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Growth rate and fatty acid composition of orange mud crab instars, Scylla olivacea, reared at different temperatures
Egyptian Journal of Aquatic Research xxx (xxxx) xxx
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Growth rate and fatty acid composition of orange mud crab instars, Scylla olivacea, reared at different temperatures Mohamad N. Azra a,⇑, Camila Prestes Dos Santos Tavares b, Ambok Bolong Abol-Munafi a, Mhd Ikhwanuddin a,c,⇑ a b c
Institute of Tropical Aquaculture and Fisheries, Universiti Malaysia Terengganu, 21030, Kuala Nerus, Terengganu, Malaysia Integrated Group of Aquaculture and Environmental Studies, Federal University of Paraná, 80035-050 Curitiba, Parana, Brazil STU-UMT Joint Shellfish Research Laboratory, Shantou University, Guangdong, China
a r t i c l e
i n f o
Article history: Received 4 May 2019 Revised 7 November 2019 Accepted 21 November 2019 Available online xxxx Keywords: Aquaculture Crustacean Fatty acids Molt Scylla olivacea Temperature
a b s t r a c t The objective of the present study was to examine the effect of different rearing temperatures on growth, survival, instar development, and fatty acid (FA) composition of orange mud crab instars, Scylla olivacea. The experiment was conducted at four different water temperatures of 24 °C, 28 °C, 32 °C and 36 °C using instar stage 1 (C1) in temperature-controlled recirculating rearing systems and lasted for 30 days. The study findings revealed that higher temperatures (32 °C) significantly reduced intermolt duration, whereby most crabs reached instar stages C6-C7, although 10.3% were still in the C2C3 developmental stage. In addition, at temperature 36 °C all crabs were dead after three days of culture. Surprisingly, at constant temperature of 28 °C, total Polyunsaturated Fatty Acid were lower (0.52) compared to those at lower, 24 °C (0.71) and higher temperature of 32 °C (0.74). However, the direct effects of temperature on total fatty acids composition in crabs could not be distinguished from indirect effects of variable crab size and molt number. In conclusion, high temperature of 32 °C negatively affects instars growth and may induce molting, especially in S. olivacea. Ó 2019 National Institute of Oceanography and Fisheries. Hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction Information on instar growth and survival of brachyuran crabs is essential for enhancing seed production in nursery conditions, which will indirectly increase hatchery profitability. Mud crabs, genus Scylla, are used as a model organism in most studies because of their convenient size, easy availability, and the fact that they are commercially important in fisheries and aquaculture industries. The instar mud crab’s carapace size, and especially those pertaining to genus Scylla, ranges from 1.5 to 4.0 cm, requiring three to four weeks of rearing in the nursery (Quinitio and Estepa, 2011). Studies of the effects of climate change-mediated water temperature on crustacean growth and development are increasingly being conducted with the aim of increasing production and developing sustainable culture management practices Peer review under responsibility of National Institute of Oceanography and Fisheries. ⇑ Corresponding authors at: Institute of Tropical Aquaculture and Fisheries, Universiti Malaysia Terengganu, Mengabang Telipot, 21030, Kuala Nerus, Terengganu, Malaysia. E-mail addresses: [email protected] (M.N. Azra), [email protected] (M. Ikhwanuddin).
(Abol-Munafi and Azra, 2018). Evidence yielded by such investigations indicates that water temperature plays a vital role in the physiology, growth, and maturation of brachyuran crabs (Fischer and Thatje, 2008; Penha-Lopes et al. 2006). The effects of water temperature on mud crabs were examined by Nurdiani and Zeng (2007) and Ruscoe et al. (2004), whereby crabs were reared from the early larval stage up to instar C3. However, as crabs are capable of adapting to temperature changes in the subsequent growth stages, more comprehensive studies are needed spanning the entire C1C7 range. Recently, Ikhwanuddin et al. (2019) investigated the effects of water temperature on instar crabs during their culture and development. Biochemical parameters, such as lipid and fatty acid (FA) composition, have been consistently used as nutritional status of aquaculture species (El-Ghafour et al. 2018; Dridi et al. 2017; El-Karim et al. 2016; Li et al., 2014; El-Kassas, 2013). In general, FAs are essential for marine crustacean development, as they provide the structural membrane components, as well as serve as a source of energy, especially in the early lifecycle stages (Stoner et al. 2013). Thus, data on FA composition might be a useful tool for the formulation of most optimal crab diet, especially in the early developmental stages.
https://doi.org/10.1016/j.ejar.2019.11.006 1687-4285/Ó 2019 National Institute of Oceanography and Fisheries. Hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article as: M. N. Azra, C. P. D. S. Tavares, A. B. Abol-Munafi et al., Growth rate and fatty acid composition of orange mud crab instars, Scylla olivacea, reared at different temperatures, Egyptian Journal of Aquatic Research, https://doi.org/10.1016/j.ejar.2019.11.006
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M.N. Azra et al. / Egyptian Journal of Aquatic Research xxx (xxxx) xxx
On the other hand, the effect of water temperature on lipid and FA composition in brachyuran crabs has been previously explored (Matozzo et al., 2011). To the best of our knowledge, no information is presently available on the growth of S. olivacea instars reared at different temperatures and the link between the growth conditions and the whole-body FA composition. In fact, only few studies on the instar stages of portunid crabs have been conducted to date, while extensive research exists on the larval and broodstock stages (Tavares et al. 2018; Ghazali et al. 2017a; Taufik et al. 2016). We have been motivated by this gap in the extant knowledge to conduct the present study with the main objective to determine the effects of four rearing temperatures (24, 28, 32 and 36 °C) on the survival, individual growth, intermolt duration, instar development, and fatty acid composition of the orange mud crab, S. olivacea. Materials and methods
(2018). The complete experimental setup was based on a modification from previous study by Azra et al. (2018). A controlled chiller system cooled and maintained the water temperature at 24 °C. While 28 °C, 32 °C and 36 °C were achieved by using one, three, and four thermostatic heaters, respectively. Fifteen cylindrical compartments (one compartment per crab instar) in each of twelve RMASs were used to culture the crabs individually. Four simultaneous replicates were utilized for each temperature treatment (n = 180). Instar crabs were fed chopped Selaroides spp. ad libitum for one hour twice daily, at 08:00 and 20:00 h. Before the start of each trial (single experiment with all treatments), the water temperature in the instar culture tank was gradually increased or decreased by 1 °C per hour (from 26 °C) until the desired experimental temperature was reached. During the experiment, water salinity was kept at 26.0–27.0‰. The water quality parameters were evaluated twice daily before the feeding times, and were maintained at the following levels: ammonia-N < 0.5 mg L-1, nitrite < 0.15 mg L-1, DO > 4 mg L-1, and pH at 7.3–8.1.
Sampling and production of berried females
Growth and survival
Sixty mature female (carapace width > 9.5 cm) S. olivacea crabs were collected from Terengganu coastal waters and were transported to the Institute of Tropical Aquaculture and Fisheries’s Hatchery, Universiti Malaysia Terengganu, Terengganu, Malaysia. Water salinity and temperature were maintained at 25.0–27.0 ppt and 26.5–28.3 °C, respectively. Crabs were fed raw squid (Loligo spp.) ad libitum twice daily (at 08:00 and 20:00 h) until they spawned (Azra and Ikhwanuddin, 2015). Every day, uneaten food and waste was removed an hour after feeding. The spawning crabs were transferred into 100 L tanks and were reared individually, without feeding, until the eggs hatched. To ensure that all the instars were of the same age, only females whose eggs hatched on the same day (within 12 hours) were included in the sample for further analyses.
Instar crabs were checked daily for molting. If an individual molted, its carapace width (CW) was assessed after the carapace had fully hardened using a Vernier caliper (Mitutoyo Model CD600 CSX). Crab survival was defined as the percentage of crabs that survived for 30 days, and the survival rates for each day were calculated as day x = (crab abundance at day 0 – cumulative mortality by day x) / crab abundance. Survival was determined by direct observation, with minimal potential for error, as the crabs were reared in individual cylindrical compartments.
Larval rearing and instar preparation Newly hatched phototactic larvae were siphoned off from the hatching tanks to a 500 L larvae tank and were reared from zoea stage 1 (Z1) until they reached the megalopa stage. The specimens that reached the megalopa stage were cultured individually in 0.5 L plastic containers until they reached the first instar stage, C1. On average, it took 30 days for the larvae at Z1 to reach the first day of C1. The mean survival rate from Z1 to C1 was 3.5%. During larval culture, the water salinity was maintained at 26.0–27.0%, the pH was kept within the 7.55–8.10 range, while ensuring that dissolved oxygen (DO) content was above 6 mg L-1, and the temperature remained within 26.8 °C to 28.1 °C. The water culture was exchanged at the rate of 10% every day during the larval rearing culture. The zoea feeding regimes were based on the previous study by Ikhwanuddin et al. (2012).
Fatty acid analysis Prior to the final (30th) day of culture, and after survival had been established, whole-body FA composition was determined by the one-step method described by Abdulkadir and Tsuchiya (2008). A pooled sample of five to six crabs from each replicate was randomly collected, cryo-anesthetized for 5 min and cryopreserved at 80 °C for subsequent FA analysis. This was principally carried out by combining the extraction and esterification processes. After adding 2 mL 14% boron trifluoride (BF3) in methanol and a magnetic stirring bar, the tubes were flushed with nitrogen gas and sealed. The capped tubes incubated at 100 °C for 120 min with continuous stirring. After the solutions cooled to room temperature, 1 mL hexane was added, followed by 2 mL distilled water. The tube was vortexed until it was fully blended and then centrifuged for 3 min at 2,500 rpm (650g). The upper hexane layer containing the fatty acid methyl esters (FAMEs) was subjected to gas chromatography, using GC-2010 Shimadzu coupled to the AOC-20i + s detector, allowing the FAME peaks to be separated and quantified. The FAMEs were identified by comparing their retention times with the data available in the NIST08 Mass Spectral Library database.
Experimental design Statistical analysis Only healthy, active, and intact crabs at C1 stage were randomly chosen for the experiment, with the initial mean carapace length (CL) of 3.23 ± 0.07 mm. These crabs were cultured for 30 days in twelve Re-circulating Marine Aquaculture System (RMAS) with aerated, filtered seawater equipped with thermostatically controlled chillers and immersion heaters at four rearing temperatures, namely 24 °C, 28 °C, 32 °C and 36 °C. The selected temperatures and the setup adopted for cooling and heating the water were based on the methodology adopted by Azra et al.
One-way ANOVA was conducted to ascertain if the differences are statistically significant in CW and intermolt duration at different instar stages after 30 days of culture. Whereas a post-hoc test (Duncan’s multiple range tests) was performed for comparisons. Appropriate data transformations were performed as necessary, following Levene’s test for homogeneity of variance. The analysis results were presented as means ± standard deviations, and p < 0.05 was adopted as the indicator of statistical significance.
Please cite this article as: M. N. Azra, C. P. D. S. Tavares, A. B. Abol-Munafi et al., Growth rate and fatty acid composition of orange mud crab instars, Scylla olivacea, reared at different temperatures, Egyptian Journal of Aquatic Research, https://doi.org/10.1016/j.ejar.2019.11.006
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M.N. Azra et al. / Egyptian Journal of Aquatic Research xxx (xxxx) xxx
Results Growth and survival After 30 days of culture, no major mortality was noted at any of the tested temperatures (except the 36 °C with 100% mortality after three days of culture). Overall, > 74.3% of the specimens survived from C1 to C7. Higher mortality (87.5%) occurred during the early stages (C1 to C2, and C3 to C4) compared to the later stages (12.5%). Once crabs reached C4 or C5, their survival rapidly increased reaching > 90% until the crabs reached the C6C7 stage. The lowest survival rate was observed at 32 °C (74.3%), whereas rearing crabs at 24 °C resulted in the highest survival rate of 87.8%. At 28 °C, 80.5% of crabs survived, and at 36 °C, all crabs were dead after three days of culture, which precluded the use of data pertaining to this temperature in further analyses. There are significant differences in the survival among the culture temperature (p < 0.05). The CW increased throughout the culture period and
the effects of water temperature on CW were statistically significant (p < 0.05) (Table 1). However, ANOVA results showed the effects of the three treatments (24, 28, and 32 °C) on CW sizes at the early culture stage. C1C2 were not statistically significant, whereas statistical significance (p < 0.05) was established at the later culture stages of C2C7 (Table 1). Specifically, culture at 32 °C significantly reduced intermolt duration, and crabs subjected to this treatment successfully reached instar stages C6 to C7 after 29–30 days (Table 1). Moreover, while the CWs decreased at early stages and increased at later stages at 32 °C, no significant differences were seen at 28 °C (Table 1). Instar development At the end of 30 days of culture, the majority of crabs reared at 24 °C reached C2 to C5, while crabs at 32 °C were at the C5 to C7 developmental stage (Fig. 1). Crabs reared at 28 °C exhibited a high
Table 1 Mean carapace width increment, mean intermolt duration, and percentage increase in different instar stages, of orange mud crab, Scylla olivacea at three water temperatures (24, 28, and 32 °C) based on 30 days of cultivation. Water Tempe-rature (°C)
Mean number of days until the next molting C1 C2
C2 C3
C3 C4
C4 C5
C5 C6
C6 C7
24 28 32
6 5 3 Mean carapace width in mm (mean ± SD) 4.41 ± 0.20a 4.39 ± 0.15a 4.47 ± 0.21a
7 6 4 Percentage increase
8 7 5
9 8 5
N/A N/A 6
N/A N/A 7
29–30 26–30 29–30
5.65 ± 0.21a 5.43 ± 0.22b 5.66 ± 0.29a
7.08 ± 0.33a 6.73 ± 0.25b 6.91 ± 0.28c
9.08 ± 0.20a 8.26 ± 0.40b 8.42 ± 0.39b
N/A N/A 10.15 ± 0.41
N/A N/A 13.01 ± 0.23
28.11»25.31»28.25 (C1 to C5) 23.69»23.94»22.73 (C1 to C5) 26.62»22.08»21.85»20.55»28.18 (C1 to C7)
24 28 32
Total days
SD = standard deviation. Superscript letters indicate significant difference at p < 0.05.
Fig. 1. Instar development of orange mud crab, Scylla olivacea, by moult stage at the end of the 30-day growth period at three experimental temperatures. Histograms represent total counts for all replicates.
Please cite this article as: M. N. Azra, C. P. D. S. Tavares, A. B. Abol-Munafi et al., Growth rate and fatty acid composition of orange mud crab instars, Scylla olivacea, reared at different temperatures, Egyptian Journal of Aquatic Research, https://doi.org/10.1016/j.ejar.2019.11.006
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Table 2 Fatty acid compositions (mg g 1 dry weight) of whole body compositions of orange mud crab, Scylla olivacea at different culture temperatures. Fatty acids
24 °C
28 °C
32 °C
C4:0 C6:0 C8:0 C10:0 C14:0 C15:0 C16:0 C17:0 C18:0 C20:0 C21:0 C22:0 C23:0 Total SFA1 C14:1 C15:1 C16:1 C20:1 C24:1 C18:1N9T C22:1N9 Total MUFA2 C18:2N6T C18:2N6C C18:3N6 C20:3N6 C20:4N6 n-6 C18:3N3 C20:3N3 C20:5N3 C22:6N3 n-3 Total PUFA3 Total LC-PUFA4 Total Fatty Acids (n-3/n-6) ratio DHA:EPA concentration (ratio)
1.40 ± 0.23a 0.13 ± 0.15a 0.65 ± 0.16a – 0.11 ± 0.15 0.06 ± 0.08a 0.64 ± 0.18a 0.09 ± 0.01a 0.35 ± 0.07a 0.05 ± 0.04a 0.05 ± 0.09a 0.36 ± 0.51a 0.03 ± 0.03a 3.92 0.05 ± 0.06a 0.20 ± 0.03a 0.07 ± 0.04a 0.18 ± 0.10a 1.08 ± 0.90a 1.25 ± 0.08a 0.06 ± 0.09a 2.89 0.01 ± 0.02a 0.11 ± 0.04a 0.02 ± 0.02a 0.03 ± 0.06a 0.07 ± 0.03a 0.24 0.01 ± 0.01a 0.04 ± 0.07a 0.19 ± 0.25a 0.23 ± 0.15a 0.47 0.71 0.56 7.53 ± 0.85 1.96 0.23: 0.19 (1.21)
0.86 ± 0.15b 0.09 ± 0.03b 0.44 ± 0.01b – – 0.03 ± 0.01b 0.42 ± 0.02b 0.08 ± 0.01a 0.32 ± 0.04a 0.01 ± 0.01a 0.01 ± 0.01b 0.10 ± 0.08b 0.01 ± 0.01a 2.37 0.04 ± 0.03a 0.21 ± 0.14a 0.04 ± 0.04b 0.07 ± 0.05b 0.76 ± 0.94b 0.98 ± 0.07b – 2.10 0.04 ± 0.05b 0.06 ± 0.01b 0.02 ± 0.03a 0.02 ± 0.03a 0.06 ± 0.02a 0.20 0.01 ± 0.01a 0.08 ± 0.07b 0.09 ± 0.03b 0.14 ± 0.05b 0.32 0.52 0.39 5.01 ± 0.21 1.60 0.14: 0.09 (1.56)
0.82 ± 0.15b 0.02 ± 0.02c 0.50 ± 0.07c 0.05 ± 0.06 – 0.02 ± 0.01b 0.05 ± 0.02c 0.58 ± 0.04b 0.11 ± 0.03b 0.40 ± 0.03b 0.04 ± 0.08a 0.13 ± 0.02b 0.19 ± 0.09b 2.91 – 0.22 ± 0.05a 0.11 ± 0.03c 0.11 ± 0.07c 0.52 ± 0.43c 1.23 ± 0.11a 0.03 ± 0.01b 2.22 0.06 ± 0.04b 0.07 ± 0.01b 0.02 ± 0.01a 0.07 ± 0.03b 0.06 ± 0.01a 0.28 0.02 ± 0.01a 0.02 ± 0.02c 0.24 ± 0.15c 0.18 ± 0.05c 0.46 0.74 0.57 5.87 ± 0.57 1.64 0.18: 0.24 (0.75)
Within rows for each fatty acids class, means with common letters are not significantly different. 1Saturated fatty acids – sum of all FA without double bonds.2Monounsaturated fatty acids – sum of all FA with a single double bond. 3 Polyunsaturated fatty acid – sum of all FA with 2 double bonds. 4Long-chain PUFA – sum of all FA with chain length 20 carbon atoms and 3 double bonds.
synchronous molting rate, with >96% measured at C5 (Fig. 1). At 32 °C, crabs reached the C6C7 stage earlier than those reared at 24 and 28 °C. In contrast, crabs reared at 24 °C reached only C4C5 and their CW was significantly reduced. Fatty acid (FA) composition The dominant fatty acid (FA) components identified in these specimens were saturated fatty acids (SFAs) with percentage from 47% to 52% of Total Fatty Acids (TFA). The ratio of omega-3 to omega-6 content was higher in crabs reared at 24 °C and than in those subjected to 28 °C and 32 °C treatment. The results also showed that total FAs were higher in cooler temperature compared to all experimental temperature (Table 2). Discussion Authors of previous study in this field have established that high survival rate, high synchronous molting rate, and short molting period are the optimal crustacean growth parameters, especially for crabs and shrimp (Figueiredo et al. 2008). The analyses conducted as part of the present investigation demonstrated that
the lowest temperature tested, 24 °C, resulted in the highest survival rates, while the moderate temperature of 28 °C led to high synchronous molting rates, and the highest temperature, 32 °C, shortened the percentage of CW increment. The present study supported the recent review by Azra et al. (2019), which stated that water temperature is among the most important environmental factors affecting the survival and development of brachyuran crabs. Effects of water temperature on growth, survival, and instar development The lowest and the highest survival rate of 74.3% and 87.8% was observed at 32 °C and 24 °C, respectively. As crabs were reared in individual culture vessels to eliminate the potential for intraspecific competition and cannibalism, this likely contributed to the higher survival rates (Yang et al. 2018) from stage C4 to C5. Moreover, survival rates subsequently increased and >90% of instars were maintained alive until the crabs reached C6C7, while higher mortality rates were recorded during the early stages, C1 to C3. After three days of culture at 36 °C, 100% mortality was noted, which precluded analyses based on this temperature. Similar observations were made by Gong et al. (2015), who studied the green mud crab, Scylla paramamosain, as well as by Penha-Lopes et al. (2006) in their investigation of the spider crab, Mithraculus forceps. Moreover, Gong et al. (2015) showed that culture at temperature of 39 °C led to 100% mortality of all C1 crabs and no successful molting was recorded. This suggests that 39 °C might be near the upper limit of the larvae’s ability to acclimatize (Azra et al. 2018). A slight delay of instar development, at the lower temperature, may further increase their chance of survival, which may be attributed to the increasing body thickness of the integument with growth. The thickening of integument in the later stage may have provided a protective mechanical protection to lower temperature (Baylon, 2009). A decrease in intermolt duration with increased temperature has been reported for other brachyuran and anomuran crabs (Gong et al., 2015), and our findings likewise showed that S. olivacea crabs reared at 32 °C had a shorter average molting duration. This was expected, as metabolic processes in most poikilotherms are expedited at higher temperatures (Hartnoll, 2001). After 30 days, the crabs cultured at 32 °C had molted six times, while those reared at 24 °C had only molted four times. The limited amount of published data on instar larval development, especially at different rearing temperatures related to the nursery culture of portunid crabs, makes the comparisons with the findings reported by other authors difficult. However, the evidence yielded by previous studies on the blue king crab, Paralithodes platypus, and the red king crab, Paralithodes camtschaticus, indicates that the majority of instars reared at 1.5 °C remained at C1 to C2. However, crabs reared at a higher temperature of 12 °C reached C5 to C6 after 60 days of culture (Stoner et al., 2010; Stoner et al., 2013). Our results were similar to those pertaining to lithodid crabs, reported by Stoner et al. (2013), who noted that, after 30 days of culture (60 days after hatching) at 24 °C, hatchlings reached C2 to C3 developmental stage, whereas crabs reared at 32 °C were at stage C6 to C7. The present investigation also shows that after 30 days of culture period, only crabs reared in 32 °C reached the C7 stage, compared to others tested temperatures where crabs reached the C4 to C5 stage only. Effects of temperature on crab fatty acid composition The biochemical changes at various stages in the crustacean lifecycle, especially the FA composition, have been the subject of
Please cite this article as: M. N. Azra, C. P. D. S. Tavares, A. B. Abol-Munafi et al., Growth rate and fatty acid composition of orange mud crab instars, Scylla olivacea, reared at different temperatures, Egyptian Journal of Aquatic Research, https://doi.org/10.1016/j.ejar.2019.11.006
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M.N. Azra et al. / Egyptian Journal of Aquatic Research xxx (xxxx) xxx Table 3 Effect of water temperature on fatty acid composition in crabs. Common and species name
Temperature
Effect on FAs
Reference
Mud crab, Scylla serrata
Acclimation temperature of 5 °C and 10 °C, control at 27 °C 1.5, 4.5, 8 and 12 °C
Significant difference in FA composition at low temperatures Warmer temperature produced higher docosahexaenoic acid (DHA): eicosapentaenoic acid (EPA) ratio and elevated the monounsaturated FA amounts Total fatty acid composition varied under low temperature adaptation, whereby long-chain FA levels increased at lower temperatures Lower quantities of long-chain FAs at the lowest experimental temperatures of 24 °C and the highest temperature of 32 °C compared to the constant temperature of 28 °C and a higher EPA:DHA ratio at the lowest (24 °C) and the highest temperature (32 °C)
Kong et al. (2007)
Red king crab, Paralithodes camtschaticus Black mud crab, Scylla serrata
Adaptation temperature of 5, 10 and 15 °C, control at 27 °C
Orange mud crab, Scylla olivacea
Nursery temperature of 24, 28 and 32 °C
extensive studies over the past decade due to the fact that FAs play a vital role in the development of marine crustaceans as sources of energy and structural components of membranes (Suprayudi et al. 2004). However, there is a paucity of studies focusing specifically on the instar/juvenile and early adult stages, and most extant researches have been conducted on the anomuran rather than brachyuran crabs. In addition, authors of the limited number of studies pertaining to brachyurans focused on adults and the early life history stages of embryonic development (Ghazali et al 2017b; Ikhwanuddin et al. 2018). Moreover, changes in FA composition as a function of water temperature at which crabs are reared are commonly used as an indicator of their ability to acclimatize to a sudden change in water temperature through the adjustment of membrane fluidity (Stoner et al., 2010). Given the limited understanding of the link between FA composition in the instar/juvenile stages and rearing temperature, the present study has yielded some valuable findings. For example, growth and development in warmer temperatures resulted in higher levels of SFAs and fewer unsaturated FAs, as well as higher levels of monounsaturated FAs (MUFA) with a higher EPA:DHA ratio (Table 3). The DHA and EPA fatty acids play important roles in the early survival and growth of marine larvae (Suprayudi et al., 2004). However, a direct comparison of EPA:DHA ratios at different temperatures was not possible as a part of this investigation because most crabs were at different stages in the lifecycle. However, since crabs reared at 24 °C and 28 °C reached the same stage (C4C5) in the final days of the 30-day culture period, an indirect comparison was made. The obtained findings indicate that the higher EPA:DHA ratios obtained at 28 °C seem to be associated with a smaller carapace size (8.26 mm) and shorter intermolt duration (26–30 days) compared to those obtained at 24 °C, where crabs had an average CW of 9.08 mm and a longer intermolt duration of 29–30 days. These results are in line with those reported by Takeuchi et al. (1999), who concluded that both EPA and DHA were important in shortening the intermolt duration as well as increasing the CW of Portunus trituberculatus. The present study also showed that total Polyunsaturated Fatty Acid (PUFA) and Long-Chain Polyunsaturated Fatty Acids (LC-PUFA) were lower at constant temperature of 28 °C, compared to the lower (24 °C) and higher (32 °C) temperature. This might caused by the fact that most of the crabs at constant temperature produce the same stage of instar development (Fig. 1). By day 30, the most dominant FA components in S. olivacea crablets were SFAs, a type of FA with no double bonds, which was found to be the most critical FA group for growth, survival, and reproduction in most marine aquaculture species (Glencross, 2009). Crabs reared at all three temperatures had lower concentrations of LC-PUFAs relative to other FAs. This finding is in line with the observation made by Glencross (2009), who noted that, in most animals,
Stoner et al. (2010)
Wang et al. (2007)
Present study
especially mud crabs, the LC-PUFAs cannot be synthesized de novo, and must therefore be obtained from the diet or as precursors present in the food. The omega-3 and omega-6 FAs are essential dietary components because they affect the larval growth rate, the CW, and the flesh quality of species raised by aquaculture (Glencross, 2009). In the present study, a direct comparison of omega-3 and omega6 concentrations in crabs raised at the three experimental temperatures was not possible since the crabs were at different stages of their lifecycle during culture. Equally, indirect comparison cannot be made of crabs cultured at 24 °C with those at 28 °C even though some crabs reached the same stage of growth, C4C5. The obtained results are in line with those obtained by Wang et al. (2007), who reported increased concentrations of LC-PUFAs in crabs cultured at a lower temperature. Furthermore, Purac et al. (2011) reported that the crab’s metabolic rates tend to decline at low temperatures, which alters the rate of FA biosynthesis or utilization. The ratio of omega-3 to omega-6 was higher in crabs reared at 32 °C, indicating that most crabs reached C4 to C7 developmental stage. Thus, we propose the use of the ratio of omega-3 to omega-6 in future studies to determine the development status of marine animals, especially crabs. Conclusion and implications In conclusion, higher temperatures of 32 °C negatively affected instars growth and might have induced molting. The present results suggest a constant temperature of 28 °C for synchronized molting of S. olivacea. The scynchronization of molted crabs might give huge advantages to avoid any cannibalism chance of culturing the instar crab during the nursery stage. Water temperature is a fundamental and dominant determinant of the instar development phase of portunid crabs. Findings yielded by the extant studies in this field showed that growout culture of portunid crabs, especially genus Scylla, is only possible through the establishment of hatcheries as a seed-stock source. Thus, developing rearing methods that would consistently ensure high production and survival of instar crabs in the hatchery would be highly advantageous for profitable commercial aquaculture of portunids. Acknowledgment This work was supported by Malaysia’s Ministry of Education under Fundamental Research Grant Scheme (Vot. No. 59518) and the first author is grateful for support from the same organization to his Post-Doctoral program in Institute of Tropical Aquaculture and Fisheries, Universiti Malaysia Terengganu.
Please cite this article as: M. N. Azra, C. P. D. S. Tavares, A. B. Abol-Munafi et al., Growth rate and fatty acid composition of orange mud crab instars, Scylla olivacea, reared at different temperatures, Egyptian Journal of Aquatic Research, https://doi.org/10.1016/j.ejar.2019.11.006
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Please cite this article as: M. N. Azra, C. P. D. S. Tavares, A. B. Abol-Munafi et al., Growth rate and fatty acid composition of orange mud crab instars, Scylla olivacea, reared at different temperatures, Egyptian Journal of Aquatic Research, https://doi.org/10.1016/j.ejar.2019.11.006
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Growth rate and fatty acid composition of orange mud crab instars, Scylla olivacea, reared at different temperatures Mohamad N. Azra a,⇑, Camila Prestes Dos Santos Tavares b, Ambok Bolong Abol-Munafi a, Mhd Ikhwanuddin a,c,⇑ a b c
Institute of Tropical Aquaculture and Fisheries, Universiti Malaysia Terengganu, 21030, Kuala Nerus, Terengganu, Malaysia Integrated Group of Aquaculture and Environmental Studies, Federal University of Paraná, 80035-050 Curitiba, Parana, Brazil STU-UMT Joint Shellfish Research Laboratory, Shantou University, Guangdong, China
a r t i c l e
i n f o
Article history: Received 4 May 2019 Revised 7 November 2019 Accepted 21 November 2019 Available online xxxx Keywords: Aquaculture Crustacean Fatty acids Molt Scylla olivacea Temperature
a b s t r a c t The objective of the present study was to examine the effect of different rearing temperatures on growth, survival, instar development, and fatty acid (FA) composition of orange mud crab instars, Scylla olivacea. The experiment was conducted at four different water temperatures of 24 °C, 28 °C, 32 °C and 36 °C using instar stage 1 (C1) in temperature-controlled recirculating rearing systems and lasted for 30 days. The study findings revealed that higher temperatures (32 °C) significantly reduced intermolt duration, whereby most crabs reached instar stages C6-C7, although 10.3% were still in the C2C3 developmental stage. In addition, at temperature 36 °C all crabs were dead after three days of culture. Surprisingly, at constant temperature of 28 °C, total Polyunsaturated Fatty Acid were lower (0.52) compared to those at lower, 24 °C (0.71) and higher temperature of 32 °C (0.74). However, the direct effects of temperature on total fatty acids composition in crabs could not be distinguished from indirect effects of variable crab size and molt number. In conclusion, high temperature of 32 °C negatively affects instars growth and may induce molting, especially in S. olivacea. Ó 2019 National Institute of Oceanography and Fisheries. Hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction Information on instar growth and survival of brachyuran crabs is essential for enhancing seed production in nursery conditions, which will indirectly increase hatchery profitability. Mud crabs, genus Scylla, are used as a model organism in most studies because of their convenient size, easy availability, and the fact that they are commercially important in fisheries and aquaculture industries. The instar mud crab’s carapace size, and especially those pertaining to genus Scylla, ranges from 1.5 to 4.0 cm, requiring three to four weeks of rearing in the nursery (Quinitio and Estepa, 2011). Studies of the effects of climate change-mediated water temperature on crustacean growth and development are increasingly being conducted with the aim of increasing production and developing sustainable culture management practices Peer review under responsibility of National Institute of Oceanography and Fisheries. ⇑ Corresponding authors at: Institute of Tropical Aquaculture and Fisheries, Universiti Malaysia Terengganu, Mengabang Telipot, 21030, Kuala Nerus, Terengganu, Malaysia. E-mail addresses: [email protected] (M.N. Azra), [email protected] (M. Ikhwanuddin).
(Abol-Munafi and Azra, 2018). Evidence yielded by such investigations indicates that water temperature plays a vital role in the physiology, growth, and maturation of brachyuran crabs (Fischer and Thatje, 2008; Penha-Lopes et al. 2006). The effects of water temperature on mud crabs were examined by Nurdiani and Zeng (2007) and Ruscoe et al. (2004), whereby crabs were reared from the early larval stage up to instar C3. However, as crabs are capable of adapting to temperature changes in the subsequent growth stages, more comprehensive studies are needed spanning the entire C1C7 range. Recently, Ikhwanuddin et al. (2019) investigated the effects of water temperature on instar crabs during their culture and development. Biochemical parameters, such as lipid and fatty acid (FA) composition, have been consistently used as nutritional status of aquaculture species (El-Ghafour et al. 2018; Dridi et al. 2017; El-Karim et al. 2016; Li et al., 2014; El-Kassas, 2013). In general, FAs are essential for marine crustacean development, as they provide the structural membrane components, as well as serve as a source of energy, especially in the early lifecycle stages (Stoner et al. 2013). Thus, data on FA composition might be a useful tool for the formulation of most optimal crab diet, especially in the early developmental stages.
https://doi.org/10.1016/j.ejar.2019.11.006 1687-4285/Ó 2019 National Institute of Oceanography and Fisheries. Hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article as: M. N. Azra, C. P. D. S. Tavares, A. B. Abol-Munafi et al., Growth rate and fatty acid composition of orange mud crab instars, Scylla olivacea, reared at different temperatures, Egyptian Journal of Aquatic Research, https://doi.org/10.1016/j.ejar.2019.11.006
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On the other hand, the effect of water temperature on lipid and FA composition in brachyuran crabs has been previously explored (Matozzo et al., 2011). To the best of our knowledge, no information is presently available on the growth of S. olivacea instars reared at different temperatures and the link between the growth conditions and the whole-body FA composition. In fact, only few studies on the instar stages of portunid crabs have been conducted to date, while extensive research exists on the larval and broodstock stages (Tavares et al. 2018; Ghazali et al. 2017a; Taufik et al. 2016). We have been motivated by this gap in the extant knowledge to conduct the present study with the main objective to determine the effects of four rearing temperatures (24, 28, 32 and 36 °C) on the survival, individual growth, intermolt duration, instar development, and fatty acid composition of the orange mud crab, S. olivacea. Materials and methods
(2018). The complete experimental setup was based on a modification from previous study by Azra et al. (2018). A controlled chiller system cooled and maintained the water temperature at 24 °C. While 28 °C, 32 °C and 36 °C were achieved by using one, three, and four thermostatic heaters, respectively. Fifteen cylindrical compartments (one compartment per crab instar) in each of twelve RMASs were used to culture the crabs individually. Four simultaneous replicates were utilized for each temperature treatment (n = 180). Instar crabs were fed chopped Selaroides spp. ad libitum for one hour twice daily, at 08:00 and 20:00 h. Before the start of each trial (single experiment with all treatments), the water temperature in the instar culture tank was gradually increased or decreased by 1 °C per hour (from 26 °C) until the desired experimental temperature was reached. During the experiment, water salinity was kept at 26.0–27.0‰. The water quality parameters were evaluated twice daily before the feeding times, and were maintained at the following levels: ammonia-N < 0.5 mg L-1, nitrite < 0.15 mg L-1, DO > 4 mg L-1, and pH at 7.3–8.1.
Sampling and production of berried females
Growth and survival
Sixty mature female (carapace width > 9.5 cm) S. olivacea crabs were collected from Terengganu coastal waters and were transported to the Institute of Tropical Aquaculture and Fisheries’s Hatchery, Universiti Malaysia Terengganu, Terengganu, Malaysia. Water salinity and temperature were maintained at 25.0–27.0 ppt and 26.5–28.3 °C, respectively. Crabs were fed raw squid (Loligo spp.) ad libitum twice daily (at 08:00 and 20:00 h) until they spawned (Azra and Ikhwanuddin, 2015). Every day, uneaten food and waste was removed an hour after feeding. The spawning crabs were transferred into 100 L tanks and were reared individually, without feeding, until the eggs hatched. To ensure that all the instars were of the same age, only females whose eggs hatched on the same day (within 12 hours) were included in the sample for further analyses.
Instar crabs were checked daily for molting. If an individual molted, its carapace width (CW) was assessed after the carapace had fully hardened using a Vernier caliper (Mitutoyo Model CD600 CSX). Crab survival was defined as the percentage of crabs that survived for 30 days, and the survival rates for each day were calculated as day x = (crab abundance at day 0 – cumulative mortality by day x) / crab abundance. Survival was determined by direct observation, with minimal potential for error, as the crabs were reared in individual cylindrical compartments.
Larval rearing and instar preparation Newly hatched phototactic larvae were siphoned off from the hatching tanks to a 500 L larvae tank and were reared from zoea stage 1 (Z1) until they reached the megalopa stage. The specimens that reached the megalopa stage were cultured individually in 0.5 L plastic containers until they reached the first instar stage, C1. On average, it took 30 days for the larvae at Z1 to reach the first day of C1. The mean survival rate from Z1 to C1 was 3.5%. During larval culture, the water salinity was maintained at 26.0–27.0%, the pH was kept within the 7.55–8.10 range, while ensuring that dissolved oxygen (DO) content was above 6 mg L-1, and the temperature remained within 26.8 °C to 28.1 °C. The water culture was exchanged at the rate of 10% every day during the larval rearing culture. The zoea feeding regimes were based on the previous study by Ikhwanuddin et al. (2012).
Fatty acid analysis Prior to the final (30th) day of culture, and after survival had been established, whole-body FA composition was determined by the one-step method described by Abdulkadir and Tsuchiya (2008). A pooled sample of five to six crabs from each replicate was randomly collected, cryo-anesthetized for 5 min and cryopreserved at 80 °C for subsequent FA analysis. This was principally carried out by combining the extraction and esterification processes. After adding 2 mL 14% boron trifluoride (BF3) in methanol and a magnetic stirring bar, the tubes were flushed with nitrogen gas and sealed. The capped tubes incubated at 100 °C for 120 min with continuous stirring. After the solutions cooled to room temperature, 1 mL hexane was added, followed by 2 mL distilled water. The tube was vortexed until it was fully blended and then centrifuged for 3 min at 2,500 rpm (650g). The upper hexane layer containing the fatty acid methyl esters (FAMEs) was subjected to gas chromatography, using GC-2010 Shimadzu coupled to the AOC-20i + s detector, allowing the FAME peaks to be separated and quantified. The FAMEs were identified by comparing their retention times with the data available in the NIST08 Mass Spectral Library database.
Experimental design Statistical analysis Only healthy, active, and intact crabs at C1 stage were randomly chosen for the experiment, with the initial mean carapace length (CL) of 3.23 ± 0.07 mm. These crabs were cultured for 30 days in twelve Re-circulating Marine Aquaculture System (RMAS) with aerated, filtered seawater equipped with thermostatically controlled chillers and immersion heaters at four rearing temperatures, namely 24 °C, 28 °C, 32 °C and 36 °C. The selected temperatures and the setup adopted for cooling and heating the water were based on the methodology adopted by Azra et al.
One-way ANOVA was conducted to ascertain if the differences are statistically significant in CW and intermolt duration at different instar stages after 30 days of culture. Whereas a post-hoc test (Duncan’s multiple range tests) was performed for comparisons. Appropriate data transformations were performed as necessary, following Levene’s test for homogeneity of variance. The analysis results were presented as means ± standard deviations, and p < 0.05 was adopted as the indicator of statistical significance.
Please cite this article as: M. N. Azra, C. P. D. S. Tavares, A. B. Abol-Munafi et al., Growth rate and fatty acid composition of orange mud crab instars, Scylla olivacea, reared at different temperatures, Egyptian Journal of Aquatic Research, https://doi.org/10.1016/j.ejar.2019.11.006
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Results Growth and survival After 30 days of culture, no major mortality was noted at any of the tested temperatures (except the 36 °C with 100% mortality after three days of culture). Overall, > 74.3% of the specimens survived from C1 to C7. Higher mortality (87.5%) occurred during the early stages (C1 to C2, and C3 to C4) compared to the later stages (12.5%). Once crabs reached C4 or C5, their survival rapidly increased reaching > 90% until the crabs reached the C6C7 stage. The lowest survival rate was observed at 32 °C (74.3%), whereas rearing crabs at 24 °C resulted in the highest survival rate of 87.8%. At 28 °C, 80.5% of crabs survived, and at 36 °C, all crabs were dead after three days of culture, which precluded the use of data pertaining to this temperature in further analyses. There are significant differences in the survival among the culture temperature (p < 0.05). The CW increased throughout the culture period and
the effects of water temperature on CW were statistically significant (p < 0.05) (Table 1). However, ANOVA results showed the effects of the three treatments (24, 28, and 32 °C) on CW sizes at the early culture stage. C1C2 were not statistically significant, whereas statistical significance (p < 0.05) was established at the later culture stages of C2C7 (Table 1). Specifically, culture at 32 °C significantly reduced intermolt duration, and crabs subjected to this treatment successfully reached instar stages C6 to C7 after 29–30 days (Table 1). Moreover, while the CWs decreased at early stages and increased at later stages at 32 °C, no significant differences were seen at 28 °C (Table 1). Instar development At the end of 30 days of culture, the majority of crabs reared at 24 °C reached C2 to C5, while crabs at 32 °C were at the C5 to C7 developmental stage (Fig. 1). Crabs reared at 28 °C exhibited a high
Table 1 Mean carapace width increment, mean intermolt duration, and percentage increase in different instar stages, of orange mud crab, Scylla olivacea at three water temperatures (24, 28, and 32 °C) based on 30 days of cultivation. Water Tempe-rature (°C)
Mean number of days until the next molting C1 C2
C2 C3
C3 C4
C4 C5
C5 C6
C6 C7
24 28 32
6 5 3 Mean carapace width in mm (mean ± SD) 4.41 ± 0.20a 4.39 ± 0.15a 4.47 ± 0.21a
7 6 4 Percentage increase
8 7 5
9 8 5
N/A N/A 6
N/A N/A 7
29–30 26–30 29–30
5.65 ± 0.21a 5.43 ± 0.22b 5.66 ± 0.29a
7.08 ± 0.33a 6.73 ± 0.25b 6.91 ± 0.28c
9.08 ± 0.20a 8.26 ± 0.40b 8.42 ± 0.39b
N/A N/A 10.15 ± 0.41
N/A N/A 13.01 ± 0.23
28.11»25.31»28.25 (C1 to C5) 23.69»23.94»22.73 (C1 to C5) 26.62»22.08»21.85»20.55»28.18 (C1 to C7)
24 28 32
Total days
SD = standard deviation. Superscript letters indicate significant difference at p < 0.05.
Fig. 1. Instar development of orange mud crab, Scylla olivacea, by moult stage at the end of the 30-day growth period at three experimental temperatures. Histograms represent total counts for all replicates.
Please cite this article as: M. N. Azra, C. P. D. S. Tavares, A. B. Abol-Munafi et al., Growth rate and fatty acid composition of orange mud crab instars, Scylla olivacea, reared at different temperatures, Egyptian Journal of Aquatic Research, https://doi.org/10.1016/j.ejar.2019.11.006
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Table 2 Fatty acid compositions (mg g 1 dry weight) of whole body compositions of orange mud crab, Scylla olivacea at different culture temperatures. Fatty acids
24 °C
28 °C
32 °C
C4:0 C6:0 C8:0 C10:0 C14:0 C15:0 C16:0 C17:0 C18:0 C20:0 C21:0 C22:0 C23:0 Total SFA1 C14:1 C15:1 C16:1 C20:1 C24:1 C18:1N9T C22:1N9 Total MUFA2 C18:2N6T C18:2N6C C18:3N6 C20:3N6 C20:4N6 n-6 C18:3N3 C20:3N3 C20:5N3 C22:6N3 n-3 Total PUFA3 Total LC-PUFA4 Total Fatty Acids (n-3/n-6) ratio DHA:EPA concentration (ratio)
1.40 ± 0.23a 0.13 ± 0.15a 0.65 ± 0.16a – 0.11 ± 0.15 0.06 ± 0.08a 0.64 ± 0.18a 0.09 ± 0.01a 0.35 ± 0.07a 0.05 ± 0.04a 0.05 ± 0.09a 0.36 ± 0.51a 0.03 ± 0.03a 3.92 0.05 ± 0.06a 0.20 ± 0.03a 0.07 ± 0.04a 0.18 ± 0.10a 1.08 ± 0.90a 1.25 ± 0.08a 0.06 ± 0.09a 2.89 0.01 ± 0.02a 0.11 ± 0.04a 0.02 ± 0.02a 0.03 ± 0.06a 0.07 ± 0.03a 0.24 0.01 ± 0.01a 0.04 ± 0.07a 0.19 ± 0.25a 0.23 ± 0.15a 0.47 0.71 0.56 7.53 ± 0.85 1.96 0.23: 0.19 (1.21)
0.86 ± 0.15b 0.09 ± 0.03b 0.44 ± 0.01b – – 0.03 ± 0.01b 0.42 ± 0.02b 0.08 ± 0.01a 0.32 ± 0.04a 0.01 ± 0.01a 0.01 ± 0.01b 0.10 ± 0.08b 0.01 ± 0.01a 2.37 0.04 ± 0.03a 0.21 ± 0.14a 0.04 ± 0.04b 0.07 ± 0.05b 0.76 ± 0.94b 0.98 ± 0.07b – 2.10 0.04 ± 0.05b 0.06 ± 0.01b 0.02 ± 0.03a 0.02 ± 0.03a 0.06 ± 0.02a 0.20 0.01 ± 0.01a 0.08 ± 0.07b 0.09 ± 0.03b 0.14 ± 0.05b 0.32 0.52 0.39 5.01 ± 0.21 1.60 0.14: 0.09 (1.56)
0.82 ± 0.15b 0.02 ± 0.02c 0.50 ± 0.07c 0.05 ± 0.06 – 0.02 ± 0.01b 0.05 ± 0.02c 0.58 ± 0.04b 0.11 ± 0.03b 0.40 ± 0.03b 0.04 ± 0.08a 0.13 ± 0.02b 0.19 ± 0.09b 2.91 – 0.22 ± 0.05a 0.11 ± 0.03c 0.11 ± 0.07c 0.52 ± 0.43c 1.23 ± 0.11a 0.03 ± 0.01b 2.22 0.06 ± 0.04b 0.07 ± 0.01b 0.02 ± 0.01a 0.07 ± 0.03b 0.06 ± 0.01a 0.28 0.02 ± 0.01a 0.02 ± 0.02c 0.24 ± 0.15c 0.18 ± 0.05c 0.46 0.74 0.57 5.87 ± 0.57 1.64 0.18: 0.24 (0.75)
Within rows for each fatty acids class, means with common letters are not significantly different. 1Saturated fatty acids – sum of all FA without double bonds.2Monounsaturated fatty acids – sum of all FA with a single double bond. 3 Polyunsaturated fatty acid – sum of all FA with 2 double bonds. 4Long-chain PUFA – sum of all FA with chain length 20 carbon atoms and 3 double bonds.
synchronous molting rate, with >96% measured at C5 (Fig. 1). At 32 °C, crabs reached the C6C7 stage earlier than those reared at 24 and 28 °C. In contrast, crabs reared at 24 °C reached only C4C5 and their CW was significantly reduced. Fatty acid (FA) composition The dominant fatty acid (FA) components identified in these specimens were saturated fatty acids (SFAs) with percentage from 47% to 52% of Total Fatty Acids (TFA). The ratio of omega-3 to omega-6 content was higher in crabs reared at 24 °C and than in those subjected to 28 °C and 32 °C treatment. The results also showed that total FAs were higher in cooler temperature compared to all experimental temperature (Table 2). Discussion Authors of previous study in this field have established that high survival rate, high synchronous molting rate, and short molting period are the optimal crustacean growth parameters, especially for crabs and shrimp (Figueiredo et al. 2008). The analyses conducted as part of the present investigation demonstrated that
the lowest temperature tested, 24 °C, resulted in the highest survival rates, while the moderate temperature of 28 °C led to high synchronous molting rates, and the highest temperature, 32 °C, shortened the percentage of CW increment. The present study supported the recent review by Azra et al. (2019), which stated that water temperature is among the most important environmental factors affecting the survival and development of brachyuran crabs. Effects of water temperature on growth, survival, and instar development The lowest and the highest survival rate of 74.3% and 87.8% was observed at 32 °C and 24 °C, respectively. As crabs were reared in individual culture vessels to eliminate the potential for intraspecific competition and cannibalism, this likely contributed to the higher survival rates (Yang et al. 2018) from stage C4 to C5. Moreover, survival rates subsequently increased and >90% of instars were maintained alive until the crabs reached C6C7, while higher mortality rates were recorded during the early stages, C1 to C3. After three days of culture at 36 °C, 100% mortality was noted, which precluded analyses based on this temperature. Similar observations were made by Gong et al. (2015), who studied the green mud crab, Scylla paramamosain, as well as by Penha-Lopes et al. (2006) in their investigation of the spider crab, Mithraculus forceps. Moreover, Gong et al. (2015) showed that culture at temperature of 39 °C led to 100% mortality of all C1 crabs and no successful molting was recorded. This suggests that 39 °C might be near the upper limit of the larvae’s ability to acclimatize (Azra et al. 2018). A slight delay of instar development, at the lower temperature, may further increase their chance of survival, which may be attributed to the increasing body thickness of the integument with growth. The thickening of integument in the later stage may have provided a protective mechanical protection to lower temperature (Baylon, 2009). A decrease in intermolt duration with increased temperature has been reported for other brachyuran and anomuran crabs (Gong et al., 2015), and our findings likewise showed that S. olivacea crabs reared at 32 °C had a shorter average molting duration. This was expected, as metabolic processes in most poikilotherms are expedited at higher temperatures (Hartnoll, 2001). After 30 days, the crabs cultured at 32 °C had molted six times, while those reared at 24 °C had only molted four times. The limited amount of published data on instar larval development, especially at different rearing temperatures related to the nursery culture of portunid crabs, makes the comparisons with the findings reported by other authors difficult. However, the evidence yielded by previous studies on the blue king crab, Paralithodes platypus, and the red king crab, Paralithodes camtschaticus, indicates that the majority of instars reared at 1.5 °C remained at C1 to C2. However, crabs reared at a higher temperature of 12 °C reached C5 to C6 after 60 days of culture (Stoner et al., 2010; Stoner et al., 2013). Our results were similar to those pertaining to lithodid crabs, reported by Stoner et al. (2013), who noted that, after 30 days of culture (60 days after hatching) at 24 °C, hatchlings reached C2 to C3 developmental stage, whereas crabs reared at 32 °C were at stage C6 to C7. The present investigation also shows that after 30 days of culture period, only crabs reared in 32 °C reached the C7 stage, compared to others tested temperatures where crabs reached the C4 to C5 stage only. Effects of temperature on crab fatty acid composition The biochemical changes at various stages in the crustacean lifecycle, especially the FA composition, have been the subject of
Please cite this article as: M. N. Azra, C. P. D. S. Tavares, A. B. Abol-Munafi et al., Growth rate and fatty acid composition of orange mud crab instars, Scylla olivacea, reared at different temperatures, Egyptian Journal of Aquatic Research, https://doi.org/10.1016/j.ejar.2019.11.006
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M.N. Azra et al. / Egyptian Journal of Aquatic Research xxx (xxxx) xxx Table 3 Effect of water temperature on fatty acid composition in crabs. Common and species name
Temperature
Effect on FAs
Reference
Mud crab, Scylla serrata
Acclimation temperature of 5 °C and 10 °C, control at 27 °C 1.5, 4.5, 8 and 12 °C
Significant difference in FA composition at low temperatures Warmer temperature produced higher docosahexaenoic acid (DHA): eicosapentaenoic acid (EPA) ratio and elevated the monounsaturated FA amounts Total fatty acid composition varied under low temperature adaptation, whereby long-chain FA levels increased at lower temperatures Lower quantities of long-chain FAs at the lowest experimental temperatures of 24 °C and the highest temperature of 32 °C compared to the constant temperature of 28 °C and a higher EPA:DHA ratio at the lowest (24 °C) and the highest temperature (32 °C)
Kong et al. (2007)
Red king crab, Paralithodes camtschaticus Black mud crab, Scylla serrata
Adaptation temperature of 5, 10 and 15 °C, control at 27 °C
Orange mud crab, Scylla olivacea
Nursery temperature of 24, 28 and 32 °C
extensive studies over the past decade due to the fact that FAs play a vital role in the development of marine crustaceans as sources of energy and structural components of membranes (Suprayudi et al. 2004). However, there is a paucity of studies focusing specifically on the instar/juvenile and early adult stages, and most extant researches have been conducted on the anomuran rather than brachyuran crabs. In addition, authors of the limited number of studies pertaining to brachyurans focused on adults and the early life history stages of embryonic development (Ghazali et al 2017b; Ikhwanuddin et al. 2018). Moreover, changes in FA composition as a function of water temperature at which crabs are reared are commonly used as an indicator of their ability to acclimatize to a sudden change in water temperature through the adjustment of membrane fluidity (Stoner et al., 2010). Given the limited understanding of the link between FA composition in the instar/juvenile stages and rearing temperature, the present study has yielded some valuable findings. For example, growth and development in warmer temperatures resulted in higher levels of SFAs and fewer unsaturated FAs, as well as higher levels of monounsaturated FAs (MUFA) with a higher EPA:DHA ratio (Table 3). The DHA and EPA fatty acids play important roles in the early survival and growth of marine larvae (Suprayudi et al., 2004). However, a direct comparison of EPA:DHA ratios at different temperatures was not possible as a part of this investigation because most crabs were at different stages in the lifecycle. However, since crabs reared at 24 °C and 28 °C reached the same stage (C4C5) in the final days of the 30-day culture period, an indirect comparison was made. The obtained findings indicate that the higher EPA:DHA ratios obtained at 28 °C seem to be associated with a smaller carapace size (8.26 mm) and shorter intermolt duration (26–30 days) compared to those obtained at 24 °C, where crabs had an average CW of 9.08 mm and a longer intermolt duration of 29–30 days. These results are in line with those reported by Takeuchi et al. (1999), who concluded that both EPA and DHA were important in shortening the intermolt duration as well as increasing the CW of Portunus trituberculatus. The present study also showed that total Polyunsaturated Fatty Acid (PUFA) and Long-Chain Polyunsaturated Fatty Acids (LC-PUFA) were lower at constant temperature of 28 °C, compared to the lower (24 °C) and higher (32 °C) temperature. This might caused by the fact that most of the crabs at constant temperature produce the same stage of instar development (Fig. 1). By day 30, the most dominant FA components in S. olivacea crablets were SFAs, a type of FA with no double bonds, which was found to be the most critical FA group for growth, survival, and reproduction in most marine aquaculture species (Glencross, 2009). Crabs reared at all three temperatures had lower concentrations of LC-PUFAs relative to other FAs. This finding is in line with the observation made by Glencross (2009), who noted that, in most animals,
Stoner et al. (2010)
Wang et al. (2007)
Present study
especially mud crabs, the LC-PUFAs cannot be synthesized de novo, and must therefore be obtained from the diet or as precursors present in the food. The omega-3 and omega-6 FAs are essential dietary components because they affect the larval growth rate, the CW, and the flesh quality of species raised by aquaculture (Glencross, 2009). In the present study, a direct comparison of omega-3 and omega6 concentrations in crabs raised at the three experimental temperatures was not possible since the crabs were at different stages of their lifecycle during culture. Equally, indirect comparison cannot be made of crabs cultured at 24 °C with those at 28 °C even though some crabs reached the same stage of growth, C4C5. The obtained results are in line with those obtained by Wang et al. (2007), who reported increased concentrations of LC-PUFAs in crabs cultured at a lower temperature. Furthermore, Purac et al. (2011) reported that the crab’s metabolic rates tend to decline at low temperatures, which alters the rate of FA biosynthesis or utilization. The ratio of omega-3 to omega-6 was higher in crabs reared at 32 °C, indicating that most crabs reached C4 to C7 developmental stage. Thus, we propose the use of the ratio of omega-3 to omega-6 in future studies to determine the development status of marine animals, especially crabs. Conclusion and implications In conclusion, higher temperatures of 32 °C negatively affected instars growth and might have induced molting. The present results suggest a constant temperature of 28 °C for synchronized molting of S. olivacea. The scynchronization of molted crabs might give huge advantages to avoid any cannibalism chance of culturing the instar crab during the nursery stage. Water temperature is a fundamental and dominant determinant of the instar development phase of portunid crabs. Findings yielded by the extant studies in this field showed that growout culture of portunid crabs, especially genus Scylla, is only possible through the establishment of hatcheries as a seed-stock source. Thus, developing rearing methods that would consistently ensure high production and survival of instar crabs in the hatchery would be highly advantageous for profitable commercial aquaculture of portunids. Acknowledgment This work was supported by Malaysia’s Ministry of Education under Fundamental Research Grant Scheme (Vot. No. 59518) and the first author is grateful for support from the same organization to his Post-Doctoral program in Institute of Tropical Aquaculture and Fisheries, Universiti Malaysia Terengganu.
Please cite this article as: M. N. Azra, C. P. D. S. Tavares, A. B. Abol-Munafi et al., Growth rate and fatty acid composition of orange mud crab instars, Scylla olivacea, reared at different temperatures, Egyptian Journal of Aquatic Research, https://doi.org/10.1016/j.ejar.2019.11.006
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Please cite this article as: M. N. Azra, C. P. D. S. Tavares, A. B. Abol-Munafi et al., Growth rate and fatty acid composition of orange mud crab instars, Scylla olivacea, reared at different temperatures, Egyptian Journal of Aquatic Research, https://doi.org/10.1016/j.ejar.2019.11.006