Effects of different dietary organic acids on the survival, growth, and hepatopancreatic histopathology of the blue swimmer crab Portunus pelagicus.
Organic acids and their salts are slightly acidic compounds, often associated with a carboxyl group, which have been used for many years in livestock feeds as preservatives. Organic acids are "generally regarded as safe" that can be naturally found in various plant and animal sources as well as some being a byproduct of bacterial metabolism within the gastrointestinal tract of animals (Lim et al. 2015, Romano et al. 2016). Their acidifying properties may impart certain benefits to the host animal such as acting as antimicrobials to pathogens and/or improving nutrient utilization as demonstrated with various fish species (Ringo 1991, Sugiura et al. 1998, Baruah et al. 2007, Hossain et al. 2007, Hernandez et al. 2012, Khajepour & Hosseini 2012, Castillo et al. 2014, Koh et al. 2016). A review of the literature suggested that the success of dietary organic acid often depends on the organic acid type and host aquatic species (Ng & Koh 2016).
For example, in an early study, Ringo (1991) showed that Arctic char Salvelinus alpinus fed with sodium propionate- or sodium lactate-treated diets had reduced and increased growth, respectively. More recently, it was shown that dietary additions of calcium lactate, potassium diformate, or citric acid improved the growth of red drum Sciaenops ocellatus, which was suggested to be due, in part, by enhanced digestive enzyme activity (Castillo et al. 2014). On the other hand, dietary sodium citrate was shown to damage the liver of tilapia, as well as decreasing their growth and overall health status (Romano et al. 2016).
Fish have received the most focus of research on dietary organic acids, although there has been increasing interest on the applications to shrimp in the last 5 y (Kiihlmann et al. 2011, Nuez-Ortin 2011, Silva et al. 2013, 2015, Khalil et al. 2014, Su et al. 2014, Chuchird et al. 2015, Ng et al. 2015, Romano et al. 2015). Silva et al. (2013) showed that dietary sodium propionate and sodium butyrate significantly increased feed intake in white shrimp Litopenaeus vannamei, but sodium acetate decreased feed intake and phosphorus availability. It was later demonstrated that among different organic acid salts in the diets of L. vannamei, which included sodium formate, sodium acetate, sodium lactate, sodium propionate, sodium butyrate, sodium fumarate, sodium succinate, or sodium citrate, there was significantly improved growth for those fed with the sodium propionate-, sodium butyrate-, sodium fumarate-, and sodium succinate-treated diets (Silva et al. 2015). Meanwhile, when using different dietary levels of an organic acids blend (OAB) at 0%, 1%, 2%, or 4%, the growth of L. vannamei was significantly improved for those fed with the 2% OAB diet compared with the control (Romano et al. 2015). Enhanced phosphorus utilization and more hepatopancreatic nutrient reserves were suggested as likely contributors to this finding (Romano et al. 2015). Moreover, when the same dietary OAB and concentration was used in a commercial farming situation and with the black tiger shrimp Penaeus monodon, the dry matter, crude protein, ash, and phosphorus utilization were significantly enhanced (Ng et al. 2015).
On the other hand, diets containing 0.6% formic acid were shown to reduce the growth of Litopenaeus vannamei compared with an untreated diet and were significantly lower than those fed with astaxanthin additions (Chuchird et al. 2015). Meanwhile, dietary sodium lactate at 1% reportedly improved the growth of the freshwater prawn Macrobrachium rosenbergii, but levels of 2%, 3%, or 4% caused hepatopancreatic damage (Khalil et al. 2014). To the best of our knowledge, there are no published reports on the effects of organic acid additions in the diets of the blue swimmer crab Portunus pelagicus.
Although Portunus pelagicus is a relatively new species to the aquaculture industry, there is great commercial interest in this portunid crab and other closely related species, since they can grow fast, have a good taste, and are in high demand (Noordin et al. 2015). Currently, portunid crabs are often fed with trash fish and/or diets manufactured for shrimps (Jin et al. 2013). As indicated earlier, there is increasing research that has reported on the beneficial effects of dietary organic acids in shrimp feeds. Therefore, knowing the productivity implications of crabs when fed with different organic acids will likely be important if organic acids are added to commercial shrimp feeds. The aim of this study was to examine the effects of different organic acid salts in the diets of P. pelagicus early juveniles on their survival, growth, and hepatopancreatic histopathology after 20 days.
MATERIALS AND METHODS
Five isonitrogenous diets were formulated to contain different organic acid salts, which included the control (no added organic acid salt), sodium citrate, sodium acetate, sodium propionate, or sodium butyrate added at 2%. This dose was chosen because it was optimal based on a previous study on marine shrimp (Romano et al. 2015). The dietary formulation is presented in Table 1. The main protein sources were soybean meal (40%) and fishmeal (10%), and since their crude protein levels were similar, their contributions to the dietary crude protein level of 30% would be approximately 24% and 6%, respectively. Meanwhile, agar was added as a binder and the phospholipid and cholesterol were supplemented at 6% and 1%, respectively, to satisfy the requirements for portunid crabs (Sheen 2000, Li et al. 2014). The diets were made according to slightly modified methods from those by Romano et al. (2012). Briefly, all dry and wet ingredients were thoroughly mixed separately in a blender, and then combined and mixed together. Meanwhile, 500 ml of distilled water was heated to 80[degrees]C, and then 12 g of agar powder was added while this solution was continuously mixed with a magnetic stirrer. The solution was cooled, and when the temperature reached 40[degrees]C, the ingredients were slowly added, mixed, and once all the ingredients were added, the mixture was then poured into a large blender and mixed again. This mixture was the poured on metal trays, kept in a refrigerator until gelled, and then placed in an oven at 55[degrees]C overnight. It was then broken into small pieces and kept at -20[degrees]C in airtight plastic bags until use.
The proximate composition of the diets was measured using standard AOAC (1997) methods and was generally similar among the diets. The pH was measured according to the method of Romano et al. (2015), in which 5 g of each diet were immersed in 50 ml distilled water and after 1 h, the pH was measured with a pH probe after prior calibration with precision buffers. The pH was the lowest for the sodium citrate diet, whereas the other diets had a pH similar to the control diet (Table 1).
Source of Experimental Animals and Setup
The crabs were larvicultured according to the method by Romano and Zeng (2006); however, instead of 300 1 tanks, the larvae were kept in 1,000 1 tanks. Once the crabs settled to the first crab stage (within 12 h of metamorphosis), they were siphoned out, and a total of 225 intact and apparently healthy crabs were placed individually within 500-ml clear plastic containers to prevent cannibalism. These newly settled juvenile crabs (C1 stage) had an initial mean ([+ or -]SD) wet weight, carapace width, and length of 5.9 [+ or -] 1.8 mg, 2.74 [+ or -] 0.49 mm, and 2.03 [+ or -] 0.37 mm, respectively. Once all the crabs were in the containers, they were then randomly assigned one of the five treatments, which yielded 45 replicate crabs in each treatment.
The crabs were fed with their respective diets, and in the afternoon, any remaining uneaten feed was siphoned out and new diets were provided. Each day, the crabs received a 30% water exchange and the water source was natural seawater (salinity of 32) after prior mechanical filtration (to 1 [micro]m) and ultraviolet sterilization. The water ambient temperature was 26-28[degrees]C. Each day, the presence of molts or any mortality was recorded. If a case of mortality was found, its carapace length and width was measured using a digital caliper (0.01 mm; Mitutoyo, Japan). After 10 days, when the majority of the crabs reached the third juvenile stage (C3), they were all transferred to 5-1 containers filled with 2 1 seawater.
After 20 days, all the crabs were individually measured for their carapace length, width, and wet weights. For the wet weights, the crabs were blotted dry with a tissue and placed on a zeroed electronic balance (0.01 g). The four crabs were fixed in a 4% formaldehyde, 5% acetic acid, and 1.3% calcium chloride formalin solution for 3 days and then 70% ethanol until processing to observe the histopathology. This procedure softened the crab shell, which allowed the entire crab to be sectioned without the need for dissection. The remaining crabs were measured for dry weights by drying in an oven at 50[degrees]C until constant weight.
For histology, the crabs were processed at increasing amounts of ethanol, cleared in xylene and finally in paraffin wax. After the crabs were embedded, sections (5-6 [micro]m) were made and then stained with hematoxylin and eosin (H&E) or Periodic-acid Schiff (PAS). At least eight sections (four for each stain) were made for each crab in all treatments. In all cases for PAS staining, an equal amount of sections from each treatment were stained at the same time and duration to prevent any potential bias for PAS staining intensity. In addition, separate sections were pretreated in [alpha]-amylase and stained with PAS as a control as described by Karami et al. (2016). After mounting, sections were left to dry, examined under a microscope (Leica DM750) at 40X magnification, and photographs were taken using a mounted digital camera (Leica ICC50). Particular focus was made on the hepatopancreas.
To quantify the number of epithelial cells in the hepatopancreas, 20 randomly selected tubules from four replicate crabs in each treatment were counted for the B-cells ("blasenzellen" cells), E-cells ("embryonlzellen" cells), R-cells ("restzellen" cells), and F-cells ("fibrenzellen" cells).
The specific growth rate (SGR) for wet weights, carapace lengths, and carapace widths as well as the dry weights, intermolt durations, and prevalence of epithelial cells were reported as mean [+ or -] SE. After prior confirmation of homogeneity of variance as well as normality, the data were subjected to one-way analysis of variance, and if any significant differences (P < 0.05) were detected, Duncan's post hoc test was used to identify difference among treatments. Since each crab was one replicate, a one-way analysis of variance on the survival data could not be performed.
Survival and Growth
The survival was the highest for crabs in the sodium acetate treatment (86.6%) and lowest in the control treatment (43.3%) along with the highest percentage of molt death syndrome (MDS; Table 2). The SGR for wet weights was significantly higher (P < 0.05) for crabs fed with the sodium acetate- or sodium propionate-supplemented diets, whereas the SGR for carapace length and width were significantly higher in all organic acid treatments compared with the control. Similarly, the dry weights were also significantly higher for crabs fed with the organic acid diets compared with the control (Table 2).
For the intermolt durations, there were no significant differences (P > 0.05) from the first (C1-C2) or second molt (C2-C3); however, by the third molt (C3-C4), significant differences were detected in which the crabs in all the dietary organic acid treatments had significantly shorter intermolt durations compared with the control (Table 3). Meanwhile, the total amount of molts was highest in the sodium citrate treatment, followed by the sodium propionate treatment, and the lowest was in the control.
The hepatopancreatic tubules are distributed throughout the posterior and lateral regions of the crab and consisted of predominately B- and E-cells with occasional R- and F-cells. The overall tubule shape varied from "star-like" to oval/rounded. Based on the PAS staining, the hepatopancreatic tubules of Portunus pelagicus were stained more intensely compared with the gills, connective tissue, or muscle (Fig. 1). The histological sections of the hepatopancreas from crabs in the control or the sodium acetate treatment stained with H&E are shown in Figure 2A and B, respectively. No significant differences in the epithelial cell prevalence or overall structure among all the treatments were found (Table 4). The hepatopancreatic sections from crabs in the control or sodium acetate treatments stained with PAS are shown in Figure 3A and B, respectively. No noticeable difference in the hepatopancreatic PAS staining intensity was observed among the different treatments.
Results of the current study showed that the survival, development, and growth of Portunus pelagicus juveniles were substantially improved by addition of all the tested organic acid salts in their diets. Significant improvements to the SGR for wet weights were found for crabs fed with the sodium acetate- or sodium propionate-treated diets, whereas all the tested dietary organic acids led to significantly higher SGR for carapace width and length. Similarly, the dry weights were significantly higher in all the dietary organic acid treatments compared with the control indicating a lower and higher water and meat content, respectively. Although this study had a relatively short duration of 20 days, the experiment began when the crabs reached the first crab stage and was of a sufficient duration for the majority of crabs to undergo at least three molts to reach the C4 stage. During the last transition from C3 to C4 stage, the intermolt duration was significantly shortened for the crabs in all the organic acid-treated diets indicating growth acceleration.
The benefits of including dietary organic acids have been demonstrated in several studies on crustaceans (Silva et al. 2013, Ng et al. 2015, Romano et al. 2015), although to the best of our knowledge, this is the first report on a crab species. Therefore, as a first step, different organic acid salts were chosen as additives in the diets of Portunus pelagicus and at a similar level used by other studies (Silva et al. 2013, 2015, Ng et al. 2015, Romano et al. 2015). When different organic acids were comprehensively evaluated, it was shown that dietary sodium propionate, sodium butyrate, sodium fumarate, or sodium succinate enhanced the growth of Litopenaeus vannamei, but sodium citrate, sodium formate, sodium acetate, or sodium lactate had no effect (Silva et al. 2015). In the same study, it was also found that dietary sodium citrate and sodium lactate inhibited the digestive enzymes, trypsin and chymotrypsin, of L. vannamei, whereas sodium acetate and sodium propionate enhanced these activities (Silva et al. 2015). Meanwhile, Silva et al. (2013) showed that sodium propionate and sodium butyrate acted as feed attractants and, in a follow-up experiment, significantly increased feed intake in L. vannamei. These latter findings could have important implications to the portunid crab aquaculture industry since, if organic acids can similarly act as feed attractants, cannibalism might be mitigated. This appears to warrant further research since cannibalism is recognized as a significant limitation to the nursery and grow-out farming of portunid crabs, including P. pelagicus (Ravi & Manisseri 2012).
One of the ways to determine the nutritional or overall health status of crustaceans, particularly on small samples, is by observing the histopathology of their hepatopancreas (Longo & Diaz 2015). In the context of dietary organic acids, results on the histopathology of digestive organs have been mixed. For example, Khalil et al. (2014) showed that dietary sodium lactate of 2% caused hepatopancreatic damage to Macrobrachium rosenbergii, whereas increasing sodium citrate levels from 1% to 4% in the diets of tilapia caused increased liver damage that included necrosis and hemorrhaging (Romano et al. 2016). In contrast, when Litopenaeus vannamei and Penaeus monodon were fed with an OAB in the diets at 2%, this improved the overall hepatopancreatic condition, as evidenced by significantly more R-cells, which are responsible for lipid storage (Ng et al. 2015, Romano et al. 2015). In the current study, the epithelial cells within the hepatopancreas were not significantly altered, and similarly the overall hepatopancreatic structure or PAS staining intensity did not appear noticeably different among treatments suggesting that energy storage was similar. Considering that growth and development were improved for the crabs fed with the organic acid-treated diets, it seems plausible that excess energy and nutrients could have been consumed at a faster rate to support the accelerated molting of these young crabs and/or the relatively short duration that minimized excess nutrient storage. In the latter case, the R-cells were the least prevalent compared with the other epithelial cells that consisted of B-cells, which are responsible for digestive enzyme secretion/nutrient absorption, F-cells, which synthesize enzymes, and E-cells, which are undifferentiated. This is in contrast to the R- and E-cells reportedly being the most and least abundant cells, respectively, in the hepatopancreas of juvenile/adult crabs (Longo & Diaz 2015), and such a discrepancy may be related to the age difference of the crabs.
The relatively low survival in the control treatment of 43.3%, which was often attributed to MDS, appears to imply that the control diet was nutritionally suboptimal. The dietary protein was indeed relatively low at approximately 29%; however, a similar level of 31%, which was mostly derived from fishmeal, had no effect on the survival of Portunus trituberculatus (Jin et al. 2013). It appears more likely that the higher mortalities were related to the use of soybean meal as the dominant protein source, since this may reduce nutrient utilization to aquatic animals. For example, soybean meal is well known to contain phytic acid, which is an antinutritional factor that stores phosphorus in an inaccessible form, but may also bind with minerals and proteins (Ng & Romano 2013). Dietary organic acids have been shown to improve nutrient digestibility, particularly for phosphorus (Silva et al. 2013, 2015, Ng et al. 2015, Romano et al. 2015), which could potentially explain the drastically higher survival of the crabs (>72%) with dietary organic acid inclusions. Some of the modes of action include chelating minerals as well as acting as an energy source, enhancing digestive enzyme activity, and/or improving feed attractiveness (Ng & Koh 2016), and requires further investigations. Moreover, the antimicrobial and immune-stimulating actions of some dietary organic acids have been observed in fish and shrimp (Ng & Koh 2016), and also warrants further research in portunid crabs as it relates to health and increased survival.
In conclusion, sodium propionate and sodium acetate in the diets of Portunus pelagicus appeared to provide the best results in terms of survival and growth; however, all the tested organic acid types that included sodium acetate, sodium citrate, sodium propionate, and sodium butyrate substantially improved the survival, growth, and development of P. pelagicus early juveniles. Despite such an improvement, there appeared to be no difference in the hepatopancreatic structure, prevalence of epithelial cells, or staining intensity for glycogen. Based on the results of the current study, the inclusion of these organic acids in the diets of P. pelagicus would likely benefit this industry. More investigations are needed to understand the underlying mechanisms for such an improvement as well as potential use of these organic acid salts as feed attractants to cannibalistic crustacean species.
This study was funded by a grant from Universiti Putra Malaysia (UPM), project no. GP-IPB/2014/9440403.
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SUHAILA ABDUL SUKOR, (1) SOFEA TAHER, (1) FARIBORZ EHTESHAMEI, (1) AZIZ ARSHAD, (1) WING-KEONG NG (2) AND NICHOLAS ROMANO (1) *
(1) Department of Aquaculture, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia; (2) Fish Nutrition Laboratory, School of Biological Sciences, Universiti Sains Malaysia, Penang 11800, Malaysia
* Corresponding author. E-mails: firstname.lastname@example.org or email@example.com
TABLE 1. Ingredient formulation and proximate composition (% dry weight) of the experimental diets. Sodium Sodium Ingredients Control citrate acetate Fishmeal * 10 10 10 Soybean meal 40 40 40 ([dagger]) Lecithin 6 6 6 ([double dagger]) Cholesterol 1 1 1 ([double dagger]) Vitamin and mineral 8 8 8 premix ([section]) Tapioca starch 12 12 12 Dicalcium phosphate 0.5 0.5 0.5 ([double dagger]) Choline chloride 1 1 1 ([double dagger]) Agar ([double dagger]) 12 12 12 Fish oil ([paragraph]) 7.5 7.5 7.5 Sodium citrate 0 2 0 ([parallel]) Sodium acetate 0 0 2 ([parallel]) Sodium propionate 0 0 0 ([parallel]) Sodium butyrate 0 0 0 ([parallel]) Cellulose 2 0 0 Proximate composition Dry matter 96.42 96.67 96.29 Crude protein 29.15 28.36 29.18 Crude lipid 5.57 5.50 5.48 Crude fiber 3.28 3.40 2.78 Crude ash 9.79 9.73 10.86 pH 6.37 5.58 6.54 Sodium Sodium Ingredients propionate butyrate Fishmeal * 10 10 Soybean meal 40 40 ([dagger]) Lecithin 6 6 ([double dagger]) Cholesterol 1 1 ([double dagger]) Vitamin and mineral 8 8 premix ([section]) Tapioca starch 12 12 Dicalcium phosphate 0.5 0.5 ([double dagger]) Choline chloride 1 1 ([double dagger]) Agar ([double dagger]) 12 12 Fish oil ([paragraph]) 7.5 7.5 Sodium citrate 0 0 ([parallel]) Sodium acetate 0 0 ([parallel]) Sodium propionate 2 0 ([parallel]) Sodium butyrate 0 2 ([parallel]) Cellulose 0 0 Proximate composition Dry matter 96.03 96.00 Crude protein 29.12 28.92 Crude lipid 6.10 5.74 Crude fiber 2.68 2.20 Crude ash 10.83 10.58 pH 6.49 6.35 * Local fishmeal (54.07% crude protein, 4.93% crude lipid on as is basis), ([dagger]) Soybean meal (54.48% crude protein, 1.75% crude lipid on as is basis). ([double dagger]) Sigma Aldrich Co.: lecithin, 30% phosphatidylcholine P3644; cholesterol, 92.5% powder C8503; dicalcium phosphate, C7263; choline chloride, 98% powder C7527; and agar, A7002. ([section]) Same composition as given in the work of Romano et al. (2016). ([paragraph]) From menhaden source. ([parallel]) Sigma Aldrich Co.: sodium citrate, 71497; sodium acetate, S2889; sodium propionate. P1880; and sodium butyrate, 303410. TABLE 2. Survival (%), MDS (%), mean ([+ or -] SE) SGRs for wet weights, carapace length, carapace width, and final dry weights of early juvenile blue swimmer crabs Portunus pelagicus after being fed diets containing different organic acids for 20 days. Survival MDS SGR weight (%) (%) (%/day) Control 43.3 20.7 4.83 [+ or -] 0.84 (b) Sodium citrate 75.1 11.9 6.46 [+ or -] 0.41 (ab) Sodium acetate 86.6 10.4 7.89 [+ or -] 0.85 9 (a) Sodium propionate 78.0 11.5 7.21 [+ or -] 0.48 (a) Sodium butyrate 72.2 12.5 6.45 [+ or -] 0.68 (ab) P values na na 0.032 SGR width SGR length (%/day) (%/day) Control 3.02 [+ or -] 0.21 (b) 2.43 [+ or -] 0.31 (b) Sodium citrate 3.66 [+ or -] 0.16 (a) 2.99 [+ or -] 0.18 (ab) Sodium acetate 3.76 [+ or -] 0.13 (a) 3.04 [+ or -] 0.18 (a) Sodium propionate 3.72 [+ or -] 0.14 (a) 3.18 [+ or -] 0.15 (a) Sodium butyrate 3.62 [+ or -] 0.20 (a) 3.14 [+ or -] 0.19 (a) P values 0.044 0.047 Dry weights (mg) Control 1.5 [+ or -] 0.1 (b) Sodium citrate 2.4 [+ or -] 0.3 (a) Sodium acetate 2.3 [+ or -] 0.2 (a) Sodium propionate 2.6 [+ or -] 0.1 (a) Sodium butyrate 2.8 [+ or -] 0.3 (a) P values 0.009 "na" for survival and MDS since each crab acted as a replicate and could not be included in statistical analysis. TABLE 3. Mean ([+ or -] SE) intermolt duration (days) of the first three crab stages and the total number of molts from Portunus pelagicus early juveniles after being fed diets containing different organic acids for 20 days. Treatment C1-C2 C2-C3 Control 3.16 [+ or -] 0.36 (31) 7.98 [+ or -] 0.46(11) Sodium citrate 3.53 [+ or -] 0.38 (38) 7.57 [+ or -] 0.45 (30) Sodium acetate 3.09 [+ or -] 0.31 (32) 7.50 [+ or -] 0.47 (18) Sodium propionate 3.47 [+ or -] 0.42 (33) 6.91 [+ or -] 0.61 (25) Sodium butyrate 3.11 [+ or -] 0.37 (34) 7.89 [+ or -] 1.03(14) P values 0.853 0.265 Total number Treatment C3-C4 of molts Control 10.03 [+ or -] 0.83a (3) 45 Sodium citrate 7.16 [+ or -] 0.70b (11) 79 Sodium acetate 8.52 [+ or -] 0.64b (7) 57 Sodium propionate 8.65 [+ or -] 0.86b (8) 66 Sodium butyrate 7.75 [+ or -] 0.77b (5) 53 P values 0.012 The values in parentheses are "n" values. TABLE 4. Mean ([+ or -] SE) prevalence of B-, R-, E-, and F-cells in the hepatopancreatic tubules of Portunus pelagicus early juveniles after being fed diets containing different organic acid types for 20 days. Treatment B-cells R-cells Control 4.87 [+ or -] 0.54 3.55 [+ or -] 0.58 Sodium citrate 5.38 [+ or -] 1.25 3.18 [+ or -] 0.75 Sodium acetate 5.16 [+ or -] 0.52 3.93 [+ or -] 0.28 Sodium 5.74 [+ or -] 1.05 3.99 [+ or -] 0.61 propionate Sodium butyrate 4.91 [+ or -] 1.22 4.15 [+ or -] 0.83 Treatment F-cells E-cells Control 7.97 [+ or -] 2.20 5.34 [+ or -] 1.21 Sodium citrate 8.45 [+ or -] 2.41 7.16 [+ or -] 2.04 Sodium acetate 6.36 [+ or -] 1.92 6.29 [+ or -] 1.54 Sodium 7.24 [+ or -] 1.73 6.81 [+ or -] 2.39 propionate Sodium butyrate 6.92 [+ or -] 2.54 7.08 [+ or -] 3.11 No significant differences among the treatments were detected (P > 0.05).
Please note: Some tables or figures were omitted from this article.
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|Author:||Sukor, Suhaila Abdul; Taher, Sofea; Ehteshamei, Fariborz; Arshad, Aziz; Ng, Wing-Keong; Romano, Nich|
|Publication:||Journal of Shellfish Research|
|Date:||Aug 1, 2016|
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