps for controlling ¯ow-rate and silicon sealant ®lled ports for sampling was used during trials. Chambers were sealed, trials run and samples processed using the same method as for P. charcoti. The specimens were held at 11.58C for 10 days with trials every 2±3 days before being transferred to a room at 4.58C. They were transferred in their holding tank, which was allowed to cool to the ambient temperature overnight. 202 H.I. Daly, L.S. Peck / J. Exp. Mar. Biol. Ecol. 245 (2000) 197 ±214

After a further 48 h acclimation time, respiration trials were carried out every 3 days for 9 days. The specimens were then transferred to 08C, but both died within 24 h of transfer. Body mass and volume were recorded at the beginning and end of the trials. Specimens usually remained inactive during trials but on occasion showed some increased activity in arm movements, which was noted. Trials where considerable activity, such as jetting, was recorded were abandoned. Throughout the experiment the E. cirrhosa were not fed. The exact time that the specimens last fed before respiration trials began is unknown, but, assuming that they fed regularly in the wild, it would have been approximately 4 days prior to experimenta- tion. Attempts were made to provide live food (Carcinus maenas) at the start of the trial to establish a known feeding time, but this proved unsuccessful due to high prey mortality during transportation. The joule has been used here as the energy unit, and the conversion factor to calories is 1 calorie54.184 J (Schmidt-Nielsen, 1991).

3. Results

The cumulative food consumption for the P. charcoti specimens varied in relation to body size (Fig. 2). The mean feeding rates over the trial were 31.44 mg day21 dry mass

Fig. 2. Cumulative food consumption C (mg dry wt) for Pareledone charcoti feeding on Mytilus edulis over the 50 days when measurements were taken. H.I. Daly, L.S. Peck / J. Exp. Mar. Biol. Ecol. 245 (2000) 197 ±214 203

Fig. 3. Growth for Pareledone charcoti, as wet mass (g) increase excluding water released from the mantle cavity or web. for specimen 1, 22.78 mg day21 dry mass for specimen 2 and 14.23 mg day21 dry mass for specimen 3. The growth of the three specimens over the trial was very small (Fig. 3). The period over which growth was recorded in the P. charcoti specimens was relatively brief (up to 120 days), and the best ®t to the observed growth over this period was linear. The slope of the regression line for each specimen was positive. When each slope was tested for signi®cance of difference from zero, the slope for specimen 1 was signi®cantly different from zero (t510.79, p,0.001), while the slopes for specimens 2 and 3 were not (t52.00, p.0.05 and t51.76, p.0.05, respectively). These results indicate that growth by specimens 2 and 3 over the trial was very slow. Instantaneous growth rates (G), expressed as the percent increase in body mass per day, were calculated from the equation: ln W 2 ln W G 53]]]21] 100 t212 t where W12and W are body weights at times t 12and t , respectively (Forsythe and Van Heukelem, 1987). Daily growth rates were 0.13% for specimen 1, 0.08% for specimen 2 and 0.12% for specimen 3. Respiration rates either decreased gradually with time (specimens 1 and 2), or 204 H.I. Daly, L.S. Peck / J. Exp. Mar. Biol. Ecol. 245 (2000) 197 ±214

21 Fig. 4. Respiration rate R (mg O2 h ) for Pareledone charcoti with increasing time after last feeding event. 21 Points labelled with the respiration in mg O2 h indicate trials where increased activity of the specimen was recorded. remained constant (specimens 3) (Fig. 4). Activity was recorded as low in most trials, and on the occasions where increased activity was noted (Fig. 4), there was not a noticeable elevation in metabolism. The mean respiration rates for P. charcoti are shown in Table 1, which includes comparative values from O'Dor and Wells (1987) for temperate and tropical octopods. All values were calculated for a standard sized animal of 1 kg wet mass using a scaling coef®cient 0.75, obtained as a mean of the exponents for octopods listed in O'Dor and Wells (1987). The respiration rates listed are for inactive animals. Nitrogen excretion increased with specimen size (Fig. 5). The mean excretion rates 21 21 recorded were 0.170 (S.D.50.007) mg atoms NH3 -N g h for specimen 1, 0.167 21 21 (S.D.50.004) mg atoms NH3 -N g h for specimen 2 and 0.203 (S.D.50.055) mg 21 21 atoms NH3 -N g h for specimen 3. Production of faeces were at the rates of 2.742, 2.700 and 1.950 mg day21 dry mass for specimens 1, 2 and 3, respectively, over a period of around 80 days. The rates of production of faeces for specimens 1 and 2 were very similar although specimen 1 had a higher mean consumption rate, 31.440 mg day21 dry mass compared to 22.781 mg day21 dry mass for specimen 2. Faeces from specimen 1 may have broken up quickly in the tank before it could be collected, possibly by movement of the octopus, and may be underestimated.

Using mean VO2 and ammonia excretion rates, the atomic O:N ratio for each P. charcoti specimen was calculated. O:N ratios did not vary signi®cantly throughout the H.I. Daly, L.S. Peck / J. Exp. Mar. Biol. Ecol. 245 (2000) 197 ±214 205

Table 1 Respiration rates calculated for three Pareledone charcoti specimens and comparative mean values from O'Dor and Wells (1987) for a standard sized animal of 1 kg wet mass Species Wet body Temperature Respiration rate Sourcea 21 21 mass (g) (8C) (mg O2 kg h ) Pareledone charcoti 73 0 10.82 Present study 51 0 11.59 Present study 29 0 8.05 Present study Eledone cirrhosa 312 4.5 18.53 Present study E. cirrhosa 312 11 40.58 Present study Octopus do¯eini 9600 11 70.41 1 Bathypolypus arcticus 3 13 46.80 2 E. cirrhosa 564 18 61.37 3 Octopus briareus 345 20 45.64 4 Octopus vulgaris 817 21 84.07 5 O. vulgaris 42 22 73.40 6 Octopus cyanea 1000 25 125.71 7 O. briareus 348 30 99.94 4 a Sources: (1) Johansen (1965), (2) O'Dor (unpublished) from O'Dor and Wells (1987), (3) Milne (pers. commun. 1981), (4) Borer and Lane (1971), (5) Wells et al. (1983), (6) Wells and Wells (1995), (7) Van Heukelem (1976).

21 Fig. 5. Ammonia excretion rate, U (mg atoms NH3 -N h ) for Pareledone charcoti, in relation to calculated specimen dry weights. 206 H.I. Daly, L.S. Peck / J. Exp. Mar. Biol. Ecol. 245 (2000) 197 ±214

Table 2 Components of the energy balance equation for Pareledone charcoti in J day21a (mW), for mean dry mass Parameter Specimen 1 Specimen 2 Specimen 3 (9.51 g dry mass) (6.87 g dry mass) (4.01 g dry mass) C 723.51 (8.37) 524.23 (6.07) 327.59 (3.79) R 496.08 (5.74) 405.96 (4.70) 184.76 (2.14) U 0.029 (3.3931024 ) 0.022 (2.5731024 ) 0.018 (2.0431024 ) F 21.39 (0.25) 21.06 (0.24) 15.21 (0.18) P 1 G 279.48 (3.23) 101.00 (1.17) 103.43 (1.20) Scope for growth: C 2 (R 1 U 1 F) 206.01 (2.38) 97.19 (1.12) 127.61 (1.48) Difference between P 1 G and scope for growth 73.47 (0.85) 3.81 (0.04) 224.18 (0.28) a The mean dry mass of P. charcoti specimens were estimated using data for Pareledone turqueti. trial and were 24.6 (range 22±27) for specimen 1, 27.2 (range 24±30) for specimen 2 and 13.3 (range 9±19) for specimen 3. The previous results for C, R, U, F and P 1 G were used in the construction of an energy budget for each of the P. charcoti specimens using conversion factors detailed below, and results are listed in Tables 2 and 3. Energetic input from consumption (C) was calculated using the conversion 23.01 J mg21 dry mass M. edulis ¯esh quoted in Thompson and Bayne (1974). The coef®cient 21 used to determine the energetic cost of respiration (R), was 13.598 J mg O2 . This was based on fuelling metabolism using the relative percentage of protein, carbohydrate and lipid present in the M. edulis (Giese, 1969), which were fed to the P. charcoti. The 21 21 values 13.347 J mg O22 for protein respiration, 14.770 J mg O for carbohydrate 21 respiration and 13.723 J mg O2 for lipid respiration from Bra®eld and Llewellyn (1982) were used for the calculation. In calculating the amount of energy lost through excretion of nitrogenous waste products (U ), the coef®cient 288.278 J mol21 (Bra®eld and Soloman, 1972) was used. Only losses through ammonia excretion were considered, which were small in relation to other components in the energy balance equation. The energetic content of octopus faeces (F), determined by bomb calorimetry in Van Heukelem (1976) as 7.799 J mg21 dry mass, was used in the calculation. The value 22.259 J g21 dry mass was used as the energy composition for octopus tissues, combining somatic (P) and gonadal (G) growth. This ®gure was obtained as a

Table 3 Components of the energy budget for P. charcoti expressed as a percentage of consumption (C) Parameter Specimen 1 Specimen 2 Specimen 3 R 68.57 77.44 56.40 U 0.004 0.004 0.005 F 2.96 4.02 4.64 P 1 G 38.63 19.27 31.57 Total 110.16 100.73 92.61 H.I. Daly, L.S. Peck / J. Exp. Mar. Biol. Ecol. 245 (2000) 197 ±214 207 mean value from O. vulgaris (Wissing et al., 1973) and O. cyanea (Van Heukelem, 1976), based on bomb calorimetry. As no specimens of P. charcoti were available for estimation of water content, estimates obtained from Pareledone turqueti, a similar Antarctic species, were used to calculate dry mass (Daly, 1996). Energy budgets for all three P. charcoti specimens were approximately in balance (Table 2) Each energetic component is also shown in milliwatts. The scope for growth calculation underestimated the recorded growth (P 1 G) by 73.469 J day21 in specimen 1, overestimated it by 24.180 J day21 in specimen 3, and almost balanced in specimen 2, underestimating by only 3.810 J day21 . Table 3 illustrates the results expressed as a percentage of C and shows that the proportions calculated for each specimen were broadly consistent. Assimilation ef®ciency for P. charcoti was calculated as:

C 2 F Assimilation ef®ciency (%) 53]] 100 C

The values 97.04, 95.98 and 95.36% were obtained for specimens 1, 2 and 3, respectively.

3.1. Assessment of cold adaptation in P. charcoti by comparison with E. cirrhosa

Respiration rate values for both species were corrected to standard wet animal mass of 150 g using the weight exponent 0.75 (O'Dor and Wells, 1987), as this was approximately half way between the masses of the P. charcoti and E. cirrhosa specimens used (Table 4). A Q10 of 3.063 was calculated for E. cirrhosa between 11.5 and 4.58C. As Q10 calculations require respiration rates at two temperatures, a value could not be obtained for P. charcoti alone as respiration was only measured at one temperature (08C). In order to compare results for P. charcoti and E. cirrhosa,a combined Q10 value was calculated including the respiration rates for P. charcoti with the result for E. cirrhosa at 4.58C. This produced a Q10 of 3.807. The combined respiration rate data for the two species ®ts the curved Q10 relationship of 3 (Fig. 6), suggesting that P. charcoti respire at a rate consistent with extrapolation from E. cirrhosa rates.

Table 4 Comparative respiration rates for individual Pareledone charcoti specimens at 08C and Eledone cirrhosa at 4.5 and 11.58C for a standard animal of 150 g wet mass Specimen Wet body Temperature Respiration rate 21 mass (g) (8C) (mg O2 h ) P. charcoti (1) 73 0 2.61 P. charcoti (2) 51 0 2.79 P. charcoti (3) 29 0 1.94 E. cirrhosa 323 4.5 4.21 E. cirrhosa 300 4.5 4.73 E. cirrhosa 323 11.5 11.05 E. cirrhosa 300 11.5 8.51 208 H.I. Daly, L.S. Peck / J. Exp. Mar. Biol. Ecol. 245 (2000) 197 ±214

Fig. 6. Comparative respiration rate for Pareledone charcoti at 08C and Eledone cirrhosa at 4.5 and 11.58C for a standard animal of 150 g wet mass. The curve, Q1053, was calculated using the mean respiration rate of E. cirrhosa at 4.58C.

4. Discussion

Low temperature is a major factor affecting metabolic and growth rates in polar organisms (Clarke, 1998; Somero, 1998). Clarke (1983, 1991a) also argued that the observed slow growth rates in most marine polar invertebrates studied may be due to a strongly seasonal brief period of growth, which, when averaged over a year appears slow. Thus, the limitation of resources during winter, rather than suppression by low temperature would be responsible for slow growth. However, signi®cant winter growth or year round growth have been found in bryozoans (Barnes, 1995), brachiopods (Peck et al., 1997), and the bivalve Yolida eightsi (Peck, unpubl. data). The impact of reduced resources in the form of low primary production is likely to be greatest on species low in the food chain, such as suspension feeders, with organisms higher in the food chain, such as predatory cephalopods, buffered from the effect to some extent. Temperature has also been shown to be the main factor controlling development in marine invertebrate larvae (Hoegh-Guldberg and Pearse, 1995), where very long development periods have been found in echinoderms (Bosch et al., 1987; Pearse et al., 1991; Stanwell-Smith and Peck, 1998), brachiopods, (Peck and Robinson, 1994), nemerteans (Peck, 1993), isopods (White, 1970; Wagele,È 1987) and molluscs (Hain, 1991). Despite this, seasonality can have an important effect on growth, with some Antarctic species exhibiting periods of fast growth in summer (Dayton, 1989; Rauschert, 1991; Barnes, 1995) and low temperature should not be accepted as the only important factor. H.I. Daly, L.S. Peck / J. Exp. Mar. Biol. Ecol. 245 (2000) 197 ±214 209

One of the striking biological characteristics in cephalopods is rapid growth, which was shown to vary from 2.00 to 4.65% per day at around 100 g wet body mass in a range of octopod species at 15±258C (Forsythe and Van Heukelem, 1987). The slow growth of 0.13, 0.08 and 0.12% increase in body mass per day recorded for the P. charcoti specimens (Fig. 3), demonstrate that a potentially different life history strategy may be adopted by polar cephalopods. Antarctic Pareledone may have a similar strategy to the Arctic octopod Bathypolypus arcticus, which had a growth rate of 0.18% body mass increase per day, estimated using linear regression over an 18-month period (O'Dor and Macalaster, 1983). In B. arcticus a 3.7 g increase over a month was recorded, with a ®nal mass of 70 g, and a proposed life-span of up to 4 years (O'Dor and Macalaster, 1983). The life-span of P. charcoti is not known, but the male specimen, which was held in the aquarium at BAS, Cambridge survived for 18 months, and was collected at least 3 months prior to this. Respiration rates for a number of octopus species have been summarised in Table 1 21 and mean VO2 for a 312 g wet mass E. cirrhosa fell from 40.58 to 18.53 mg O2 kg h21 at 11.5 and 4.58C, respectively. Respiration in a small Bathypolypus arcticus 21 21 specimen was recorded as 46.80 mg O2 kg h at 138C (Table 1). This temperature is considerably higher than their norm of 4628C (Macalaster, 1976) and is comparable to the rates recorded for E. cirrhosa in this study. In P. charcoti, respiration fell very slowly from routine to a low constant rate, which is close to standard levels (Fig. 4), 21 21 from 10.47 to 9.52 mg O2 kg h in 40 days in specimen 1, and 13.92 to 8.91 mg O2 kg21 h21 in 30 days in specimen 2, compared with the rapid decline recorded by Wells and Wells (1995) for O. vulgaris (approx. 44 g wet body mass), dropping from 162 to 80 21 21 mg O2 kg h after 5 days of fasting at around 228C. Similar slow reductions in metabolism to standard levels were recorded in other Antarctic benthic invertebrates by Peck (1989) where the limpet Nacella concinna and the brachiopod Liothyrella uva took 25±50 days to return to standard or basal levels. As octopods have well developed nervous and visual systems, they can be easily stressed by handling and external movement. When metabolic rates are raised after disturbance by tank cleaning or handling, they can remain increased for around an hour in temperate species (Wells et al., 1983). It is possible that the time required for the metabolic rate of a polar cephalopod to return to normal after the stress of handling is considerably longer (or shorter) than an hour. However, in the absence of previous data a settling period of one hour between placing the animals in the chamber and taking samples was used to reduce the effect of the initial stress. The P. charcoti usually settled quickly in the chambers and often remained motionless during the trial, suggesting that any stresses were small. Although ammonia is the dominant excretory product in most marine invertebrates (Bayne et al., 1985; Regnault, 1987), it is not the only nitrogenous compound excreted. Hoeger et al. (1987), reported that urea comprised 30% of the nitrogen excretion in Octopus rubescens. As we only measured ammonia excretion, the ®nal energetic cost of excretion may be underestimated. However, nitrogenous waste comprises ,0.01% of the total energy budget, so the error created by recording only ammonia levels is probably small. The excretion rates in the starved P. charcoti at 08C were similar, with a 21 21 mean rate of 0.180 mg atoms NH3 -N g h , and are lower than rates reported by 210 H.I. Daly, L.S. Peck / J. Exp. Mar. Biol. Ecol. 245 (2000) 197 ±214

21 21 Boucher-Rodoni and Mangold (1985) of 0.560 mg atoms NH3 -N g h in O. vulgaris 21 21 at 15±168C, which had been recently feeding, and 0.307 mg atoms NH3 -N g h in starved animals. Atomic O:N ratios are linked to the availability of energy stores and the utilisation of body protein. This ratio of oxygen consumption to nitrogen excretion by atomic equivalents produces an index of the relative amounts of protein, compared to carbohydrate and lipid that are being catabolised by the organism. Theoretically, the minimum value for an O:N ratio is around 7, when only protein is being catabolised. Mayzaud and Conover (1988) calculated that organisms catabolising equal proportions of lipid and protein would have an O:N ratio of 24, with values more than this indicating lipid or carbohydrate as the major component in catabolism, and values less indicating increased protein catabolism. The values for specimens 1 and 2 indicate protein accounting for around half of the catabolic substrate, while specimen 3 is catabolising a higher proportion of protein. O:N ratios can be variable, and may fall or rise in response to starvation, depending on the biochemical composition of the organism (Mayzaud and Conover, 1988). In fed O. vulgaris at 15±168C mean O:N ratios of 7.19 and 14.15 were recorded, indicating mainly protein catabolism, while ratios of 9.62 and 24.76 were recorded in starved animals, denoting increased utilisation of lipid or carbohydrate stores (Boucher-Rodoni and Mangold, 1985), which is consistent with the O:N ratios obtained for P. charcoti. The results in Table 2 represent an approximately balanced energy budget for P. charcoti, although the scope for growth underestimates the recorded somatic and gonadal growth (P 1 G) for specimens 1 and 2, and overestimates growth in specimen 3. As only resting metabolism was recorded, the activity component of energy utilisation was not estimated. Its inclusion would increase the imbalance in the budgets for specimens 1 and 2, but decrease the underestimation for specimen 3. However, observations indicated that natural long term levels of activity were low. Other possible errors include the loss of tissues in holding systems. Octopods kept in static water holding tanks are often observed to shed skin, mucus and sucker rings, which contribute to energetic loss. In Octopus cyanea, where shed sucker rings have been observed to accumulate in the water, Van Heukelem (1976) recorded the loss as 1.5% of the total energy budget. However, in the static water holding tanks of the P. charcoti specimens in this study, shed skin, mucus and sucker rings were not frequently observed, and the associated energetic loss is likely to be small, and probably less than the 1.5% observed in O. cyanea. Mucus production may also be important in mollusc energy budgets and accounted for nearly 30% of consumed energy in the abalone Haliotis tuberculata (Peck et al., 1987). However, there are no reports of cephalopods producing large amounts of mucus and this error is also likely to be small. When the energy budgets are expressed as a percentage of consumption (C) (Table 3), it is possible to compare the relative components of the equation. Assimilation ef®ciencies were surprisingly high at 95±97%, but they are consistent with the 96.5% found for O. cyanea by Van Heukelem (1976). In comparison with the energy budget calculated for O. cyanea by Van Heukelem (1976), there are a number of differences. The mean proportions comprising growth and respiration were 48.6 and 48.2%, respectively, for fed O. cyanea, and were 29.8,% and 67.5%, respectively, for P. charcoti H.I. Daly, L.S. Peck / J. Exp. Mar. Biol. Ecol. 245 (2000) 197 ±214 211 which refused food for part of the trial. This suggests that, although P. charcoti are respiring at a lower level than O. cyanea (Table 1), the proportion of the energy budget required for maintenance is greater, even when periods of reduced food intake are included, when there is a reduced effect of SDA. This increased proportion of energy for basal metabolism in a polar organism could be interpreted as evidence for metabolic cold adaptation. However, numbers involved in these trials were small and such conclusions are dif®cult to apply with con®dence. Growth would also have been slow if the animals were nearing the end of their normal life-span.

Using results from Milne, A.H. (pers. commun.) for E. cirrhosa at 18.58C, a Q10 was also calculated for E. cirrhosa between 18 and 11.58C, producing a value of 1.89. which is well within the normal range. Coef®cients for other octopods of 2.14 for Octopus cyanea around 308C (Van Heukelem, 1976), and 2.18 for Octopus briareus between 20 and 308C (Borer and Lane, 1971) have been recorded, and the Q10 for E. cirrhosa between 11.5 and 4.58C of 3.063 is also within the expected range. The inclusion of P. charcoti at 08C and E. cirrhosa at 4.58C in a combined Q10 calculation produces the value 3.807. This higher value may indicate that P. charcoti has a metabolic rate even slower than that extrapolated for E. cirrhosa if they could survive at 08C. If P. charcoti was displaying metabolic cold adaptation (MCA) and functioning with an elevated metabolic rate in comparison with E. cirrhosa, the Q10 would be lower than 3.063. However, as the Q10 function describes a curve (Fig. 6), it appears that P. charcoti are respiring at a rate predicted by E. cirrhosa rates extrapolated along this curve to low temperatures. Precht (1958) describes an increase in Q10 values with an increase in the temperature range as `inverse' compensation, i.e. rather than adjusting metabolism to maintain it constant, the changes make it more disparate. O'Dor and Wells (1987) termed this `paradoxical' adaptation, and discuss its bene®t to squid making daily vertical migrations from cold deep water to warm surface water for feeding. This is when animals go into torpor at low temperatures or when they increase their activity level to increase the feeding rate to meet increased energy demands at higher temperatures.

The P. charcoti±E. cirrhosa Q10 value is within the range found for other Antarctic invertebrates; Peck (1989) recorded Q10 values of 2.41 in acclimated Nacella concinna from 21.5 to 14.58C, and Luxmoore (1984) calculated values of 2.00 for Serolis polita and 4.21 for Serolis cornuta between 21.5 and 14.58C. The low respiration rates in P. charcoti support recent conclusions that MCA does not occur in polar marine invertebrates (Luxmoore, 1984; Clarke, 1991a,b, 1993).

Acknowledgements

Thanks to Dr Paul Rodhouse (British Antarctic Survey) and Professor Peter Boyle (University of Aberdeen), who supervised H.I.D. during her doctorate when this work was carried out, and for their comments on the manuscript. Thanks also to Linda Key of Aberdeen University for her assistance in supplying Eledone cirrhosa for the study. This work was funded under NERC (CASE) studentship GT4/92/104 between the British Antarctic Survey and the University of Aberdeen. [SS] 212 H.I. Daly, L.S. Peck / J. Exp. Mar. Biol. Ecol. 245 (2000) 197 ±214

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