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Physiological responses of brown crab (Cancer pagurus Linnaeus 1758) to dry storage under conditions simulating vitality stressors.

ABSTRACT Knowledge of the brown crab's (Cancer pagurus Linnaeus 1758) tolerance to emersion related to air temperatures and duration is scarce. In the current study, the condition of the brown crab was examined during emersion in laboratory experiments simulating dry storage and transport at air temperatures between 2-20[degrees]C. Samples of hemolymph were taken at different intervals simultaneously as the crab was classified using a vitality index based on behavior and responses. Individual variances in hemolymph pH, total ammonia (TA), lactate, and glucose were large; but, in general, pH decreased and TA and lactate increased during emersion, most at the highest temperatures. When hemolymph samples were grouped according to vitality index, deterioration was found for pH, TA, and lactate as the vitality dropped, whereas no trend was found for glucose. Vitality of crabs was reduced during emersion, with the greatest reduction being crabs exposed to high temperatures. To secure strong and healthy crabs, exposure at 20[degrees]C and 15[degrees]C should not exceed 5 h and 10 h, respectively. For crabs exposed at 10[degrees]C and 5[degrees]C, emersion for 36 h and 72 h, respectively, did not seem to have negative consequences for the animals. A general increase in vitality occurred when crabs were reimmersed. Delayed mortality occurred for weak and moribund crabs, highest during the first 24 h of reimmersion, and continuing over the next day, suggesting that such crabs should not be transported in the live value chain.

KEY WORDS: dry storage, temperature, vitality index, hemolymph parameters, tolerance, Cancer pagurus

INTRODUCTION

The European brown crab (Cancer pagurus L.) is mainly harvested in the English Channel, around the British Isles and along the Norwegian coast, with landings of 46,000 tons in 2006 (EUROSTAT 2007). The fishery is conducted in two principal ways according to the location and time of year. Offshore vessels fish year-round using specialized vivier boats with the catch stored in flow-through systems and the landings mainly distributed to the live market in vivier trucks (Uglow et al. 1986, Barrento et al. 2008, Lorenzon et al. 2008). Smaller inshore boats usually store the catch onboard, out of water (dry). In a vivier system, the crabs' claws are inactivated to avoid injuries to other crabs (Chartois et al. 1994, Lawler 2001). However, in the Norwegian inshore fishery, the claws are not inactivated. To avoid injuries, the crabs are stacked tightly in the storage units. Inshore boats prefer to land the catch daily, which is then transported dry in refrigerated trucks to be sold live or on to a processing plant. The crabs are revitalized either by exposing them to flowing water or just storing them in a cool room. The time in air from catching to the destination may, in some regions, be as much as 2-3 days (Woll et al. 2006).

On emersion, brown crabs exhibit a general reduction in aerobic metabolism of about 13.6% of immersed levels (Uglow et al. 1986). However, when exposed to air, the gills tend to collapse and clump together, reducing the ability and effective area for exchange of respiratory gases and ions (Johnson & Uglow 1985). This causes a switch to anaerobic pathways of energy production with increased utilization of carbohydrate, resulting in the production of lactic acid that cannot be rapidly mobilized or excreted (Taylor & Whiteley 1989, Spicer et al. 1990, Lorenzon et al. 2007, Lorenzon et al. 2008). In combination with the increased levels of carbon dioxide, the lactic acid leads to a decrease in hemolymph pH. As a result of the reduced ability of the gills, the main waste of the nitrogenous pathways ammonia--accumulates in the hemolymph. High levels of ammonia damage the gills and ultimately kill the animal (Regnault 1992, Hosie 1993, Danford 2001).

The use of dry storage is beneficial as a result of the reduced transportation of water, which reduces the risk to the local environmental (from introduction of nonindigenous species or spread of pathogens) and reduced transportation costs (Robson et al. 2007). However, the physiological stress associated with extended air exposure can be irreversible if the animals are weakened and cannot reestablish homeostasis. It is possible to ensure a good survival rate and for the crabs to retain a healthy condition by ensuring environmental conditions are kept within critical limits during all stages of the live market chain (Gomez-Jimenez et al. 2001). Exposure to atmospheric air can cause an increase in blood glucose levels. This is related to the mobilization of energy stores, under conditions of low oxygen availability, for use as a fuel in anaerobic metabolism, resulting in the production and accumulation of lactate (Durand et al. 2000). Circulating glucose is frequently used as an indicator of immediate stress in crustaceans, and glucose may increase very quickly as a result of a hyperglycemic response to handling (Telford 1974). Studies have shown that the changes in the hemolymph composition during emersion are good indicators of reduced viability resulting from handling in rock lobster (Panulirus cygnus) (Paterson & Spanoghe 1997, Paterson et al. 2005).

Knowledge of the brown crab's tolerance to dry storage related to air temperatures and duration is scarce. This study examined how the hemolymph parameters pH, lactate, total ammonia (TA), and glucose varied with duration of air exposure at different temperatures typically encountered by the crabs during transportation. Findings are compared with a vitality index in the quest for an easy and reliable method to determine the health status of individual crabs. Furthermore, the study aims to give a rough guideline for the time available for dry storage of crabs at various air temperatures that can be used by the crab industry. Because there is an increasing market for live crabs, we followed the crabs during reimmersion to gain further information on delayed mortality caused the emersion.

MATERIALS AND METHODS

The effect of emersion was investigated through two laboratory experiments starting October 10, 2005 (trial 1; i.e., in the peak season of the crab fishery), and August I, 2007, (trial 2; i.e., in the beginning of the Norwegian crab season; Table 1). A preliminary trial was conducted to get indications of tolerance time in a cool room (4[degrees]C) and on deck during a warm summer day (17[degrees]C). The result was used to plan trial 1 in order to allow the experiments to run long enough to predict the tolerance time for crabs immersed at low and high temperatures.

Pretreatment of Animals

The crabs used in the experiments were captured in traps by commercial fishermen in Romsdal, in the northwestern part of Norway, all within a distance of 20 nautical miles from the experimental facility at Moreforsking Marin. Only strong female crabs in the intermolt stage were collected, with preference for crabs between 140 mm and 160 mm carapace width (CW). The crabs' claws were inactivated by rubber bands (Woll & Berge 2007) before they were stacked in 68-L plastic boxes, 70-80 crabs in each. The boxes were covered with a layer of wet paper before dry transport (3-5 h) at ambient air temperature (10 14[degrees]C) to the laboratory.

In the laboratory, the boxes were stacked and the rack supplied with flow-through seawater 100 L/min from 5 m depth at ambient salinity (31-33 [per thousand]) and temperature (Table 2). The flow was sufficient to fill the boxes and keep all crabs immersed. Oxygen saturation was higher then 80% in each of the boxes. This lasted for 2 days, which was considered sufficient for them to be revitalized (i.e., get rid of accumulated waste in the hemolymph and tissue that built up during catch and transport to the facility). The crabs were not fed during this treatment.

Experimental Design

Strong revitalized crabs without any fractures were used for the dry storage experiments. The onset of emersion was conducted between 9:00-12:00 AM (i.e., the time of the day when the crabs are supposed to have a low activity level) (Ansell 1973, Danford 2001). For each temperature group, crabs were randomly selected from the revitalized crabs, and were individually marked and packed into plastic boxes covered with moist paper, with 1 box for each subsample. The number of crabs in each subsample and the number of collecting times for each temperature group was decided in advance, based on the result from the preliminary trial. The boxes were stored at the respective air temperatures where relative air humidity was between 90% and 95% for low temperatures (2[degrees]C, 5[degrees]C, and 10[degrees]C) and 75% and 85% for high temperatures (15[degrees]C and 20[degrees]C).

At each sampling point in each temperature, 1 subsample was taken for assessment of vitality and a sample of hemolymph. After this, the individual marked crabs were returned to the subsample box. The box with the crabs were then reimmersed in flow through water.

During trial 1, crabs were immersed at 2, 5, 15, and 20[degrees]C. Subsamples (n = 20) were taken every 24 h for crabs immersed at 2[degrees]C and 5[degrees]C, and every 12 h and 6 h for crabs immersed at 15[degrees]C and 20[degrees]C, respectively. There was a total of 3 sampling points for each temperature (Table 1). A reference group (n = 20) was randomly selected from the revitalized crab and bled immediately (Table 2).

During trial 2, crabs were immersed at 5, 10, 15, and 20[degrees]C. Subsamples (n = 16) were taken every 24 h for the group immersed at 5[degrees]C, and every 12 h for the group immersed at 10[degrees]C. To obtain more knowledge about the first part of emersion for these lower temperatures, subsamples were also taken after 6 h of emersion. For the groups immersed at 15[degrees]C and 20[degrees]C, subsamples were collected every 6 h, with a total of 4 sampling points at 15[degrees]C and 3 sampling points at 20[degrees]C (Table 1). The reference group (n = 64) consisted of 4 groups (n = 16) randomly selected for each of the temperature groups before emersion (Table 2).

Assessing Vitality Index

Vitality was assessed based upon a 5-point scale with 5 points being very strong and healthy animals, and 1 point being dead (Table 3). Responses used to assess the vitality were the aggressive claw response and the defensive leg and claw response (for preventing access to the abdominal area), the strength of the mouthparts, and the eyestalk response. If no eye reaction (cerebral ganglion) and no maxilliped reaction (posterior ganglion) were observed, the crab was classified as dead (Baker 1955).

Mortality during emersion was calculated from the number of crabs at the start of the emersion and the number that survived the emersion. Delayed mortality was calculated from the number of crabs that survived emersion and the number that were alive after 24 h and 48 h reimmersion. Cumulative mortality was calculated from the number of crabs in the group emersion and the number alive after 48 h of reimmersion.

Determination of Hemolymph Parameters

Hemolymph samples (1.5 mL) were taken from the sinus behind the 4th or 5th pereiopod using a 21-gauge needle attached to a 2-mL syringe (Danford 2001). The time taken for hemolymph collection was preferentially kept to less than 30 sec to minimize stress to the animals. This is especially important in experiments involving analysis of glucose to avoid a hyperglycemic response to stress (Danford 2001). Taking multiple hemolymph samples from the same individuals during the experiment was avoided.

pH was measured immediately at ambient temperature using a microelectrode and pH meter (EBRO phx 1495, EBRO Electronics, Ingolstadt, Germany). The hemolymph sample was divided for subsequent analysis. Samples prepared for measurements of TA were centrifuged at 8,000 rpm for 5 min, after which the plasma fraction was transferred to Eppendorf tubes and immediately frozen at -20[degrees]C until analyses using the fluid injection method as described by Hunter and Uglow (1990). For lactate and glucose determination, the hemolymph was transferred into tubes containing anticlotting buffer fluorid oksalat (Microtainer microgard) and mixed thoroughly before being centrifuged within 15 rain at 8,000 rpm for 5 min. The plasma fraction was then transferred in to Eppendorf tubes, one for each of the parameters, and frozen at -20[degrees]C until analyzed colorimetrically by Vitros 950 AT (Coefficient of variance, 0.02-0.05) (Ullman et al. 1996). The anticlotting buffer has no effect on the reading. The minimum value detected by this method is 0.5 mM and 1.1 mM for lactate and glucose, respectively.

The average was calculated for each of the hemolymph parameters at each sampling point and for the different temperature groups. Cumulated values were then calculated by subtracting the average values of the hemolymph samples taken of the reference group from those of the immersed crabs.

Data Analyses

Analysis of variance (ANOVA) was used to compare the levels of the hemolymph parameters for the different temperature groups. ANOVA was also used to compare the hemolymph parameters analyzed at the termination of air exposure with vitality indexes, and to compare hemolymph parameters for crabs that survived or died during reimmersion. Data sets of equal variance was analyzed using l-way ANOVA, and subsequently with Bonferroni or Tukey's pairwise tests. Data of unequal variance were compared using nonparametric Kruskal-Wallis 1-way ANOVA, with further pairwise comparisons made using the Mann-Whitney test. Significance was considered to be P < 0.05 for all tests. SYSTAT v. 10.2 (SYSTAT Software Inc., Chicago, IL) was used in the analysis.

RESULTS

Seawater temperature during revitalizing and reimmersion was 11.5 [+ or -] 0.5[degrees]C in trial 1 (October) and 15.5 [+ or -] I[degrees]C in trial 2 (August; Table 3).

Hemolymph Parameters

Mean hemolymph pH for the reference groups bled immediately after removal from the seawater was 7.85 and 7.94, and for TA was 171 taM and 200 taM, all within levels considered normal for brown crab (Table 2). The variation in hemolymph lactate between individuals was high 2.2 [+ or -] 3.4 mM and 4.8 [+ or -] 5.7 mM in trials 1 and 2, respectively. In trial l, hemolymph lactate was more than 5 mM for l crab (10%). In the next trial, hemolymph lactate was higher than 5 mM for 16 crabs (25%).

Hemolymph pH decreased during emersion and was most pronounced at higher temperatures. The lowest mean value was measured for the crabs immersed for 18 h at 20[degrees]C both in trials 1 and 2 (Fig. 1). A decrease in pH was also apparent after 6 h of emersion for crabs stored at 5[degrees]C, before returning to the approximate initial value after 48 h of exposure in both trials. A similar pattern was seen for hemolymph TA and lactate with increasing values during emersion, with the highest increase at 15[degrees]C and 20[degrees]C (Fig. 1). Some obvious differences between the trials were found for the crabs immersed at 20[degrees]C for 18 h. Hemolymph TA and lactate were 2-3 times higher in trial 2 (TA, 1,648 [+ or -] 65 [micro]M; lactate, 44 [+ or -] 2.9 mM) than in trial 1 (TA, 834 [+ or -] 86 [micro]M; lactate, 23.8 [+ or -] 1.5 mM).

Hemolymph glucose level showed a different response than the other parameters. The concentration increased during the first 12-24 h, then decreased. At the termination of trial l, there was no significant difference in glucose concentration between the experimental groups and the reference group (Fig. 1).

Vitality

Crabs in the reference groups were all strong (vitality index 5). During emersion, the crabs lost the prompt and aggressive claw response. Strong immersed crabs were therefore classified as healthy (vitality index 4; Table 3).

During both trials, most of the crabs exposed to the lower temperatures (2[degrees]C and 5[degrees]C) remained healthy (index 4) throughout an emersion period of 48 h. After 72 h, weak and moribund crabs were noted; after 96 h, 4 crabs (25%) were dead (trial 2; Table 1). At the highest temperature (20[degrees]C), the crabs appeared to be in good vitality after 6 h. However, after 12 h, 70% of the individuals in trial 1 and 100% in trial 2 were weak or moribund. After 18 h, at the termination for emersion at this temperature, 40% and 44% of the individuals were dead (Table 1).

To illustrate the differences in air temperature, the mean vitality index for the individuals was plotted against the emersion time at the different temperatures (Fig. 2). Results showed that at 2[degrees]C, 5[degrees]C, and 10[degrees]C no marked changes in vitality indexes were observed within the time of the experiment. At higher temperatures, average vitality dropped quickly with increasing time, in agreement with our earlier discussion (Fig. 1 and Table 1).

Relation between Vitality Indexes" and Hemolymph Parameters

When hemolymph samples were grouped according to vitality index, differences in the hemolymph parameters were found. Deterioration for each vitality degradation was found for pH in trial 1 (ANOVA, [F.sub.3,253] = 80.14, P < 0.001) and in trial 2 ([F.sub.3,357] = 106.9, P < 0.001 ; Fig. 3). This was also the case for TA in trial 1 ([F.sub.3,241] = 127, P< 0.001) and in trial 2 ([F.sub.3,340] = 100.0, P < 0.001; Fig. 3). Deterioration for each vitality degradation was also found for hemolymph lactate in trials 1 and 2; however, it was not significant between weak and moribund crabs (Fig. 3). No trend between vitality indexes and hemolymph glucose was found (Fig. 3).

Delayed Mortality during Reimmersion

Delayed mortality during reimmersion increased with time and was highest for the crabs that were moribund at the termination of the air exposure. Thirty-nine percent of these crabs died within 48 h in trial 1 and 24% in trial 2 (Table 4). However, the majority of the crabs were healthy at the end of emersion. Only a small portion of these died, none after 24 h reimmersion, and 1% between 24 and 48 h in both trials (Table 4).

[FIGURE 1 OMITTED]

Differences were found in hemolymph parameters measured at the termination of aerial exposure for crabs that survived or died during reimmersion. Hemolymph pH was lower, and TA and lactate levels were higher (i.e., a worse physiological condition for those crabs that died after 24 h of reimmersion; Table 5). This was also the state after 48 h of reimmersion for pH (trial 2), TA (trials 1 and 2), and lactate (trial 1 ; Table 5). For hemolymph glucose, no differences were found between the crabs that survived and those that died (Table 5).

[FIGURE 2 OMITTED]

Recommended Maximum Time for DO, Storage

Based on the previous results, an illustration was made to suggest roughly the time available before reduced vitality can be expected under the various temperatures (Fig. 4). In the Figure 4, 2 exponential lines were fitted by eye to the observations of weak and moribund crabs in the overall material. A combination of temperature and holding time that places one below the lower line is expected to keep crabs in overall good vitality. Similarly, ending above the upper line in Figure 4 at the same temperatures is likely to result in a marked reduction of overall vitality and a rapid increase in mortality. According to this figure, crabs may tolerate dry storage at 5[degrees]C for up to 72 h and still have a good vitality, whereas storage exceeding 90 h is likely to result in a marked reduction of overall vitality. At 10[degrees]C, the same interval will be 30-44 h; at 15[degrees]C, 11-18 h; and at 20[degrees]C, 5-9 h.

[FIGURE 3 OMITTED]

DISCUSSION

During dry storage and transport of live crabs, aerial exposure is an important stress factor during the value chain from catch to final destination. In the current study, several biomarkers that can be used to evaluate health and stress conditions were measured and compared with vitality indexes during laboratory experiments simulating dry storage and transport at different temperatures and durations. Two experiments were conducted: one at the beginning and one at the peak season of crab fishing. No between differences in vitality were observed within the 2 experiments.

Revitalizing of Experimental Animals and References Groups

Revitalizing the crabs before emersion was conducted by stacking the crabs tightly in boxes in a rack with flow-through water. This is often used in the Norwegian fishery, aboard fishing boats, or at the receiving stations. Unexpected high levels of hemolymph lactate (>5 mM) were found for individual crabs in the reference groups bled immediately after revitalizing, approximately 10% in trial 1 and 25% in trial 2. Lactate levels for the remaining crabs were similar to previous studies by Danford (2001) (1.49 [+ or -] 0.12 mM). Hemolymph lactate levels are an indicator of hypoxic stress for decapods (Truchot 1980, Booth et al. 1982), and it appears that such stress occurred during revitalization in this study, deduced from the large variance in lactate levels, which indicated that some crabs were not receiving an adequate oxygen supply. The most conspicuous reason for this is unequal water drainage through the boxes. However, it is not known how stacking in boxes affects the crabs. Depending on how tightly the animals were packed, being immersed, water provide lift and animals can be free to move or interact aggressively, an event increasing glycaemia. Some individuals may find this more stressful than others. We know that large variations are observed in diurnal rhythm (Ansell 1973, Aldrich 1975, Skajaa et al. 1998) and in individual crab activity levels (Skajaa et al. 1998). A few crabs in the study by Danford (2001) were also found to have high lactate levels despite 2 wk of revitalization in large tanks at 12[degrees]C.

During revitalization, the seawater temperature was 3-4[degrees]C higher in trial 2 than in trail 1. The level of dissolved oxygen in seawater is inversely correlated with temperature. The fact that higher temperatures increase activity levels and oxygen demand (Ansell 1973) may explain the higher variance in lactate levels in trial 2. A buildup in hemolymph lactate levels under hypoxic conditions was described for the velvet crab Necora puber, and the levels were found to be significantly lower in water at 10[degrees]C than at 16[degrees]C (Whyman et al. 1985).

Hemolymph Parameters

Hemolymph acidosis may, to some degree, be compensated for in different species, but it is not known how this functions in the brown crab. As seen from the drop and then rise in the pH of crabs stored at 5[degrees]C, this probably occurs when crabs are stored at cold temperatures. Two compensatory mechanisms have been postulated for crustaceans in general, both involving a change in hemolymph strong ion differences: (1) increasing strong ion differences by dissolving the calcium carbonate of the exoskeleton, releasing calcium and carbonate ions into the hemolymph (DeFur et al. 1980, Hagerman & Uglow 1982); and (2) intra- and extracellular [Na.sup.+]/[H.sup.+] (N[H.sub.4.sup.+]) and [Cl.sup.-1] /HC[O.sub.3] exchanges (Cameron 1986).

Hemolymph lactate and TA levels increased with exposure time, and the rise was greater for the crabs immersed at higher temperatures. There was, however, a difference between trials for the crabs exposed to 20[degrees]C, with the levels of both lactate and TA reaching much higher levels in crabs from trial 2. The reason for this difference is unclear.

[FIGURE 4 OMITTED]

Lactate and TA levels for crabs exposed to the lower temperatures (2[degrees]C and 5[degrees]C) never reached a level comparable with the crabs exposed to the high temperatures. They even remained relatively constant after 12-24 h until the termination of the treatment. A significant decrease in lactate levels has been recorded in the spiny lobster Panulirus interruptus after 38 h of emersion. Gdmez-Jimenez (1998) suggested that this might indicate that prolonged emersion improved oxygen uptake (gill diffusing capacity). This is supported by similar findings from Taylor and Whiteley (1989) and Taylor and Waldron (1997). This study indicates that a similar adaptation may occur for brown crab during long and cold storage.

Hemolymph glucose increased during the first 12-24 h of emersion at all temperatures, then decreased to approximate initial values in both trials. Glucose, as a frequently used indicator of immediate stress in crustaceans (Telford 1974), also gave mixed results in examination of lobsters, where some of the severely stressed animals had baseline glucose levels. This finding is consistent with other recent studies (Hall & Van Ham 1998) that questioned the orthodoxy that stressed crustaceans are necessarily hyperglycemic. A possibility is that the elevated glucose level in the hemolymph cannot be sustained indefinitely and falls back to baseline levels if the stress is prolonged (Paterson et al. 2005).

Hemolymph Parameters Compared with Vitality Indexes

The physiological variables of the hemolymph pH, lactate, and TA were significantly different between strong, healthy, weak, and moribund crabs. They were all negatively influenced when compared with strong crabs (reference group), and primarily for the moribund crabs, indicating that the vitality indexes based on the reflexes used in the experiment may be an indicator to evaluate the physiological status of the brown crab. This finding is consistent with other experiments that have been conducted to identify easy methods to determine health status in rock lobster (Paterson et al. 2005).

Expected Time Available during Air Exposure

The results obtained demonstrate that the time available for dry holding is markedly reduced if temperature increases above 10[degrees]C, if the aim is to keep the crabs in good vitality when arriving at the destination. The time shown in Figure 4 is a conservative guideline of the time available during air exposure at various air temperatures. Figure 4 is based on limited data available from this study; a more detailed study of time available during air exposure between 10[degrees]C and 15[degrees]C is recommended. In the commercial fishery, other factors are likely to be of additional importance to vitality, such as internal injuries and physical damage caused by rough handling, environmental factors like draught and low air humidity during storage and transport, and insufficient sorting practice retaining weak and low-quality crabs. In our study, only crabs that were hard-shelled, healthy, and showed no signs of damage were selected, and thus might not represent an insufficiently sorted catch. The study is also based on a limited size range of female crabs, so the effect of size and sex was not investigated. The results should, thus, be treated with caution, and vitality indexes (including mortality) based on air temperature and duration should also be verified through examination of commercial dry storage and transportation.

Delayed Mortality during Reimmersion

Delayed mortality during reimmersion was associated mostly with crabs that were assessed as moribund at the end of air exposure, but also with crabs that were assessed as weak. Only 1% of the crabs assessed as healthy were dead after 48 h of reimmersion in trials 1 and 2 compared with 39% and 24% of those that were moribund at the end of emersion. Adaptation periods from 2-14 days to stabilize mortality during reimmersion after dry transport stress have previously been reported for brown crab (Woll & Berge 2007). According to this, a higher mortality would likely appear during further reimmersion in this study.

Several experiments have been conducted to identify simple and reliable methods to determine health status and to predict mortality during reimmersion. Davis and Ottmar (2006) discovered that easily acquired observations on a suite of simple reflex actions provided excellent predictions of mortality in fishes related to both physiological stressors (e.g., thermal stress, air exposure) and physical injury. For crab (Chionoecetes spp.), Stoner et al. (2008) identified 6 reflexes that were stereotypical, repeatable, and easy to assess. The reflex impairment provided an excellent predictor of delayed mortality independent of gender, size, and shell condition, and predicted mortality in crabs with no obvious external damage. Our results are similar to previous studies (Stevens 1990, Stoner et al. 2008) that showed that reflexes associated with the eye and mouth were least sensitive, and that mouth movement was maintained in crabs close to death. Leg retraction and kick (i.e., immediate strong agitation of legs when abdominal flap was lifted) were important reflexes easily weakened or lost.

In the commercial live value chain for brown crab, the results obtained from the current study should be considered during all steps, from onboard storage until arriving at the final destination. Dry storage may improve the logistics and reduce the cost of transportation, but must be carried out within the critical limits to ensure the animals' well-being and survival.

ACKNOWLEDGMENT

This study was supported by the Research Council of Norway (no. 158944/120) and, in part, by an European Commission grant (CrustaSea, coll-CT-2006-030421). The authors extend special thanks to Roger F. Uglow (University of Hull) for collaboration at project realization. Acknowledgements also go to the staff at Moreforsking Matin; to Jan E. Dyb, who assisted with the laboratory work: and to James Kennedy, for useful comments on the manuscript.

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ASTRID K. WOLL, * WENCHE E. LARSSEN AND INGE FOSSEN ([dagger)]

Moreforsking Marin, PO Box 5075, N-6021 Alesund, Norway

* Corresponding author. E-mail: astrid@mfaa.no

([dagger]) Current address: MonAqua as, Industriveien 18, N-6517 Kristiansund, Norway
TABLE 1.

Vitality and percent mortality (M) during simulated emersion at
different temperatures and length (treatment), and during reimmersion.

                  Treatment

Trial         Temperature     h     n
              ([degrees]C)

1                   2        24     20
                             48     20
                             72     20

                    5        24     20
                             48     20
                             72     21

                   15        12     20
                             24     20
                             36     22

                   20         6     20
                             12     20
                             18     30

Sum trial 1                        253

2                   5         6     16
                             12     16
                             24     16
                             48     16
                             72     16
                             96     16

                   10         6     16
                             12     16
                             24     16
                             36     16
                             48     16

                   15         6     16
                             12     16
                             18     16
                             24     16

                   20         3     16
                              6     16
                             12     16
                             18     16

Sum trial 2                        304

                                     Vitality End Treatment (n)
                  Treatment

Trial         Temperature     h    Healthy   Weak   Moribund   Dead
              ([degrees]C)

1                  2         24        18      2       0        0
                             48        19      1       0        0
                             72        19      0       1        0

                   5         24        20      0       0        0
                             48        20      0       0        0
                             72        18      2       0        1

                   15        12        15      3       0        2
                             24        11      5       1        3
                             36         5      7       8        2

                   20         6        19      1       0        0
                             12         6      5       9        0
                             18         0      1      17       12

Sum trial 1                           170     27      36       20

2                  5          6        16      0       0        0
                             12        15      1       0        0
                             24        16      0       0        0
                             48        15      1       0        0
                             72        14      2       0        0
                             96         8      4       0        4

                   10         6        16      0       0        0
                             12        16      0       0        0
                             24        16      0       0        0
                             36        15      1       0        0
                             48        14      1       1        0

                   15         6        14      2       0        0
                             12        13      3       0        0
                             18         5      9       4        0
                             24         6      8       2        0

                   20         3        13      3       0        0
                              6        13      3       0        0
                             12         0     11       5        0
                             18         0      2       7        7

Sum trial 2                           225     51      19       11

                                         M Reimmersed (n)
                  Treatment
                                   % M
Trial         Temperature     h    Air   0-24 h   24-48 h
              ([degrees]C)

1                  2         24     0       0        0
                             48     0       0        0
                             72     0       0        2

                   5         24     0       0        0
                             48     0       0        0
                             72     5       0        1

                   15        12    10       0        1
                             24    15       2        0
                             36     9       4        0

                   20         6     0       0        0
                             12     0       1        0
                             18    40       5        3

Sum trial 1                                12        7

2                  5          6     0       0        0
                             12     0       0        0
                             24     0       0        0
                             48     0       0        0
                             72     0       1        2
                             96    25       0        0

                   10         6     0       0        0
                             12     0       0        0
                             24     0       0        0
                             36     0       0        0
                             48     0       1        0

                   15         6     0       0        1
                             12     0       0        0
                             18     0       1        2
                             24     0       0        0

                   20         3     0       0        0
                              6     0       0        0
                             12     0       3        3
                             18    44       0        2

Sum trial 2                                 6       10

                  Treatment                   % M

Trial         Temperature     h    Reimmersion     Total
              ([degrees]C)              *        ([dagger])

1                  2         24         0            0
                             48         0            0
                             72        10           10

                   5         24         0            0
                             48         0            0
                             72         5           10

                   15        12         6           15
                             24        12           25
                             36        20           27

                   20         6         0            0
                             12         5            5
                             18        44           67

Sum trial 1

2                  5          6         0            0
                             12         0            0
                             24         0            0
                             48         0            0
                             72        19           19
                             96         0           25

                   10         6         0            0
                             12         0            0
                             24         0            0
                             36         0            0
                             48         6            6

                   15         6         6            6
                             12         0            0
                             18        19           19
                             24         0            0

                   20         3         0            0
                              6         0            0
                             12        38           38
                             18        22           56

Sum trial 2

* Percent mortality (M) calculated for crabs that survived emersion.

([dagger]) Mortality calculated for start number of crabs.

n, number of crabs in each subsample.

TABLE 2.

Live weight and hemolymph parameters (mean [+ or -] SD) for reference
groups bled immediately after 2 days of revitalization in ambient
flow-through seawater (SW).

Trial      SW Temperature      n        Weight (g)
            ([degrees]C)

1         11.5 [+ or -] 0.5   20 *    504 [+ or -] 12
2         15.5 [+ or -] 1.0   64      511 [+ or -] 87
P value                                    0.781

Trial               pH              TA ([micro]M)      Lactate (mM)

1         7.94 [+ or -] 0.07 (a)   171 [+ or -] 51   2.2 [+ or -] 3.4
2         7.85 [+ or -] 0.12 (b)   201 [+ or -] 65   4.8 [+ or -] 5.7
P value           0.001                 0.059             0.373

Trial       Glucose (mM)

1         1.4 [+ or -] 0.43
2         1.2 [+ or -] 0.28
P value         0.062

* n = 10 for lactate and glucose.

Values with different superscript letters are significantly different
at the 5% level. TA, total ammonia.

TABLE 3.
Responses used to assess vitality of Cancer pagurus.

Vitality Index                      Response

5, strong        Strong claws with prompt response (aggressive)
                 Claws end legs held into abdomen and prevent
                   access to the area (defensive)
                 Grips hand when held upside down
                 Mouthparts strong
                 Eyestalk response
4, healthy       Like index 5; however, aggressive/defensive
                   response still strong but not prompt
3, weak          Claws and legs weak with slow response
                 Loose grip when held upside down
                 Mouthparts strong
                 Eyestalk response
2, moribund      No or slight movements in legs and claws
                 No grip when held upside down
                 Mouthparts stack
                 Eyestalk response
1, dead          No movement in mouthparts when touched
                 No eyestalk response

TABLE 4.
Cumulative mortality (M) during reimmersion for crabs that
were healthy, weak, or moribund at the end of emersion.

         At End of Emersion    % M at Reimmersion

Trial   Vitality      n         24 h       48 h

1       Healthy      170          0          1
        Weak          27          7         11
        Moribund      36         28         39
2       Healthy      225          0          1
        Weak          51          6         18
        Moribund      19         12         24

TABLE 5.
Hemolymph test results (Kruskal-Wallis test) at the termination of
emersion separated by crabs that survived or died during reimmersion.

                                             Hemolymph
                                             Parameter
                                         (mean [+ or -] SD)

Trial            Reimmersion       n             pH

1       0-2 4h        Survivors   221    7.64 [+ or -] 0.28
                      Dying        12    7.14 [+ or -] 0.17
                      P value                  <0.000
        24-48 h       Survivors   214    7.64 [+ or -] 0.27
                      Dying         7    7.42 [+ or -] 0.37
                      P value                    NS
2       0-24 h        Survivors   287    7.54 [+ or -] 0.21
                      Dying         6    7.18 [+ or -] 0.34
                      P value                  0.006
        24-48 h       Survivors   277    7.55 [+ or -] 0.21
                      Dying        10    7.36 [+ or -] 0.23
                      P value                  0.014

                                      Hemolymph
                                      Parameter
                                  (mean [+ or -] SD)

Trial            Reimmersion              TA

1       0-2 4h        Survivors     588 [+ or -] 350
                      Dying       1,634 [+ or -] 455
                      P value           <0.001
        24-48 h       Survivors     573 [+ or -] 336
                      Dying       1,035 [+ or -] 473
                      P value           0.012
2       0-24 h        Survivors     407 [+ or -] 257
                      Dying       1,297 [+ or -] 429
                      P value           <0.001
        24-48 h       Survivors     397 [+ or -] 247
                      Dying         742 [+ or -] 368
                      P value           0.002

                                      Hemolymph
                                      Parameter
                                  (mean [+ or -] SD)

Trial            Reimmersion           Lactate

1       0-2 4h        Survivors     23 [+ or -] 23.6
                      Dying         60 [+ or -] 11.5
                      P value           0.002
        24-48 h       Survivors   22.5 [+ or -] 23.4
                      Dying       40.4 [+ or -] 11.9
                      P value           0.052
2       0-24 h        Survivors   14.3 [+ or -] 7.7
                      Dying       22.7 [+ or -] 6.5
                      P value           0.017
        24-48 h       Survivors   14.2 [+ or -] 7.6
                      Dying       16.9 [+ or -] 9.7
                      P value             NS

                                      Hemolymph
                                      Parameter
                                  (mean [+ or -] SD)

Trial            Reimmersion           Glucose

1       0-2 4h        Survivors    2.1 [+ or -] 1.1
                      Dying        2.1 [+ or -] 1.5
                      P value             NS
        24-48 h       Survivors    2.1 [+ or -] 1.1
                      Dying        1.8 [+ or -] 1.0
                      P value             NS
2       0-24 h        Survivors    2.0 [+ or -] 0.6
                      Dying        1.8 [+ or -] 0.5
                      P value             NS
        24-48 h       Survivors    2.0 [+ or -] 0.6
                      Dying        2.0 [+ or -] 0.6
                      P value             NS

NS, not significant; TA, total ammonia.
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Author:Woll, Astrid K.; Larssen, Wenche E.; Fossen, Inge
Publication:Journal of Shellfish Research
Article Type:Report
Geographic Code:4EXNO
Date:Aug 1, 2010
Words:7369
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