Evaluation of the neutral red retention assay as a stress response indicator in cultivated mussels (Mytilus spp.) in relation to seasonal and environmental conditions.

The neutral red assay (NRA) was evaluated as an indicator of stress response in mussels that were held under various culture situations. The NRA measures retention time of neutral red dye in the hemocyte organelle, the lysosome, which can be correlated to the condition of a mussel under stressful circumstances. Shelf life and standard meat yield also provide an indication of mussel condition. The objectives of this study are to compare and evaluate mussel stress response in relation to: (1) seasonal and environmental changes and (2) postharvest handling. Neutral red retention (NRR) levels and shelf life were reduced in late summer mussels (postspawning) compared with early summer mussels (prespawning), and increased in autumn/early winter mussels, indicating a seasonal pattern of stress response associated with reproduction. Harvested mussels exhibited a decrease in NRR during extended air exposure, especially when held at air temperatures above and below air temperatures comparable to ambient water temperatures. The results demonstrated that NRA was a useful, sensitive index of physiological stress response in mussels subjected to various culture practices and conditions. The implication of this work for mussels growers is that reduced air exposure following harvest and reduced handling during certain seasons will result in less stressed mussels and hence a better quality product.

Key Words: mussel culture, neutral red assay, stress response

INTRODUCTION

The blue mussel (Mytilus spp.) culture industry in Canada has been on a rapid increase since the mid 1980s. In Newfoundland, with the onset of various programs such as the spatfall monitoring program, environmental and biological monitoring program, and product quality programs, the overall quality and quantity of blue mussels continues to improve (Clemens et al. 1999, Macneill et al. 1999). Because the market demand continues to rise for the Atlantic Canada mussel industry, a better understanding of how various production parameters (i.e., environmental and handling conditions) affect mussels need to be examined and evaluated to determine when or how conditions may become detrimental and reduce the quality of the product.

During the process of culturing mussels, there are several seasonal and short-term environmental factors that the mussels are exposed to while in the growout phase, including changes in water temperature, salinity, and food availability. Stress can be defined as any environmental stimulus that disturbs normal function in an organism, and that any reaction in the organism to this stress is the stress response (Bayne 1985). Under some circumstances conditions during the culture of mussels can have a negative impact on the mussels and may induce a stress response. Stress response in animals is dynamic, and results in the alteration of functional properties that can be quantified with the environmental stimuli (Bayne et al. 1976, Koehn & Bayne 1989). To date, stress response by mussel growers has generally been observed at the whole animal level with a reduction in performance such as growth, feeding, condition, and/or quality.

During the growth period of cultured mussels in Atlantic Canada they are completely and continuously submerged in water, and are subject to seasonal and environmental conditions associated with the reproductive cycle, hydrographic events, food availability, and fluctuating water temperatures. High mortalities have been associated with reproduction, genetics, starvation, and water temperatures, all of which may be influenced by oceanic events (i.e., tidal or wave action, currents) (Freeman & Dickie 1979, Worrall & Widdows 1984, Carver & Mallet 1991, Sephton et al. 1993, Myrand & Gaudreault 1995). As well, during harvesting, mussels can be exposed to a number of environmental factors including abrupt exposure to extreme air or water temperatures, sunlight, rain, and wind, any of which can pose stress on mussels and induce some form of stress response (Warwick 1984, Widdows & Shick 1985, Eertman et al. 1993).

The need to develop earlier and more rapid indicators of stress responses in mussels is ongoing and previous studies have shown changes in physiological characteristics under a number of conditions in feral blue mussels (Bayne 1973, Thompson et al. 1978, Moore et al. 1979, Lowe et al. 1995a, b, Tremblay et al. 1998a, b). Past research on various bivalve species subjected to a number of stressors has shown that the site of earliest detectable stress response is in the lysosome, an organelle of the hemocytes (Moore et al. 1979, Moore 1980, Bayne et al. 1981a, b, Lowe et al. 1995a, b). The NRA can be used to measure alterations in lysosomal stability induced by various stress factors. In their stable form (no stress response), lysosomes will accumulate and retain the neutral red dye for an extended period of time. However, once destabilized (stress response) following a stressor, the lysosomes will coalesce to form larger lysosomal structures and the neutral red dye will leak into the cytosol of the cell through damaged membranes (Moore 1980, Lowe et al. 1995a). The rate of lysosomal changes following the addition of the neutral red dye is indicative of the stress response and can be directly related to the degree of stress being imposed on the mussels.

The objectives of this study are to evaluate the NRA as a stress response indicator in cultivated mussels at the subcellular level (lysosomes), in relation to seasonal and environmental changes and to postharvest handling.

MATERIALS AND METHODS

Study Site and Animals

This study was carried out using market size mussels (i.e., >50 mm shell length), which were grown and harvested at Charles Arm, Notre Dame Bay, Newfoundland (49.34 N, 55.28 W) from the period of June 2001 to June 2002. Mussels from this site are >90% Mytilus edulis and <10% Mytilus trossulus or hybrids (Penhey et al. 2001). Charles Arm is a small semienclosed site of an axial length ~3.1 km and connected to the sea by a narrow channel <100 m in width (Penney et al. 2001). Mussels were randomly obtained from mussel socks located on longlines in the middle of the site and shipped in coolers with ice packs to the Marine Institute (St. John's, NL) where they were maintained in flow through raceway systems of seawater at ambient temperature and salinity for at least 2 days prior to experimental use (Harding et al. 2004b).

Experimental Design and Set-up

To assess the seasonal pattern of naturally (physical and biological) induced stress response, mussels were sampled monthly from the culture site from June 2001 to June 2002, with the exception of April 2002 due to ice conditions at the site. Seasonal measurements took into account that there was a natural algae bloom in the late summer to early autumn and in the spring of the year, the normal spawning period in June (assessed by a decline in condition index), and that the water temperature was in the range of 2[degrees]C (winter) to 15[degrees]C (summer). Measurements of food supply (Chlorophyll-a [Chl-a]), condition index (meat yield), and temperature at the Charles Arm site were monitored on a monthly basis up until November 2001 (see Fig. 2 later) (Nichols et al. 2002). The NRA (n = 12 for each monthly sampling), shelf life (3 replicates of 50 mussels for each monthly sampling), and condition index (standard meat yield) were measured against seasonal changes.

To assess the effects of air temperature and extended air exposure on the mussels' subcellular stress response, animals were sampled following harvesting, and following a 2-day recovery in the laboratory (ambient seawater, temperature, and salinity) at 4 times over a year; early summer (June 2001, prespawn), late summer (August 2001, postspawn), early winter (January 2002, recovered), and spring (May 2002, overwinter / early prespawn). In one experiment, unprocessed mussels were exposed to air temperatures below, equal to, or above ambient water temperatures, at various times of year. Thermal differentials were in the range of 5[degrees]C to 10[degrees]C (Table 1). In a second experiment, unprocessed mussels were exposed to air temperatures below, equal to, or above the ambient water temperature of 5[degrees]C as observed for that time of the year, with differentials in air temperature of 5[degrees]C to 20[degrees]C. Mussels were exposed to each temperature for an 8-h period with samples (n = 12 mussels) for the NRA being taken at hour 0 as a control, and at hours 4 and 8 for each temperature exposure.

Sampling Procedures

The NRA was performed using the method described by Lowe et al. (1995a) with minor modifications; poly-L-lysine and neutral red stock solutions were increased from 10 [micro]L to 20 [micro]L to enhance cell adhesion to microscope slides and dye intensity in the cells, respectively. Briefly, 0.3 mL of hemolymph was drawn from the posterior adductor muscle into an equal amount of physiological saline using a 21-gauge needle, with the mixture placed in a siliconized Eppendorf tube and gently inverted. A 40-[micro]L sample of the hemolymph/saline mixture was then placed onto a microscope slide that had been treated with poly-L-lysine. The cells were then left for 15 min to adhere to the slide. A neutral red working solution was prepared from 20 [micro]L of neutral red stock solution and 5 mL of physiological saline, and 40 [micro]L was added to the cell layer and incubated for 15 min in a humidity chamber. After 15 min of incubation, a cover slip was added to the slide and the cells were examined under low light intensity using a compound microscope at x400 magnification. Cells were examined every 15 min for the first 60 rain, then at 90, 120, and 180 min.

For each examination, 25 hemocytes from each mussel sample were examined and given a rating based on 1 of the 4 following characteristics: No stress response = characterized by appearance of tiny pink dots, which are intact lysosomes containing neutral red dye; moderately low stress response = increase in the size of lysosomes due to lysosomal membrane fusion; moderately high stress response = lysosomes appear larger and more faint in color due to fusion of lysosomal membranes and leakage of neutral red dye into the cytosol; high stress response = cytosol is completely tinged pink due to leakage of neutral red dye out of the lysosomes, and only remnants of the lysosomal membranes may be still visible. When higher than 50% of the 25 ceils exhibiting high stress response at a particular time point, then the assay was stopped for that mussel and the previous examination time period recorded as the score for NRR.

Shelf life was defined as the time when 5% of mussels in a sample were dead or moribund in a lot. For each monthly sample, 3 replicates of 50 mussels were assessed. Mussels were placed in chilled dry storage of 2[degrees]C to 4[degrees]C at high humidity, the accepted industry method, each month. Shelf life trials were terminated at day 20, by which time mussels had a strong odor, which would make them unmarketable by industry standards, from initiation of the experiment regardless whether 5% mortality had been reached. Mussel mortality or morbidity (shells fail to remain closed) was assessed daily.

Standard meat yield (CI = condition index) was determined by the following calculation based on 1 kg of live mussels, which were steamed for 10 min (Ibarra et al. 2000):

CI = cooked meat weight/(cooked meat weight + shell weight) x 100

Data analysis

Data were analyzed using the SPSS (version 10) statistical software. The means ([+ or -]SE), ANOVA, and post hoc tests (Tukey's b) were calculated. The level of significance was set [alpha] = 0.05.

RESULTS

Seasonal Variation

Monthly sampling of mussels showed a seasonal pattern in stress response (Fig. 1A). NRR, a measure of subcellular (lysosome) stress response, showed a significant difference among the months (one-way ANOVA; [F.sub.(11,130)] = 21.327, P < 0.001), with significant decreases in NRR following spawning events in June 2001, September 2001, and June 2002 (Tukey's b, P < 0.05) followed by gradual increases in NRR in the months following spawning.

[FIGURE 1 OMITTED]

The average shelf life of mussels examined during each of the monthly samplings showed that there was a significant difference among the months (one-way ANOVA; [F.sub.(11,24)] = 15.675, P < 0.001) (see Fig. 1B). The average time mussels can survive before they reach 5% mortality was reduced during certain times of the year, especially following the major spawning period in June (see Fig. 1A, B).

The condition of the mussels, as determined by a standard meat +yield, showed meat yields decreased following spawning events and gradually increased in months following spawning (see Fig. 1C). Comparison of NRR, shelf life, meat yield and parameters of food availability (Chl-a), temperature, and salinity indicate that NRR and shelf life are influenced by various combinations of these parameters at various times of the year (Fig. 2). NRR was shown to be most affected by the reproductive cycle (determined using standard meat yield) with a significant correlation, r = 0.87, and shelf life being influenced inversely by water temperature with a correlation of r = -0.74 (Table 2). Regression analysis on the correlation between NRR and meat yield showed significance with [R.sup.2] = 0.75 (P = 0.025), however between temperature and shelf life significance was lower with [R.sup.2] = 0.54 (P = 0.9) (Fig. 3 A,B).

[FIGURES 2-3 OMITTED]

Air Exposures

Experiments examining the rapid, short term exposure of mussels to various air temperatures showed that the NRR response was affected by variation in air temperature, duration of the exposure during harvesting (<8 h), and seasonal factors (Fig. 4). There was a significant interaction between the seasons and the air temperatures (ANOVA; [F.sup.(6,371)] = 5.786, P < 0.001), between the seasons and the hours of sampling (ANOVA; [F.sup.(6,371)] = 6.164, P < 0.001), and between the air temperatures and the hours of sampling (ANOVA; [F.sup.(4,371)] = 6.084, P < 0.001).

[FIGURE 4 OMITTED]

A seasonal pattern of mussel stress response was apparent as NRR (observing time zero for each season) was reduced in months surrounding the spawning period (see Fig. 4). Mussels had low NRR in the postspawning period but this was significantly higher in all other sampling periods. Seasonally, the NRR scores showed that there was no significant difference between mussels in June 2001 (prespawned) and January 2002 (recovered), but that there was a significant difference in mussels in August 2001 (postspawned) and in mussels in May 2002 (overwintered) than all the other seasons respectively (Tukey's b, P < 0.05).

In general, results showed that NRR scores of normally sub-merged mussels subjected to rapid, short term air exposure and held at an air temperature equal to ambient water temperature were significantly higher than any other air exposure temperature. The air temperatures used in this experiment are given in Table 1. Furthermore, NRR scores of mussels held at air temperatures that were below the ambient water temperature were significantly higher than NRR scores of mussels held at an air temperature that was higher than the ambient water temperature, with the exception of when air below the ambient water temperature was at subzero temperature (Tukey's b, P < 0.05). Mussels held at air temperatures that were equal to ambient water temperatures had the highest NRR scores (and less stress) regardless of season or period of time of exposure compared with NRR scores of mussels held at air temperatures either below or above the ambient water temperature (see Figs. 4A, B, and D).

In addition, this experiment showed that short term extended air exposure on mussels reduced NRR in all seasons and temperatures, as NRR scores were significantly different among the hours (ANOVA; [F.sub.(2,371)] = 136.857, P < 0.001, Tukey's b, P < 0.05) (see Fig. 4D). Further examination of air temperature exposure showed that NRR declined more rapidly when mussels were exposed to greater differentials in air temperatures in relation to the ambient water temperature of 5[degrees]C (Fig. 5).

[FIGURE 5 OMITTED]

DISCUSSION

Seasonal and Monthly Variations

The objective of this study is to examine effects of seasonal influences on cultured mussels using and evaluating the NRA as a stress response indicator. The significant difference in NRR among the months and seasons indicates that seasonally related factors were influencing natural and normally induced levels of stress response in mussels. This was an expected occurrence because previous studies have shown that there are differences in a number of physiological factors among the seasons, especially between the warmer and colder months (i.e., summer and winter) (Bayne & Thompson 1970, Moore 1976, Hofmann & Somero 1995, Chapple et al. 1998). Metabolic rate has been shown to differ from summer and autumn to the winter, lysosomal responses have been reported to differ from winter to the summer, and protein levels vary seasonally in feral bivalves (Bayne & Thompson 1970, Moore 1976, Hofmann & Somero 1995, Chapple et al. 1998). Conditions that change with the seasons, including reproductive patterns, water temperatures, and food availability, can lead to major alterations in physiologic mechanisms because the animal strives to maintain homeostasis (Bayne & Thompson 1970, Moore 1976, Hofmann & Somero 1995, Chapple et al. 1998).

The seasonal pattern of natural stress response levels in cultured mussels was shown to be closely related to the reproductive cycle as determined by the standard meat yield (condition index). Spawning events were a factor each time NRR decreased. NRR decreases were observed to significantly correlate with a decrease in condition index. Previous research has shown changes in physiological activities occur during the reproductive cycle in feral mussels including high lysosomal destabilization, higher metabolic demands, lower survival, reallocation of energy, lower oxygen consumption, fluctuations in plasma components, and changes in body glycogen and byssal thread production (Bayne & Thompson 1970, Bayne 1973, Thompson et al. 1978, Worrall & Widdows 1984, Young 1985, Eertman et al. 1993, Myrand & Gaudreault 1995, Tremblay et al. 1998b). The present findings on cultivated mussels support these earlier studies on wild mussel populations.

Naturally induced stress response levels continually decreased throughout the winter months as NRR increased, and only began to decline again in late spring/early summer (June 2002), which was comparable to stress response levels in the previous year (June 2001). Previous studies that examined the reproductive cycle in wild mussels have shown that gametogenesis occurs very slowly during the winter and that it its progression is rapid in the spring and early summer, followed by spawning in July (Thompson 1984), thus suggesting a link between stress responses and the breeding cycle.

To further understand seasonal variation of natural stress response levels in mussels, shelf life was also examined as an indicator of stress response at the whole animal level. The shelf life varied little throughout the year with the exception of July 2001 through to September 2001 following the major spawning period. Furthermore, the condition index of mussels was highest prior to spawning and lowest following spawning. These findings support the results of previous studies that showed mussels are in poor condition and have low meat yields following spawning, and they undergo a number of physiological changes during this time that can impede survival ability (Bayne & Thompson 1970, Bayne 1973, Dare & Edwards 1975, Slabyj & Hinkle 1976, Warwick 1984).

Seasonal variations as illustrated by the NRA, shelf life and meat yields can be affected not only by reproduction, but also by seasonal changes in temperature and/or food availability. Seasonal trends in water temperature and food availability (leading to nutritive stress) have been shown to affect byssal production, lysosomal stability, disease (bacterial challenges), and metabolic activity in wild mussels and other bivalve species (Bayne 1973, Young 1985, Hauton et al. 1998, Hauton et al. 2001).

In October and November following spawning, NRR and condition index were significantly lowered, however shelf life continued to increase. Although the NRA measured a high stress response in the months of October and November, rapidly declining water temperatures going into these months, and increased food availability from the autumn phytoplankton bloom prior to these months at the sampling site, Charles Arm, enhanced the survival ability of mussels by reducing the impact of high stress response levels at the subcellular level up to the whole animal level. This study showed that water temperature and shelf life were strongly linked. Previous studies have shown that mussels are unable to cope with seasonally elevated water temperatures, particularly postspawn, thus a decrease in water temperature would be a benefit to the survivability of the mussels (Sephton et al. 1993, Myrand & Gaudreault 1995). Koehn and Bayne (1989) indicated that with an abundance of energy reserves available, physiological performance of individuals may not be significantly affected by differences in their metabolic efficiency. As suggested by Tremblay et al. (1998), individuals with lower maintenance requirements (related to low water temperature and increased food) would have better survival. In Newfoundland, total particulate matter is highest in summer in more open areas, but is higher in the autumn and spring in small semienclosed inlets such as Charles Arm (Thompson 1984, Penney et al. 2001), thus suggesting that food availability was a factor in enhanced, sustained shelf life in the fall, as were seasonally lower water temperatures.

In general, the combination of thermal, nutritive, and reproductive stress influenced the outcome of stress response as assessed by NRA, shelf life and meat yield of mussel. Experiments that evaluated the effects of air temperatures as found during harvesting on barge decks on stress response in mussels corroborated the impacts of these seasonal influences.

By examining stress response using the NRA, in conjunction with the assessment of shelf life and condition index, late autumn (December) through to the spring (May) of the year is the time period when mussels are in the best physiological condition and are least stressed in this subarctic farming site.

Air Exposures

The effects of ambient air temperatures, especially over extended exposure periods, have been shown to be detrimental to mussels (Moore et al. 1979, Widdows & Shick 1985). Understanding the influence of sudden short term air exposure on continuously submerged mussels is important during harvesting on barge decks so as to minimize the impact it may have on the quality of the mussel product. During harvesting mussels are exposed to and maintained in air of various temperatures for relatively short periods of time (<8 h), and they enter an anoxic state bringing about changes in physiological parameters (Moore et al. 1979, Widdows & Shick 1985). These changes are an immediate response to a new environment and in the case of harvesting are a reaction to a short term event that does not allow the mussels time to adapt physiological processes that would later be apparent if time in this condition was extended.

As expected, the results of our experiments showed that exposure to air temperatures equivalent to seasonal ambient water temperatures were least detrimental to mussels, as assessed by the NRA. In general, during summer months, subcellular stress response, as measured by NRR, was high at any exposure temperature and can be attributed to seasonal influences of reproduction. Overall, air temperatures within 5[degrees]C of ambient seawater temperatures had little additional effect on mussel stress response, but those mussels exposed to a greater thermal change had a proportionally greater response. Air temperatures that exceeded ambient water temperature by 15[degrees]C induced the highest stress response in mussels. Air temperatures vary throughout the year and may harm mussels if they are exposed for extended periods (Widdows & Shick 1985). Moore et al. (1979) demonstrated that in mussels, Mytilus edulis, and cockles, Cerastoderma edule, an exposure to an increase in air temperature of 10[degrees]C induced no change in lysosomal activity, but did with a 20[degrees]C increase, which is in accordance with the present findings. Our study also found that mussels were under high stress during air exposure at subzero temperatures. Past studies have shown that there is a high incidence of mortality in mussels harvested during subzero temperature, and that although Mytilus edulis can tolerate freezing up to about -10[degrees]C, tissues become injured when 64% of the cellular water is removed to form ice (Williams 1970, Slabyj & Hinkle 1976, Slabyj 1980). In this work, exposure of winter harvested mussels to -3[degrees]C air temperature (5[degrees]C below ambient water temperature), induced a significant reduction in NRR compared with air temperature equivalent to the ambient water temperature of 2[degrees]C.

Experiments revealed that extended exposure to air, no matter what the temperature, increased stress response in mussels during every sampling season. Previous studies support this finding, because aerial exposure has been shown to cause physiological changes, such as altered lysosomal stability in wild Mytilus edulis and the clam, Mya arenaria, ammonia accumulation in plasma, and increases in the number of hemocytes in mussels (Thompson et al. 1978, Eertman et al. 1993, Tremblay & Pellerin-Massicotte 1997).

Examinations of stress response associated with air exposure suggest that harvesting on barge decks should occur when air temperatures are close to being equivalent to ambient water temperatures, and prolonged period of air exposure should be avoided when air temperatures are below 0[degrees]C. If air temperatures exceed a 5[degrees]C differential from ambient water temperatures, mussels should be covered to avoid undue stress responses and subsequent reduction in shelf life that may be induced by heating in the sun in summer or freezing in the winter. As well, extended holding of mussels on harvesting decks should be minimized to less than 4 h in duration during any season, unless alternative approaches, such as icing the mussels, can be arranged. The present work suggests that by manipulating or altering standard culture practices we may improve mussel quality and production, and in the end profits for growers.

Shelf Life and Industry Practices

The findings of air exposure on barge decks do not address shelf life issues. Shelf life is a long process extending days as opposed to just hours and provides time for mussels to adapt to anoxic conditions. Exposure to air during harvesting induces initial physiological changes with the onset of anoxia in mussels and to the air temperatures in which mussels are being harvested, and are not reflective of the survivability of mussels during extended, storage conditions. Concurrent studies with this work examined the effect of various storage conditions, including storage in air, and duration of storage on stress response (i.e., NRA) and shelf life of mussels during the same sampling periods (Harding et al. 2004a).

The NRA can be used successfully to examine stress response levels in lysosomes of cultured mussels, and to determine if various conditions to which cultured mussels are exposed, such as air temperatures, will induce a stress response at the subcellular level in the lysosomes. The NRA can provide information about culture conditions and their effects on mussels, and there are several questions remaining to be answered. For example, can the NRA be used to predict how non market-sized mussels respond to certain conditions while in the water, or during times of grading and socking. As well, the question of genetics and stress response need to be addressed. Different species may have different adaptive abilities and thresholds of tolerance to a variety of conditions. Studies are needed to quantify stress response in mussels of different genetic makeup.

The question of how physiological parameters correlate with the lysosomal stress response changes needs to be examined. The lysosome has been identified as the site of earliest "detectable" change to the state of the mussel, and presuming that the role of lysosomes is largely immunological in hemocytes, it is conceivable that the stress response, as reflected by changes in protein concentrations, [O.sub.2] consumption, ammonia levels, and hemocyteactivity may be triggered by lysosomal changes. Hence, there is a need to examine the integration of all these processes. Understanding these interactions may lead to development of earlier indicators that the animal has been exposed to stressful conditions and allow for earlier manipulations of conditions to avoid expression of the stress response at the whole animal level.

TABLE 1.

Air temperature exposures (below, equal to, and above ambient
water temperature) during the various seasons of early summer
(June 2001), summer (August 2001), early winter (January 2002),
and spring (May 2002).

               Below Ambient                              Above Ambient
                   Water                                      Water
                Temperature         Ambient Water          Temperature
   Season      ([degrees]C)    Temperature ([degrees]C)   ([degrees]C)

Early Summer         2                     7                   17
Summer               6                    16                   21
Early winter        -3                     2                   12
Spring               0                     5                   15

TABLE 2.

Correlation values (Pearson r value) for the comparisons among
neutral red retention (NRR), standard meat yield (MY), shelf life
(SL), chlorophyll-a (chl-a), water temperature (T), and salinity (S)
for the period of June 2001 to November 2001 (n = 6).

        NRR     MY        SL     Chl-a       T         S

NRR      1    0.867 *   -0.167   -0.011    0.178     -0.124
MY            1          0.32    -0.266   -0.167     -0.213
SL                       1       -0.178   -0.738 *   -0.091
Chl-a                             1        0.192      0.162
T                                          1         -0.102
S                                                     1

* Significant correlation, P < 0.05.

ACKNOWLEDGMENTS

The authors thank AquaNet, the Canadian Network Centres of Excellence for Aquaculture for partial funding of this project, Memorial University School of Graduate Studies, Marine Institute of Memorial University (especially members of the staff of Centre for Aquaculture and Seafood Development--A. Struthers, J. Nichols, and S. Macneill), Canadian Centre for Fisheries Innovation, National Research Council--Institute for Marine Biosciences and to the staff and management of Thimble Bay Farms.

LITERATURE CITED

Bayne, B. L. 1973. Physiological changes in Mytilus edulis L. induced by temperature and nutritive stress. J. Mar. Biol. Assoc. U.K. 53:39-58.

Bayne, B. L. 1985. Responses to environmental stress: tolerance, resistance and adaptation. In: Marine Biology of Polar Regions and Effects of Stress on Marine Organisms. In: J.S. Gray & M.E. Christiansen, editors. London: John Wiley and Sons Ltd. pp. 331-349.

Bayne, B. L., K. R. Clarke & M. N. Moore. 1981a. Some practical considerations in the measurement of pollution effects on bivalve molluscs, and some possible ecological consequences. Aquat. Toxicol. 1:159-174.

Bayne, B. L., D. R. Livingstone, M. N. Moore & J. Widdows. 1976. A cytochemical and biochemical index of stress in Mytilus edulis L. Mar. Poll. Bull. 7:221-224.

Bayne, B. L., M. N. Moore & R. K. Koehn. 1981b. Lysosomes and the response by Mytilus edulis L. to an increase in salinity. Mar. Biol. Lett. 2:193-204.

Bayne, B. L. & R. J. Thompson. 1970. Some physiological consequences of keeping Mytilus edulis in the laboratory. Helgoland. Weiss Meersunt. 20:526-552.

Carver, C. E. & A. L. Mallet. 1991. Summer mortality of the blue mussel in Prince Edward Island. Bull. Aquacul. Assoc. Canada 91:30-36.

Chapple, J. P., G. R. Smerdon, R. J. Berry & A. J. S. Hawkins. 1998. Seasonal changes in stress-70 protein levels reflect thermal tolerance in the marine bivalve Mytilus edulis L. J. Exp. Mar. Biol. Ecol. 229:53-68.

Clemens, T., C. Couturier, G. J. Parsons & P. Dabinen. 1999. Newfoundland Aquaculture Industry Association environmental monitoring program of shellfish farms. Bull Aquacul. Assoc. Canada 99(3):29-34.

Dare, P. J. & D. B. Edwards. 1975. Seasonal changes in flesh weight and biochemical composition of mussels (Mytilus edulis L.) in the Conway estuary, North Wales. J. Exp. Mar. Biol. Ecol. 18:89-97.

Eertman, R. H. M., A. J. Wagenvoort, H. Hummel & A. C. Smaal. 1993. "Survival in air" of the blue mussel Mytilus edulis L. as a sensitive response to pollution-induced environmental stress. J. Exp. Mar. Biol. Ecol. 170:179-195.

Freeman, K. R. & L. M. Dickie. 1979. Growth and mortality of the blue mussel (Mytilus edulis) in relation to environmental indexing. J. Fish. Res. 36:1238-124.

Harding, J. M., C. Couturier, G. J. Parsons & N. W. Ross. 2004a. Evaluation of the neutral red assay as a stress response indicator in cultivated mussels (Mytilus spp.) in relation to post-harvest processing activities and storage conditions. Aquaculture 231:315-326.

Harding, J. M., C. Couturier, G. J. Parsons & N. W. Ross. 2004b. Evaluation of short-term transport conditions on stress response of cultivated blue mussels (Mytilus spp.). Proceedings of the International Stress Indicators Workshop, Quebec City, Quebec. (In press).

Hauton, C., L. E. Hawkins & S. Hutchinson. 1998. The use of the neutral red retention assay to examine the effects of temperature and salinity on haemocytes of the European flat oyster Ostrea edulis (L). Comp. Biochem. Physiol. 119:619-623.

Hauton, C., L. E. Hawkins & S. Hutchinson. 2001. Response of haemocyte lysosomes to bacterial inoculation in the oysters Ostrea edulis L. and Crassostrea gigas (Thunberg) and the scallop Pecten maximus (L.). Fish. Shellfish Immun. 11:143-153.

Hofmann, G. E. & G. N. Somero. 1995. Evidence for protein damage at environmental temperatures: seasonal changes in levels of ubiquitin conjugates and HSP70 in the intertidal mussel Mytilus trossulus. J. Exp. Biol. 198:1509-1518.

Ibarra, D., C. Couturier & T. Mills. 2000. Calculation of meat yields by mussel growers in Newfoundland. In: F. Shahidi, editor. Seafood in health and nutrition transformation in fisheries and aquaculture: global perspectives. St. John's, NL: Canada Science Tech. Publishing Company. pp. 261-269.

Koehn, R. K. & B. L. Bayne. 1989. Towards a physiological and genetical understanding of the energetics of the stress response. Biol. J. Linn. Soc. 37:157-171.

Lowe, D. M., V. U. Fossato & M. H. Depledge. 1995a. Contaminant-induced lysosomal membrane damage in blood cells of mussels Mytilus galloprovincialis from Venice Lagoon: an in vitro study. Mar. Ecol. Prog. Ser. 129:189-196.

Lowe, D. M., C. Soverchia & M. N. Moore. 1995b. Lysosomal membrane responses in the blood and digestive cells of mussels experimentally exposed to fluoranthene. Aquat. Toxicol. 33:105-112.

Macneill, S., M. Pryor, C. Couturier & G. J. Parsons. 1999. Increasing spat collection for mussel culture: Newfoundland Aquaculture Industry Association larval and spatfall monitoring program. Bull. Aquacul. Assoc. Canada 99:24-28.

Moore, M. N. 1976. Cytochemical demonstration of latency of lysosomal hydrolyses in digestive cells of the common mussel, Mytilus edulis, and changes induced by thermal stress. Cell Tissue Res. 175:279-287.

Moore, M. N. 1980. Cytochemical determination of cellular responses to environmental stressors in marine organisms. In: A. D. McIntyre & J. B. Pearce, editors. Biological effects of marine pollutants of monitoring organisms. Rapports et Proces-Verbaux des R6unions. Cons. Int. Explor. Mer. pp. 7-15.

Moore, M. N., D. M. Lowe & S. L. Moore. 1979. Induction of lysosomal destabilization in marine bivalve molluscs exposed to air. Mar. Biol. Lett. 1:47-57.

Myrand, B. & J. Gaudreault. 1995. Summer mortality of blue mussels (Mytilus edulis Linneaus, 1758) in the Magdalen Islands (Southern Gulf of St. Lawrence, Canada). J. Shellfish Res. 14:395-404.

Nichols, J., C. Couturier, M. Pryor & S. Macneill. 2002. Environmental and biological characteristics of Newfoundland shellfish farms for 1993-2001. ACERA Mussels Project Final Report. Grand Falls, NL: Department of Fisheries and Aquaculture. pp. 38-59.

Penney, R. W., C. H. McKenzie & T. J. Mills. 2001. Assessment of the particulate food supply available for mussel (Mytilus spp.) farming in a semi-enclosed northern inlet. Estuarine, Coastal Shelf Sci. 53:107-121.

Sephton, T. W., T. Landry & C. F. Bryan. 1993. Summer mortality of cultured blue mussels in Prince Edward Island, Canada. Bull, Aquacul. Assoc. Canada 93:59-51.

Slabyj, B. M. 1980. Storage and processing of mussels. In: R. A. Lutz, editor. Mussel culture and harvest: a North American perspective. Amsterdam, Oxford, New York: Elsevier Scientific Publishing Company. pp. 247-262.

Slabyj, B. M. & C. Hinkle. 1976. Handling and storage of blue mussels in shell. Res. Life Sci. 23(4):1-13.

Thompson, R. J. 1984. The reproductive cycle and physiological ecology of the mussel Mytilus edulis in a subarctic, non-estuarine environment. Mar. Biol. 79:277-288.

Thompson, R. J., C. J. Bayne, M. N. Moore & T. H. Carefoot. 1978. Haemolymph volume, changes in the biochemical composition of the blood, and cytological responses of the digestive cells in Mytilus californianus Conrad, induced by nutritional, thermal and exposure stress. J. Comp. Physiol. 127:287-298.

Tremblay, R., B. Myrand & H. Guderley. 1998a. Thermal sensitivity of organismal and mitochondrial oxygen consumption in relation to susceptibility of blue mussels, Mytilus edulis (L.), to summer mortality. J. Shellfish Res. 17:141-152.

Tremblay, R., B. Myrand & H. Guderley. 1998b. Temporal variation of lysosomal capacities in relation to susceptibility of mussels, Mytilus edulis, to summer mortality. Mar. Biol. 132:641-649.

Tremblay, R. & J. Pellerin-Massicotte. 1997. Effect of the tidal cycle on lysosomal membrane stability in the digestive gland of Mya arenaria and Mytilus edulis L. Comp. Biochem. Physiol. 117A:99-104.

Warwick, J. 1984. A code of practice for mussel processing. Wellington, New Zealand: New Zealand Fishing Industry Board. pp 2-32.

Widdows, J. & J. M. Shick. 1985. Physiological responses of Mytilus edulis and Cardium edule to aerial exposure. Mar. Biol. 85:217-232.

Williams, R. J. 1970. Freezing tolerance in Mytilus edulis. Comp. Biochem. Physiol. 35:145-161.

Worrall, C. M. & J. Widdows. 1984. Investigation of factors influencing mortality in Mytilus edulis L. Mar. Biol. Lett. 5:85-97.

Young, G. A. 1985. Byssus-thread formation by the mussel Mytilus edulis: effects of environmental factors. Mar. Ecol. Prog. Ser. 24:261-271.

JOANNE M. HARDING, (1) CYR COUTURIER, (1) G. JAY PARSONS (1) * AND NEIL W. ROSS

* Corresponding author. E-mail: [e-mail]

(1) Fisheries and Marine Institute, Memorial University of Newfoundland, P.O. Box 4920, St. John's, Newfoundland and Labrador, Canada A1C 5R3; (2) National Research Council, Institute for Marine Biosciences, 1411 Oxford Street, Halifax, Nova Scotia, Canada B3H 3Z1