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Limiting the use of rotifers to the first zoeal stage in mud crab (Scylla serrata Forskål) larval rearing
Aquaculture 231 (2004) 517 – 527 www.elsevier.com/locate/aqua-online
Limiting the use of rotifers to the first zoeal stage in mud crab (Scylla serrata Forska˚l) larval rearing Ian M. Ruscoe *, Graham R. Williams, Colin C. Shelley Darwin Aquaculture Centre, Fisheries Group, Department of Business, Industry and Resource Development, GPO Box 3000, Darwin, NT 0801, Australia Received 14 August 2003; received in revised form 18 November 2003; accepted 18 November 2003
Abstract Commercial mud crab (Scylla spp.) aquaculture in many countries has been stifled to some degree by a lack of seed stock. A commercially viable larval rearing protocol for Scylla serrata has yet to be established. Two experiments were conducted to assess the requirement for rotifers in the larval feeding regime for S. serrata. These examined the timing of introduction and cessation of rotifers, and co-feeding protocols with Artemia. In the first experiment, it was shown that rotifers were necessary in the feeding regime for acceptable growth and survival, and when used as the only food source during the first zoeal stage (Z1), gave better survival than when cofed with Artemia. It was also found that when rotifers were removed from the co-feeding regime at Z3, no detrimental effect on growth or survival occurred. Final survival to megalop of 58.67 F 7.35% was achieved when rotifers were fed singly up to zoea 2 and then co-fed with Artemia through to megalop. Crab larvae fed only Artemia throughout the rearing period took significantly longer to reach the megalop stage and suffered higher levels of mortality. The second experiment investigated the effects on survival, when rotifer feeding was discontinued at Z2, Z3, Z4, Z5 and when rotifers were fed up to the megalop stage (M). Artemia were offered to these treatments from Z2 onwards. A no-rotifer (NR) control that was fed Artemia-only from stocking, was also tested. It was again shown that rotifers, especially in the early stages, promoted acceptable levels of survival and growth. Without rotifers, moulting of crab larvae was delayed and survival was significantly lower during the first two zoeal stages. There was also a significant difference in survival to megalop, and a significant negative relationship between duration of rotifer feeding and survival to megalop. The best survival of 78.00 F 5.54% was achieved when rotifers were removed from the feeding regime at Z2. The NR treatment had the lowest survival of 32.00 F 7.51%. These trials established that while rotifers are a valuable
* Corresponding author. Tel.: +61-8-8924-4265; fax: +61-8-8924-4277. E-mail address: [email protected] (I.M. Ruscoe). 0044-8486/$ - see front matter. Crown Copyright D 2004 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2003.11.021
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inclusion in the feeding regime for larval S. serrata, their use should be limited to the first zoeal stage only to maximise growth and survival. Crown Copyright D 2004 Published by Elsevier B.V. All rights reserved. Keywords: Rotifers; Mud crab; Larval rearing; Scylla serrata; Feeding regime
1. Introduction There are four species of mud crab within the genus Scylla which inhabit tropical to warm temperate inshore zones and which form the basis of relatively small, yet important commercial fisheries. The most widespread of these, Scylla serrata, occurs throughout the indo-west Pacific from southern Africa to Tahiti, including the northern half of Australia, north to Okinawa, and south to the bay of Islands in New Zealand (Keenan, 1999). They are usually found in intertidal and subtidal zones of estuaries and in mangrove systems. They are easily caught using simple baited traps, grow to a size in excess of 1.5 kg, and have an excellent tolerance to air exposure (Gillespie and Burke, 1992). For these reasons, they are easily marketed and are an important source of income throughout their range. Several countries have investigated farming of mud crabs, however, the major constraint to further expansion of mud crab aquaculture is the current lack of a regular supply of seed stock (Cholic, 1999; Fortes, 1999; Keenan, 1999). In Australia, the only species of mud crab being investigated for culture is S. serrata. At the Darwin Aquaculture Centre, this species has been stocked at densities of approximately 10 larvae/l for larviculture. Typically a combination of microalgal species has been introduced at the time of stocking. Rotifers have been fed throughout the zoeal stages at approximately 10/ml in conjunction with freshly hatched Artemia nauplii from the second day of the second zoeal stage (Z2/2) (Williams et al., 1999). Researchers in other Asian countries have utilised much higher larval stocking and live food densities. Baylon and Failaman (2001) stocked larvae at up to 100 individuals/l in a greenwater system, where rotifers were included at 30/ml, and Artemia were added at 0.5 –5/ml as the larvae grew from Z1 to Z5. Baylon et al. (2001) also suggested a combined feeding regime of rotifers and Artemia to Z4, and a switch to Artemia alone at Z5. Nghia et al. (2001) reviewed larval rearing practises for S. paramamosian and made several recommendations including algal densities of 0.1 – 3 million cells/ml, an enriched rotifer density of 45/ml for the first 6 days and an Artemia density of 20/ml from day 4 onwards. Recently, the nutritional quality of Artemia as a live feed for mud crab larvae has also been investigated. The results of Takeuchi et al. (2000) suggest that the n 3 highly unsaturated fatty acids (n 3 HUFA) should be present in Artemia to promote survival and growth. Kobayashi et al. (2000) reported that EPA was important for survival, whereas DHA was important in promoting growth of S. tranquebarica. Quinito et al. (1999) suggested that artificial supplements could be used to enrich traditional live feeds with the view of improving larval survival. There seems to be a wide consensus between research institutes in feeding rotifers initially, and introducing Artemia nauplii and sub-adults as the larvae grow. However, no
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consensus has been reached on enrichment protocols, larval food densities, or the stages where the different species of live food are most beneficial or needed. Rotifers are commonly utilised as the first feeding organism for many marine and estuarine species. Their production however is often seen as a major expense in marine hatchery management due to its requirement for substantial floor space, microalgal or artificial feeds, and personnel time in culture management. If rotifer use can be eliminated or substantially reduced in larval mud crab rearing, labour, resource requirements and therefore production costs can be reduced. The following two experiments were designed to assess at which stage, if at all, rotifers are required in the feeding regime of S. serrata larvae, and for how long; approximately when Artemia should be introduced; and whether enrichment of live food, or artificial food is beneficial.
2. Materials and methods 2.1. Broodstock The broodstock females, whose progeny were used in these experiments, were identified as S. serrata according to the description of Keenan et al. (1998). Mature females were collected from the wild (Elizabeth river, Darwin Harbour) and held communally in a 10,000-l environmentally controlled recirculating system inside the hatchery at the Darwin Aquaculture Centre (DAC). Temperature and salinity were adjusted to 30jC and 30x, respectively. Ambient light regimes were used. When the females were observed to be carrying eggs, they were transferred to a separate recirculating incubation system and were held individually in 100-l tanks with reduced light levels and were not fed. After 9 days of incubation, the females were transferred to a 600-l hatching tank with flow-through water supply (approx. 3 l/ min), after a small sample of eggs were examined for development and health assessment. The following morning, the strongly phototactic and schooling first stage zoea (Z1) were concentrated with a torch light and were captured in a 5-l plastic jug. Incubation and hatching tank temperature and salinity was the same as the broodstock tank. 2.2. Rearing facilities The larvae used in both experiments were reared using the same facilities with treatments being imposed through the live food. Facilities common to both experiments are described first. The rearing containers used were 5-l hemispherical acrylic bowls containing 3 l of culture water. These clear acrylic bowls were then placed inside an identical bowl which had the outer surface painted black, and therefore provided a darkened background. The bowls were held on floating ‘pontoons’ (8 –10 bowls each) within a 7000-l constantly exchanging water bath underneath a shade structure at the DAC. Gentle aeration was provided through a single glass 1 ml pipette in each bowl.
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Water used to fill the bowls was settled and fractionated for 10 days before stocking. This water was stored overnight in a 100-l plastic tub submerged in the same water bath as the bowls. In addition, two 2-l capacity submerged carbon filters powered by a single 15-mm diameter airlift in each were run for approximately 18 h prior to use. New water for each day was treated as above. The salinity of the rearing water was adjusted from 30xat the beginning of the experiments down to approximately 25xat the megalop stage. The larvae for each bowl were individually counted and stocked at 10 larvae/l (30 larvae/bowl) on day 0. The larvae were slowly acclimated to the bowl by floating in a shallow tray with frequent small additions of the culture water. Each subsequent morning, the larvae were individually removed from the bowl using a large bore plastic 3 ml pipette, were counted, and any mortalities removed. Each bowl was emptied, cleaned in freshwater and new culture water and live food was added, together with the remaining crab larvae. Megalops were removed from the bowls as they appeared, and were included in the survival calculations after that time. When all remaining larvae had moulted to megalop, the experiment was terminated as previous research from the DAC had developed a suitable feeding regime for the megalop to crablet stage (Williams et al., 1999). All treatments in both experiments received equal cell numbers of three species of microalgae to a final density of 5 104 cells/ml. The algae used were Nannochloropsis oculata, Chaetoceros mulleri and an endemic unnamed Isochrysis-like species titled PS11 (Thinh et al., 1999). Rotifers (Colurella adriatica) were mass cultured in microalgae, usually a combination of N. oculata and PS11 at a density of approximately 100 –150/ml. Each day, rotifers were harvested by draining through a submerged 64-Am screen, were rinsed in 5-Am filtered seawater and concentrated to approximately 5000/ml in a 20-l bucket containing a combination of laboratory cultured N. oculata and PS11 for 60 – 120 min. These were then further rinsed and fed at a rate of 10 rotifers/ml in a single daily feed. Artemia (INVE, AF grade : 430 Am) used in these experiments were newly hatched first instar nauplii, except where stated, and these were fed at a rate of 0.5 nauplii/ml in a single daily feed. For both experiments, where there is a change in the feeding protocol, for example, a cessation of rotifer feeding on day 2 of Z3 (Z3/2), the change occurs 1 day after >50% of the larvae have moulted to Z3. 2.3. Experiment 1 Five experimental treatments were imposed. Each treatment consisted of a different zooplankton feeding regime, as summarised in Table 1. There were five replicates of each treatment arranged in a completely randomised design. Artificially enriched rotifers for use in treatment 5 were held in a 20-l bucket containing 5 g of Dry Selco (INVE) for between 60 and 120 min. These were also further rinsed and fed at 10 rotifers/ml in a single daily feed. Artemia (INVE, AF grade : 430 Am) for treatment 5 were instar I nauplii from days 4 to 9, and 24 h enriched nauplii were fed from day 10 onwards. These were enriched overnight in a 15-l bucket containing 1 g of Frippakk CD2 Ultra larval shrimp food.
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Table 1 Treatment identifiers and descriptions of the feeding regimes for experiment 1 Name
Rotifer feeding from – to (inclusive)
Artemia feeding from – to (inclusive)
T1 T2 T3 T4 T5*
nil Stocking – megalop Stocking – Z3/2 Stocking – megalop Stocking – megalop enriched with INVE Dry Selco
Stocking – megalop Stocking – megalop Stocking – megalop Z2/2 – megalop Z2/2 – megalop enriched with Frippackk CD 2 Ultra larval shrimp food
Z3/2 refers to the second day of the third zoeal stage. * T5 experimental units also received artificial food consisting of Spirulina (SP + kINVE) and Frippackk CD2 Ultra larval shrimp food (INVE), each to a final concentration of 1 mg/l.
Artemia were fed at a rate of 0.5/ml in a single feed. Treatment 5 also received artificial food as detailed in Table 1. 2.4. Experiment 2 Six experimental treatments were imposed which again differed in relation to their zooplankton feeding regimes, which are summarised in Table 2. There was no artificial enrichment of live food, and only instar 1 Artemia were fed. There were five replicates of each treatment arranged in a completely randomised design. 2.5. Water quality Temperature of random bowls from each experiment was measured each morning at 0800 h. Ammonia was also measured every second day from a single randomly chosen bowl from each treatment. 2.6. Data analysis Daily survival was expressed as the percentage of zoea alive at the time of counting, and duration to megalop was recorded in terms of days. These variables were analysed by ANOVA (Systat V7, SPSS, Chicago, USA), and where significant differences were observed, they were separated using a post-hoc least significant Table 2 Treatment identifiers and descriptions of the feeding regimes for the experiment 2 Name
Rotifer feeding from – to (inclusive)
Artemia feeding from – to (inclusive)
NR Z2 Z3 Z4 Z5 M
nil Stocking – Z2/1 Stocking – Z3/1 Stocking – Z4/1 Stocking – Z5/1 Stocking – megalop
Stocking – megalop Z2/2 – megalop Z2/2 – megalop Z2/2 – megalop Z2/2 – megalop Z2/2 – megalop
Z2/1 refers to the first day of the second zoeal stage.
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difference comparison of means test. Residuals were examined to determine a requirement for data transformation. Final survival to megalop in experiment 2 was also regressed against days of rotifer feeding (for those treatments receiving rotifers) in order to determine if there was a significant relationship between these two variables.
3. Results 3.1. Experiment 1 At 0800, the temperature of the experimental units was between 20.8 and 27.4 jC and averaged 22.4 jC throughout the experiment. Ammonia – nitrogen ranged between < 0.05 and 0.3 mg/l. Examination of residuals revealed that all data was homogeneous and transformation was not required. Table 3 and Fig. 1 show survival data for the five treatments in this experiment. At day 2, there was a highly significant difference (P < 0.001) between treatments for survival, with treatments T4 and T5 which were receiving rotifers only, at that point, having higher survival than those treatments receiving rotifers in combination with Artemia (T2 and T3), or Artemia only (T1). Survival in T1 was also significantly lower than T2 and T3. Day 2 was the first day that a significant difference was established between the treatments. There was also a significant difference in survival to megalop ( P < 0.05) with T1 performing poorest overall. T5 that was receiving enriched live food and artificial food performed no better than treatment 4, which was fed un-enriched live food only. Also, there was no significant difference between T2 that was fed rotifers throughout, and T3 where rotifer feeding was discontinued at Z3. In combination with the poorest survival, T1 also took significantly ( P < 0.0001) longer to reach the megalop stage (Table 1). This extended larval development was initiated at Z1, as larvae in this treatment did not moult to Z2 until day 7, compared to day 4 when rotifers were offered. By day 18, all treatments except T1 had megalops present. Megalops did not appear in this treatment until day 21.
Table 3 Mean survival (%) (F S.E.) at day 2, final survival to megalop (%) (F S.E.) and duration to megalop (F S.E.) for the five treatments in experiment 1 Treatment
Survival day 2 (%)
Survival to megalop (%)
Duration to megalop (days)
T1 T2 T3 T4 T5
76.00 F 3.56c 87.33 F 1.94b 84.66 F 2.91b 98.67 F 0.82a 96.00 F 1.94a
25.33 F 5.92c 36.00 F 5.31bc 44.67 F 9.98abc 52.95 F 8.23ab 58.67 F 7.35a
23.88 F 0.57b 19.76 F 0.30a 20.25 F 0.21a 19.81 F 0.26a 19.43 F 0.16a
Values in columns with the same superscripts are not significantly different ( P V 0.05). For treatment descriptions, refer to Table 1.
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Fig. 1. Daily survival for the five treatments in experiment 1. Error bars omitted for clarity. For treatment descriptions, refer to Table 1.
3.2. Experiment 2 At 0800, the temperature of the experimental units was between 24.8 and 27.8 jC and averaged 26.3 jC throughout the experiment. Ammonia – nitrogen ranged between < 0.05 and 0.2 mg/l. Data relating to survival and duration to megalop for the various treatments in the second experiment is presented in Table 4 and Fig. 2. The no-rotifer (NR) treatment exhibited significantly (P = 0.005) lower survival at day 7, when the larvae in this treatment first moulted to Z2. All other treatments were at the Z3 stage by this time. There were also significant differences in survival to megalop ( P < 0.01) with the NR treatment performing poorest overall. The Z2 treatment had the highest survival of 78.00 F 5.54%. There was a highly significant difference in the mean duration to megalop ( P < 0.001) with the NR treatment being approximately 4 days slower. All remaining treatments were Table 4 Mean survival (%) (F S.E.) and duration to megalopa (days) (F S.E.) for the various treatments in experiment 2 Treatment
Survival day 7 (%)
Survival to megalop (%)
Duration to megalop (days)
NR Z2 Z3 Z4 Z5 M
68.67 F 7.35b 89.33 F 3.23a 90.00 F 2.79a 96.00 F 0.67a 92.00 F 1.33a 84.00 F 7.18a
32.00 F 7.51b 78.00 F 5.54a 70.67 F 3.40a 69.33 F 3.23a 62.67 F 12.45a 54.00 F 12.4ab
22.28 F 0.33b 17.32 F 0.06a 17.37 F 0.11a 17.22 F 0.01a 17.23 F 0.08a 17.21 F 0.08a
Values in columns with the same superscripts are not significantly different ( P V 0.05). For treatment descriptions, refer to Table 2.
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Fig. 2. Daily survival for the various treatments in experiment 2. Error bars omitted for clarity. For treatment descriptions, refer to Table 2.
not significantly different from one another. Again the extended larval development was initiated at Z1. For those treatments receiving rotifers, there was a weak yet significant ( P < 0.01; R2 = 0.18) negative correlation between duration of rotifer feeding and survival to megalop.
4. Discussion The timing of the introduction of a prey organism into a culture system is important for a number of reasons. Firstly, live food organisms are provided as prey, and therefore need to be easily captured, digested and assimilated by the cultured species. Various prey organisms have varying sizes, swim speeds, digestibility’s and assimilation efficiencies all of which may change with ontogeny of the prey and the larvae. It is generally through research such as this that appropriate feeding regimes are elucidated. While the provision of various species of prey organisms with differing physical and nutritional characteristics throughout the larval rearing period may ensure the larvae have a suitable prey at all times, the difficulties in terms of culture and hatchery management may make the overall hatchery unprofitable. Secondly, prey organisms compete with the larvae for specifically provided microalgae and dissolved oxygen, which can be in limited supply in intensive hatchery systems. Some live feeds can produce toxic metabolites, including ammonia, that can compromise water quality (Samocha et al., 1989). The methods utilised in these experiments, specifically moderate larval and live food densities, as well as complete daily water exchanges, go some way to negating these potential problems. However, in a commercial hatchery operation, competition for resources and the influence of live food on
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water quality may impact on larval health, feeding ability, and ultimately growth and survival. For these reasons, timing of live prey provision is considered an important issue. The main aim of these experiments was to evaluate the timing of introduction, and cessation of feeding, of the two main live food organisms, rotifers and Artemia. The results of experiment 1 demonstrate that when only rotifers were fed during Z1 (T4 and T5), survival was higher than when rotifers were offered in conjunction with Artemia (T2 and T3). Furthermore, when Artemia were fed alone during Z1 (T1), survival was again significantly poorer. Baylon et al. (2001) observed that zoea 1 S. serrata larvae were passive feeders that did not actively pursue prey, so the capture of a highly mobile Artemia nauplii was less likely than a slower swimming and higher density rotifer. For treatments T2 and T3, Artemia were supplied in addition to the normal rotifer ration, so the reason for the poorer survival of the larvae could not be due to a lack of food, but may have been due to visual confusion of the larvae resulting in decreased rotifer consumption. The Artemia-only treatment (T1) in the first experiment sustained losses of greater than 50% before the Z3 stage. As well as influencing survival, the lack of rotifers in the early stages of this treatment slowed the development process to megalop by approximately 4 days. The reason for the poor performance of mud crab larvae when fed Artemia alone can only be speculated, but may have been the result of Artemia being too difficult to catch and ingest, or they may have been lacking certain essential nutrients such as DHA, as was lacking from the Artemia in the experiments of Quinito et al. (1999). Kobayashi et al. (2000) suggested that DHA is important for growth of mud crab larvae, and this nutrient is known to be in very low concentrations in newly hatched Artemia nauplii (Evjemo and Olsen, 1997). The feeding of DHA enriched Artemia from day 1 may overcome this problem. Whether the Z1 larvae could catch and consume an instar II (or later), Artemia nauplii may also be important, but increasing Artemia density in the early stages at least may overcome this problem. T5 yielded the best survival to megalop, but the enriched Artemia and artificial diets provided seemed to have little effect as these did not confer significantly better survival than T4. This then lends further weight to the argument that the poor larval survival and development when fed Artemia-only during Z1 is most likely a prey capture problem rather than a nutritional issue. The presence of rotifers in experiment 1 significantly and directly improved survival of mud crab larvae in early stages up to Z3, however, when rotifers were removed from the feeding regime at this stage, there was no effect on survival. This is apparent when comparing T2 that received rotifers throughout, against T3 where rotifers were discontinued at day 5. There was no immediate effect on survival and in fact T2, that were still receiving rotifers, tended to show poorer survival towards the end of the experiment. Similarly, Nghia et al. (2001) suggested that survival of the mud crab S. paramamosain was higher when rotifer feeding was discontinued after 6 days as opposed to 9 and 12 days. If rotifers need only be supplied during the first 5 days of culture, as opposed to continual feeding throughout, then substantial savings in terms of algal production, floor space and labour can be achieved. It will also result in a more easily managed larval rearing system. The results of experiment 2 reinforced that when rotifers were not fed in the first zoeal stage (NR), moulting to the second zoeal stage was delayed, and survival was reduced. Even though there was no significant difference in survival to megalop, or duration to megalop for the remaining treatments, it can be argued that co-feeding of rotifers and
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Artemia should not be practised after zoea 2, as rotifers seem not to provide any benefit beyond this stage. Furthermore, the significant negative regression between the period of rotifer feeding and survival would suggest that providing rotifers past Z2 would tend to decrease the survival of mud crab larvae. It may be possible to feed rotifers for the first few days and then switch to Artemia while the larvae are still at the Z1 stage, although this was not tested and further experimentation is required. It should also be noted that the results may vary under different feeding densities and culture conditions. Interestingly, the newly hatched Artemia nauplii used throughout the second experiment supported good survival and development to the megalop stage. These nauplii were not nutritionally enriched in any way prior to feeding. The nauplii would probably have picked up trace amounts of DHA in the greenwater culture. Kobayashi et al. (2000) found that mud crab (S. tranquebarica) larvae fed enriched Artemia containing 1.3% EPA (% of total lipids) and trace amounts of DHA showed high survival, whereas DHA concentrations above 0.46% resulted in lower survival even when EPA was maintained at 1.3%. The results of the experiment 1 showed that rotifers were essential for acceptable survival and development of early larval stages, but can be removed from the feeding regime as early as Z3. It was also concluded that Artemia should only be fed from the Z2 stage, as providing Artemia before Z2 reduced survival. Artificial food, enrichment media, and artificial diets did not significantly benefit the cultures. The second experiment refined the need for rotifers to the first zoeal stage only, and showed that newly hatched INVE AF 430 Artemia nauplii were sufficient to grow S. serrata larvae through to the megalop stage, from zoeal stage 2. There is however scope for further experimentation on the benefits of enrichment of rotifers and Artemia on the growth and survival of S. serrata larvae.
Acknowledgements This work was funded by the Fisheries Research and Development (FRDC) under grant number 2000/210. The authors would like to thank Rachel Naylor and Cameron Moir for the valuable technical assistance throughout this project.
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Forum Held in Darwin, Australia. 21 – 24 April, 1997. ACIAR Proceedings, vol. 78. ACIAR Publishing, Canberra, Australia, pp. 27 – 32. Gillespie, N.C., Burke, J., 1992. Mud crab storage and transport in Australian commerce. In: Angell, C.A. (Ed.), Report on the Seminar on the Mud Crab Culture and Trade, Surat Thani, Thailand, November 5 – 8 1991. Bay of Bengal Program, BOBP/REP/5 1, Madras, India, pp. 207 – 209. Keenan, C.P., 1999. Aquaculture of the mud crab, genus Scylla—past, present and future. In: Keenan, C.P., Blackshaw, A. (Eds.), Mud Crab Aquaculture and Biology. Proceedings of an International Scientific Forum Held in Darwin, Australia. 21 – 24 April, 1997. ACIAR Proceedings, vol. 78. ACIAR Publishing, Canberra, Australia, pp. 9 – 13. Keenan, C.P., Davie, P., Mann, D.L., 1998. A revision of the genus Scylla DeHaan, 1883 (Crustacea: Decapoda: Brachyura: Portunidae). Raffles Bulletin of Zoology 46, 217 – 245. Kobayashi, T., Takeuchi, T., Arai, D., Seikya, S., 2000. Sustainable dietary levels of EPA and DHA for larval mud crab during the Artemia feeding period. Nippon Suisan Gakkaishi 66, 1006 – 1013 (in Japanese with English abstract). Nghia, T.T., Wille, M., Sorgeloos, P., 2001. Overview of larval rearing techniques for mud crab (Scylla paramamosian), with special attention to the nutritional aspect, in the Mekong delta, Vietnam. Book of Abstracts. 2001 Workshop on Mud Crab Rearing, Ecology and Fisheries. Can Tho University, Vietnam, p. 13. 8 – 10 January. Quinito, E.T., Parado-Estepa, F., Alava, V., 1999. Development of hatchery techniques for the mud crab Scylla serrata: 1. Comparison of different feeding schemes. In: Keenan, C.P., Blackshaw, A. (Eds.), Mud Crab Aquaculture and Biology. Proceedings of an International Scientific Forum Held in Darwin, Australia. 21 – 24 April, 1997. ACIAR Proceedings, vol. 78. ACIAR Publishing, Canberra, Australia, pp. 125 – 130. Samocha, T.M., Uzeil, N., Browdy, C.L., 1989. The effect of feeding two prey organisms, nauplii of Artemia and rotifers, Brachionus plicatilis (Muller), upon survival and growth of larval marine shrimp, Penaeus semisulcatus (de Haan). Aquaculture 77, 11 – 19. Takeuchi, T., Kobayashi, T., Shimizu, T., Sekiya, S., 2000. The necessity and sustainable feeding schedule of Artemia nauplii for larval mud crab. Nippon Suisan Gakkaishi 66, 984 – 992 (in Japanese with English abstract). Thinh, L.V., Renaud, S.M., Parry, D.L., 1999. Evaluation of recently isolated Australian tropical microalgae for the enrichment of the dietary value of brine shrimp, Artemia nauplii. Aquaculture 170, 161 – 173. Williams, G.R., Wood, L., Dalliston, B., Shelley, C., Kuo, C., 1999. Mud crab (Scylla serrata) megalopa larvae exhibit high survival rates on Artemia-based diets. In: Keenan, C.P., Blackshaw, A. (Eds.), Mud Crab Aquaculture and Biology. Proceedings of an International Scientific Forum Held in Darwin, Australia. 21 – 24 April, 1997. ACIAR Proceedings, vol. 78. ACIAR Publishing, Canberra, Australia, pp. 131 – 140.
Limiting the use of rotifers to the first zoeal stage in mud crab (Scylla serrata Forska˚l) larval rearing Ian M. Ruscoe *, Graham R. Williams, Colin C. Shelley Darwin Aquaculture Centre, Fisheries Group, Department of Business, Industry and Resource Development, GPO Box 3000, Darwin, NT 0801, Australia Received 14 August 2003; received in revised form 18 November 2003; accepted 18 November 2003
Abstract Commercial mud crab (Scylla spp.) aquaculture in many countries has been stifled to some degree by a lack of seed stock. A commercially viable larval rearing protocol for Scylla serrata has yet to be established. Two experiments were conducted to assess the requirement for rotifers in the larval feeding regime for S. serrata. These examined the timing of introduction and cessation of rotifers, and co-feeding protocols with Artemia. In the first experiment, it was shown that rotifers were necessary in the feeding regime for acceptable growth and survival, and when used as the only food source during the first zoeal stage (Z1), gave better survival than when cofed with Artemia. It was also found that when rotifers were removed from the co-feeding regime at Z3, no detrimental effect on growth or survival occurred. Final survival to megalop of 58.67 F 7.35% was achieved when rotifers were fed singly up to zoea 2 and then co-fed with Artemia through to megalop. Crab larvae fed only Artemia throughout the rearing period took significantly longer to reach the megalop stage and suffered higher levels of mortality. The second experiment investigated the effects on survival, when rotifer feeding was discontinued at Z2, Z3, Z4, Z5 and when rotifers were fed up to the megalop stage (M). Artemia were offered to these treatments from Z2 onwards. A no-rotifer (NR) control that was fed Artemia-only from stocking, was also tested. It was again shown that rotifers, especially in the early stages, promoted acceptable levels of survival and growth. Without rotifers, moulting of crab larvae was delayed and survival was significantly lower during the first two zoeal stages. There was also a significant difference in survival to megalop, and a significant negative relationship between duration of rotifer feeding and survival to megalop. The best survival of 78.00 F 5.54% was achieved when rotifers were removed from the feeding regime at Z2. The NR treatment had the lowest survival of 32.00 F 7.51%. These trials established that while rotifers are a valuable
* Corresponding author. Tel.: +61-8-8924-4265; fax: +61-8-8924-4277. E-mail address: [email protected] (I.M. Ruscoe). 0044-8486/$ - see front matter. Crown Copyright D 2004 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2003.11.021
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inclusion in the feeding regime for larval S. serrata, their use should be limited to the first zoeal stage only to maximise growth and survival. Crown Copyright D 2004 Published by Elsevier B.V. All rights reserved. Keywords: Rotifers; Mud crab; Larval rearing; Scylla serrata; Feeding regime
1. Introduction There are four species of mud crab within the genus Scylla which inhabit tropical to warm temperate inshore zones and which form the basis of relatively small, yet important commercial fisheries. The most widespread of these, Scylla serrata, occurs throughout the indo-west Pacific from southern Africa to Tahiti, including the northern half of Australia, north to Okinawa, and south to the bay of Islands in New Zealand (Keenan, 1999). They are usually found in intertidal and subtidal zones of estuaries and in mangrove systems. They are easily caught using simple baited traps, grow to a size in excess of 1.5 kg, and have an excellent tolerance to air exposure (Gillespie and Burke, 1992). For these reasons, they are easily marketed and are an important source of income throughout their range. Several countries have investigated farming of mud crabs, however, the major constraint to further expansion of mud crab aquaculture is the current lack of a regular supply of seed stock (Cholic, 1999; Fortes, 1999; Keenan, 1999). In Australia, the only species of mud crab being investigated for culture is S. serrata. At the Darwin Aquaculture Centre, this species has been stocked at densities of approximately 10 larvae/l for larviculture. Typically a combination of microalgal species has been introduced at the time of stocking. Rotifers have been fed throughout the zoeal stages at approximately 10/ml in conjunction with freshly hatched Artemia nauplii from the second day of the second zoeal stage (Z2/2) (Williams et al., 1999). Researchers in other Asian countries have utilised much higher larval stocking and live food densities. Baylon and Failaman (2001) stocked larvae at up to 100 individuals/l in a greenwater system, where rotifers were included at 30/ml, and Artemia were added at 0.5 –5/ml as the larvae grew from Z1 to Z5. Baylon et al. (2001) also suggested a combined feeding regime of rotifers and Artemia to Z4, and a switch to Artemia alone at Z5. Nghia et al. (2001) reviewed larval rearing practises for S. paramamosian and made several recommendations including algal densities of 0.1 – 3 million cells/ml, an enriched rotifer density of 45/ml for the first 6 days and an Artemia density of 20/ml from day 4 onwards. Recently, the nutritional quality of Artemia as a live feed for mud crab larvae has also been investigated. The results of Takeuchi et al. (2000) suggest that the n 3 highly unsaturated fatty acids (n 3 HUFA) should be present in Artemia to promote survival and growth. Kobayashi et al. (2000) reported that EPA was important for survival, whereas DHA was important in promoting growth of S. tranquebarica. Quinito et al. (1999) suggested that artificial supplements could be used to enrich traditional live feeds with the view of improving larval survival. There seems to be a wide consensus between research institutes in feeding rotifers initially, and introducing Artemia nauplii and sub-adults as the larvae grow. However, no
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consensus has been reached on enrichment protocols, larval food densities, or the stages where the different species of live food are most beneficial or needed. Rotifers are commonly utilised as the first feeding organism for many marine and estuarine species. Their production however is often seen as a major expense in marine hatchery management due to its requirement for substantial floor space, microalgal or artificial feeds, and personnel time in culture management. If rotifer use can be eliminated or substantially reduced in larval mud crab rearing, labour, resource requirements and therefore production costs can be reduced. The following two experiments were designed to assess at which stage, if at all, rotifers are required in the feeding regime of S. serrata larvae, and for how long; approximately when Artemia should be introduced; and whether enrichment of live food, or artificial food is beneficial.
2. Materials and methods 2.1. Broodstock The broodstock females, whose progeny were used in these experiments, were identified as S. serrata according to the description of Keenan et al. (1998). Mature females were collected from the wild (Elizabeth river, Darwin Harbour) and held communally in a 10,000-l environmentally controlled recirculating system inside the hatchery at the Darwin Aquaculture Centre (DAC). Temperature and salinity were adjusted to 30jC and 30x, respectively. Ambient light regimes were used. When the females were observed to be carrying eggs, they were transferred to a separate recirculating incubation system and were held individually in 100-l tanks with reduced light levels and were not fed. After 9 days of incubation, the females were transferred to a 600-l hatching tank with flow-through water supply (approx. 3 l/ min), after a small sample of eggs were examined for development and health assessment. The following morning, the strongly phototactic and schooling first stage zoea (Z1) were concentrated with a torch light and were captured in a 5-l plastic jug. Incubation and hatching tank temperature and salinity was the same as the broodstock tank. 2.2. Rearing facilities The larvae used in both experiments were reared using the same facilities with treatments being imposed through the live food. Facilities common to both experiments are described first. The rearing containers used were 5-l hemispherical acrylic bowls containing 3 l of culture water. These clear acrylic bowls were then placed inside an identical bowl which had the outer surface painted black, and therefore provided a darkened background. The bowls were held on floating ‘pontoons’ (8 –10 bowls each) within a 7000-l constantly exchanging water bath underneath a shade structure at the DAC. Gentle aeration was provided through a single glass 1 ml pipette in each bowl.
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Water used to fill the bowls was settled and fractionated for 10 days before stocking. This water was stored overnight in a 100-l plastic tub submerged in the same water bath as the bowls. In addition, two 2-l capacity submerged carbon filters powered by a single 15-mm diameter airlift in each were run for approximately 18 h prior to use. New water for each day was treated as above. The salinity of the rearing water was adjusted from 30xat the beginning of the experiments down to approximately 25xat the megalop stage. The larvae for each bowl were individually counted and stocked at 10 larvae/l (30 larvae/bowl) on day 0. The larvae were slowly acclimated to the bowl by floating in a shallow tray with frequent small additions of the culture water. Each subsequent morning, the larvae were individually removed from the bowl using a large bore plastic 3 ml pipette, were counted, and any mortalities removed. Each bowl was emptied, cleaned in freshwater and new culture water and live food was added, together with the remaining crab larvae. Megalops were removed from the bowls as they appeared, and were included in the survival calculations after that time. When all remaining larvae had moulted to megalop, the experiment was terminated as previous research from the DAC had developed a suitable feeding regime for the megalop to crablet stage (Williams et al., 1999). All treatments in both experiments received equal cell numbers of three species of microalgae to a final density of 5 104 cells/ml. The algae used were Nannochloropsis oculata, Chaetoceros mulleri and an endemic unnamed Isochrysis-like species titled PS11 (Thinh et al., 1999). Rotifers (Colurella adriatica) were mass cultured in microalgae, usually a combination of N. oculata and PS11 at a density of approximately 100 –150/ml. Each day, rotifers were harvested by draining through a submerged 64-Am screen, were rinsed in 5-Am filtered seawater and concentrated to approximately 5000/ml in a 20-l bucket containing a combination of laboratory cultured N. oculata and PS11 for 60 – 120 min. These were then further rinsed and fed at a rate of 10 rotifers/ml in a single daily feed. Artemia (INVE, AF grade : 430 Am) used in these experiments were newly hatched first instar nauplii, except where stated, and these were fed at a rate of 0.5 nauplii/ml in a single daily feed. For both experiments, where there is a change in the feeding protocol, for example, a cessation of rotifer feeding on day 2 of Z3 (Z3/2), the change occurs 1 day after >50% of the larvae have moulted to Z3. 2.3. Experiment 1 Five experimental treatments were imposed. Each treatment consisted of a different zooplankton feeding regime, as summarised in Table 1. There were five replicates of each treatment arranged in a completely randomised design. Artificially enriched rotifers for use in treatment 5 were held in a 20-l bucket containing 5 g of Dry Selco (INVE) for between 60 and 120 min. These were also further rinsed and fed at 10 rotifers/ml in a single daily feed. Artemia (INVE, AF grade : 430 Am) for treatment 5 were instar I nauplii from days 4 to 9, and 24 h enriched nauplii were fed from day 10 onwards. These were enriched overnight in a 15-l bucket containing 1 g of Frippakk CD2 Ultra larval shrimp food.
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Table 1 Treatment identifiers and descriptions of the feeding regimes for experiment 1 Name
Rotifer feeding from – to (inclusive)
Artemia feeding from – to (inclusive)
T1 T2 T3 T4 T5*
nil Stocking – megalop Stocking – Z3/2 Stocking – megalop Stocking – megalop enriched with INVE Dry Selco
Stocking – megalop Stocking – megalop Stocking – megalop Z2/2 – megalop Z2/2 – megalop enriched with Frippackk CD 2 Ultra larval shrimp food
Z3/2 refers to the second day of the third zoeal stage. * T5 experimental units also received artificial food consisting of Spirulina (SP + kINVE) and Frippackk CD2 Ultra larval shrimp food (INVE), each to a final concentration of 1 mg/l.
Artemia were fed at a rate of 0.5/ml in a single feed. Treatment 5 also received artificial food as detailed in Table 1. 2.4. Experiment 2 Six experimental treatments were imposed which again differed in relation to their zooplankton feeding regimes, which are summarised in Table 2. There was no artificial enrichment of live food, and only instar 1 Artemia were fed. There were five replicates of each treatment arranged in a completely randomised design. 2.5. Water quality Temperature of random bowls from each experiment was measured each morning at 0800 h. Ammonia was also measured every second day from a single randomly chosen bowl from each treatment. 2.6. Data analysis Daily survival was expressed as the percentage of zoea alive at the time of counting, and duration to megalop was recorded in terms of days. These variables were analysed by ANOVA (Systat V7, SPSS, Chicago, USA), and where significant differences were observed, they were separated using a post-hoc least significant Table 2 Treatment identifiers and descriptions of the feeding regimes for the experiment 2 Name
Rotifer feeding from – to (inclusive)
Artemia feeding from – to (inclusive)
NR Z2 Z3 Z4 Z5 M
nil Stocking – Z2/1 Stocking – Z3/1 Stocking – Z4/1 Stocking – Z5/1 Stocking – megalop
Stocking – megalop Z2/2 – megalop Z2/2 – megalop Z2/2 – megalop Z2/2 – megalop Z2/2 – megalop
Z2/1 refers to the first day of the second zoeal stage.
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difference comparison of means test. Residuals were examined to determine a requirement for data transformation. Final survival to megalop in experiment 2 was also regressed against days of rotifer feeding (for those treatments receiving rotifers) in order to determine if there was a significant relationship between these two variables.
3. Results 3.1. Experiment 1 At 0800, the temperature of the experimental units was between 20.8 and 27.4 jC and averaged 22.4 jC throughout the experiment. Ammonia – nitrogen ranged between < 0.05 and 0.3 mg/l. Examination of residuals revealed that all data was homogeneous and transformation was not required. Table 3 and Fig. 1 show survival data for the five treatments in this experiment. At day 2, there was a highly significant difference (P < 0.001) between treatments for survival, with treatments T4 and T5 which were receiving rotifers only, at that point, having higher survival than those treatments receiving rotifers in combination with Artemia (T2 and T3), or Artemia only (T1). Survival in T1 was also significantly lower than T2 and T3. Day 2 was the first day that a significant difference was established between the treatments. There was also a significant difference in survival to megalop ( P < 0.05) with T1 performing poorest overall. T5 that was receiving enriched live food and artificial food performed no better than treatment 4, which was fed un-enriched live food only. Also, there was no significant difference between T2 that was fed rotifers throughout, and T3 where rotifer feeding was discontinued at Z3. In combination with the poorest survival, T1 also took significantly ( P < 0.0001) longer to reach the megalop stage (Table 1). This extended larval development was initiated at Z1, as larvae in this treatment did not moult to Z2 until day 7, compared to day 4 when rotifers were offered. By day 18, all treatments except T1 had megalops present. Megalops did not appear in this treatment until day 21.
Table 3 Mean survival (%) (F S.E.) at day 2, final survival to megalop (%) (F S.E.) and duration to megalop (F S.E.) for the five treatments in experiment 1 Treatment
Survival day 2 (%)
Survival to megalop (%)
Duration to megalop (days)
T1 T2 T3 T4 T5
76.00 F 3.56c 87.33 F 1.94b 84.66 F 2.91b 98.67 F 0.82a 96.00 F 1.94a
25.33 F 5.92c 36.00 F 5.31bc 44.67 F 9.98abc 52.95 F 8.23ab 58.67 F 7.35a
23.88 F 0.57b 19.76 F 0.30a 20.25 F 0.21a 19.81 F 0.26a 19.43 F 0.16a
Values in columns with the same superscripts are not significantly different ( P V 0.05). For treatment descriptions, refer to Table 1.
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Fig. 1. Daily survival for the five treatments in experiment 1. Error bars omitted for clarity. For treatment descriptions, refer to Table 1.
3.2. Experiment 2 At 0800, the temperature of the experimental units was between 24.8 and 27.8 jC and averaged 26.3 jC throughout the experiment. Ammonia – nitrogen ranged between < 0.05 and 0.2 mg/l. Data relating to survival and duration to megalop for the various treatments in the second experiment is presented in Table 4 and Fig. 2. The no-rotifer (NR) treatment exhibited significantly (P = 0.005) lower survival at day 7, when the larvae in this treatment first moulted to Z2. All other treatments were at the Z3 stage by this time. There were also significant differences in survival to megalop ( P < 0.01) with the NR treatment performing poorest overall. The Z2 treatment had the highest survival of 78.00 F 5.54%. There was a highly significant difference in the mean duration to megalop ( P < 0.001) with the NR treatment being approximately 4 days slower. All remaining treatments were Table 4 Mean survival (%) (F S.E.) and duration to megalopa (days) (F S.E.) for the various treatments in experiment 2 Treatment
Survival day 7 (%)
Survival to megalop (%)
Duration to megalop (days)
NR Z2 Z3 Z4 Z5 M
68.67 F 7.35b 89.33 F 3.23a 90.00 F 2.79a 96.00 F 0.67a 92.00 F 1.33a 84.00 F 7.18a
32.00 F 7.51b 78.00 F 5.54a 70.67 F 3.40a 69.33 F 3.23a 62.67 F 12.45a 54.00 F 12.4ab
22.28 F 0.33b 17.32 F 0.06a 17.37 F 0.11a 17.22 F 0.01a 17.23 F 0.08a 17.21 F 0.08a
Values in columns with the same superscripts are not significantly different ( P V 0.05). For treatment descriptions, refer to Table 2.
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Fig. 2. Daily survival for the various treatments in experiment 2. Error bars omitted for clarity. For treatment descriptions, refer to Table 2.
not significantly different from one another. Again the extended larval development was initiated at Z1. For those treatments receiving rotifers, there was a weak yet significant ( P < 0.01; R2 = 0.18) negative correlation between duration of rotifer feeding and survival to megalop.
4. Discussion The timing of the introduction of a prey organism into a culture system is important for a number of reasons. Firstly, live food organisms are provided as prey, and therefore need to be easily captured, digested and assimilated by the cultured species. Various prey organisms have varying sizes, swim speeds, digestibility’s and assimilation efficiencies all of which may change with ontogeny of the prey and the larvae. It is generally through research such as this that appropriate feeding regimes are elucidated. While the provision of various species of prey organisms with differing physical and nutritional characteristics throughout the larval rearing period may ensure the larvae have a suitable prey at all times, the difficulties in terms of culture and hatchery management may make the overall hatchery unprofitable. Secondly, prey organisms compete with the larvae for specifically provided microalgae and dissolved oxygen, which can be in limited supply in intensive hatchery systems. Some live feeds can produce toxic metabolites, including ammonia, that can compromise water quality (Samocha et al., 1989). The methods utilised in these experiments, specifically moderate larval and live food densities, as well as complete daily water exchanges, go some way to negating these potential problems. However, in a commercial hatchery operation, competition for resources and the influence of live food on
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water quality may impact on larval health, feeding ability, and ultimately growth and survival. For these reasons, timing of live prey provision is considered an important issue. The main aim of these experiments was to evaluate the timing of introduction, and cessation of feeding, of the two main live food organisms, rotifers and Artemia. The results of experiment 1 demonstrate that when only rotifers were fed during Z1 (T4 and T5), survival was higher than when rotifers were offered in conjunction with Artemia (T2 and T3). Furthermore, when Artemia were fed alone during Z1 (T1), survival was again significantly poorer. Baylon et al. (2001) observed that zoea 1 S. serrata larvae were passive feeders that did not actively pursue prey, so the capture of a highly mobile Artemia nauplii was less likely than a slower swimming and higher density rotifer. For treatments T2 and T3, Artemia were supplied in addition to the normal rotifer ration, so the reason for the poorer survival of the larvae could not be due to a lack of food, but may have been due to visual confusion of the larvae resulting in decreased rotifer consumption. The Artemia-only treatment (T1) in the first experiment sustained losses of greater than 50% before the Z3 stage. As well as influencing survival, the lack of rotifers in the early stages of this treatment slowed the development process to megalop by approximately 4 days. The reason for the poor performance of mud crab larvae when fed Artemia alone can only be speculated, but may have been the result of Artemia being too difficult to catch and ingest, or they may have been lacking certain essential nutrients such as DHA, as was lacking from the Artemia in the experiments of Quinito et al. (1999). Kobayashi et al. (2000) suggested that DHA is important for growth of mud crab larvae, and this nutrient is known to be in very low concentrations in newly hatched Artemia nauplii (Evjemo and Olsen, 1997). The feeding of DHA enriched Artemia from day 1 may overcome this problem. Whether the Z1 larvae could catch and consume an instar II (or later), Artemia nauplii may also be important, but increasing Artemia density in the early stages at least may overcome this problem. T5 yielded the best survival to megalop, but the enriched Artemia and artificial diets provided seemed to have little effect as these did not confer significantly better survival than T4. This then lends further weight to the argument that the poor larval survival and development when fed Artemia-only during Z1 is most likely a prey capture problem rather than a nutritional issue. The presence of rotifers in experiment 1 significantly and directly improved survival of mud crab larvae in early stages up to Z3, however, when rotifers were removed from the feeding regime at this stage, there was no effect on survival. This is apparent when comparing T2 that received rotifers throughout, against T3 where rotifers were discontinued at day 5. There was no immediate effect on survival and in fact T2, that were still receiving rotifers, tended to show poorer survival towards the end of the experiment. Similarly, Nghia et al. (2001) suggested that survival of the mud crab S. paramamosain was higher when rotifer feeding was discontinued after 6 days as opposed to 9 and 12 days. If rotifers need only be supplied during the first 5 days of culture, as opposed to continual feeding throughout, then substantial savings in terms of algal production, floor space and labour can be achieved. It will also result in a more easily managed larval rearing system. The results of experiment 2 reinforced that when rotifers were not fed in the first zoeal stage (NR), moulting to the second zoeal stage was delayed, and survival was reduced. Even though there was no significant difference in survival to megalop, or duration to megalop for the remaining treatments, it can be argued that co-feeding of rotifers and
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Artemia should not be practised after zoea 2, as rotifers seem not to provide any benefit beyond this stage. Furthermore, the significant negative regression between the period of rotifer feeding and survival would suggest that providing rotifers past Z2 would tend to decrease the survival of mud crab larvae. It may be possible to feed rotifers for the first few days and then switch to Artemia while the larvae are still at the Z1 stage, although this was not tested and further experimentation is required. It should also be noted that the results may vary under different feeding densities and culture conditions. Interestingly, the newly hatched Artemia nauplii used throughout the second experiment supported good survival and development to the megalop stage. These nauplii were not nutritionally enriched in any way prior to feeding. The nauplii would probably have picked up trace amounts of DHA in the greenwater culture. Kobayashi et al. (2000) found that mud crab (S. tranquebarica) larvae fed enriched Artemia containing 1.3% EPA (% of total lipids) and trace amounts of DHA showed high survival, whereas DHA concentrations above 0.46% resulted in lower survival even when EPA was maintained at 1.3%. The results of the experiment 1 showed that rotifers were essential for acceptable survival and development of early larval stages, but can be removed from the feeding regime as early as Z3. It was also concluded that Artemia should only be fed from the Z2 stage, as providing Artemia before Z2 reduced survival. Artificial food, enrichment media, and artificial diets did not significantly benefit the cultures. The second experiment refined the need for rotifers to the first zoeal stage only, and showed that newly hatched INVE AF 430 Artemia nauplii were sufficient to grow S. serrata larvae through to the megalop stage, from zoeal stage 2. There is however scope for further experimentation on the benefits of enrichment of rotifers and Artemia on the growth and survival of S. serrata larvae.
Acknowledgements This work was funded by the Fisheries Research and Development (FRDC) under grant number 2000/210. The authors would like to thank Rachel Naylor and Cameron Moir for the valuable technical assistance throughout this project.
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