The effect of extended wet-storage on the condition, physiology and stress response of cultured blue mussels (Mytilus edulis L. 1758) during summer and fall in northeastern Newfoundland

Aquaculture 372–375 (2013) 111–118

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The effect of extended wet-storage on the condition, physiology and stress response of cultured blue mussels (Mytilus edulis L. 1758) during summer and fall in northeastern Newfoundland Jessica Wyatt a, b, Sharon Kenny a, Kimberly D. Hobbs a, Terry Mills c, H. Dawn Marshall d, Harry M. Murray a,⁎ a

Fisheries and Oceans Canada, 80 White Hills Road, P.O. Box 5667, St. John's, NL, Canada, A1C 5X1 School of Fisheries, Marine Institute of Memorial University of Newfoundland, St. John's, NL, Canada, A1C 5R3 c NorLantic Processors Ltd., P.O. Box 381, Botwood, NL, Canada, A0H 1E0 d Department of Biology, Memorial University of Newfoundland, St. John's, NL, Canada, A1C 5S7 b

a r t i c l e

i n f o

Article history: Received 18 June 2012 Received in revised form 30 October 2012 Accepted 1 November 2012 Available online 9 November 2012 Keywords: Mytilus edulis Extended holding Physiology Condition index Innate immunity

a b s t r a c t In order to determine the effects of extended wet-storage under ambient conditions we investigated the physiological response of blue mussels held for up to 3 months in a commercial holding facility. During the summer and the fall there was a significant decline in dry weight and condition of mussels held over time when compared to field controls. Neutral red retention time decreased significantly in holding for both seasons but not in the field controls, indicating an increase in stress response for held mussels. Little variation was noted in the expression of the oxidative stress genes superoxide dismutase and GSH-peroxidase during the summer or fall regardless of treatment. Expression of the antimicrobial peptide MGD2 was evident in the summer holding samples as early as 1 week but did not occur in the fall holding samples until 3 months. This suggests that holding conditions have stimulated the immune response in a manner potentially related to time in holding and environmental temperature. Based on our results we recommend that one month should be the maximum time allowable in ambient extended holding during warm water seasons. During the warmest part of the summer this could be reduced to two weeks. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.

1. Introduction In Newfoundland, Canada, Mytilus edulis (L. 1758) is farmed on the northeast and south coasts of the island. Since 2001 production has increased from 1452 to 2500 t in 2010 (Statistics Canada, 2011) and the industry is continuing to expand. In Atlantic Canada mussels are cultured using the long line suspended culture system, in contrast to the bottom culture practices used in the Netherlands (Mallet et al., 1990) or the raft-system used in Spain. Mussels generally reach market size (55 mm to 60 mm) in 20 months and can be harvested year-round depending on meat quality and yield, which vary seasonally (Mallet et al., 1990). Newfoundland mussels are shipped to local, national, and international markets; unfortunately fresh product may not ship directly to market immediately following harvest and processing due to unforeseen transportation or weather conditions. As a result mussels may spend extended time in ambient wet storage. In fact, it is a common occurrence for mussels to be held in a wet-holding facility for up to one week before processing and shipping and in more extreme cases they could spend up to one month or longer. Short-term storage ⁎ Corresponding author. Tel.: +1 709 772 2302; fax: +1 709 772 5315. E-mail address: [email protected] (H.M. Murray).

in ambient water results in the lowest degree of stress response compared to storage on ice or in chilled air (Harding et al., 2004). Extended time in wet holding may cause an increase in stress response and consequently a decrease in condition and meat yield. Determining the maximum time spent in holding before there is a significant decrease in meat yield and shelf life is crucial to the industry. Several factors can affect condition and physiological stress response in mussels during holding; these include temperature, population density, reproductive effort, and food availability (Karayücel and Karayücel, 2000; Smaal and van Stralen, 1990). Each of these factors can also be affected by seasonality (e.g., Dare and Edwards, 1975; Okumuş and Stirling, 1998; Orban et al., 2002). Few studies have examined the effects of extended wet-storage on the physiology of mussels however some recent studies have investigated the influence of environmental parameters on immune system function and oxidative stress response. Nunez-Acuna et al. (2012) evaluated variations in the expression of Mytilin B, Defensin, SOD and Catalase in Mytilus chilensis as a response to environmental stressors. It was noted that changes in expression could be correlated with temperature, chlorophyll levels and the influx of fresh water. Similarly, Li et al. (2009) found that while not quantitative, variation in expression of some immune related genes in Mytilus galloprovincialis could be correlated with fluctuations in temperature (i.e. MGD2, Myticin B, and lysozyme) and salinity (i.e. MGD2). These

0044-8486/$ – see front matter. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquaculture.2012.11.002

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studies suggest that physiological parameters related to oxidative and immune system stress can be influenced by changes in environmental conditions. It is the objective of the present study to determine whether length of time in a holding facility and its associated environment have an effect on the condition and physiology of the blue mussel, M. edulis during the summer and fall seasons. Stress response was evaluated by monitoring changes in lysosomal membrane stability over time in holding through the neutral red assay compared to similar changes in mussels under field conditions. Changes in mRNA expression of oxidative stress genes (superoxide dismutase and GSH peroxidase) and the antimicrobial peptide MGD2 over time were evaluated for mussels in holding and directly from the harvest site (field controls) using reverse transcriptase PCR. 2. Materials and methods 2.1. Study site, experimental setup, and sampling protocol Mussels from the 2008 year-class were collected from Site 13 in Bulley's Cove, Newfoundland and Labrador, Canada, and transported to a commercial processing facility in nearby Pleasantview. At the beginning of each experiment unprocessed mussels were placed into two 1000 l holding tanks at an industry standard stocking density of 0.362 kg/l with a continuous flow-through of water. Water was pumped into the tanks directly from the bay at a distance of 145 m from the facility and at a depth of approximately 13 m from the surface. Water temperatures in the holding tanks were recorded daily by workers in the facility. Temperature checks with water from the main intake showed that there was no difference between this and water in holding, so for the purpose of the present experiment only those temperatures in tanks were recorded and summarized. For both summer and fall experimental seasons, mussels were sampled at four time points; initially upon introduction into the processing facility, then after 1 week, 1 month and 3 months in holding. At each sample time mussels were sampled at random from one of the two holding tanks and simultaneously from the original grow-out site. Subsamples were taken for morphometric analysis, neutral red assay, and gene expression analysis. 2.2. Morphometric analysis At each sampling period, 150 mussels were randomly sampled for morphometric and condition index analysis. Mussels were placed in coolers on ice and transported back to St. John's, Newfoundland and Labrador, for measurement and analysis. For each individual, the total weight (g) was measured to the nearest 0.001 g. Length (mm; maximum anterior–posterior axis), depth (mm; maximum dorso-ventral axis), and width (mm; maximum lateral axis) were measured using a digital caliper (MasterCraft) to the nearest 0.1 mm. Following morphometric measurements, the meat was carefully dissected away from the shell and placed in preweighed aluminum trays for dry weight analysis. Meats were dried to a constant weight at 80 °C for 48–72 h (modified from Lutz et al., 1980) and weighed to the nearest 0.001 g. Shells were allowed to air dry for 48–72 h and their weight was measured to the nearest 0.001 g. Condition index was calculated as the ratio of dry tissue weight to dry shell weight × 100%. 2.3. Neutral red assay The Neutral red protocol was modified from Lowe et al. (1995a, 1995b) and Harding et al. (2004). Briefly, 12 mussels were randomly sampled from the holding tank and simultaneously from the grow-out site for neutral red analysis. Initially, 0.1 ml of hemolymph was extracted from the posterior adductor muscle into a 1 ml syringe with a 22.5 gauge needle containing 0.1 ml of mussel physiological saline

(4.77 g HEPES, 25.48 g NaCl, 13.06 g MgSO4, 0.75 g CaCl2 in 1 l Millipore water; pH 7.3). Once hemolymph was extracted the needle was removed to reduce shear stress on the cells and the solution was pipetted into a 1.5 ml siliconized Eppendorf® tube. Once the 12 mussels had been bled the tubes were carefully inverted to mix the suspension and 40 μl was pipetted onto the center of a poly-L lysine coated slide (20 μl in 100 μl distilled water). The slides were then transferred to a lightproof humidity chamber and incubated for 15 min to allow cells to adhere. Subsequently, excess fluid was drained off the slide by gently tapping the long edge on a paper towel. A stock solution of neutral red was prepared (28.8 mg of neutral red in 1 ml of dimethyl sulfoxide (DMSO)). Working solution was made by diluting 20 μl in 5 ml of physiological saline. An aliquot was then added onto the middle of the slide and the slides were incubated in a lightproof humidity chamber for an additional 15 min before a cover slip (18 × 18 mm) was added and initial observations were recorded. Using a compound microscope observations were made after 15 min and then at 30 minute intervals up to 180 min. Cells were first located at 10× and then examined in detail under 40× objective. Observations were recorded by assigning a four point numerical score (1–4) to 25 cells per field of view. The scoring scale was assigned so that (1) represented low stress indicated by the appearance of tiny pink dots (lysosomes) in the cytosol, (2) represented moderate stress as indicated by an increase in lysosome size, (3) represented moderate high stress as indicated by leakage of dye from lysosomes to the cytosol, and (4) represented high stress response indicated by increased membrane degradation, lysosomal vacuolation, or rounding up of cells. Observations were terminated for a given slide, once 50% of the cells showed a high degree of stress response. Retention times were compared with reference to sample time and treatment only. Direct comparisons were not drawn between treatments (field control and holding) due to the variation in field sample origin. As an alternative, One-Way ANOVA was applied to the calculated difference between initial observation and final. Differences were reported based on significant p values. 2.4. Gene expression 2.4.1. Tissue samples Gill tissues were dissected from both the holding and freshly harvested mussels and placed in a 1.5 ml Eppendorf tube containing 1 ml of RNAlater (Ambion, Austin, TX). Tissues were stored on ice and transported back to St. John's, Newfoundland and Labrador, where they were placed at −20 °C. Hemocyte samples for RNA analysis were also collected. Approximately, 1–2 ml of hemolymph was extracted from the posterior adductor muscle, transferred to a 2 ml Eppendorf tube and centrifuged at 800 ×g for 15 min at room temperature. Following centrifugation the supernatant was carefully decanted, the pellet resuspended in RNAlater, and the tube placed on ice for transport to the laboratory whereupon samples were stored at −20 °C until RNA extraction. 2.4.2. RNA extraction and cDNA synthesis Total RNA was extracted from mussel gill tissue using the RNeasy Mini Kit (Qiagen, Mississauga, ON, Canada) according to the manufacturer's instructions. RNA was quantified using Nanodrop 2000 (Thermo Fisher Scientific Inc., Wilmington, DE). Hemocyte total RNA was extracted from hemocyte pellets by resuspending the pellet and transferring it to a 15 ml RNase-free tube. The resuspended pellet was further diluted with at least 2–3 × the volume of mussel physiological saline and centrifuged at 4 °C for 30 min at 2000 ×g. Once the majority of the supernatant was removed, 600 μl of lysis buffer with β-mercaptoethanol was added and the lysate transferred to a 1.5 ml RNase-free Eppendorf tube for homogenization and extraction using the RNeasy Mini Kit with the manufacturer's recommended instructions for animal cells. Total

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Table 1 Primers used in cDNA synthesis and PCR analysis. Primer

%GC

Tm (°C)

Amount of oligo (nM)

Sequence

Product size (bp)

18s rRNA MT F 18s rRNA MT R 18s rRNA HE F 18s rRNA HE R SOD F SOD R GSH peroxidase MT F GSH peroxidase MT R MGD2 F MGD2 R

45 55 50 55 50 50 40 45 55 50

53.0 56.8 54.5 55.9 55.2 55.2 51.2 52.8 57.1 53.9

30.6 31.6 26.3 24.4 28.9 31.2 39.3 28.2 18.7 33.6

5′-CACGCCTGGGAATTTTCTTA-3′ 5′-ACCCACACCGTGATTCTCTC-3′ 5′-GCTTTGCCTTTCGGTACTTG-3′ 5′-CGGAGTAGTCGATCGTGGAT-3′ 5′-ACTGCCAAACCTTCCGTATG-3′ 5′-AATCCTCCACCGTTGTTGAC-3′ 5′-TTCATCAATTTTCGGCCTTC-3′ 5′-GGGCATTCAGGATTTCTTCA-3′ 5′-GATGCACGGGAGGTTACTGT-3′ 5′-CTGCAGCCTGCTGGTTATGATTG-3′

238 212 240 237 211

RNA was eluted into a 30 μl final volume and quantified using Nanodrop 2000 (Thermo Fisher Scientific Inc., Wilmington, DE). First strand cDNA was synthesized from 1 μg of purified total RNA using the QuantiTect Reverse Transcription Kit (Qiagen, Mississauga, ON, Canada) according to the manufacturer's instructions. Oxidative stress and immune gene expression were examined using PCR primers designed from MytiBase sequences (http://mussel.cribi.unipd.it/) and Primer 3 software (http://primer3.sourceforge.net/) (Table 1). Expression profiles of genes of interest were compared to the expression of a normalizer gene (18S ribosomal RNA). PCR reactions were carried out on 2.5 μl of the cDNA using the Taq PCR Core Kit (Qiagen, Mississauga, ON, Canada) in a total reaction volume of 25 μl. The amplification conditions were: 3 min at 95 °C; 30 cycles of 1 min at 95 °C, 1 min at 50 °C, 30 s at 72 °C; and 10 min at 72 °C. Amplification products were resolved on a 2% agarose gel containing ethidium bromide (500 μg/ ml) and run at 100 V for 1 h and compared to a 1 kb+ DNA ladder (Invitrogen).

seasons the change in measured parameter (wet weight, dry weight, and condition index) was calculated for each experiment and then the mean change was tested for differences using One-Way Analysis of Variance.

2.5. Statistical analysis

3.1. Morphometric analysis

Morphometric and neutral red data were analyzed using Sigmaplot 11.0 statistical and graphical software (Systat software). Data were tested for normality and means (± SE) were calculated. One-way ANOVA and the appropriate post hoc tests were conducted. Significance was set at α = 0.05. Since the assumptions of equivalence and normality were not met, the Kruskal–Wallis One Way Analysis of Variance based on ranks was used. Additionally due to the large amount of variability in field controls over time and season, it was difficult to compare holding and controls directly from a statistical perspective. To alleviate this problem and compare holding and field control growth and condition data directly within and between

Morphometric measurements during the summer for mussels in holding and field controls are shown in Tables 2 and 3 respectively. Mussels in holding showed little change in mean shell length, depth, or width during the summer. Mean shell length remained between 56 mm and 60 mm for the holding mussels while there was a significant increase in mean shell length from 58.83 mm to 70.53 mm for the fresh harvest mussels. Morphometric measurements for fall holding and fresh harvest mussels are shown in Tables 3 and 4 respectively. Mussels that remained in extended holding during the fall also showed little significant variation in mean shell length remaining between 69 mm and 71 mm. The mean shell length for fall fresh field control mussels varied at each sample time however there was a significant decrease from 70.53 mm initially to 65.64 mm at the final sample. Changes in condition and meat weight for holding and field control mussels during both the summer and fall are shown in Fig. 2. Mean wet tissue weight decreased significantly for mussels held

3. Results The average monthly water temperatures in holding during the experiment from June to December 2010 are shown in Fig. 1. Average water temperatures during the summer ranged from 6.85 °C in June to 15.66 °C in August. Due to high water temperatures both of the experimental holding tanks suffered massive mortalities during the summer holding trial (in mid-August) however a sample was saved and frozen for analysis. The final fresh harvest mussels were collected at the scheduled three-month sample time in September 2010. During the fall initial average water temperatures were approximately 15 °C in September decreasing to 2.0 °C in December.

Table 2 Morphometric characteristics of Mytilus edulis during the summer holding trial (2008 year class).

Fig. 1. Average monthly water temperature (°C) recorded in holding tanks from June to December 2010.

Parameter

Initial

One week

One month

Two months

Length (mm) Width (mm) Depth (mm) Wet tissue weight (g) Dry tissue weight (g) Dry shell weight (g) Condition index

58.83±5.35a 27.94±2.60a 20.85±2.02a 7.47±3.15a 0.78±0.33a 5.00±1.16a 16.86±4.81a

56.15±6.40b 26.96±2.58ab 20.00±2.16b 6.90±2.16b 0.68±0.23b 4.56±1.11b 15.11±4.67b

58.76±6.244a 27.60±2.16a 20.80±1.78a 8.88±2.20c 0.79±0.41ab 5.09±1.00a 15.46±6.79b

60.14±4.56ab 28.82±2.20c 21.34±1.85a 4.82±1.36d 0.32±0.10c 5.52±1.14c 5.89±1.46c

Values represent mean ± SD (n = 150). Different letter superscripts in the same row represent statistical significance (pb 0.05). Sampled initially, and at one week, one month, and two months in holding.

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Table 3 Morphometric characteristics of field control Mytilus edulis for the summer trial (2008 year class). Parameter

Initial

One week

One month

Three months

Length (mm) Width (mm) Depth (mm) Wet tissue weight (g) Dry tissue weight (g) Dry shell weight (g) Condition index

58.83±5.35a 27.94±2.60a 20.85±2.02a 7.47±3.15a 0.78±0.33a 5.00±1.16a 16.86±4.81a

64.66±10.31b 31.00±3.47b 23.53±3.31b 13.43±5.46b 1.32±0.52b 7.09±2.13b 19.00±5.67b

63.48±7.23b 29.82±3.00c 22.47±2.90c 11.48±4.05c 1.24±0.53ab 6.64±2.033b 18.80±5.74b

70.53±5.96c 34.45±2.44d 25.39±2.36d 16.74±4.76d 1.45±0.38c 9.36±1.78c 15.60±3.44a

Values represent mean ± SD (n = 150). Different letter superscripts in the same row represent statistical significance (p b 0.05). Sampled simultaneously with those in holding from initial grow out site initially, and at one week, one month, and three months.

during the summer from 7.47 g initially to 4.82 g at the final sample. There was a significant increase in wet tissue weight during the fall from 16.73 g to 20.59 g (Fig. 2A). The comparative change in wet weight between seasons was also found to be significant (p b 0.05). Similarly, there was a significant difference between summer holding and field controls, where the change in control wet weight increased slightly during summer (p b 0.05). Mean dry tissue weight decreased significantly over time in extended holding for both the summer (from 0.78 g to 0.32 g) and the fall (from 1.46 g to 1.05 g) (Fig. 2B). Comparative changes in dry weight between summer and fall holding indicated a greater significant change during summer (p b 0.05). Condition also decreased significantly over time in extended holding for these two seasons although the decline was more drastic during the summer (Fig. 2C). Comparative changes in condition for holding for the two seasons also showed a significant difference with the highest mean decrease in summer holding (p b 0.05). Wet tissue weight varied significantly at each sample time for the field control mussels. During the summer mean wet tissue weight ranged from 11.48 g to 16.47 g at the final sample. During the fall the field control mussels had greater mean wet tissue weight ranging from a minimum of 14.85 g to 19.39 g (Fig. 2D). Comparative changes over time indicated a significant difference between seasons with summer showing the greater mean change in wet weight (p b 0.05). Dry tissue weight increased significantly throughout the summer trial from 0.78 g initially to 1.45 g at the final sample. During the fall however there was a significant decrease from 1.46 g to 1.05 g (Fig. 2E). Average change in dry weight over time was significantly greater in summer versus fall (p b 0.05). Condition index increased significantly at the one week and one month sample during the summer followed by a significant decrease at the final sample. During the fall there was little variation in condition remaining at approximately 15% (Fig. 2F). When one compares average change in condition for both seasons, the summer shows a slightly higher but nonsignificant change compared to fall (p > 0.05) (Table 5).

Table 4 Morphometric characteristics of Mytilus edulis during the fall holding trial (2008 year class). Parameter

Initial

One week

One month

Three months

Length (mm) Width (mm) Depth (mm) Wet tissue weight (g) Dry tissue weight (g) Dry shell weight (g) Condition index

70.53±5.96a 34.45±2.44a 25.39±2.36a 16.73±4.75a 1.46±0.38a 9.36±1.78 15.64±3.42a

68.94±5.58b 34.14±4.68b 24.68±2.29b 17.13±4.81a 1.48±0.50a 8.58±1.90a 17.42±4.84b

69.98±5.15ab 33.55±5.15b 15.15±2.22a 19.91±4.72b 1.25±0.30b 9.04±2.07 13.95±2.21c

70.85±5.09a 33.81±2.43ab 25.45±2.01ab 20.59±4.85b 1.05±0.23c 9.01±1.78 11.62±2.30d

Values represent mean ± SD (n = 150). Different letter superscripts in the same row represent statistical significance (p b 0.05). Sampled initially, and at one week, one month, and three months in holding.

3.2. Gene expression analysis During summer the expression of superoxide dismutase (SOD) in gill was the weakest in the initial and one-week holding samples. Both one-week and one-month field control samples showed similar levels of expression relative to 18S rRNA, with one week looking stronger (Fig. 3). During the fall SOD was expressed in the gill at all sample times and both treatments. Expression of glutathione s-peroxidase (GSHpx) also appeared the weakest in the initial summer gill sample compared to the subsequent sample times and treatments (Fig. 4). During summer sampling the one week and one month holding samples showed comparable GSHpx expression levels to that of the field controls. GSHpx was expressed at every sample time for fall holding including the initial. GSHpx was evident in all field control samples during both sample seasons. Expression of M. galloprovincialis defensin-like peptide 2 (MGD2) in hemocytes was only apparent at one week and one month holding during the summer and in the three-month holding and one-week field control in the fall (Fig. 5). 3.3. Neutral red assay There was a significant decline in NR retention time during the summer holding trial. Mean retention time decreased from 95 min initially to 19.5 min after one month (Fig. 6a; one-way ANOVA p b 0.05). The field control mussels also showed a significant decline in NR retention time to 13.5 min (Fig. 6a; one-way ANOVA p b 0.05). Comparative analysis of change in neutral red retention over the time of the experiment for summer holding and field control samples indicated no significant difference between groups (p> 0.05). During the fall holding trial, neutral red retention time decreased from 100 min initially to 30 min after three months in holding (Fig. 6b; one-way ANOVA, p b 0.05). The field control mussels showed no significant changes in NR retention over time (Fig. 6b one-way ANOVA, p > 0.05). Comparative analysis of change in neutral red retention over time for fall holding and field controls indicated a significant difference. The overall change in dye retention was significantly greater in the holding treatment than that of the field controls (pb 0.05). The change in neutral retention time was not significant between summer and fall holding treatments but was significant between summer and fall field controls. The greater significant change in neutral red retention was observed in summer field samples (p b 0.05). 4. Discussion Mussel condition is known to be affected by factors such as water temperature, food availability, and reproductive status (e.g. Dare and Edwards, 1975; Okumuş and Stirling, 1998; Orban et al., 2002). A low condition index could indicate the presence of a stressor such as poor environmental conditions, disease, or a recent spawning event (Lucas and Beninger, 1985). Stressful environmental conditions including accumulation of waste or crowding may be responsible for the low condition index. Here we assessed the condition of mussels in extended wet holding using the ratio of dry meat weight to dry shell weight to eliminate bias due to fluctuations in water content. The significant decline in condition for mussels held during both summer and fall suggests that extended holding had a negative effect on the overall physiology of the blue mussel, M. edulis. During the holding trials we also observed an increase in mean wet tissue weight despite a decline in mean dry tissue weight signifying a state of energy depletion or starvation (Lucas and Beninger, 1985). The significant changes in dry meat weight and condition index occurred only after two (summer) or three (fall) months in holding, suggesting that one month should be the maximum time in ambient wet-storage for these seasons. Although there is a continuous flow-through of water directly from the surrounding bay, limited food availability, crowding, and

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Fig. 2. Change in mean (±SE) wet tissue weight (A), dry tissue weight (B) and condition (C) for mussels in wet storage during summer and fall compared to change in wet tissue weight (D), dry tissue weight (E), and condition (F) for summer and fall field control samples.

the accumulation of waste products could have a negative impact on mussel health due to higher stocking density in the holding tanks compared to the grow-out site. Mussels grown in high density and low-food have been shown to have lower condition indices and tissue weight (Alluno-Bruscia et al., 2001). The standard density at which the mussels are stored in processing facilities may be too high for extended holding. During the summer when water temperatures are increased the combination of density and high water temperatures may have precipitated the mass mortalities experienced in the holding. Hemocytes are invertebrate cells that mediate molluscan immunity (Donaghy et al., 2009; Mitta et al., 2000a, 2000b, 2000c). Various forms of antimicrobial activity as well as a variety of environmental

stress responses have been shown to be associated with these cells in bivalves (Ciacci et al., 2009; Malagoli et al., 2007; Mayrand et al., 2005; Mitta et al., 1999). Lysosomes are hydrolytic enzyme-containing organelles that receive and degrade macromolecules from various cellular trafficking pathways (Luzio et al., 2007). They play a critical role in detoxification and defence in shellfish with specific association with hemocytes (Lowe et al., 1995a,b). These processes are membrane dependent and thus the stability of the lysosomal membrane can be used to determine efficiency in performing these functions (Zhao et al., 2011). Neutral red is a lipophilic chemical dye that can passively diffuse across the lysosomal membrane but depends on the pH of the lysosome and the efficiency of the proton pump (Zhao et al., 2011). If the

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Table 5 Morphometric characteristics of field control Mytilus edulis during the fall trial (2008 year class). Parameter

Initial

One week

One month

Three months

Length (mm) Width (mm) Depth (mm) Wet tissue weight (g) Dry tissue weight (g) Dry shell weight (g) Condition index

70.53 ± 5.96a 34.45 ± 2.44a 25.39 ± 2.36a 16.73 ± 4.75a 1.46 ± 0.38a 9.36 ± 1.78a 15.64 ± 3.42a

66.89 ± 6.17b 32.21 ± 3.20b 23.80 ± 2.43b 14.85 ± 5.74b 1.15 ± 0.31b 14.30 ± 2.98b 14.30 ± 2.98b

69.04 ± 5.62a 33.44 ± 3.39b 24.48 ± 2.38b 19.39 ± 5.28c 1.38 ± 0.36a 8.98 ± 2.01a 15.48 ± 3.11a

65.64 ± 4.23b 31.67 ± 3.24c 23.27 ± 1.83c 14.25 ± 4.04b 1.05 ± 0.29c 7.00 ± 1.34c 15.00 ± 2.99ab

Values represent mean ± SD (n = 150). Different letter superscripts in the same row represent statistical significance (p b 0.05). Sampled simultaneously with those in holding from initial grow out site initially, and at one week, one month, and three months.

membrane is destabilized or the pump compromised due to damage or stress, then neutral red will leak into the cytosol more quickly. The rate at which this happens can be correlated with stress level in the organism. Zhao et al. (2011) provided a very comprehensive review of lysosomal membrane response to various physiological stressors including environmental conditions using the neutral red assay. During the summer season a significant decline in lysosomal neutral red retention time for mussels from both holding and field control groups was observed. This observation indicated that extended holding as well as the natural environment both provided stressful environmental conditions, most likely as a result of high water temperatures at this time of year (Harding et al., 2004; Mallet et al., 1990). Because the water is pumped into the facility directly from the surrounding bay the mussels in the facility should be subjected to similar environmental conditions as those in the field. During the fall, mussels in holding also showed a significant increase in stress response over time. In contrast field control mussels showed no significant difference in neutral red retention time. The average lysosomal retention time was also greater in the fall compared to the summer months. These results are consistent with Harding et al. (2004), who reported that neutral red retention time was the lowest in the summer, particularly evident following a spawning event. Harding et al. (2004) also found that extended storage caused a significant decline in neutral red retention independent of storage condition. Antimicrobial peptide expression in mussel hemocytes is well documented and has been associated with both pathogen and environmental challenges (Cheng-Hua et al., 2009; Li et al., 2009; Mitta et al., 1999, 2000a, 2000b, 2000c). In the current study, during the summer trial, expression of mussel defensin (MGD2) was apparent after one week and

three months in holding, and not at all in the field control mussels. During the fall trial, expression was apparent after three months in holding and at a low level in the one-week field control sample. These results are in accordance with results from Roche (2002) who found that MGD2 expression did not occur until the winter months (February), but in contrast to Li et al. (2009) who found that expression of defensin as well as Myticin B in M. galloprovincialis was more pronounced in the spring–summer under farm conditions. Defensin expression has also been shown to be induced after bacterial challenge (Mitta et al., 1999, 2000a, 2000b, 2000c; Roche, 2002) and the increase of MGD2 expression after time in holding may suggest the accumulation of bacteria in the holding tanks as a result of waste accumulation or mortalities. The low level of MGD2 expression in the fall one-week field control sample could be the result of adverse weather conditions that occurred at that time and location and may have caused an increase of freshwater input, runoff, and mixing. Other varieties of antimicrobial peptide (i.e. Mytilin B) have been shown to increase expression during periods of freshwater influence (Nunez-Acuna et al., 2012). When one compares the comparative response of hemocytes (defensin expression and lysosomal stability) from mussels sampled from both holding and field control conditions, it appears that

Fig. 3. Expression of superoxide dismutase (240 bp) and 18s rRNA MT (238 bp) in Mytilus edulis gill tissue for summer (A) and fall (B) holding experiments. Visualized on a 2% agarose gel (20 μl 500 μl EtBr) displaying semi-quantitative rt-PCR amplification products. 1 kb + DNA ladder, initial (I), one week (1 W), one month (1 M), three month (3 M), and negative control (NTC).

Fig. 5. Expression of MGD2 (211 bp) and 18s rRNA HE (212 bp) in Mytilus edulis hemocytes for summer (A) and fall (B) holding experiments. Visualized on a 2% agarose gel (20 μl 500 μl EtBr) displaying semi-quantitative rt-PCR amplification products. 1 kb + DNA ladder, initial (I), one week (1 W), one month (1 M), three months (3 M), and negative control (NTC).

Fig. 4. Expression of GSH-peroxidase (237 bp) and 18s rRNA MT (238 bp) in Mytilus edulis gill tissue for summer (A) and fall (B) holding experiments. Visualized on a 2% agarose gel (20 μl 500 μl EtBr) displaying semi-quantitative rt-PCR amplification products. 1 kb + DNA ladder, initial (I), one week (1 W), one month (1 M), three months (3 M), and negative control (NTC).

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GSH-peroxidase to be at their lowest from February to April and increase during the late spring and early summer. Seasonal variations in mollusc oxidative stress can arise from complex interactions between temperature, food supply, and pollution. It is therefore difficult to conclude if the expression patterns alone found in these gill samples are the result of extended storage conditions or natural seasonal variations. Comparison of enzyme activity may be warranted in future studies. Taken as a whole, the molecular markers for stress, chosen for this investigation suggest that holding under ambient conditions does affect the overall physiology and condition of mussels and that these effects can be reflected in the decrease in hemocyte lysosomal membrane stability and loss of dry weight and condition over time. The expression of the antimicrobial peptide MGD2 at later time points and various oxidative stress markers also suggests a physiological response to holding when compared to field control responses, although further work will be necessary to dissect the role of these players in the overall story. Under the conditions evaluated in this study, we recommend a one month maximum holding time of mussels during the fall and a one week maximum during the summer when water temperatures are high.

Acknowledgments This study was supported by the Aquaculture Collaborative Research and Development Program (ACRDP). We would also like to thank the Research and Development Corporation of Newfoundland and staff at NorLantic Processors Ltd.

References Fig. 6. Change in lysosomal neutral red retention time for summer (A) and fall (B) holding and field control mussels. H = holding; C = field control. Lower case letters are considered non-significant and upper case letters are significant within treatments (holding vs control). Asterisk denotes a significant change over time for the fall holding samples versus field controls. Bars represent mean ± SE (n = 12).

antimicrobial response is potentially more related to environmental quality where as lysosomal stability as shown through the neutral red assay is more influenced by variation in environmental temperature or a combination of both. Li et al. (2009) noted, after evaluating the influence of temperature, salinity and bacterial tissue content on immune gene expression (defensin and Mytilin B) in M. galloprovincialis, that there was no definitive relationship between environmental condition (temperature and salinity) and immune gene expression. In contrast, Nunez-Acuna et al. (2012) did find that Mytilin B expression was more prominent in areas with high fresh water influence. These contrasting results indicate a need for evaluating the overall physiological response. Oxidative stress occurs when antioxidant defence mechanisms are overcome by prooxidant forces causing a variety of physiological anomalies. In bivalves antioxidant defence systems are affected by environmental parameters such as temperature, pollution, or seasonality (Power and Sheehan, 1996; Soldatov et al., 2007; Valavanidis et al., 2006; Verlecar et al., 2007; Viarengo et al., 1991). Levels of mixed function oxidase antioxidants such as SOD and GSH-peroxidase tend to be the lowest during the winter when lipid peroxidation levels increase (Manduzio et al., 2004; Power and Sheehan, 1996). Although levels and seasonal changes of these enzymes have been extensively studied in the digestive gland, the gill is the major feeding organ of bivalves and as such is likely to be a major site of oxidative stress (Power and Sheehan, 1996). This study evaluated expression levels of antioxidant genes in gill tissue of mussels held in extended wet-storage. Relative expression levels of the SOD and GSH-peroxidase were the lowest in the initial summer sample and increased over time for holding and field control samples. Power and Sheehan (1996) found enzyme levels of

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