J therm Biol Vol 6 pp 257 |o 265 1981 0306-4565/81 040"57-09102Q0/0 Printed in Great Britain Pergamon Press Lid

RESPONSES OF THE MUSSEL MYTZLUS EDULIS L. TO SIMULATED SUBARCTIC TIDE POOL CONDITIONS

John Davenport and Miguel Carrion-Cotrina

N.E.R.C. Unit of Marine Invertebrate Biology, Marine Science Laboratories, Menal Bridge,

Gwynedd, North Wales, UK.

ABSTRACT

Mussels in subarctic Norway commonly occur in shallow intertidal pools. By living xn pools (a habitat rarely inhabited in lower latitudes) they are insulated against low air tempera- tures but exposed to high salinities (<700/00) beneath overlying ice. Mussels avold exposing their tissues to such high salinltles because a shell valve closure response to low temperature operates at about -1.5°C before ice sheets form and bottom water salinities rise. Shell valve closure coincides with very low cardiac and ciliary actlvlty.

KEYWORDS

Cold tolerance~ low temperatures! high salinityj mussels; Mytilus edulis~ shell valve closurej subarctic tide pools~ heart ratesj ciliary activity.

INTRODUCTION

In temperate latitudes the mussel M~tilus edulis L. is essentially an epifaunal bivalve attached by its byssus to rocks or stonesj intertidal specimens living above low water neap tide level are normally exposed to air on each low tide. In estuaries mussels are sometimes found half buried on muddy shores and, on the coast, occasional specimens are found ~n inter- tidal pools, but animals living in either of these situations make up a quite insignlflcant proportion of the population. In contrast, at high latitudes in subarctic Norway, large numbers of small mussels, about I-2 cm in length, may be found buried in coarse gravel at the bottom of shallow middle and upper shore intertidal pools with only a small portion of the posterior shell margin protruding above the substrata. At Troms~, Norway, such pools have previously been studied by Davenport (1979a) who showed that in winter, when the pools b@come frozen over at low tide when air temperatures may fall to -20oc, high salinlties (50-650/00) develop in the unfrozen water beneath the ice sheets! temperatures as low as -5 to -8°C have also been recorded from these saline water layers. Besides Mytilus, the pools contaln great densities of the amphipod Gammarus duebeni LilJeborg and an unidentlfied flatworm. Because of the presence of so many animals in such small pools it appears likely that the sallne layers will also become deoxygenated rapidly, but no data are available to confirm this point. The conditions encountered by tide pool Troms~ mussels at low tide are summarlzed in Fig. I. When the tide rises the pools are rapidly flushed out by water of normal salinity (c 330/00) and relatively warm temperature (c +5oc). It is clear, therefore, that subarctic tldepool mussels are exposed to very changeable conditlons of temperature and salinity over short periods during winterz the study reported here was designed to establish how mussels reacted to such fluctuations and to compare the responses of Norwegian and Britlsh specimens of Mytilus.

MATERIALS AND METHODS

Collection and Maintenance

Specimens of Mytilus edulls were collected intertldally on the foreshore close to Troms~ Aquarium (Norway, c 6~Y~N) and from Bangor Pier on the southern shore of the Menal Strait. The Norwegian mussels were flown from Troms~ to Manchester and transported thence to Menai Bridge by road. All mussels were held in water of 32-33.50/00 salinity at +5°C for at least two weeks before use in experiments.- They were fed daily on a mixed culture of the following algae:- Rhodc~onas baltlca, Skeletonema costatum, Pavlova lutheri and Chaetoceros calcitrans, and their seawater medium was changed every 48 hours since quarantine considera- tions precluded the use of flowing seawater with the Troms~ specimens.

Survival limits

There is same dispute about the lower lethal temperature of Mytilusj Kanwlsher (1955)

257

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o

o ,~

JOHN D~,VENPORT and MIGUEL CARRION-COTRI~,A

air: -10 to -20°C

258

s e m i - b u r i e d

m u s s e l s

Fig. 1. Tlde pool conditions.

suggested a temperature of -15°C while more recently Williams (1970) found some mortality after 70 hours at -10°C. However, both of these temperatures are well below the lowest temperatures found at the bottom of intertidal pools at Troms~. Consequently it was not thought that experiments to determine lower thermal limits were desirable. Similarly it is well known that mussels can withstand anoxic conditions for many days, and are able to sur- vive exposure to low salinities for several hours by recourse to closure of the shell valves (e.g. Milne, 1940). A survey of the literature revealed no comparable data concerning high salinity survivalj in consequence the following experiment was performed upon both Welsh and Norwegian mussels. Large quantities of high salinity water were prepared by heating filte- red seawater (340/0o) to 50oc and allowing it to evaporate. Once it had reached a salinity of 680/oo the water was stored in covered plastic dustbins and constantly aerated until used in experiments. 15 Norwegian mussels were exposed to each of the following salinlties at +5°C: - 34 (control), 40.8, 47.6, 54.4, 61.2 and 680/0o. Animals were held in 2-1itre plastic vessels, 15 mussels per vessel, and their media were changed every 48 hours. They were observed daily for 30 days for mortalityj death was assumed if a mussel was gaping and showed no reaction to tactile stimuli. The experiment was repeated on Welsh specimens of Mytilus.

Behavioural responses to rising salinities

Siphonal and shell valve responses to rising salinity levels were observed in a 500 ml perspex vessel supplied with water at +5°C and 300 ml min -! by the apparatus of Davenport, Gruffydd and Beaumont (1975). In the past this apparatus has been used to supply water of low or falling salinity to experimental anlmals~ in that mode it functioned by mlxing sea- water and fresh water. In the present study seawater (340/00) was mixed with high salinity water (680/00) prepared in the manner described above. 12 Norwegian and 12 Welsh mussels were each exposed in turn to a linear salinity reqime (see Davenport, 1979b) rising from 34 to 680/00 over a period of 15 mln. For each animal, samples of the medium were collected by hypodermic syringe when the following events occurred:- closure of the exhalant siphon, closure of the Inhalant siphon and shell valve closure. After shell valve closure had occurred a further water sample was wi£hdrawn from the mantle cavity of the mussel. The salinity of all the samples taken was immediately determined on a small Goldberg refracto- meter accurate to about IO/oo.

Behaviour in an artificial tide pool

To investigate the behavlour of mussels exposed to low temperatures and high salinltles beneath ice sheets the apparatus shown in Fig. 2 was constructed and sited within a cold

M. eduhs m a simulated subarcttc Ude pool 259

room held at +5°C. A cubical plywood box was lined with polystyrene sheet and fitted with a removable but insulated lid. A low voltage electric motor was mounted on top of the lid; a shaft connected to the motor passed through the lid and drove a lightweight plastic fan which circulated the air within the box. Set in one wall of the box was a transparent panel consisting of two perspex sheets held apart on three sides by 2 mm thick perspex stripsj this panel acted as a double glazed viewing window which, since it was not sealed on the upper edge, was immune to condensation. Behind the lower portion of the viewing panel, and attached to it, was a cubical perspex chamber open at the top and insulated on three sides by sheet polystyrene (the fourth side being insulated by the viewing panel and the bottom being protected by the general insulation of the ply box). In all experiments, mussels were held in 270 ml of seawater within this chamber which simulated a small pool. The assembly of viewing panel and chamber was easy to remove from or insert within the box, complete wlth water and animals. The temperature within the apparatus was controlled by a Churchill thermoregulator which circulated glycol of any temperature down to -20Oc through a 10 metre length of narrow bore copper pipe, which was coiled to occupy much of the free space within the ply box and therefore omitted from Fig. 2 for clarity. Also omitted are two thermo- meters, one registering air temperatures just above the "pool" surface and the other monit- oring temperature in the water close to the mussels being observed, and a small torch bulb supplied from the same low voltage supply as the fan motor. The torch bulb was mounted just above the "pool" and provided adequate illumination for clear viewing without causing heating problems. To monitor sallnltles within the "pool" a I mm diameter plastic catheter passed through the viewing panel to the water at the bottom of the perspex chamber. Normally the catheter was blocked off with a hypodermic syringe and needle, but at will a drop of water could be removed for salinity measurement on a Goldberg refractometer, or a small amount of methylene blue dye could be injected into the pool to see if mussels were pumping.

connect ions to copper col (10 metres Ion

cold ( - 2 0 % ) glycol

/ \

Jlated spex box cm cube)

insulated ply box (38cm cul

double glazed perspex plate

g or dye delivery catheter

Fig. 2. Artificial tide pool.

260 JOHN DAVENPORT and MIGUEL CARRION-COTRINA

9 Norwegian and 9 Welsh mussels were exposed three at a tlme in turn to the temperature and salznity regime shown in Fig. 3. Thls regime was inltlated by ad3ustlng the ply box tempera- ture to +5°C and inserting the perspex chamber complete with seawater (at +5°C) and the three mussels. After 12 hours the Churchill thermoregulator was adjusted to -20°C; the alr temperature above the "pool" rapidly fell to about -13°C. Cooling was maintained for 17 hours, by which time there was a thick ice sheet on the "pool" overlaying unfrozen water at -4°C whlch had a salinity of 68°/oo. At thls Point the viewing panel/chamber assembly was placed in seawater at +5Oc to thaw. The cycle was closely repeatable; 3 mussels from each locality were exposed to one cycle, 3 to two cycles and 3 to three cycles. Animals were observed during cooling and thawlng to see if the shell valves/siphons were open and methylene blue was occasionally injected into the "pool" to assess pumping actlvlty. A further 8 Norwegian mussels were exposed to 3 cycles and when in 68o/00 for the last time were removed from the "pool" qulckly, (before the shell valves opened) and mantle fluid and haemolymph samples were collected and measured for osmolarity in the fashion descrlbed by Shumway (1977). During the development of the regime and technique some "failed" experi- ments yielded additional information to be presented in the results section.

. . . . . air temperature ( ° C )

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water temperature

water salinity ( % 0 )

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single cycle F i g . 3 . Artificial pool regime.

Heart ratesand ciliary activity

Norwegian and Welsh mussels, acclimated to +5°C, were exposed to temperatures declining over many hours (not repeatable) from +18 to -1.5°C. Their heart rates were continuously monitored by the impedance technique of Trueman (1967). To assess the effect of temperature on the ciliary activity of mussels from the two localities the following experiment was performed. 6 mussel gill preparations from the Norwegian stock and 6 from the Welsh animals were prepared as described by Davenport and Fletcher (1978). Particle transport rates by the frontal gill cilia were assessed by using carbowax particles - white, opaque, wax covered diatomaceous earth material which gives repeatable results without damage to the cilia. From each locality 3 preparations were exposed to rising temperatures until ciliary

M eduhs m a s=mulated subarctic Ude pool 26i

activity ceasedj 3 preparations were cooled until ice crystals appeared in the seawater surrounding the preparations.

RESULTS

High sallnit~ survival

No mortality at all occurred in mussels exposed to 34, 40.8 or 47.6o/00 durlng the experi- mental period. The remainder of the results are presented in Fig. 4. No mortality was observed in any of the higher salinity media (54.4, 61.2, 680/00) until at least 9 days had elapsed. During this period the animals kept their shell valves tightly closed for most of the time. After 9-10 days deaths started to occur in media of 61.2 and 680/00 and all animals were dead after 24-25 days at these sallnitles. 54.40/00 was much less damaglng| only I Norwegian and 2 Welsh mussels failed to survive the experimental perzod at thzs salinity. No significant differences in salinity tolerance between Norwegzan and Brltzsh mussels were detected.

100-- 100 --

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Norwegian mussels Co

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e- - in 54.4%0, o - - in 61.2%o, o- - in 68%0 Fig. 4. Mortality in high salinities.

Behavioural responses to rising salinit~

The results derived from Norwegian and Welsh mussels are shown zn Table I:-

TABLE I Responses of M~tilus edulis to Rising Supranormal Salinzties

(34 to 680/00 in 15 min. Temperature: +5Oc)

Event

I. Exhalant Siphon closure 2. Inhalant Siphon Closure 3. Shell Valve Closure

Retained Water Concentration (mantle fluid)

Corresponding Salinity (°/oo; m~an value with 95% confidence intervals)

_ A. Norwegian Mussels B. Welsh mussels P

52.8 + 2.3 46.2 + 2.8 ~0.001 55.8 + 2.3 50.3 ~ 3.6 ~0.050 58.5 + 2.6 53.4 ~ 3.7 ~0.050 49.1 ÷ 1.8 42.7 + 2.1 ~0.010

T . 6/4--~

262 JOHN DAVENPORT and MIGUEL CARRION-COTRINA

From these results it may be seen that mussels close xn response to hlgh sal~n%tles Just as they do to low ones. As Davenport (1979b) demonstrated for the low salznity response, the reaction to high salinities proved to be a three part sequence starting with closure of the exhalant siphon and progressing vla inhalant siphon shutting to complete closure of the shell valves. Retained water sallnities were always close to, but slightly lower than, those which induced closure of the exhalant siphon. Cessation of pumping and closure of the exhalant siphon appear to be linked (Sleigh, 1962), and It seems that shutting the exhalant siphon determines the subsequent retained water salinity more than does tight shell valve closure. From Table I it may be seen that all observed salinitles were higher for Norweq~an mussels than for Welsh M~tilus, the differences being statistically significant at the 95% level or better.

Behaviour in an artificial tide pool

When exposed to the temperature/salinity cycles shown in Fig. 3, mussels usually closed their shell valves at about 0°C when temperatures fell for the first timer during subsequent cycles (if encountered) shell closure was delayed until between -1.5 and -2.0°C though siphons were closed and pumping absent below 0°C. Clearly mussels Isolated their tissues and mantle fluid from the environment before ice started to form and salinities started to rise. During thawing in the normal sequence (-4 to +5°C in about 10 mln) mussels all opened at 0°C and were pumping vigorously at +1°C. No evidence for differences in response between Norwe- gian and Welsh mussels was found. The osmotic data for the 8 mussels exposed to three "pool" cycles are shown in Table 2:-

TABLE 2 Osmotic Data for Specimens of M~tilus from Trome~, (exposed to

3 simulated tldal cyc.~es and sampled when the external salinity was 680/00).

Animal Retained Water Salinity Retained Water O.P. Haemolymph O.P. NO. (o/0o) (mO~ Kg-1) (mO~ Kg -1)

I 35 1020 1010 2 33 960 960 3 36 1049 1075 4 37 1078 1065 5 33 961 1055 6 36 1020 1005 7 38 1120 1115 8 33 970 1110

Mean 35.1°/oo 1022 mOem Kg-I 1041 mOsm Kg -I

These results again suggest that mussels isolate themselves in ~esponse to temperatures around 0 to -1.5°C rather than to rising salinltles.

During preliminary "falled" experiments the followlng observations were made. On one oCcasion thawing from -4 to +5°C oCcurred over 2 hours (rather than I0 min). In this experiment mussels resumed activity at -1.5°C, both pumping and protruding the foot. During another run thawing was stopped suddenly with water of 59o/00 and -1.5°C lying beneath a thick ice layerJ after 12 hours mussels were wide open and pumping. Finally, on one occasion mussels were left in the artificial pool and cooling p~ogressed for 20 hoursj after this time water of 104°/oo and -8°C surrounded the animals. In this case, the only one recorded in the present study, the retained water (c 340/0o) was frozen but the animals re- covered rapidly in seawater at +5°C.

Heart/qillar~ rates

The effect of temperature on mussel heart rates is demonstrated in Fig. 5! ciliary responses are displayed in Fig. 6. Heart rates declined in almost linear fashion from about 30 beats mln -I at +18°(2 to around 3 heats m4n-I at'-l.5°C. At -1.5OC shell valve closure oCCUrred and heart action became very irrlgular a~d often absent after a few minutes. No difference between Norwegian and Welsh mussels was evident.

Ciliary activity at all temperatures was similar in mussels from both localities. Particle transport velocity, which peaked at around 1.0 mm eec-I (at about 25°C), fell to less than 0.1 mm sec -I at eubzero temperatures so ciliary activity clearly becomes negllglble as the freezing point of seawater (c -1.9°C) is reached. At high temperatures ciliary activity stopped (irreversibly) at 30-32°C.

M eduhs m a simulated subarcttc ude pool 263

I C g m

E ca

m @

I @

m L - -

L -

Q @

3 0 -

2 0 -

1 0 -

•

0

0 o

Q

Norwegian m u s s e l s - •

We lsh fnusse ls - o

0

o O

o

0

m

if

I I I I I I

+ 2 0 ÷10 , . 0 - 5 "c

Fig. 5. Effect of temperature on heart rate.

DISCUSSION

Field measurements at Troms# and experience with the artificial tide pool used in the present study show that mussels living in tide pools gain considerable insulation (by at least 8-10 deg C in some cases} from low air temperatures. This will clearly be of advantage to Mytilus living intertidally in Northern Norway where air temperatures below the lower lethal temperature of the species are not uncommon. Mussels appear to gain further advantage in that their body fluids do not freeze even though they are surrounded by water at -4°C because the water is hypersaline and unfrozen! it therefore does not seed the mantle fluid with ice crystals. Although this study shows that mussels are capable of detecting rising salinities and isolating themselves from the environment at about 500/00, and despite the ability of Mytilus to survive salinltles as high as 68o/00 for about 10 days, it appears that these attributes are irrelevant to mussels living in subarctic tide pools. The reason for this is that Mytilus apparently possesses a direct closure response to low temperature which operates in the 0 to -1.5°C region; consequently mussels will close their shell valves Just before ice sheets form on rock pools and long before salinitles rise sufficiently to trigger a salinity reaction. This pattern of behaviour, which is presumably repeated on virtually every low tide during severe weather, will ensure that the tissues of mussels are exposed to little or no omaotic stress. The shell valve closure response to low temperature operates at a temperature (c -1.5°C) at which the physiological processes of mussels (e.g. heart rate, ciliary activity} practically cease (see Figs. 5 and 6); there would consequently be no advantage in keeping the shell valves open at lower temperatures as no benefit in terms of food intake or gaseous exchange would accrue.

The results of this study do give rise to one problem, however. If it is advantageous for quite large numbers of subarctic Norwegian mussels to live in intertidal pools, why then are so few found in pools in British waters? Air temperatures as low as those encountered at

264 JOHN DAVENPORT and MIGUEL CARRION-COTRINA

(n

E E I

W

¢U Im

_e 0 . I i._ al Q.

1.0--

0 . 5 - -

O _

Norwegian mussels -- solid symbols, Welsh - -open symbols.

o eo & .

0 A •

O O Q

e

A o ~qo o o

o A O o | =

, o l l l ' ~ • •

0 1 , ~oo

0 0 A

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I I I I I I -2 I +10 + 2 0 ÷ 3 0 + 4 0

0 °c

Fig. 6. Effect of temperature on ciliary activity.

Troms~ do occur on British coasts, albeit infrequently (e.g. 1947, 1963), so the insulation provided by a pool existence would occaslonally be useful to Mytilus. The reason for the difference in mussel habit may well reflect a difference in predatory fauna. On British coasts similar pools on rocky or gravelly shores are characterlsed by large numbers of fairly big mobile predators (e.g. blennles, shore crabs, hermit crabs) which are probably concen- trated in pools by the receding tide. Mussels, especially those gaping in warmer weather, would make tempting targets for such animals. However, on the shore of subarctic Norway these predators are absent, probably because of the routine severity of the winters, and it is suggested that this absence allows mussels to exploit a niche denied to them at lower latitudes.

ACKNOWLEDGEMENTS

The authors wish to thank Dr. B. Gulliksen of the Aquarium, Troms~ for arranging for the transfer of mussels to the U.K. One of us (M.C.C.} was in receipt of a British Councll grant during the course of the investigation.

REFERENCES

Davenport, J. (1979a). Cold tolerance in Gammarus 8uebenl LilJeborg. Astarte, 12, 31-34. Davenport, J. (1979b). The isolation response of au4ssels, MytI~US ~ulis (L.), exposed to

falling seawater concentrations. J. mar. biol. ASs. U~K. ~ 59, 123-132. Davenport, J. and J. S. Fletcher (1978}. The effects of sL~ulated estuarine mantle cavity

conditions upon the actlvltyof the frontal gill cilia of Mytilue edulls (L.). J. mar. blol. Ass. U.K.r 58, 671-681.

Davenport, J., LI. D. Gruffydd and A. R. Beaumont (1975). An apparatus to supply water of fluctuating salinlty and its use in a study of the salinity tolerance of larvae of the scallop Pecten maxlmus L. J. mar. blol. Ass. U.K., 55, 391-409.

Kanwlsher, J. W. (1955). Freezing in Intertldal animals. Biol. Bull. mar. biol. Lab., Woods Hole, 109, 56-63.

M eduhs m a simulated subarctic tide pool 265

Milne, A. (1940). Some ecological aspects of the intertidal area of the estuary of the Aberdeenshire Dee. Trans. Roy. Soc. Edin.~ 60, Part I, No. 4, 107-139.

Shumway, S. E. (1977). Effect of salinity fluctuation on the osmotic pressure and Na +, Ca ++ and Mg ++ ion concentrations in the haemolymph of bivalve molluscs. Mar. Biol., 41, 153- 177.

Trusman, E. R. (1967). Activity and heart rate of bivalve molluscs in their natural habitat. Nature, 214, 832-833.

Williams, R. J. (1970). Freezing tolerance in Mytilus edulis. Comp. Biochsm. Physiol., 35, 145-161.