Effects of Extended-Rotifers Inclusion and Live Food- Enrichment with Probiotics on the Survival, Metamorphosis, Development Time and Growth of Mud Crab, Scylla paramamosain (Estampador) Larvae

Corresponding Author: Quy Moc Ong School of Molecular and Life Science, Curtin University, Kent Street, Bentley, Perth, Western Australia 6102, Australia Email: mocquyts@yahoo.com.vn mocquy.ong@student.curtin.edu.au Abstract: The present larval nutrition study was conducted to evaluate the effects of feeding probiotic-enriched rotifers inclusion mixed with Artemia until zoeal stages 4 (Z4) and Z5 on the survival, metamorphosis, development time and growth of mud crabs, Scylla paramamosain larvae. The efficiency of the feeding regime was tested by two-factor analysis. A 4×2 factorial experimental design with eight combinations of enriched or un-enriched or a mixture of enriched and un-enriched live food (factor 1) in each of two feeding regimes (factor 2) in triplicate was setup. After 24 days of larval cultivation, there were no significant (p>0.05) interactive effects between the two selected factors on survival and metamorphosis rate of larvae. Larval survival in all treatments, under both factors, decreased gradually (p<0.05) during the cultivation period. From 15 Days After Hatching (DAH), the survival and metamorphosis rates of larvae fed both enriched rotifers and Artemia were higher (p<0.05) than those of the larvae without enriched live food. Similarly, from 18 DAH onwards, the extension of rotifers inclusion mixed with Artemia until Z5, irrespective of enrichment, showed higher (p<0.05) survival, metamorphosis and growth of megalopa than when the rotifers were included only until Z4 stage. Overall, an amalgamation of extending rotifer feeding and enriching them at the same time with Artemia can result in considerable improvements in survival and metamorphosis which can in turn, have beneficial impact on the technical feasibility of a commercial crab hatchery.


Introduction
Mud crab, Scylla paramamosain, is a commercially important source of income for coastal fishers in the Asia-Pacific region (Keenan, 1999). In Vietnam, S. paramamosain is the most prevalent cultured species in coastal areas (Le Vay et al., 2001;Lindner, 2005) and the second most common crustacean species after the shrimp (Penaeus monodon and Litopenaeus vannamei) (Nghia et al., 2007). The pond-based crab culture system totally relies on wild-caught seed (Keenan, 1999), which has become limited by over-exploitation and diminishing mangrove habitats (Le Vay et al., 2001). Although mud crab seed production has been studied extensively (Brick, 1974;Heasman and Fielder, 1983;Marichamy, 1996;Quinitio et al., 1999;Mann et al., 1999;Davis, 2003;Nghia et al., 2007;Yi et al., 2009;Noorbaiduri and Ikhwanuddin, 2015), the survival of hatchery-produced crablets is still unreliable and inconsistent (Baylon and Failaman, 1999;Hamasaki et al., 2011), mainly due to mass mortality during the zoeal (Z) and megalopa stages, especially during metamorphosis from Z5 to megalopa . Mass mortalities are mainly linked to poor larval nutrition Suprayudi et al., 2004) caused by selection of improper feeding regimes.
Nutritional deficiencies and inappropriate feeding regime, for example, feeding either rotifers alone (Nghia, 2004;Baylon, 2009), or exclusively Artemia (Ruscoe et al., 2004;Zeng et al., 2004), or excessive or starved Artemia Dan et al., 2016) to mud crab larvae resulted in low survival and prolonged larval development time. On the contrary, Artemia enriched with lecithin and cholesterol (Holme et al., 2007) as well as feeding a mixture of rotifers and Artemia to crab larvae (Zeng and Li, 1999;Nghia, 2004;Ruscoe et al., 2004) improves survival. Zeng and Li (1999), Ruscoe et al. (2004) and Nghia (2004) have recommended that the inclusion of rotifers should be extended until Z2 or Z3 stages to increase the survival of crab larvae. Similarly, our previous study (Quy et al., 2018) showed that including rotifers until the late zoeal stages (Z3 to Z5), mixed with Artemia, increased in a higher metamorphosis of megalopa, thus, confirming that including the extension of rotifers in the feeding regime of mud crab larvae can contribute additional nutrients essential for moulting of most of the larval stages, as Artemia alone are incapable of providing these additional nutrients to the larvae (Baylon, 2009).
Similar to larval mal-nutrition, fungal, parasites and bacterial infections contribute to poor survival; however, enriching live feeds with probiotics increases survival rates (Dan and Hamasaki, 2015). Probiotics are defined as bio-friendly agents that can control and compete with pathogenic bacteria as well as stimulate the growth of aquatic animals without undesirable side effects (Farzanfar, 2006). The beneficial effects of dietary inclusion of probiotics are displayed through competition for habitat and nutrients to displace harmful bacteria (Moriarty, 1998), provision of nutrients and enhanced digestive enzyme activities (Ziaei-Nejad et al., 2006;Wang, 2007;Zhou et al., 2009;Gupta et al., 2016), improvement of water quality (Nimrat et al., 2012), enhancing immune responses (Rengpipat et al., 2000;Tseng et al., 2009;Kumar et al., 2013;Ambas, 2015) and producing antiviral substances (Lakshmi et al., 2013;Pandiyan et al., 2013). Although the beneficial uses of probiotics, including bacteria of the genus Bacillus, have been made in larval, post-larval and juvenile stages of shrimp (Rengpipat et al., 1998;Ziaei-Nejad et al., 2006;Balcázar and Rojas-Luna, 2007;Yu et al., 2008;Guo et al., 2009;Liu et al., 2010;Nimrat et al., 2012;Vinoj et al., 2013;Dong et al., 2014;Jamali et al., 2015) and in mud crab larvae and juvenile (Wu et al., 2014;Dan and Hamasaki, 2015;Talib et al., 2017), no information is available on the effects of supplementing probiotics and extending the inclusion of rotifers on the survival, metamorphosis, development time and growth of mud crab larvae. Thus, the aim of this study was to evaluate the effects of enriching live feeds (Artemia and rotifers) with commercially available Bacillus spp. as probiotics and extending the inclusion of rotifers until late zoeal stages of S. paramamosain.

Water Source
Seawater was pumped from the river and left one night in a reservoir tank before being disinfected with calcium hypochlorite at the rate of 20 mg L −1 . After two day of chlorination, when no chlorine residue detected, the disinfected seawater was transferred into a storage tank, where the water was treated again with 2 mg L −1 Vikor-S (Bayer, Germany) for further use in the experiment.

Live Food Culture
According to the method described by Lind (2014), L-strain rotifers (Brachionus plicatilis) originated from a marine fish hatchery were stocked into indoor 4,000-L square concrete tank. The tank was filled 60% of disinfected seawater (30‰) and provided gently aeration by air-stone. Rotifers were fed a mixture diet of commercially available rotifer feed (S. parkle, INVE Aquaculture, Thailand) and baker's yeast (50:50) at a rate of 0.4 g for one million rotifers as recommended by Lind (2014) at 6 am, 12 pm and 6 pm daily. When density of rotifers achieved approximately 500 individuals mL −1 , they were partially harvested to feed to mud crab larvae.
Artemia franciscana cysts were incubated by an incubation container. Before incubating, the cyst were disinfected with sodium hypochlorite solution at a concentration of 20 mg L −1 for 10 min to avoid any infection of harmful bacteria and fungi and then washed with seawater. Salinity and pH of disinfected seawater using for Artemia incubation were 30‰ and 8.3, respectively. Strong aeration and light were also require. At the temperature of 29°C, the newly hatched Artemia nauplii appeared after 15 h of incubation and they were harvested to enrich or directly feed to mud crab larvae.

Method of Enriching the Live Foods
Bacillus spp. is known as an effective probiotic bacteria in shrimp culture (Ambas et al., 2013), so a commercial probiotic (Lymnozyme FT-2B, Cisbay, USA) including Bacillus subtilis, B. licheniformis, B. amyloliquefaciens and B. Pumilus was used to enrich rotifers and Artemia in the current study. The rotifers, partially harvested from mass production in the 4,000-L square concrete tank, were washed with fresh seawater and stocked in a 10-L plastic bucket containing 50% volume of disinfected seawater with gentle aeration. Probiotics and S. parkle at a rate of 1 mg g −1 of rotifer feed were placed together into a blender (HR2195/00, Philips, China) and blended with 100 mL of deionized water. This blended mixture was then poured into the bucket containing rotifers. The feeding rate of this mixture was adjusted following the rotifer stocking densities in the rearing medium which was 0.4 g of rotifer commercial feed meant to feed one million rotifers (for rotifer density of 1,000-1,500 rotifers mL −1 ). This was equivalently to 40 mL of the mixture of probiotics and rotifer commercial feed. This dose was based on the recommendation of feed company. After 6 hours of enrichment, rotifers were harvested and fed to crab larvae.
In contrast, newly hatched Artemia were only soaked in the commercial probiotic suspension and disinfected seawater at a rate of 1 mg L −1 , with strong aeration. This enrichment process lasted only for 1 h in order to avoid nutrient losses in Artemia due to their quick assimilation efficiencies. The enrichment protocol was strictly followed and the supplemented weight of the probiotic powder was measured by an analytical balance (PA 214-Ohaus, USA) with 0.0001 g precision. The newly hatched Artemia (instar 1) is not fully developed and thus are unable to ingest the food from water, though, they can absorb water containing Bacillus. As a result, the Bacillus could be resident of both gut and the external body of Artemia. To verify the final concentration of the probiotics, homogenised sample taken from whole rotifers or Artemia was used to determine total Bacillus spp. on Tryptic Soy Agar (TSA) plates.Total Bacillus spp. concentration in enriched rotifers and Artemia were counted periodically and also at the end of the trial in plates containing TSA medium, according to the method described by Tomasiewicz et al. (1980).

Mud Crab Brood Stock and Larval Source
Mature and berried females of S. paramamosain were fattened and managed as described by Quy et al. (2018). During the fattening period, the salinity and water temperature ranged from 28 to 30‰ and 28 to 29°C, respectively, while photoperiod was exposed to 12: 12 of light: Dark cycle following natural lights. One day before hatching, the berried female was transferred and individually reared in a 200-L round tank for hatching.
Newly hatched Z1 larvae, from single spawners, were collected and bathed in 0.1 µL L −1 iodine solution for 30 seconds and they were then temporarily reared into a 100-L plastic bucket. The larvae were fed purely with the rotifers without enrichment with probiotics at a rate of 40-50 individuals mL −1 . On the next day, the larvae were used for the experiment.

Experimental Design
Based on our previous research (Quy et al., 2018), which aimed to extend the inclusion of rotifers into the feeding regime of mud crab larvae, two feeding regimes viz. extending rotifers inclusion mixed with Artemia until Z4 and Z5 stages, were selected. In the current study, rotifers and Artemia in these feeding regimes (extending rotifers until Z4 and Z5) were enriched with a commercial probiotic. The enriched/un-enriched live food was defined as a first factor and extension of rotifers inclusion as a second factor. A 4×2 factorial experimental design with eight various combinations of enriched or un-enriched or a mixture of enriched and un-enriched live food (factor 1) in each of two feeding regimes (factor 2) in triplicate was tested (Table 1). Enriched or unenriched rotifers were fed from Z1 crab larvae and extended until the next day of the moulting to Z4 and Z5 stages, depending upon the selection of the extension of rotifers inclusion, whereas enriched or un-enriched Artemia nauplii were offered to these treatments from Z2 stage until the megalopa stage.
Twenty-four 1.5-L plastic beakers in which each beaker was filled with 1 L of disinfected seawater (30‰) and stocked with 30 mud crab Z1 collected from the 100-L plastic bucket. The beakers were moderately aerated to prevent settlement of live food and larvae. All the experimental beakers were maintained at a constant temperature (28±1°C) by immersing in a 5,000-L square concrete tank. To improve ability to catch the prey and possibly reduce any mortality by cannibalism or infection, the photoperiod was maintained constantly for 24 h by a 40-W fluorescent lamp. The pH of disinfected seawater was balanced approximately to 8.3±0.2 by adding CaCO 3 and/or NaHCO 3 before being distributed into every experimental beaker.  Mud crab larvae were fed only rotifers at a rate of 20 individuals (ind.) mL −1 from Z1 to Z2 and then fed a mixture of rotifers and Artemia at a rate of 10 and 5 ind. mL −1 , respectively, until Z4 and Z5, depending upon the treatment in factor 2. After rotifers inclusion was stopped, the density of Artemia in the feeding regime was increased from 5 to 10 ind. mL −1 until the end of the experiment. The desired densities of rotifers and Artemia nauplii in every beaker was prepared following the method described by Baylon (2009). Feeding time was applied only once a day after all crab larvae were transferred into a newly prepared beaker. The newly beakers were filled with disinfected seawater of the same environmental parameters of the replaced beakers and the larvae were carefully and quickly transferred with the help of a large bore pipette. At the same time, larval metamorphosis and mortality was recorded in each beaker. When megalopa stage appeared, they were immediately moved out to avoid cannibalism. Ten megalopa were collected and preserved in 10% formalin solution for further measurements of carapace widths, body lengths and wet weights. The experiment was ended at 24 days after hatching. The survival of the larvae, consisting of zoea and megalopa was computed using the following equation: Final larvae Survival rate zoae stocked = × Megalopa carapace width and body length were measured using a scale inserted into the eyepiece of a microscope (Olympus CX21, USA), then they were dried on tissue paper for wet weight. The wet weight of megalopa was weighted by an analytical balance (PA 214-Ohaus, USA) with 0.0001g precision.

Statistical Analysis
All data in the treatments presented in percentages, such as survival of larvae and percentage of successfully metamorphosed megalopa, were arcsine-square root transformed (Zar, 2010) before analysis. Two-way ANOVA was used to test for significant differences among treatments. In the case of no significant interactive effects between enriched/un-enriched live food and extension of rotifers inclusion, two-way ANOVA was continuously employed to test for various enrichment/non-enrichment and two levels of extensions of rotifers inclusion. If there were significant interactive effects between enriched/un-enriched live food and extension of rotifers inclusion, one-way ANOVA was employed to test for various effects of enrichment/nonenrichment, extension of rotifers inclusion and interaction between enrichment/non-enrichment and extension of rotifers inclusion. Significant differences between the treatments were detected by Tukey's multiple range tests at the 0.05 level of significance. Kruskal-Wallis non-parametric tests was used to compare megalopa carapace width, body length and wet weight because of unequal sample size (amount of megalopa of some replicate in treatments were not enough); significant differences between the treatments were detected by Mann-Whitney Test at the 0.05 level of significance. All statistical analyses were performed using IBM SPSS Statistics 24.0.

Environmental Parameters and Concentration of Total Bacillus spp. of Enriched Live Food
During the experiment, the salinity and pH in all experimental beakers were stable at 30‰ and 8.6, respectively. Alkalinity was constant at 140 mg CaCO 3 L −1 . While water temperature was 27.9±0.5°C throughout the experimental period.

Larval Survival
There were no significant (p>0.05) interactive effects between enriched/un-enriched live food with probiotics (factor 1) and extension of rotifers inclusion (factor 2) on mud crab larval survival; the main effect was examined independently for two factors (Table 2). Larval survival in all treatments (in both factors) decreased gradually (p<0.05) during the culture period. The survival of larvae fed both enriched rotifers and Artemia was higher (p<0.05) than that of larvae fed a diet without enrichment (R & A) from 15 Days After Hatching (DAH) onwards and a diet enriched with rotifers only from 15 to 18 DAH. However, the larval survival at the end of the feeding experiment was not altered (p>0.05) by enrichment when enrichment was carried out individually either to rotifers or Artemia. Similarly, extension of rotifers inclusion influenced (p<0.05) the survival of larvae from 18 DAH onwards, in which the larvae fed rotifers mixed with Artemia until Z5 showed significantly higher survival than those fed rotifers inclusion until Z4.
There was a negative correlation between larval survival and culture period (Table 3), with regression indices (R 2 ) ranging from 0.96 to 0.99. These indices were, however, independent (p>0.05) of enrichments and extension of rotifers inclusion in diets with Artemia.

Rate of Metamorphosis and Development Time of Megalopa
Mud crab larvae fed both enriched rotifers and Artemia (En-R &En-A) showed a higher (p<0.05) rate of metamorphosis of megalopa (37.2%) than those fed a diet without enrichment (R & A) (12.8%) and a diet enriched with rotifers only (En-R & A) (17.2%). Similarly, the larvae fed rotifers inclusion mixed with Artemia until Z5 showed a significantly higher (p<0.05) metamorphosis rates (27.5%) than larvae fed rotifers inclusion until Z4 (15.8%) (Fig. 1). Morphological variation was also less seen in the feeding treatments that had high successful metamorphosis of megalopa. However, the duration to metamorphose from Z1 to the megalopa stage (development time) did not completely depend (p>0.05) on enrichments and feeding duration with rotifers (Fig. 2).

Growth Performance of Megalopa
Enrichment of live food did not significantly (p>0.05) influence carapace width, body length and wet weight of megalopa, but extension of rotifers inclusion mixed with Artemia until Z5 improved (p<0.05) carapace width and wet weight of megalopa (1.66 mm and 4.02 mg, respectively) compared to the treatment in which rotifers were included until Z4 (1.62 mm and 3.77 mg, respectively; Table 4).
To our knowledge, this is the first study investigating the effects of extending rotifer feeding in an Artemia-based diet with enriched live foods, both Artemia and rotifers, with a commercial probiotic based on Bacillus spp. in mud crab (S. paramamosain) larvae. In the current study, a diet that included both enriched rotifers and Artemia with probiotics increased survival and metamorphosis rates of megalopa compared to a diet without enriched live food. The same results were obtained in L. vannamei fed both enriched rotifer and Artemia (Jamali et al., 2015). Total bacteria and probiotic Bacillus increased in the host (Jamali et al.,, 2015) that resulted in increasing activity of digestive enzymes (Ziaei-Nejad et al., 2005) and inhibiting the growth of pathogens (Decamp et al., 2008), which in turn improved larval survival. In addition, Nogami et al. (1997) reported that the bacterial strain PM-4 (Thalassobacterutilis), which was added daily into rearing water increased the survival of swimming crab (Portunus trituberculatus) larvae by repressing the growth of harmful bacteria and fungi. Dan and Hamasaki (2015) also found that suppressing larval necrosis symptoms and improving survival of early larvae of S. serrata were done when probiotics was inoculated in the larval rearing water. Although the mode of probiotic administration differed between our study and the previously mentioned studies, the beneficial outcomes of probiotics were not significantly different (Ziaei-Nejad et al., 2005). Similar to the factorial 1 outcome, the inclusion of rotifers mixed with Artemia until Z5 improved megalopa survival, metamorphosis rate and growth compared to the treatment where rotifers were included only until Z4.
Previous studies have also shown that a mixed diet of rotifers and Artemia until the late zoeal stages improved larval survival of S serrata (Baylon and Failaman, 1999), P. Pelagicus (Redzuari et al., 2012), Upogebia pusilla (Faleiro and Narciso, 2009) and Panopeus herbstii (Harvey and Epifanio, 1997), but these studies failed to mention the benefits that achieved as extending of rotifers inclusion. In the present study, morphological variation and formation of immature megalopa, resulting from accumulation of insufficient nutrients (Dan et al., 2013) were less seen in the crab larvae fed rotifers inclusion mixed with Artemia until Z5 than in those fed rotifers inclusion only until Z4. This could be due to the starvation of Artemia that can be reduced to a certain extent by feeding them rotifer excrements (Dan et al., 2016). Crab larvae can be exposed to less starved Artemia when rotifers are present until Z5 in a mixed diet with Artemia.
Additionally, morphological variation and formation of immature megalopa, which are caused by quantity of available food (Pestana and Ostrensky, 1995), may be less appearance as rotifers and Artemia, both are available in rearing water in our study. According to Genodepa et al. (2004), the feeding behaviour of mud crab larvae is raptorial. The late zoeal stages showed a strong preference for Artemia, which supplied the majority of energy intake required for moulting to the next stage (Harvey and Epifanio, 1997); however, after moulting, they preferred rotifers, which provided energy for survival (Baylon et al., 2004). This indicates that extending rotifers inclusion, mixed with Artemia, until Z5 can provide additional benefits in reducing the morphological variation and the formation of immature megalopa, which would result in mass mortality of crab larvae (Dan et al., 2013). For cost-effective aspect, Ruscoe et al. (2004) and Baylon (2009) found that extending rotifers inclusion until late zoeal stages with Artemia does not provide economic benefits due to the use of expensive resources in terms of floor space, microalgae and labour (Nghia, 2004;Ruscoe et al., 2004). On the contrary, rotifers used in the present study produced according to the procedures described by Lind (2014) were of high daily productivity and low production cost. Furthermore, extending the period of rotifer inclusion in the feeding regime could save the usage of Artemia (Quy et al., 2018). Therefore, extending rotifers inclusion mixed with Artemia until Z5 is possible to reduce cost production for mud crab hatchery.
Probiotics may rapidly alter the intestinal microbiota of the larvae. Gatesoupe (1999) showed that the intestinal microbiota of early larval stages was easily altered by the invasion of the microorganisms from water and food, even when their digestive tract was not fully developed and feeding had not yet commenced. In the present study, a mixture of Bacillus spp. was inoculated in live food at the beginning of the experiment; therefore, any harmful bacteria that could invade the cultured species through the food chain (Van Stappen, 1996, p.114) may have been reduced or eliminated. Enriched live food can also become a vector for bringing desirable microbes into the digestive system of the host (Lavilla-Pitogo et al., 2002), thereby potentially improving the properties of the indigenous microflora of the larvae as well as interfering with the development of harmful bacteria. For example, when the probiotic Bacillus spp. was used, Vibrio numbers were reduced and no luminous Vibrio responsible for losses in shrimp hatcheries existed (Moriarty, 1998;. Similarly, Rengpipat et al. (1998) and Far et al. (2009) have reported that B. subtilis proliferated and replaced Vibrio spp. in the digestive tract of shrimp (P. monodon) treated with probiotics that increased in survival and growth of the shrimp. In agreement with these results, our study showed that the survival and metamorphosis rates of the larvae fed both enriched rotifers and Artemia with probiotics were higher than those fed a diet without enrichment. The reduction in the number of probiotic bacteria (Dan and Hamasaki, 2015) or low concentrations of probiotics in the diet (Tseng et al., 2009) induced individual differences among the replicated dietary treatments in any feeding trial. This may explain the fact that in the current study, enrichment of only one type of live feed could not significantly improve survival and rate of metamorphosis in crab larvae.
A study by Ruscoe et al. (2004) on S. serrata, as well as our earlier study (Quy et al., 2018) on S. parasamosain, showed that feeding a mixture of unenriched rotifers and Artemia until Z4 and Z5 stages obtained similar megalopa survival. On the contrary, in the present study feeding a mixture of rotifers and Artemia, irrespective of enrichment, until Z5, rather than Z4, improved megalopa survival, metamorphosis rates and growth. It is apparent that enrichment can be considered as a factor contributing to these differences. However, there were no significant interactive effects between enrichment/non-enrichment with probiotics and extension of rotifers inclusion on mud crab larval survival and metamorphosis rates, probably because the beneficial effect of the two factors studied was more of an additive affect rather than a synergistic affect.

Conclusion
In conclusion, inclusion of enriched rotifers mixed with enriched Artemia until the stage Z5 improved survival, metamorphosis rate and growth of megalopa. Further, the enrichment of both rotifers and Artemia as a live feed is crucial to achieve beneficial outcomes in crab hatcheries. However, these results needs to be validated under commercial farming operations, wherein other natural and/or anthropogenic factors interplay. However, current study can be used as a baseline data on the use of appropriate feeding regimes to improve the survival of mud crab larvae. There is also a need to compare the bio economics between the use of exclusive formulated feed and the feeding regime used in the current study.