Crustacean Biology

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Journal of

Crustacean Biology

The Crustacean Society

Journal of Crustacean Biology, 37(3), 303–314, 2017. doi:10.1093/jcbiol/rux033

The impact of harvesting location on the physiological indicators of the American lobster (Homarus americanus H. Milne Edwards, 1837) (Decapoda: Nephropidae) during live storage K. Fraser Clark1,2,3, Jie Yang4, Adam R. Acorn1,2, John J. Garland4,5, Sarah E. Stewart-Clark4 and Spencer J. Greenwood1,2 1Department

of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, P.E.I. C1A 4P3, Canada; Lobster Science Centre, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, P.E.I., C1A 4P3, Canada; 3Present address: Department of Chemistry and Biochemistry, Mount Allison University, Sackville, NB, E4L 1E2, Canada; 4Department of Plant and Animal Sciences, Faculty of Agriculture, Dalhousie University, Bible Hill, Nova Scotia, B2N 5E3, Canada; and 5 Clearwater Seafoods Limited Partnership, Bedford, Nova Scotia, B4A 3Z7, Canada 2AVC

Correspondence: K.F. Clark; email: [email protected] (Received 12 July 2016; accepted 14 March 2017)

ABSTRACT Shore-based live holding is a required step for post-harvest economic success in the American lobster (Homarus americanus H. Milne Edwards, 1837) fishery. This study evaluated and quantified nutritional condition and gene expression of hepatopancreas during live storage of American lobsters harvested from inshore and offshore locations in Nova Scotia, Canada in early winter. Crude-fat percentage of lobster hepatopancreas and plasma biochemical indicators in haemolymph were used to evaluate the nutritional status of lobsters during live storage. Hepatopancreas crude-fat levels were significantly higher in lobsters from inshore in contrast to offshore locations. Similarly, inshore lobsters were easily distinguished from offshore lobsters because of higher haemolymph Brix and plasma triglyceride, cholesterol, and total protein concentrations. Elevated plasma aspartate aminotransferase activity in inshore lobsters when compared to offshore lobsters suggests that handling practices in the inshore fishery have more impact on harvested lobsters than offshore fishing practices. Inshore lobsters were nevertheless able to recover by four weeks in storage with no obvious mid- and long-term effects. Microarray analysis of hepatopancreas tissue resulted in gene expression profiles that routinely separated inshore from offshore lobsters at all storage time points. The biochemistry of haemolymph and the crude-fat and molecular gene expression analysis of hepatopancreas proved to be useful tools for differentiating lobster harvesting locations. Lobsters harvested inshore in January and stored at the wharf for one month in contrast to lobsters harvested fresh in February had similar gene expression and biochemical profiles; indicating that there is no disadvantage to the livestorage of lobsters caught early in the winter, compared to fresh caught lobsters a month later. Key Words: American lobster storage, haemolymph biochemistry, hepatopancreas gene expression, hepatopancreas crude-fat contents, plasma enzyme activity

INTRODUCTION The American lobster, Homarus americanus H.  Milne Edwards, 1837, is the most economically important, wild-caught marine

species in Atlantic Canada, with landings of 74,686 tonnes in 2015 (Fisheries and Oceans Canada, 2015)  and an overall economic value exceeding CAD $1.0 billion annually (Gardner et al., 2010). Approximately 25,000 people are employed on fishing

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K. F. CL AR K ET AL. haemolymph biochemical analysis for differentiating lobster nutritional status.

vessels, with an additional 10,000 employed in shore-based live holding and processing facilities. Live-lobster holding is an essential post-harvest practice. It allows for the storage of large numbers of lobsters harvested during a fishing season to be graded, sorted, and directed towards either the live market or processing facilities to meet consumer demand over an extended time period. The incentives to store live lobsters are based on quality, which is determined by shell hardness and meat content, and that live lobsters are sold as a premium product at higher prices rather than cooked or frozen processed lobster (Gardner et  al., 2010). Live lobster sales account for about 40% of the total Canadian lobster production by value (Gardner et al., 2010). Historically, about 75% of the live lobster exports were shipped to the United States, but more recent global demand for lobster is creating new market opportunities. The harvesting and holding industry sectors must therefore ensure that a quality live product is maintained in order to meet these distant markets. Proper live-holding of lobsters involves storage at temperatures below 3 oC in clean flow-through, or recirculating, seawater without the addition of food (McLeese & Wilder, 1964). Cold water storage takes advantage of the American lobster’s ability to accumulate nutritional reserves in seasons with higher temperatures for use during periods where a decrease in temperature coincides with a decrease in food availability. These periods of colder temperature are also a time of decreased foraging and metabolic rate to conserve energy reserves until higher temperatures and food resources return. Crustaceans primarily store energy reserves in the form of lipid, and to a lesser degree glycogen, in their hepatopancreas tissue (Vazquez-Boucard et  al., 2004; Sánchez-Paz et  al., 2007; Ciaramella et  al., 2014); therefore, harvesting lobster during the periods when energy reserves are highest is an important consideration for the length of time a lobster should be kept in live storage. Stewart & Li (1969) observed that these periods of higher nutritional reserves, measured by blood protein concentrations generally coincide with the moult stage of the lobster, from late intermoult through to early premoult, and that nutritional status varied by region due to moult timing differences. Although lobster nutritional stores peak during early to late premoult, complications due to a committed shift in energy use for moult preparations regardless of colder storage temperatures make these lobsters unsuitable for long term storage (Aiken, 1973). Health assessment of lobsters prior to live storage represents an important measure to ensure that a lobster can withstand the rigours of post-harvest handling, storage, and shipping procedures. The most common practices include a physical examination combined with a determination of haemolymph refractive index (Brix) on a subsample of harvested lobsters. A  more comprehensive assessment of haemolymph biochemical composition, however, allows a much more holistic determination of the physiological status of lobster. Haemolymph biochemical analysis reveals that circulating cholesterol, total protein, and triglyceride concentrations correlate with the amount of lipid, or crude-fat, energy reserves in the lobster hepatopancreas (Ciaramella et  al., 2014). The biochemical composition of crustacean haemolymph corresponds to the overall nutritional status of the animal, but it is highly influenced by many factors, including salinity, food availability, temperature, and moult stage. Detailed analysis of the biochemical composition of haemolymph has also been found to be an excellent tool for examining, and in some cases differentiating, lobster populations (Dove et al., 2005). We examined the influence of harvest location in early winter on nutritional similarities and differences in lobsters caught offshore compared to those caught inshore and either stored at the wharf where they were landed, or shipped directly to a livestorage facility. High-throughput gene-expression assays were employed to determine how well they correlated with the more traditional examination of hepatopancreas crude-fat reserves and

MATERIALS AND METHODS Handling of lobsters Male intermoult (stage 0–1.5) lobsters from the inshore lobster fishing areas (LFA), LFA 34 and 33, were harvested in early January and early February 2011 respectively; whereas lobsters from offshore fishing area LFA 41 were harvested in early February 2011. The Canadian lobster fishery is divided into 43 geographic areas, each with its own regulations regarding legal size, fishing gear, and fishing season. Lobsters within each inshore group were acquired on the same day and wharf, after either being held at that wharf in lobster cars for thirty days (LFA 34; N  =  39; 652  g ± 28  g), or harvested that day (LFA 33; N  =  39; 653  g ± 30  g) and then shipped directly to the holding facility. Offshore lobsters (N = 43; 657  g ± 26  g) were harvested during a seven-day harvesting trip and stored in flow-through seawater systems on the harvesting vessel. Lobsters were shipped directly to the long-term holding facility once the vessel returned to shore. All lobsters arrived at the holding facility within a 24  h period and were held in an individually-segregated, trickle-down seawater system chilled to 0.5–2 oC with greater than 95% oxygen saturation and a total ammonia concentration less than 1 ppm. Lobsters were handled and stored based on protocols approved by the University of Prince Edward Island Animal Care Committee protocol #09-052.

Sampling of lobsters Lobsters from each harvest treatment (inshore fresh, inshore held, and offshore) were randomly assigned to three different storage duration groups: one week, four weeks or eight weeks. All lobsters underwent routine physical assessments which included examination for exterior physical defects and gross morphological abnormalities, and assessment of bacteriological and ciliate infections in haemolymph prior to the start of the trial (Clark et  al., 2013b). An additional 13 lobsters from each harvest treatment underwent routine physical examination and bacterial and ciliate-infection assessment after each storage duration immediately prior to necropsy. A  1  ml haemolymph sample was taken from ten of these lobsters and centrifuged at 2,000g for 5  min. The plasma was removed from the pellet and stored on ice for biochemical analysis within 24 h. Total haemocyte counts (THC) were taken from six of these lobsters by drawing 500 μl of haemolymph into a syringe filled with 4.5 ml of 0.125% formalin in artificial sea water. The solution was mixed until homogeneous and then counted in duplicate using a hemocytometer. Only successive counts within 10% were used. A drop of haemolymph was placed on a digital PAL-1 Brix refractometer (Atago, Bellevue, WA, USA) to determine the temperature-corrected Brix and refractive index. Muscle samples of hepatopancreas and abdomen, 0.5–1.0 g, were taken from six of these lobsters and homogenized in RNA-preservation reagent (1.4 M guanidine isothiocyanate, 38% phenol (pH 4), 5% glycerol and 0.1 M sodium acetate) (Clark et al., 2013b) and then snap frozen in an ethanol dry-ice bath.

Crude-fat analysis of hepatopancreas Frozen hepatopancreas tissue was thawed and weighed to obtain wet weight. Thawed samples were then freeze-dried at –20 oC for 36 h and weighed again to obtain dry weights. Dried samples were ground into fine granules, 1  g of which was placed into a new filter bag. Samples were incubated at 102 oC for 3  h to remove any residual moisture. Crude-fat percentages of dry weight were obtained using an Ankomxt15 Extractor System (Ankrom Technology, NY, USA) using its automatic operation at 90 oC 304

H A RV E STI N G LOC ATI ON I MPACT O N L I V E LO BS T ER S TO R AGE and 2 μl of 60-fold diluted cDNA. All qPCR reactions were optimized for annealing temperature using either two- or three-step qPCR reactions (Appendix 1). Two-step reactions were performed at 50 oC for 2 min, 95 oC for 2 min followed by 39 cycles of 95 oC for 7 s, and finally 20 s at the assay-specific annealing temperature, followed by a plate read. Three-step reactions were treated the same way except for the 20 s annealing step, which was followed by 20 s at 72 oC and then a plate-read step. Both protocols ended with melt-curves to confirm the presence of a single peak at the appropriate temperature. All assays were confirmed to produce a single PCR product by the combination of agarose gel-based verification of a single peak at the proper size and corresponding melt-curve analysis. Gene expression was determined for the following genes: peroxisomal membrane protein 11C (PEX11C) (CN853853), isocitrate dehydrogenase (IDH) (FC556020), phosphoenolpyruvate carboxykinase (PEPCK) (DV771445), and gamma-butyrobetaine dioxygenase (BBOX1) (FC071783). Aspartate aminotransferase (AST) (FE840988), uncharacterized protein C17orf62 homolog (FE535262) and pre-mRNA splicing factor 38B (DV774783) were used as normalization genes as they combined for a geNorm V of 0.474 and a geNorm M value of 0.189 (see Appendix 1).

for 60  min, according to the manufacturer’s instructions. Teflon inserts were removed from the extraction vessel and the samples contained in filter bags were dried in an oven at 102 oC for 30 min and weighed once they had returned to room temperature for calculation of percent crude-fat calculations.

Biochemistry of haemolymph plasma Plasma samples were stored on ice for less than 24  h and submitted to the Atlantic Veterinary College Diagnostic Laboratories (Charlottetown, PEI, Canada) for biochemical analysis on a Cobas 6000 analyser (Roche Diagnostic USA, Indianapolis, IN, USA). Plasma was analyzed for sodium, potassium, chloride, calcium, phosphate, magnesium, urea, glucose, cholesterol, triglyceride, albumin (haemocyanin), lactate, uric acid, total protein, and the enzyme activity of lipase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and glutamate dehydrogenase (gLDH) (Ciaramella et al., 2014).

RNA extraction RNA was extracted from four lobsters per harvest treatment, per time period, as previously described by Clark et al. (2013b). Briefly, 200 µl of chloroform was added to 1 ml of homogenized hepatopancreas tissue, the solution shaken and incubated at room temperature for at least 3  min. The solution was then centrifuged at 12,000g for 15  min at 4 oC, and 600  μl of the supernatant was combined with 600  μl of 70% ethanol. The RNA was purified using an RNeasy spin column (Qiagen, Toronto, ON, Canada) according to the manufacturer’s instructions with treatment on an on-column DNase I  (Qiagen, Toronto ON, Canada). RNA quality was determined using an Agilent RNA 6000 Nano kit on an Agilent 2100 Bioanalyzer (Agilent Technologies, Mississauga, ON, Canada). Only high-quality RNA was used for microarray and reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) analysis. RNA was stored at –80 oC until ready for use.

Statistical analysis Statistical differences in THC, Brix, and crude-fat percentage between different treatments were calculated using a Mann-Whitney U test (Wilcoxon, 1945). All subsequent analyses were performed using the TM4/MeV v4.9.1 software suite (Saeed et  al., 2003). Statistical analyses of haemolymph biochemical constituents were performed by transforming the raw data to the log2 expression ratio of individual raw values over the average inshore fisher held one -week value. Microarray gene expression was performed on the log2 ratio of the expression ratio of the sample intensity over the reference intensity. One-way ANOVA analysis was performed using 1000 permutations with an α  =  0.05 and a false-discovery rate (FDR) (Korn et al., 2004) of no more than 0.05 for the haemolymph biochemical data. Differential gene expression was calculated using an α = 0.001 with no FDR correction. Hierarchical Clustering (Eisen et  al., 1998), Figure of Merit (Yeung et  al., 2001), and K-Medians Clustering (Soukas et al., 2000) were performed on significantly different biochemistry parameters and gene expression ratios. All statistical analyses of RT-qPCR data were performed using qbasePLUS v2.3 qPCR analysis software (Biogazelle, Ghent, Belgium) and normalization genes were determined using geNormPLUS.

cDNA synthesis, labeling, microarray hybridization, and feature extraction A spotted oligonucleotide microarray from H. americanus was used for gene- expression analysis (Clark et  al., 2013b). Briefly, 1  μg of high-quality sample RNA was converted to Alexa Flour® 555 labeled single stranded cDNA using SuperScript™ Plus Indirect cDNA Labeling System according to the manufacturer’s instructions (Life Technologies Inc., Burlington, ON, Canada). Reference aRNA samples were converted to Alexa Flour® 647- labeled cRNA using the same kit. Labeled cRNA and cDNA were quantified using a NanoDrop ND-1000. cRNA was fragmented using 10x Fragmentation Solution according to the manufacturer’s instructions (ThermoFisher Scientific, ON, Canada). Fragmented cRNA (100 pmol) and 140  ng of labeled cDNA were used for microarray hybridizations as previously described in Clark et  al. (2013b). Microarray gene expression was measured using a GenePix 4000B scanner (Molecular devices, PA, USA) scanning at a pixel diameter of 5 µ m. Feature extraction was performed by SpotReader v1.3.1 (Niles Scientific, CA, USA). Gene expression values were normalized by LOWESS, flagged, and converted to log2 expression ratios using the MIDAS v2.2.2 software (Quackenbush, 2002).

RESULTS Monitoring of health quality of lobsters Lobsters were checked weekly to ensure that no individuals were moribund or dead. Four lobsters from the offshore harvest treatment died during the trial, whereas all lobsters from both of the inshore harvest treatments survived their intended storage durations. All lobsters tested negative for bacterial or ciliate infections throughout the trial. Lobster haemolymph Brix (oBx) was consistently different between the offshore and at least one inshore lobster treatment. There were statistical differences between the inshore and offshore lobsters when storage duration was not considered, after four weeks in storage, and after eight weeks in storage (Fig.  1A, 1C, 1D). There was a statistical difference between the offshore lobsters and the inshore fresh lobsters after one week in storage, but not between the inshore fresh and the inshore held lobsters (Fig.  1B). Strong Pearson correlations were also found between haemolymph Brix and several haemolymph biochemical parameters, including: total protein (r = 0.998), haemocyanin (r = 0.988), triglyceride (r  =  0.9445), cholesterol (r  =  0.945), and glucose

Reverse-transcriptase quantitative polymerase chain reaction One μg of high-quality total RNA was converted to cDNA using a SuperScript III First-Strand Synthesis kit (ThermoFisher Scientific, ON, Canada) as previously described by Clark et  al. (2013b). All qPCR reactions were performed using total reaction volumes of 15  μl containing 1X Express SYBR GreenER (ThermoFisher Scientific, ON, Canada), 200 nM of each primer, 305

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Figure 1.  Haemolymph refractive index (brix) from American lobsters (Homarus americanus) from three different harvest treatments stored live in a holding facility. Brix readings were compared irrespective of time in storage (N = 53) (A), after one week in storage (N = 17) (B), after four weeks in storage (N = 18) (C), and after eight weeks in storage (N = 18) (D). Significant differences are denoted using superscript letters.

clustering to determine which parameters where most correlated. Cluster 1 contains glucose, triglyceride, total protein, and haemocyanin parameters; cluster 2 calcium and cholesterol; cluster 3 total protein minus haemocyanin; cluster 4 phosphate and uric acid; and cluster 5 sodium and chloride. There were several significantly different haemolymph parameters when the harvest locations were compared after one week in storage: calcium, chloride, sodium, calculated osmolarity, gLDH, potassium, AST, uric acid, cholesterol, triglyceride, haemocyanin, total protein, and total protein minus haemocyanin (Fig. 6A). These parameters clustered into five groups, where cluster 1 contained triglyceride, cholesterol, haemocyanin, total proteins, total protein minus haemocyanin, and calcium; cluster 2 potassium and AST; cluster 3 uric acid; cluster 4 calculated osmolarity, sodium, and chloride; and cluster 5 gLDH. There were ten significantly different haemolymph parameters after four weeks in storage: magnesium, calcium, total protein minus haemocyanin, phosphate, cholesterol, triglyceride, glucose, total protein, haemocyanin, and uric acid (Fig. 6B). These parameters clustered into five groups, where cluster 1 contained cholesterol, haemocyanin, triglyceride, total protein, and glucose; cluster 2 total protein minus haemocyanin; cluster 3 uric acid and calcium; cluster 4 phosphate; and cluster 5 magnesium. After eight weeks in storage, there were eight significantly different haemolymph parameters: phosphate, calcium, glucose, triglyceride, cholesterol, total protein, albumin, and uric acid (Fig.  6C). These values clustered into three groups, where cluster 1 contained triglyceride, total protein, haemocyanin, and cholesterol; cluster 2 calcium, glucose, and uric acid; and cluster 3 phosphate.

(r  =  0.923). There was also a strong, but lower, Pearson correlation between Brix and hepatopancreas crude fat percentage at r = 0.804.

Total haemocyte counts (THC) Inshore lobsters had statistically higher THC values than the offshore lobsters when harvest treatments were compared regardless of time in storage (Fig.  2A). There was a difference in THC between the inshore fresh lobsters and the offshore lobsters only after one week (Fig.  2B). Both inshore lobster groups had higher THC than the offshore lobsters after four weeks (Fig.  2C). Only the inshore held and inshore fresh had different THC after eight weeks; although the value for the inshore fresh compared to the offshore was close at P = 0.055 (Fig. 2D).

Crude-fat analysis of hepatopancreas Inshore lobsters also had statistically higher crude-fat percentage than the offshore lobsters when all storage durations were combined, and after storage after a duration of one, four, and eight weeks (Fig.  3A, 3B, 3C, 3D). A  substantial difference in the colour of hepatopancreas tissue was noticed between the inshore and offshore lobsters during necropsy. The hepatopancreas tissue was very pale yellow or pale green in all inshore lobsters, whereas it was very dark green in all offshore lobsters (Fig. 4).

Biochemistry of haemolymph plasma There were significant differences between the different harvest treatments, when all storage durations were combined, for the following parameters: sodium, chloride, calcium, total protein minus haemocyanin, phosphate, glucose, cholesterol, triglyceride, total protein, haemocyanin, and uric acid (Fig.  5). These biochemical parameters can be clustered into five groups by K-medians

Microarray analysis of hepatopancreas gene expression Microarray analysis of hepatopancreas gene expression of all harvest treatments samples separated by harvest treatment alone, results in three differentially expressed genes at α  =  0.05 and 306

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Figure 2.  Total haemocyte counts (THC) from American lobsters (Homarus americanus) from three different harvest treatments stored live in a holding facility. Counts were compared irrespective of time in storage (N = 54) (A), after one week in storage (N = 18) (B), after four weeks in storage (N = 18) (C), and after eight weeks in storage (N = 18) (D). Significant differences are denoted using superscript letters.

Figure 3.  Hepatopancreas crude-fat percentages from three different harvest treatments of American lobsters (Homarus americanus) stored live in a holding facility. Counts were compared irrespective of time in storage (A) and after one week (B), after four weeks (C), and after eight weeks (D) in storage. Significant differences are denoted using superscript letters.

an FDR not greater than 0.05. These genes are pseudohaemocyanin, BTB-protein-VII isoform A, and adenylate cyclase-associated protein 1.  If we include two additional genes, adenylate

cyclase-associated protein 1 and S-adenosylhomocysteine hydrolase, which were flagged due to very high signal intensity, hierarchal clustering of samples results in greater separation between 307

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Figure 4.  Hepatopancreas at necropsy from inshore (A) and offshore (B) American lobsters (Homarus americanus). Hp, hepatopancreas tissue; t, testis.

Figure 5.  Hierarchical clustered heat map of significantly different haemolymph biochemical parameters between individuals of the American lobster (Homarus americanus) separated by harvest treatment alone. Statistical significance was determined with a one-way ANOVA using permutations (1000), an α = 0.05 and a false discovery rate of no greater than 0.05. Colours reflect the log2 expression ratio of the individual raw values to the average inshore fisher held values after one week in storage. Colours are on a linear gradient scale from red (–2), to black (0) to green (2).

Figure 6.  Hierarchical clustered heat map of significantly different haemolymph biochemical parameters between individuals of the American lobster (Homarus americanus) separated by harvest treatment after specific durations in storage. Statistical significance was determined with a one-way ANOVA using permutations (1000), an α = 0.05 and a false discovery rate of no greater than 0.05. Colours reflect the log2 expression ratio of the individual raw values to the average inshore fisher held values after one week in storage. Colours are on a linear gradient scale from red (–2), to black (0) to green (2). Lobster storage durations were one (A), four (B), and eight (C) weeks.

four weeks in storage revealed 13 differentially expressed genes (Fig.  8B). Hierarchical clustering produced four clusters, two of which separated the offshore samples from the inshore samples reasonably well. There were 25 differentially expressed genes after eight weeks in storage (Fig.  8C). Separation of these genes into six groups revealed two groups that separated the inshore from offshore samples reasonably well.

inshore and offshore lobsters (Fig.  7A). Analysis performed at α = 0.001 without FDR results in 41 differentially expressed genes (Fig. 7B). Hierarchical clustering of samples results in good separation of the inshore and offshore lobsters. When the differentially expressed genes are clustered into seven groups, only two groups, offshore and inshore, have moderate to good separation of the samples. Thirty-nine differentially expressed genes were found following the analysis of the harvest treatments after one week in storage using an ANOVA with α = 0.001 in the absence of FDR correction (Fig. 8A). Hierarchical clustering produced nine clusters, with good separation of offshore and inshore sample in one cluster; while two additional groups separated the inshore fresh samples from the other samples. Analysis of the harvest treatment after

RT-qPCR verification of hepatopancreas gene expression Gene expression was initially analyzed by combining all of the storage durations within the harvest treatments. Differential expression of PEX11C was found between the inshore and offshore samples, while IDH was differentially expressed only 308

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Figure 7.  Hierarchical clustered heat map of significantly different hepatopancreas gene expression from all individuals of the American lobster (Homarus americanus) separated by harvest treatment. Statistical significance was determined with a one-way ANOVA using permutations (1000) and an α = 0.05 and a false discovery rate of no greater than 0.05 (A) or an α = 0.001 and no False Discovery Rate (B). Colours reflect the log2 expression ratio of the individual raw values to the average inshore fisher held values after one week in storage. Colours are on a linear gradient scale from red (–3), to black (0) to green (3).

biochemistry and gene expression used in this study have proven valuable for studying and differentiating lobsters from different locations or harvesting practices. Haemolymph Brix is a measure of the refractive index of haemolymph. It is commonly used in the lobster industry as a measure of lobster quality because it is correlated with haemolymph protein concentration (Hortin, 2012). The later study and others (Ciaramella et  al., 2014; Wang & McGaw, 2014) found a strong correlation between haemolymph Brix, or refractive index, and haemolymph plasma protein concentration of 0.998. The correlation between haemolymph Brix and the percentage of fat in hepatopancreas was lower, but still strong, at 0.804. These strong correlations suggest that haemolymph Brix, and refractive index, are valuable methods for assessing the nutritional status of lobsters before, or during, live storage. Total haemocyte counts are a relatively easy method to compare lobster physiological status between lobster harvest treatments. There was a clear difference between inshore and offshore lobster THC when all lobsters within a harvest treatment are combined. A  difference between offshore lobsters and at least one inshore harvest treatment existed at each storage duration throughout the trial. Lower THC can be caused by haemocyte infiltration into tissues, normal cellular apoptosis, and reduced replacement from haematopoetic tissues or by bacterial (Snieszko & Taylor,

between inshore fresh and offshore lobsters (Fig. 9A). There is no differential expression between the harvest treatments for PEPCK, AST and BBOX1 genes. There was no differential expression for of any of the genes examined when the individual harvest treatments are compared after one week in storage (Fig.  9B). There was nevertheless differential expression between inshore and offshore lobsters for the BBOX1 and IDH genes after four weeks in storage (Fig. 9C). Only PEX11C has differential expression between inshore and offshore samples after eight weeks in storage (Fig. 9D).

DISCUSSION Live storage of H. americanus is a critical part of the lobster industry as 45–50% of the more than 300 million pounds of lobster landed in Canada and the United States will be sold as a live product (Thériault et al., 2013). This does not include the tens of millions of lobsters that are stored for periods of days to weeks prior to being processed into frozen or prepared products. Although there are a few guidelines for the proper handling and storage of live lobster (McLeese & Wilder, 1964; Beard & McGregor, 2004; Jacklin & Combes, 2007), very little has been published with respect to the effect of live storage on lobsters harvested from inshore or offshore waters. The analytical techniques of plasma 309

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Figure 8.  Hierarchical clustered heat map of significantly different hepatopancreas gene expression from individuals of the American lobster (Homarus americanus) separated by harvest treatment after different storage durations. Statistical significance was determined with a one-way ANOVA using permutations (1000), an α = 0.001 and no false discovery rate. Colours reflect the log2 expression ratio of the individual raw values to the average inshore fisher held values after one week in storage. Colours are on a linear gradient scale from red (–3), to black (0) to green (3). Lobster storage durations were one (A), four (B), and eight (C) weeks.

1947) and parasitic (Clark et  al., 2013b) infections. All lobsters in this trial, however, tested negative for both bacterial and scuticociliate infection. Reductions in nutritional status have been linked to a decrease in circulating haemocytes in the American lobster (Stewart et al., 1967). The most likely reason for the higher THC in inshore lobsters is therefore their superior nutritional status during the January- February harvest period as demonstrated by the haemolymph biochemical analysis. The nutritional condition of crustaceans is directly related to the size of their stored energy reserves (Moore et al., 2000), which varies based on their moult stage and/or physiological state (Mai & Fotedar, 2016; Wang & McGaw, 2014). Lipids are the major energy reserves in H.  americanus, and the hepatopancreas is the largest lipid storage site (Ciaramella et al., 2014). Hepatopancreas crude-fat content in lobsters caught offshore were consistently lower than those caught inshore. This finding demonstrates that either lobsters living offshore are more nutritionally restricted than those living inshore at the time of capture, or the timing of the moult cycles between the two fishing areas are different. All of the lobsters in this study were in the inter-moult stage but the timing of moult is generally later in the deep offshore waters then in the shallow inshore waters. This would allow the inshore lobsters a longer duration to recover from their moult than offshore lobsters caught at the same time of year. Offshore and inshore lobsters were shown to have marked differences in the colour of hepatopancreas tissue. This may be associated with the types of food that the lobsters are consuming but it requires further investigation for confirmation. There are significant differences (P  0.05). This is likely due to their adapted response to limit foraging and metabolic rate during cold temperatures to maintain their energy reserves until the higher temperature and food availability present in the spring; as well as arrested moult stage development at reduced environmental temperatures (Aiken, 1973). Hierarchical clustering of the plasma biochemical components provides insight into how their plasma concentrations are

Haemolymph aspartate aminotransferase (AST) activity is considerably higher in the inshore harvested lobster. Aspartate aminotransferasis a marker of liver damage in mammals, and potentially a marker for tissue damage in the hepatopancreas of the American lobster; although it is also found in the heart and muscle tissue of lobsters (Battison, 2006). Unfortunately, there are no good biomarkers for muscle damage in lobsters in order to properly determine this source of the plasma AST (Battison, 2006). The different haemolymph concentrations between inshore and offshore harvested lobsters could reflect a difference in the harvesting and post-harvest handling methods that were used, where higher plasma AST levels could suggest more vigorous handling. There is no difference in the haemolymph AST levels between harvesting treatments after four weeks, suggesting that the lobsters were able to recover. This recovery suggests that the differences in harvest and post-harvest handling methods do not adversely affect lobster viability during medium- and long-term live storage, but may serve as a sensitive, investigative tool for discovering post-harvest handling issues. Three haemolymph parameters were different between the harvest treatments after eight weeks in storage. These included phosphate, glucose, and uric acid, all three of which were higher in the inshore than in the offshore group. Glucose is the main circulating carbohydrate in crustacean haemolymph (Sánchez-Paz et  al., 2007), and higher haemolymph glucose concentrations may reflect better nutritional status from this harvest period, or due to a stressrelated response that can elevate haemolymph glucose in crustaceans (Paterson & Spanoghe 1997; Chang et al., 1998). Uric acid haemolymph concentrations reflect either normal physiological 311

K. F. CL AR K ET AL. C oxidase 1, a component of respiratory complex IV in the electron transport chain of mitochondrial oxidative phosphorylation. This suggests that energy production of ATP through mitochondrial oxidative phosphorylation is increasing in inshore lobsters at a much greater rate than in offshore lobsters because they had already increased their energy production from mitochondrialbased β-oxidation at an earlier time point. Several genes were chosen for RT-qPCR analysis to examine the impact of harvest treatment on metabolism. Peroxisomal biogenesis factor 11C (PEX11C) is part of the PEX11 family of peroxisomal membrane proteins, which promote peroxisome division in many eukaryotes (Li & Gould, 2002; Schrader et al., 1998). Peroxisomes are involved in many metabolic pathways, including β-oxidation of very-long-chain and long branched-chain fatty acids (Wanders & Tager, 1998). The PEX11 protein is capable of inducing peroxisomal proliferation in the absence of peroxisomal fatty acid metabolism or exogenous fatty acids (Li & Gould, 2002). The statistically significantly higher expression of PEX11C in the offshore lobsters is driven by its differential expression after eight weeks in storage, increasing the rate of peroxisome-based β-oxidation of fatty acids. This would also explain the higher COI expression found through microarray analysis in inshore lobsters, which are increasing their use of mitochondrial-based β-oxidation. Phosphoenolpyruvate carboxykinase was chosen because it is the rate-limiting enzyme in gluconeogenesis (Rognstad, 1979), and is widely viewed as a marker for hepatic gluconeogenesis (Chakravarty et  al., 2005). It is also a very important enzyme involved in the production of triglyceride found in very- ow density lipoprotein during fasting (Kalhan et al., 2001). Its expression can be altered very quickly depending on the dietary or hormonal status of the animal, where insulin release significantly inhibits PEPCK gene transcription (Granner et al., 1983). There was no significant difference in PEPCK expression between any of the harvest treatments during storage, suggesting that the level of hepatic gluconeogenesis, and storage conditions, are consistent between all of the harvest treatments. Gamma-butyrobetaine dioxygenase (BBOX1) is the final step in the production of carnitine (Lindstedt & Lindstedt, 1970). Carnitine is critical for the oxidation of fatty acids as it allows their transfer into the mitochondrial matrix where β-oxidation occurs (Tanphaichitr & Broquist, 1974). Changes in carnitine concentration affect the rate of mitochondrial β-oxidation and therefore energy metabolism (Bremer, 1997). The increased expression of BBOX1 at four weeks in the offshore lobster likely represents the increased demand for energy from hepatic crude-fat reserves in the offshore lobsters, whereas the inshore lobsters can still rely on other energy reserves due to their enhanced nutritional state. After eight weeks in storage, however, all harvest treatments have become more reliant on their crude-fat reserves for energy homeostasis. Isocitrate dehydrogenase (IDH) is a metabolic enzyme responsible for the conversion of isocitrate to α-ketoglutarate in the Krebs cycle. Its expression was measured as a proxy for carbohydrate metabolism through the Krebs cycle. The expression of IDH mirrors BBOX1 expression in that it is significantly higher in the offshore lobsters after four weeks. This is likely due to the increased production of acetyl-CoA from mitochondrial-based β-oxidation being driven into the Krebs cycle. Hepatopancreas AST gene expression was measured because plasma AST activity was elevated in inshore lobsters at the oneweek time period. The expression of AST was so steady, however, that it made an excellent reference gene. The source of the circulating AST could not be identified by plasma analysis alone, but the lack of differential expression in the hepatopancreas suggests an alternate tissue source. In this case that source is probably muscle tissue and its release likely a product of handling technique. The differential expression of PEX11C, IDH and BBOX1 suggests that the offshore lobsters are acquiring more of their energy

regulated. The concentrations of measured proteins, lipids, and carbohydrate components are consistently positively correlated to hepatopancreas crude-fat reserves, and are therefore excellent indicators of nutritional status. The concentration of haemolymph ions, however, is not correlated with these haemolymph constituents. Sodium and chloride ions are correlated with each other, whereas uric acid, calcium, phosphate, and magnesium ions are not. This indicates that plasma ion concentrations are at least dependent on a physiological mechanism other than nutritional status. A printed DNA microarray is an excellent high-throughput screening tool for biomarker discovery; however, its manufacturing process induces an inherent variability which simultaneously increases the number of false-negatives and decreases the number of true-positives. The need for FDR correction can be eliminated when using a low α because potential biomarkers will be verified with the much more precise and sensitive RT-qPCR as previously demonstrated for this microarray (Clark et al., 2013a, 2013b, 2013c). There is a clear separation between inshore and offshore lobsters during the period of study using the differentially expressed genes found when the harvest treatments are separated irrespective of time in storage. The downregulation of the pseudohaemocyanin gene in the offshore samples, when compared to the inshore lobsters, is evident. The role of pseudohaemocyanin has not been fully elucidated but it is believed to be involved in energy storage (Burmester, 1999, Tang et al., 2010). This would suggest that lower pseudohaemocyanin expression may be present in lobsters that do not have sufficient nutritional energy reserves or are at different points in their moult-related development cycle. There is clear separation of inshore- and offshore-harvested lobsters after one week in storage using microarray analysis. Hierarchical clustering suggests that this difference is related to the expression of ERCC2 (excision repair cross-complementation group 2) and legumain (asparaginyl endopeptidase; EC 3.4.22.34) and three unknown genes. Unfortunately, no clear mechanistic explanation of why these genes are differentially expressed in inshore and offshore lobsters is apparent (Appendix 2). There is also a clear separation between inshore and offshore lobsters after four weeks of storage. This separation is largely linked to the mitochondrial translational release factor-1-like (MRF1L) gene, adenylate cyclase gene, and one unknown gene. The expression of these genes is higher in offshore samples than in the inshore ones. The MRF1L gene encodes a mitochondrial translation release factor that releases mitochondrial mRNA from the translation machinery (Nozaki et al., 2008). Increased availability of mitochondrial proteins and protein complexes has been closely associated with enhanced rates of mitochondrial respiration (McKee & Grier, 1990; Takahashi & Hood, 1996) critical for enhanced mitochondrial functions such as oxidative phosphorylation (McKee & Grier, 1990; Takahashi & Hood, 1996). The offshore lobsters in this study period, which had a reduced nutritional status, would have lower muscle energy reserves and therefore may rely more on the β-oxidation of hepatic reserves for energy production earlier than the inshore lobsters that were at a higher nutritional state. Inshore and offshore lobster can still be separated based on differentially expressed genes after eight weeks in storage. The expression of crustacyanin-AC subunit and the glutamate receptor family member 7, along with two unknown genes, are good at separating the offshore and inshore held lobsters. Crustacyanin is a pigment used in the lobster epicuticle to give lobsters their characteristic colour (Cianci et al., 2002; Tlusty & Hyland, 2005) and varies with the lobster’s moult cycle. Cytochrome oxidase subunit I  and four other unknown genes are also capable of separating the offshore lobsters from the inshore lobsters. The COI gene expression decreases from inshore held to inshore fresh and offshore lobsters. The COI gene is the main subunit of cytochrome 312

H A RV E STI N G LOC ATI ON I MPACT O N L I V E LO BS T ER S TO R AGE from hepatic crude-fat reserves through mitochondrial β-oxidation after four weeks in storage than the inshore lobsters. The inshore lobsters had nevertheless made a similar switch between four and eight weeks in storage. Mitochondrial β-oxidation uses long-, medium and short chain fatty acid reserves (Eaton et  al., 1997), whereas peroxisomal β-oxidation uses long and very long chain fatty acids for energy production (Reddy & Mannaerts, 1994). There is an increase in peroxisomal β-oxidation in offshore lobsters after eight weeks in storage that is not reflected in the inshore lobsters. This is likely caused by the lower initial, or perhaps different, lipid reserves in offshore lobsters. The present study of plasma biochemical profiles, total haemocyte counts, hepatopancreas crude-fat, and the analysis of gene expression has illustrated how diverse analytical techniques can increase the understanding of nutritional status in lobsters. The greater energy reserves found in inshore lobsters during the sampling period, were clearly illustrated by all of the analytical techniques utilised. Measurements of gene expression provided additional information into the metabolic mechanisms utilized by lobsters with different nutritional reserves while in live storage. The offshore lobster group required more energy from mitochondrial β-oxidation of fatty acids, and increased energy production from peroxisome-based β-oxidation, earlier than inshore lobsters. The obvious difference in nutritional status between inshore and offshore lobsters also identifies the power of nutritional and genetic analysis in separating lobster populations. This corroborates the industry’s practice of keeping offshore lobsters in storage for shorter durations than inshore lobster during this time of year. The differences observed in initial chloride, sodium, and calculated osmolality between the offshore and inshore lobsters could have been exacerbated by temporal differences in moult timing. Early post-moult crustaceans are osmoconformers, whereas intermoult crustaceans are able to lower their plasma osmolality below that of their environment (Ferraris et  al., 1987). Further work on lobsters harvested from other times of the year is required to make definitive conclusions as to whether there are indeed nutritional state differences between harvest areas. Perhaps the observed differences are attributable to moult cycle timing differences related to the varying environmental temperatures unique to each region that are known to affect moult timing. Haemolymph Brix is one of the easiest non-lethal measurements of lobster quality that can be taken from lobsters by the industry. It is routinely used to inform decisions on whether lobsters can be stored, or if they should be processed immediately. The strong correlation between Brix and the more robust analytical methods employed in this study to assess the nutritional status of lobsters corroborates the continued use of Brix measurements in the lobster industry as a means of nutritional status assessment. The haemolymph AST activity was able to demonstrate a difference in handling conditions between the two lobster populations. These differences in AST activity, however, were transient and had little or no impact after four weeks in storage. No clear differences were also found between the two inshore harvesting treatments examined in this study. This suggests that those lobsters harvested in January and stored for one month, compared to those harvested one month later, had very little differences in their energy reserves. This demonstrates that lobsters do not significantly add to their energy reserves in the cold winter months and that harvesting and storage of lobsters in cold conditions has a similar effect on their natural winter conditions. The regional differences between lobster nutritional status, whether they be due to food availability in the environment, regional differences in environmental temperatures and their control over moult timing, or other factors, clearly highlight differences in quality and physiological status of lobsters based on most of the haemolymph biochemical measurements used in this study. Analysis of the 2011 winter fishery inshore lobsters from this study resulted

in higher nutritional-status biochemical values. The recent trend in the Canadian lobster fishery has been an increase in fishing effort in the mid to offshore fishing grounds in order to increase catches. This change in fishing effort to the offshore area at the harvest peak in December to February will pose a threat to the quality of the landed catch. Objective biochemical measures can determine nutritional state, and therefore lobster fitness for storage and shipment, and, could be an effective resource management tool. They could determine proper harvest timing based on regional differences in nutritional state in order to ensure the quality and mortality from the storage of lobsters with low nutritional status.

SUPPLEMENTARY MATERIAL Supplementary material is available in Journal of Crustacean Biology online. S1 Figure. Kmeans clustering of significantly different gene expression from all individuals separated by Harvest treatment. Differential expression calculated using a one -way ANOVA with permutations (1000) α  =  0.001. Colours reflect the log2 expression ratio of the individual raw values to the average inshore fisher held values after one week in storage. Colours are on a linear gradient scale from red (–3), to black (0) to green (3). S2 Table. RT-qPCR primers with annealing temperatures and assay efficiencies.

ACKNOWLEDGEMENTS We would like to thank the ACOA-BDF (Project No.:198382) for providing the financial resources for performing this research. Excellent technical assistance was provided by Allison Darling (AVCLSC) and Jane Silver (Clearwater Seafoods). We would also like to thank the two anonymous reviewers for their very helpful comments, which enhanced the quality of the manuscript.

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