Fatty Acid Composition of Hepatopancreas and Gonads in Both Sexes of Orange Mud Crab, Scylla olivacea Cultured at Various Water Flow Velocities

Nutritional quality of the hepatopancreas and gonads of orange portunid mud crab, Scylla olivacea was evaluated for each gender under four treatment of different water velocities (0, 20, 40 and 60 cm s−1), in terms of nutrient reserve and nutrient for reproduction. About 56 crabs were used in this study in which fatty acids composition was analysed using gas chromatography mass-spectrometry (GC-MS). For hepatopancreas analysis, monounsaturated fatty acids (MUFAs) were present in the highest fatty acids concentration, followed by polyunsaturated fatty acids (PUFAs) and, saturated fatty acids (SFAs). However, long-chain polyunsaturated fatty acids (LC-PUFAs) were displayed in low concentration in the hepatopancreas. Total fatty acid (TFAs) composition was significantly higher at moderate velocity of 20 cm s−1 compared to other water velocity treatments. For gonad analysis, 20 cm s−1 showed the highest TFA concentration of 93.34 mg g−1 while, the lowest concentration of 3.90 mg g−1 occurred at 0 cm s−1. There were significant differences in male and female crab’s fatty acids contents of gonads at all flow velocities challenged (p < 0.05). PUFAs and MUFAs were dominant while, SFAs were observed at low concentration. This study revealed that, concentration of PUFAs increased as gonad maturation increased. The decreasing concentration of hepatopancreas fatty acids over the culture period indicated that nutrient was shifted from the hepatopancreas, to be used as energy reserved to gonads for further growth of eggs and offspring. The linkages between water flow strength, hepatopancreas, and gonad fatty acids concentrations, is fundamental knowledge useful in establishing efficient habitat velocities selection which will improve aquaculture production of mud crabs with high quality broodstock.


INTRODUCTION
Basic knowledge of how water velocities employed in rearing systems may impact the physiology of culture species is crucial to enhance the management of reproductive development in broodstock, especially with respect to recirculating aquaculture systems (RAS) or, indoor culture systems. The portunid crab (genus Scylla) is in high demand as a global aquaculture species because of its high price, unique flavour, high meat yield, rigid muscle and sweet flesh Kunsook & Dumrongrojwatthana 2017;Ikhwanuddin et al. 2018). In portunid crab, hepatopancreas is the main tissue where fatty acids are reserved. The hepatopancreas fatty acids change when crabs mature (Abol-Munafi et al. 2016). The hepatopancreas also reserves energy for survival, shedding and reproduction (Sarapio et al. 2017) and, also acts as a store for vitellogenin (Wang et al. 2014;Liu et al. 2018).
Most aquatic organisms undergo long distance travel and swim for reproduction purposes, encountering exhaustion under various water flow 81 conditions. Extensive investigation have been carried out to determine the exact capabilities of aquatic organisms to cope with this challenge including for example, determination of locomotion activities, reaction time for escaping from unfavorable conditions, maximum velocity tolerances and, energy in form of lipids reserved for long swimming and hardy migration (Hinch & Rand 1998;Hodgson & Quinn 2002;Lee et al. 2003;Taufik et al. 2019;Azra et al. 2019).
In natural mangrove habitats rich with food, attractive for spawning purpose are limited by water flow rates and velocities (Nagelkerken et al. 2008). How S. olivacea addresses problems posed by water velocities, whether by walking, swimming or remaining at the mercy of water current is still unclear. Investigation of this species' capabilities to maneuver and tolerate different flow velocity conditions is necessary. The main purpose of the current study was to determine the effect that various water velocities exercise on hepatopancreas and gonad maturation in both males and females, by examining changes in fatty acids concentrations. From the mud crab activity of swimming in their actively challenging offshore migration, this investigation established the modelling hypothesis that fatty acids distribution for spawning purpose in male and female S. olivacea would be controlled by the velocity of water flow.
Fatty acids have been shown to have significant effect on the physiology and spawning capacity of decapod crustaceans (Taufik et al. 2014;. Fatty acids have been suggested as the main dietary element responsible for controlling both fecundity and fertilisation of crab eggs (Alava et al. 2007). Research on fatty acids and their importance in aquatic reproduction and culture is common and includes study carried out by several authors on aspects such as nutritional tracing and, establishment of the role of physiological factors (Abdulkadir & Tsuchiya 2008;Taufik et al. 2014;Azra et al. 2018).
Experimentation on water flow rate effects, evaluating how velocity and duration of exposure can significantly alter the physiology of aquatic animals and affect their growth, contributes essential knowledge to aquaculture production of these species (Taufik et al. 2019). Environmental changes commonly cause biochemical and physiological disorder, contrary to maintenance of an ideal system (Randall & Brauner 1991). Uncertain abiotic factors such as variation in water flow can lead to changes in metabolism and, in both biochemical and morphological assimilation (Oufiero & Whitlow 2016). Higher velocities have the worst physiological impact on aquatic animals, disturbing their homeostasis and development (Hernandez et al. 2002).
Aquatic organisms cultured in flowing water systems have been suggested by researchers to be sensitive to fluctuating water velocity (Jobling et al. 1993) with high velocities influencing the physiology of some cultured aquatic species (Palstra & Planas 2011). Aquatic animals have been shown to usually move at moderate speeds under aerobic conditions (Clark et al. 2013) with excessive flow rates producing negative impact (Brown et al. 2011). The purpose of the current study is to elucidate the effect of different water velocities on S. olivacea, considering sex of the animal, including the linkage with hepatopancreas and gonad fatty acids analysis.

Description of the Methodology
The crabs used in the experiments were sampled from Setiu Wetlands, Terengganu, adjacent to the Southern South China Sea, Malaysia. Immature male and female mud crabs at 50% reach maturity, with measurement carapace width of male (♂: < 8.97 cm) and female (♀: < 9.06 cm) were chosen according to the sampling method of Ikhwanuddin et al. (2010a;. The samples were brought live to the hatchery of Institute of Tropical Aquaculture (AKUATROP), Universiti Malaysia Terengganu for culture. The carapace widths (CWs) were taken at the blunt point of the 9th antero-lateral spine of the carapace, scaled by vernier caliper (precision 0.01 mm). Body weight (BW) was measured by digital balance (precision 0.01 g). The crabs were acclimatised in a culture tank in normal light and ambient temperature conditions for two days before they were introduced into the treatment developed to generate velocity conditions, shown in Figs. 1 and 2. The crabs were acclimatised and experiments processed in salinity 20 ppt . The maintenance of the crabs was based on the previous published methods Abol-Munafi et al. 2017). In brief, water temperature was controlled at an average 24°C-28°C. The mud crab culture PVC pipe system and tanks were washed every day during the experiment. Washing included removal of fecal matter and, uneaten fishes and, was conducted each morning. Feeding proceeded after cleaning where raw fish (Decapterus spp) were given metabolic waste was managed through 50% daily exchange of water. The treatment apparatus used in this study comprised of 30 units of PVC Pipe System, two culture tanks (3000 L) and, one treated (1 tons of seawater mixed with 30 g of calcium hypochlorite and aerated 24 h under vigorous aeration before ready to use) water (20 ppt) and one holding tank (3000 L). Four different water flow velocity treatment were employed, T1, T2, T3 and T4 applying 0, 20, 40, and 60 cm s -1 , respectively. An electric pump generated flow at 6000 L h -1 . Inside the mangrove ecosystem, an average velocity pertains of approximately 10 cm s -1 (Zhang et al. 2012). In this study, copying natural free stream characteristic, velocities of 0, 20, 40 and 60 cm s -1 were generated for 60 days of specimen culture. The handy flow meter was used (Flo-Mate, model 2000, USA) which employs the Faraday method of measurement. The measurement units were assigned as centimeters per second (cm s -1 ) with accuracy of ± 5 cm s -1 . Fig. 1 displays the PVC Pipe system conceptually in cross-section and top view and Fig. 2 displays the conceptual arrangement of the full system.

One Step Method Procedure
After each treatment reached 0, 30 and 60 days rearing period, each of the 56 crabs were dissected. The stomach, outer carapace, gills and internal tissue, except gonads and hepatopancreas, were disposed of. The gonad and hepatopancreas were sampled and kept at -80°C for 48 h, before fatty acid (FA) extraction and esterification by the method of Abdulkadir and Tsuchiya (2008). Approximately, 200 mg to 300 mg of sample with three replicates were diluted with 4 mL of hexane, 2 mL of 14% BF 3 in methanol and 1 mL of C19:0 solution in a 50 mL tube. A magnetic stirring bar was used as mixer and, gas space of the tubes was replaced with N 2 over 30 s. The tube caps were closed tightly and tubes incubated for 2 h at 100°C. Upon completion, 1 mL of hexane with 2 mL H 2 O were mixed into the tubes and tubes shaken vigorously for 1 min. Subsequently, the tubes were centrifuged for 3 min at 2500 rpm (650×g). Two layers formed with the top section consisting of FAMEs. For FAME quantification, 50 mL of the FAME top layer was injected into the gas chromatograph mass spectrometer (GCMS-QP2010 Ultra). Subsequent isolation was done with a ppms-5 column (OPTIMA® 5 HT, 15 m × 0.32 mm ID, 0.25 μm film) with helium gas at pressure 50.0 kPa, velocity 0.96 mL min -1 and linear flow of 35.5 cm s -1 . The total of 1 mL of FAME was inserted at 50°C for 1 min until temperature reached 300°C at 5°C min -1 then, the sample was held for 5 min. The concentration of fatty acids from the GCMS were recorded and analysed. In this study, student t-test statistical analysis was carried out using SPSS with differences shown in graph form, which were considered significant for P < 0.05. All data were displayed as means ± SD.

RESULTS
The average TFAs profiles of the hepatopancreas and gonad samples of S. olivacea among four treatments are displayed in Figs. 3 to 6. Tables 1 to 8 show the fatty acids distribution in hepatopancreas and gonads at different flow velocities. In the hepatopancreas results, LC-PUFAs was found at only a small percentage for all treatments. Velocity of 20 cm s -1 displayed the highest concentration of TFAs compared to other velocity treatments (Fig. 3). Fig. 3 displays that TFAs levels found in the female hepatopancreas were higher than male for all treatments. For all of the four velocities evaluated, MUFAs showed highest concentration of all fatty acids. LC-PUFAs contributed only a small proportion of the fatty acids in hepatopancreas samples (Fig. 3). Male crab showed very low hepatopancreas TFAs, which is interpreted to mean that more fatty acids are used for locomotion in males. Both male and female crabs, at velocity of 20 cm s -1 displayed high concentration of TFAs. Significant differences were found between male crabs in Day 30 for omega-3 and omega-6 in S. olivacea (Fig. 4). These results displayed that fatty acids concentration in crab hepatopancreas is diverse for culture at various water velocities.
With respect to the gonad sample results, PUFAs represented the highest fraction of all classes of fatty acids. Velocity of 20 cm s -1 contributed peak TFA content, compared to other treatment velocities (Fig. 5). In Fig. 5, the analysis of TFAs of male crabs (♂: 79.6 mg g -1 ) and female crabs (♀: 93.34 mg g -1 ) were the highest recorded at day 30 for 20 cm s -1 , compared with other velocities. Male crab displayed only a small fraction of fatty acids composition at treatment velocity of 85 40 cm s -1 (Fig. 5). Higher composition fractions were found of omega-3 and omega-6 for treatment velocity of 20 cm s -1 in both sexes of crabs, compared to other treatments (Fig. 6). These investigations have shown that appropriate velocity in culture habitat will accelerate the gonad maturation of S. olivacea while, strong velocities will retard the gonad maturation process.

DISCUSSION
Nutritional value of fatty acids is crucial to the diet of cultured species and, they exert a major impact on other vital requirements such as survival, spawning, and repair of damaged tissue (Amran et al. 2018). The current investigation determined the fluctuation of fatty acids inside the hepatopancreas and gonads of the target species at different water velocities. Fatty acids profiling between sexes of S. olivacea at different water velocities is displayed in Tables 1 to 8. In hepatopancreas, at velocity 20 cm s -1 , MUFAs was highest with the range of 0.70 to 35.07 mg g -1 , SAFAs with a range of 1.30 to 16.70 mg g -1 and, PUFAs with a range of 0.90 to 9.27 mg g -1 . The TFAs levels recorded by sex of crabs showed significant difference, especially at treatment velocities 40 and 60 cm s -1 . Both PUFAs and MUFAs were found to be significantly higher (P < 0.05) in female crabs compared with male crabs. Teshima and Kanazawa (1983) claimed that fatty acids concentration in the hepatopancreas reduced as ovarian maturation increased and, that female crabs more quickly achieve gonadal maturation than male crabs.
The variation in concentration of specific fatty acids recorded every 30 day, may have been due to the different velocities involved in study. The current work reviews specific functions for specific kinds of fatty acids in aquatic animals but, the specific role of individual fatty acids still continues to unravel. For examples, PUFAs profiles, especially omega-3 and omega-6 increased at days 0 and 30. However for days 60, the concentration of PUFAs in the hepatopancreas decreased drastically. This effect might have been the PUFAs was transferred, to tissue muscle to sustain individual position in different flow condition and, to gonads for maturation. Study by Ravid et al. (1999) claimed the building blocks of PUFAs were mainly constructed by triacylglycerols, phospholipid (PL) and, and cholesterols where these lipid types were importantly responsible for plasma membrane construction and, hormone sites. Omega-3 and omega-6 are the most important class of PUFAs involved in transformation of MUFAs to other forms of PUFAs (Lee et al. 2016). Omega-3 commonly involved in integral component in cell membrane and precursors of eicosanoids (Hurtado et al. 2007). Transformation enzymes serve to change linoleic acids (LA) to other forms of long chain PUFAs, such as docosahexaenoic acid (DHA) and eicosapentanoic acid (EPA), which play a vital role of increasing tissue development, that is important in aquatic animal culture and, is also essential for larval survival (Naceur et al. 2008).
In this study's consideration of mud crab gonad, TFAs concentration at treatment velocity of 20 cm s -1 was significantly greater than at other velocities (P < 0.05). The analysis of 35 (minimum) to 36 (maximum) types of fatty acids in crab gonads showed major components of 16:0, 18:0, 24:1 and 22:6n-3 (Tables  4-8). The concentrations of EPA and DHA were higher for treatment velocity of 20 cm s -1 (Fig. 4). DHA plays important role in fertility by mobilised from hepatopancreas to ovary for vitellogenesis and oogenesis (Tantikitti et al. 2015). The major component of gonadal fatty acids for both sexes were 16:0, 18:0 and 18:1n-9. These fatty acids are important nutrition for female mud crab during ovarian development (Ying et al. 2006) and provide migration energy to offshore for spawning. Highest PUFAs accumulated in the ovary at Day 60 for treatment velocity 20 cm s -1 and, was about 25.80 mg g -1 greater than the concentration recorded for the male testis (16.26 mg g -1 ). These results of the current study are in agreement with Ying et al. (2006) and Alava et al. (2007). It can be concluded that PUFA are crucial in gonad development. Composition of fatty acids such as 18:2n-6c, 20:4n-6, and 22:6n-3 in the ovary increased with duration of the culture period, most obviously for treatment velocity of 20 cm s -1 . These fatty acids act as structural component of phospholipid which involved in cell membranes and lipoprotein transportation (Lytle et al. 1990). Previous studies have elaborated that fatty acids are fraction of PL and, the mainstay of lipoprotein transition and cell membrane structure and also, that they are crucial for prawn spawning, insemination and hatching (Lytle et al. 1990). The fatty acids 18: 2n-6c might be assumed to essential for embryonic growth of Portunus pelagicus crab (Taufik et al. 2016).
Under moderate velocity (20 cm s -1 ), crab were observed to swim freely and at 60 days of culture fatty acids accumulated highest in the gonads, compared with other treatment. This importantly, energy from dietary intake was allocated for gonad development rather than for locomotive activities. However, in high velocity (60 cm s -1 ), fatty acids accumulated lowest in the gonad, seemly more energy was used to tolerate in high velocity rather than used for gonad development. It is concluded that suitable habitat velocities can accelerate gonadal growth and maturation. In the current study, females were also observed to reach earlier maturation for treatment velocity of 20 cm s -1 than for other treatments.
This current study represents flow velocity as an additional parameter which, if appropriately selected will accelerate broodstock maturation period. The application of velocity treatment to enhance biochemical and gonad maturation in crab is not well studied, which is not the case for fish (Arbelaez-Rojas & Moraes 2013). No research has been conducted on the effect of manipulation of velocities on transfer of lipids and/or proteins, by which to enhance egg development. It has been studied for a range of aquatic organisms that exercise, brought on by habitat velocity, breaks down fatty acids accumulation (Hernandez et al. 2002) but, the same type of study has not been carried out for the mud crab. Habitat velocity has been shown to improve the accumulation of fatty acids in white flesh at the development stage. Female S. olivacea, at treatment velocity of 20 cm s -1 , had stopped feeding and lipids reserves were decreased (Fig. 4).

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The results suggest that velocities trigger vitellogenesis synthesis and gonad development. Vitellogenesis, the process of formation and accumulation of yolk, is central to oogenesis and ovarian maturation. Estrogen hormones, particularly 17β-estradiol (E 2 ), increase the synthesis of vitellogenin (Vtg) in by enhancing the transcription of the Vtg gene. An estrogen receptor (ER) and heat shock protein 90 (Hsp90) (Fliss et al. 2000;Wu & Chu 2008) are used to facilitate this process. Previous work has shown that E 2 can stimulate the development of vitellogenin synthesis and oocyte in kuruma prawn, Marsupenaeus Japanese (Yano & Hoshino 2006). A research of Metapenaeus ensis also showed that Hsp90 played a role in controlling the synthesis of vitellogenin (Wu & Chu 2008). Therefore, further research should be concentrated on hormonal manipulation by manipulate velocity.
The novelty of this study has suggested that hepatopancreas and gonad in both sexes of S. olivacea were more developed at treatment velocity of 20 cm s -1 , compared to other treatment velocities. This moderate velocity can support optimum development of the hepatopancreas. As moderate velocity stimulates good growth for crab, it should serve as suitable culture water velocity for this species. Moreover, females presented higher fatty acids content than males, with female using fatty acids for ovarian maturation, while males utilise fatty acids for seeking shelter, fighting, looking for food and, mating. Biochemical fatty acids assessment showed that the component fractions of the fatty acids, for both sexes accumulated strongly across maturation stages, in suitable water velocity of 20 cm s -1 , compared to other treatment velocities can induced high gonad capacity. The results obtained from this work will hopefully serve as a foundation for further physiological experiments in order to establish broodstock maturation development of S. olivacea, especially at grow-out stage.               Notes: Different letter showed significant different among days. C:16 and C:18 responsible for energy source (Amran et al. 2018).