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Brachionus vs Artemia duel: Optimizing first feeding of Upogebia pusilla (Decapoda: Thalassinidea) larvae
Aquaculture 295 (2009) 205–208
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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e
Brachionus vs Artemia duel: Optimizing first feeding of Upogebia pusilla (Decapoda: Thalassinidea) larvae Filipa Faleiro ⁎, Luís Narciso Laboratório Marítimo da Guia, Departamento de Biologia Animal, Faculdade de Ciências da Universidade de Lisboa, Avenida Nossa Senhora do Cabo 939, 2750-374 Cascais, Portugal
a r t i c l e
i n f o
Article history: Received 4 March 2009 Received in revised form 8 July 2009 Accepted 9 July 2009 Keywords: Upogebia pusilla Mud shrimp Larval rearing Artemia Brachionus Survival Growth
a b s t r a c t Larval rearing of many marine organisms is dependent on the availability of live food. The aim of this study was to optimize larval first feeding for the mud shrimp Upogebia pusilla, by comparing the effectiveness of the two most commonly used live feeds: Brachionus plicatilis and Artemia sp. nauplii. Survival, larval duration, molt synchronism and megalop size were compared using five feeding treatments: Artemia from zoea I to IV (B0), Brachionus during zoea I and Artemia from zoea II to IV (B1), Brachionus during zoea I and II and Artemia during zoea III and IV (B2), Brachionus from zoea I to III and Artemia during zoea IV (B3) and Brachionus from zoea I to IV (B4). The proportion of larvae that reached the megalop stage was 0.00% in treatment B0, 3.33% in treatment B1, 33.33% in treatment B2, 66.67% in treatment B3 and 76.67% in treatment B4. Larvae fed on rotifers until zoea III or zoea IV stages had a higher survival but no differences were found either in time to reach megalop or in megalop size. This study demonstrates that rotifers are essential for the survival and development of U. pusilla early larval stages but that rotifers can be successfully replaced by Artemia nauplii in the zoea IV stage. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Larviculture, more particularly the first feeding of early larval stages, appears to be the major bottleneck of marine aquaculture (Agh and Sorgeloos, 2005). Larval rearing of many fish and crustacean species is dependent upon the availability of live food, which can be complicated and expensive to obtain on a commercial scale. Several reasons can justify why live food is so essential for larval growth. In nature, the larvae of most fish and crustaceans feed on motile prey organisms and encounter problems in accepting inert diets in captivity. Moreover, the presence of enzymes (Kolkovski, 2001) and several essential biochemical compounds such as polyunsaturated fatty acids (Sargent et al., 2002) in phytoplankton and zooplankton, which are not synthesized by the physiological system of the larvae, are also important cues for the future development of marine larviculture. Brachionus spp. (rotifers) and Artemia sp. (brine shrimp) are the two live feeds most commonly used in marine aquaculture. These planktonic organisms tolerate a wide range of environmental conditions, can be cultured at high densities and can be easily enriched with nutritional supplements, antibiotics or probiotics. While some species are preferentially cultured with Artemia nauplii (e.g. Zhang et al., 1998; Villalta and Estévez, 2005), larval rearing of other organisms can be improved using rotifers as the first feed (e.g. Ruscoe
⁎ Corresponding author. Tel.: +351 214869211; fax: +351 214869720. E-mail address: fi[email protected] (F. Faleiro). 0044-8486/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2009.07.008
et al., 2004). Therefore, different factors must be considered in order to find out the best live feed for a certain species, including prey size, digestibility and nutritional quality. The nutritional effectiveness of a live feed is in the first place determined by its ingestibility and, as a consequence, by its size (Agh and Sorgeloos, 2005). Feeding an oversized prey can cause larvae to grow poorly or even starve (Treece and Davis, 2000). Prey size is particularly important for marine fish larvae that have a very small mouth and ingest their prey completely. In these cases, larvae should be fed rotifers as first food because Artemia nauplii are too large. In contrast, crustacean larvae can capture and break the prey with their feeding appendages and therefore prey size, although important, is not so critical. Besides prey size, nutritional quality is also an important aspect to consider in larval rearing. Lipids represent the most important energy source during embryonic development of most marine organisms (Sargent et al., 2002) and essential fatty acids, such as the highly unsaturated fatty acids (HUFA) EPA (20:5n-3, eicosapentaenoic acid) and DHA (22:6n-3, docosahexaenoic acid), are extremely important for larval development, especially in improving neural functions (Bell et al., 1995). Although nutritional deficiencies in prey quality can be overcome through enrichment with essential fatty acids, high HUFA levels are difficult to accumulate in Artemia nauplii due to their inherent catabolism of DHA. In contrast, rotifers are not selective for the uptake or catabolism of HUFA and high levels of DHA are easily incorporated in these organisms (Dhert et al., 1993). The aim of this study was to optimize larval first feeding for the mud shrimp Upogebia pusilla (Petagna, 1792) (Decapoda: Thalassinidea)
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culture. The growing demand of this species as live bait has led to a decline in natural populations, which can be overcome with the establishment of a successful protocol for larvae production. Considering the relatively small size of newborn larvae, the effectiveness of Artemia nauplii as a first feed was questioned and the need of a smaller prey like rotifers was investigated. Survival and growth of Upogebia pusilla larvae fed on Brachionus and Artemia nauplii were compared during the larval development.
distinguish both stages without affecting the health of the larvae. Metamorphosis to megalop stage was checked daily and experiments lasted until megalop stage was reached. Megalops were measured under a stereomicroscope with a calibrated micrometer eyepiece to the nearest 0.02 mm. Total length (TL) and carapace length (CL) were measured in the dorsal view from the top of the eyes to the tip of the telson and to the posterior margin of the carapace, respectively. 2.2. Statistical analysis
2. Materials and methods 2.1. Experimental methodology Wild ovigerous females were collected at Ria Formosa lagoon, south Portugal, in September 2008, and maintained in laboratory conditions until larval hatching. Newly hatched larvae (total length of X ± SE = 1.717 ± 0.050 mm) were individually reared in inert plastic boxes with 10 compartments (15 ml each). Water baths were used to maintain temperature constant (21–22 °C). Salinity was maintained at 34–35 PSU and photoperiod was set on 14 L:10 D. Larvae were transferred to new rearing boxes everyday. Five feeding treatments were compared: Artemia from zoea I to IV (B0), Brachionus during zoea I and Artemia from zoea II to IV (B1), Brachionus during zoea I and II and Artemia during zoea III and IV (B2), Brachionus from zoea I to III and Artemia during zoea IV (B3) and Brachionus from zoea I to IV (B4). Three replicate boxes with larvae from three different females (N = 30) were used for each treatment. Brachionus plicatilis was cultured in green water (Nannochloropsis oculata) at 25 °C and salinity 35 PSU. Artemia franciscana cysts were descapsulated and hatched at 28 °C and salinity 28 PSU, as described by Van Stappen (1996). Larvae were fed to excess (6 newly hatched Artemia nauplii ml− 1 or 42 Brachionus ml− 1) and both preys were replaced every day. Newly hatched Artemia nauplii had a total length of 0.488 ± 0.023 mm (X ± SE), while rotifers were 0.120 ± 0.026 mm (X ± SE) in length. Survival was analysed daily and the developmental stage was determined every two days according to dos Santos and Paula (2003). Zoea III and zoea IV stages were analysed together due to difficulties to
A survival analysis was used to compare survival curves between treatments. Comparisons between treatments (B0 and B1, B1 and B2, B2 and B3, B3 and B4) were made using the Cox's F-test. Larval duration, molt synchronism and megalop size were compared between treatments using ANOVA and a posteriori test (Tukey or Unequal N HSD). Larval duration was analysed based on the number of days necessary to zoea I metamorphose to zoea II, zoea III and megalop. Molt synchronism was evaluated as the period between the first and the last metamorphosis to megalop. Larval size was analysed in terms of megalop length (TL and CL) and size dispersion (ΔTL and ΔCL, determined as the difference between each megalop length and the average length). All statistical analysis was performed for a significant level of 0.05, using Statistica 8.0 software. 3. Results Survival curves for the different treatments are presented in Fig. 1. The proportion of larvae that reached the megalop stage was 0.00% in treatment B0, 3.33% in treatment B1, 33.33% in treatment B2, 66.67% in treatment B3 and 76.67% in treatment B4. Significant differences were found between treatments B0 and B1 (Cox's F-test: F = 2.361, P = 0.001), B1 and B2 (Cox's F-test: F = 2.474, P = 0.004), B2 and B3 (Cox's F-test: F = 2.587, P = 0.023), but not between B3 and B4 (Cox's F-test: F = 1.507, P = 0.214), indicating that larvae fed on Brachionus until zoea III or zoea IV stages have a higher survival rate. The succession of developmental stages for each treatment is represented in Fig. 2. The duration of zoea I stage was longer in treatment B0 (Fig. 3; ANOVA: F = 124.126, P = 0.000) and zoea II stage
Fig. 1. Survival curves for the different feeding treatments: P B0 treatment; - - - B1 treatment;
B2 treatment;
B3 treatment and
B4 treatment.
F. Faleiro, L. Narciso / Aquaculture 295 (2009) 205–208
207
Fig. 3. Duration of larval stages for the different feeding treatments.
0.627, P = 0.601; ΔTL — ANOVA: F = 0.760, P = 0.474; ΔCL — ANOVA: F = 0.865, P = 0.428). Megalops presented an average TL of 2.8 ± 0.1 mm (X ± SE) and an average CL of 1.0 ± 0.1 mm (X ± SE). 4. Discussion
Fig. 2. Succession of developmental stages for the different feeding treatments (I — zoea I; II — zoea II; III + IV — zoea III and IV; M — megalop).
was longer in treatment B1 (ANOVA: F = 5.077, P = 0.003). No differences were found in time to reach megalop between treatments (ANOVA: F = 2.576, P = 0.067) neither in molt synchronism (ANOVA: F = 3.866, P = 0.100). In what concerns megalop size, no differences were found between treatments (TL — ANOVA: F = 2.185, P = 0.601; CL — ANOVA: F =
Artemia nauplii proved to be an inadequate feed to Upogebia pusilla larvae, except for the zoea IV stage. Larvae fed on Brachionus until zoea III or zoea IV had a higher chance of survival. In fact, almost no megalops were obtained when larvae were fed Artemia until zoea III or IV and only a few number reached the megalop stage when fed Artemia until zoea II. Indeed, the switch from Brachionus to Artemia nauplii had critical consequences for larvae metamorphosis. Once zoea I, zoea II and zoea III started eating Artemia, molt synchronism decreased and mortality increased during the metamorphosis to the next stage. The importance of rotifers during the early larval stages is mainly related to predator–prey size. Although crustacean larvae can capture and break the prey with their feeding appendages, prey size should not be disregarded in the feed selection. Capture and manipulation of over large preys can be more demanding to larvae and make difficult prey ingestion, affecting feeding efficiency. However, according to Agh and Sorgeloos (2005), as long as the prey size does not interfere with the ingestion mechanism of the predator, the use of larger prey (with a higher individual energy content) will be beneficial since the predator will spend less energy in taking up a smaller number of larger prey to fulfill its energetic requirements. Although data suggest that the prey size has an important effect on the feeding efficiency of Upogebia pusilla larvae, many other factors may explain the results obtained. Besides size, Brachionus and Artemia nauplii differ in several other visual aspects, such as form, color, optical reflectivity, motion and behavior. For instance, compared to Artemia, rotifers are a less conspicuous prey but their slow swimming
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velocity makes them easier to catch. Moreover, Artemia nauplii are positively phototropic organisms that tend to be unevenly distributed in the water column, making it difficult for the larvae to capture it. Besides prey ingestibility, differences in Brachionus and Artemia nauplii digestibility and nutritional quality may also be important. Fish and crustacean larvae have a poor digestive capacity, probably due to an insufficient enzymatic activity (Hammer et al., 2000; Kolkovski, 2001), which can be improved by exogenous enzymes from preys (Kolkovski, 2001). In carnivorous larvae, prey digestibility is particularly important because they have a short gut retention time, particularly during the early larval stages, making the rapid digestion so critical (D'Abramo, 2002). The digestive capacity of a species varies with the prey and is probably related with their natural diet. Since Artemia is not a common prey in natural conditions, it is expected that many species have a poorer capacity to digest Artemia nauplii. Nutritional quality of prey such as lipids and essential fatty acids is also extremely important for larval development (Sargent et al., 2002). The nutritional value of Brachionus and Artemia as food sources for marine larvae rely mainly on the content of EPA (20:5n-3) and even more importantly of DHA (22:6n-3) (Agh and Sorgeloos, 2005), a fatty acid essential to the neural function development (Bell et al., 1995). In addition to n-3 HUFA, some attention has also been paid in recent years to n-6 HUFA, especially to ARA (20:4n-6), an important precursor of eicosanoids (Sargent et al., 1995). Although the nutritional value of both rotifers and Artemia nauplii can be enhanced through enrichment with essential fatty acids, the HUFA metabolism in these two organisms is quite different. Unlike Artemia, rotifers are not selective for HUFA catabolism and high levels of DHA and ARA can be easily incorporated, without the risk of preferential catabolism of DHA as in Artemia (Dhert et al., 1993). Although early larval stages of Upogebia pusilla need a smaller prey like rotifers, Artemia nauplii showed to be an adequate feed to zoea IV larvae. An adequate predator–prey size proportion seems to be achieved at this developmental stage. Considering that zoea IV larvae can be successfully reared either with Brachionus or Artemia nauplii, the advantages and disadvantages of the production of these live feeds should be considered before choosing one of the prey species. Although temporal and spatial fluctuations on the availability and nutritional quality of Artemia cysts occur, they are a convenient source of live feed and hatching conditions involve reduced infrastructure, manpower and labor requirements. In contrast, rotifers require more complicated culture facilities (including microalgae production) and production can be frequently affected by crashes in rotifer populations. Moreover, considering that most production costs are associated with live feed production (Treece and Davis, 2000; Waning, 2002), the higher costs of rotifer production should not be disregarded. This study demonstrated that rotifers are essential for larvae survival and for the development of early larval stages in Upogebia
pusilla culture, but also that rotifers can be successfully replaced by Artemia nauplii in the zoea IV stage. There is however a scope for further experimentation on the benefits of rotifers and Artemia nauplii enrichment and on the potential of alternative prey such as copepods to improve Upogebia pusilla larvae culture. Acknowledgments The authors would like to thank Fundação para a Ciência e a Tecnologia (scholarship SFRH/BD/28943/2006) and the Portuguese Government for providing financial support. References Agh, N., Sorgeloos, P., 2005. Handbook of Protocols and Guidelines for Culture and Enrichment of Live Food for Use in Larviculture. Artemia & Aquatic Animals Research Center, Urmia University, Urmia. 60 pp. Bell, M.V., Batty, R., Navarro, J.C., Sargent, J.R., Dick, J.R., 1995. Dietary deficiency of docosahexaenoic acid impairs vision at low light intensities in juvenile herring (Clupea harengus L.). Lipids 30, 443–449. D'Abramo, L.R., 2002. Challenges in developing successful formulated feed for culture of larval fish and crustaceans. In: Cruz-Suárez, L.E., Ricque-Marie, D., Tapia-Salazar, M., Gaxiola-Cortés, M.G., Simoes, N. (Eds.), Avances en Nutrición Acuícola VI. Memorias del VI Simposium Internacional de Nutrición Acuícola, 3–6 September 2002, Cancún, Quintana Roo, México. Dhert, P., Sorgeloos, P., Devresse, B., 1993. Contributions towards a specific DHA enrichment in the live food Brachionus plicatilis and Artemia sp. In: Reinertsen, H., Dahle, L.A., Jorgensen, L., Tvinnereim, K. (Eds.), Fish Farming Technology. Balkema, Rotterdam, Netherlands, pp. 109–115. dos Santos, A., Paula, J., 2003. Redescription of the larval stages of Upogebia pusilla (Petagna, 1792) (Thalassinidea, Upogebiidae) from laboratory-reared material. Invertebr. Reprod. Dev. 43 (1), 83–90. Hammer, H.S., Bishop, C.D., Watts, S.A., 2000. Activities of three digestive enzymes during development in the crayfish Procambarus clarkii (Decapoda). J. Crust. Biol. 20 (4), 614–620. Kolkovski, S., 2001. Digestive enzymes in fish larvae and juveniles — implications and applications to formulated diets. Aquaculture 200, 181–200. Ruscoe, I., Williams, G., Shelley, C., 2004. Limiting the use of rotifers of the first zoeal stages in mud crab (Scylla serrata Forskaal) larval rearing. Aquaculture 231, 517–527. Sargent, J.R., Bell, J.G., Bell, M.V., Henderson, R.J., Tocher, D.R., 1995. Requirement criteria for essential fatty acids. J. Appl. Ichthyol. 11, 183–198. Sargent, J.R., Tocher, D.R., Bell, J.G., 2002. The lipids. In: Halver, J.E., Hardy, D.M. (Eds.), Fish Nutrition. Elsevier Science, USA, pp. 181–257. Treece, G.D., Davis, D.A., 2000. Culture of small zooplankters for the feeding of larval fish. Southern Regional Aquaculture Center Publication No. 701, USA, p. 8. Van Stappen, G., 1996. Artemia. In: Lavens, P., Sorgeloos, P. (Eds.), Manual on the Production and Use of Live Food for Aquaculture. Fisheries Technical Paper n° 361. Food and Agriculture Organization, pp. 107–136. Villalta, M., Estévez, A., 2005. Culture of Senegal sole larvae without the need for rotifers. Aquac. Int. 13 (5), 469–478. Waning, K.M., 2002. Two Bioeconomic Studies on Haddock Culture: Live Feed and Juvenile Production. M.Sc. thesis, University of Maine, USA, 78 pp. Zhang, D., Lin, J., Creswell, R., 1998. Effects of food and temperature on survival and development in the peppermint shrimp Lysmata wurdemanni. J. World Aquac. Soc. 29 (4), 471–476.
Contents lists available at ScienceDirect
Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e
Brachionus vs Artemia duel: Optimizing first feeding of Upogebia pusilla (Decapoda: Thalassinidea) larvae Filipa Faleiro ⁎, Luís Narciso Laboratório Marítimo da Guia, Departamento de Biologia Animal, Faculdade de Ciências da Universidade de Lisboa, Avenida Nossa Senhora do Cabo 939, 2750-374 Cascais, Portugal
a r t i c l e
i n f o
Article history: Received 4 March 2009 Received in revised form 8 July 2009 Accepted 9 July 2009 Keywords: Upogebia pusilla Mud shrimp Larval rearing Artemia Brachionus Survival Growth
a b s t r a c t Larval rearing of many marine organisms is dependent on the availability of live food. The aim of this study was to optimize larval first feeding for the mud shrimp Upogebia pusilla, by comparing the effectiveness of the two most commonly used live feeds: Brachionus plicatilis and Artemia sp. nauplii. Survival, larval duration, molt synchronism and megalop size were compared using five feeding treatments: Artemia from zoea I to IV (B0), Brachionus during zoea I and Artemia from zoea II to IV (B1), Brachionus during zoea I and II and Artemia during zoea III and IV (B2), Brachionus from zoea I to III and Artemia during zoea IV (B3) and Brachionus from zoea I to IV (B4). The proportion of larvae that reached the megalop stage was 0.00% in treatment B0, 3.33% in treatment B1, 33.33% in treatment B2, 66.67% in treatment B3 and 76.67% in treatment B4. Larvae fed on rotifers until zoea III or zoea IV stages had a higher survival but no differences were found either in time to reach megalop or in megalop size. This study demonstrates that rotifers are essential for the survival and development of U. pusilla early larval stages but that rotifers can be successfully replaced by Artemia nauplii in the zoea IV stage. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Larviculture, more particularly the first feeding of early larval stages, appears to be the major bottleneck of marine aquaculture (Agh and Sorgeloos, 2005). Larval rearing of many fish and crustacean species is dependent upon the availability of live food, which can be complicated and expensive to obtain on a commercial scale. Several reasons can justify why live food is so essential for larval growth. In nature, the larvae of most fish and crustaceans feed on motile prey organisms and encounter problems in accepting inert diets in captivity. Moreover, the presence of enzymes (Kolkovski, 2001) and several essential biochemical compounds such as polyunsaturated fatty acids (Sargent et al., 2002) in phytoplankton and zooplankton, which are not synthesized by the physiological system of the larvae, are also important cues for the future development of marine larviculture. Brachionus spp. (rotifers) and Artemia sp. (brine shrimp) are the two live feeds most commonly used in marine aquaculture. These planktonic organisms tolerate a wide range of environmental conditions, can be cultured at high densities and can be easily enriched with nutritional supplements, antibiotics or probiotics. While some species are preferentially cultured with Artemia nauplii (e.g. Zhang et al., 1998; Villalta and Estévez, 2005), larval rearing of other organisms can be improved using rotifers as the first feed (e.g. Ruscoe
⁎ Corresponding author. Tel.: +351 214869211; fax: +351 214869720. E-mail address: fi[email protected] (F. Faleiro). 0044-8486/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2009.07.008
et al., 2004). Therefore, different factors must be considered in order to find out the best live feed for a certain species, including prey size, digestibility and nutritional quality. The nutritional effectiveness of a live feed is in the first place determined by its ingestibility and, as a consequence, by its size (Agh and Sorgeloos, 2005). Feeding an oversized prey can cause larvae to grow poorly or even starve (Treece and Davis, 2000). Prey size is particularly important for marine fish larvae that have a very small mouth and ingest their prey completely. In these cases, larvae should be fed rotifers as first food because Artemia nauplii are too large. In contrast, crustacean larvae can capture and break the prey with their feeding appendages and therefore prey size, although important, is not so critical. Besides prey size, nutritional quality is also an important aspect to consider in larval rearing. Lipids represent the most important energy source during embryonic development of most marine organisms (Sargent et al., 2002) and essential fatty acids, such as the highly unsaturated fatty acids (HUFA) EPA (20:5n-3, eicosapentaenoic acid) and DHA (22:6n-3, docosahexaenoic acid), are extremely important for larval development, especially in improving neural functions (Bell et al., 1995). Although nutritional deficiencies in prey quality can be overcome through enrichment with essential fatty acids, high HUFA levels are difficult to accumulate in Artemia nauplii due to their inherent catabolism of DHA. In contrast, rotifers are not selective for the uptake or catabolism of HUFA and high levels of DHA are easily incorporated in these organisms (Dhert et al., 1993). The aim of this study was to optimize larval first feeding for the mud shrimp Upogebia pusilla (Petagna, 1792) (Decapoda: Thalassinidea)
206
F. Faleiro, L. Narciso / Aquaculture 295 (2009) 205–208
culture. The growing demand of this species as live bait has led to a decline in natural populations, which can be overcome with the establishment of a successful protocol for larvae production. Considering the relatively small size of newborn larvae, the effectiveness of Artemia nauplii as a first feed was questioned and the need of a smaller prey like rotifers was investigated. Survival and growth of Upogebia pusilla larvae fed on Brachionus and Artemia nauplii were compared during the larval development.
distinguish both stages without affecting the health of the larvae. Metamorphosis to megalop stage was checked daily and experiments lasted until megalop stage was reached. Megalops were measured under a stereomicroscope with a calibrated micrometer eyepiece to the nearest 0.02 mm. Total length (TL) and carapace length (CL) were measured in the dorsal view from the top of the eyes to the tip of the telson and to the posterior margin of the carapace, respectively. 2.2. Statistical analysis
2. Materials and methods 2.1. Experimental methodology Wild ovigerous females were collected at Ria Formosa lagoon, south Portugal, in September 2008, and maintained in laboratory conditions until larval hatching. Newly hatched larvae (total length of X ± SE = 1.717 ± 0.050 mm) were individually reared in inert plastic boxes with 10 compartments (15 ml each). Water baths were used to maintain temperature constant (21–22 °C). Salinity was maintained at 34–35 PSU and photoperiod was set on 14 L:10 D. Larvae were transferred to new rearing boxes everyday. Five feeding treatments were compared: Artemia from zoea I to IV (B0), Brachionus during zoea I and Artemia from zoea II to IV (B1), Brachionus during zoea I and II and Artemia during zoea III and IV (B2), Brachionus from zoea I to III and Artemia during zoea IV (B3) and Brachionus from zoea I to IV (B4). Three replicate boxes with larvae from three different females (N = 30) were used for each treatment. Brachionus plicatilis was cultured in green water (Nannochloropsis oculata) at 25 °C and salinity 35 PSU. Artemia franciscana cysts were descapsulated and hatched at 28 °C and salinity 28 PSU, as described by Van Stappen (1996). Larvae were fed to excess (6 newly hatched Artemia nauplii ml− 1 or 42 Brachionus ml− 1) and both preys were replaced every day. Newly hatched Artemia nauplii had a total length of 0.488 ± 0.023 mm (X ± SE), while rotifers were 0.120 ± 0.026 mm (X ± SE) in length. Survival was analysed daily and the developmental stage was determined every two days according to dos Santos and Paula (2003). Zoea III and zoea IV stages were analysed together due to difficulties to
A survival analysis was used to compare survival curves between treatments. Comparisons between treatments (B0 and B1, B1 and B2, B2 and B3, B3 and B4) were made using the Cox's F-test. Larval duration, molt synchronism and megalop size were compared between treatments using ANOVA and a posteriori test (Tukey or Unequal N HSD). Larval duration was analysed based on the number of days necessary to zoea I metamorphose to zoea II, zoea III and megalop. Molt synchronism was evaluated as the period between the first and the last metamorphosis to megalop. Larval size was analysed in terms of megalop length (TL and CL) and size dispersion (ΔTL and ΔCL, determined as the difference between each megalop length and the average length). All statistical analysis was performed for a significant level of 0.05, using Statistica 8.0 software. 3. Results Survival curves for the different treatments are presented in Fig. 1. The proportion of larvae that reached the megalop stage was 0.00% in treatment B0, 3.33% in treatment B1, 33.33% in treatment B2, 66.67% in treatment B3 and 76.67% in treatment B4. Significant differences were found between treatments B0 and B1 (Cox's F-test: F = 2.361, P = 0.001), B1 and B2 (Cox's F-test: F = 2.474, P = 0.004), B2 and B3 (Cox's F-test: F = 2.587, P = 0.023), but not between B3 and B4 (Cox's F-test: F = 1.507, P = 0.214), indicating that larvae fed on Brachionus until zoea III or zoea IV stages have a higher survival rate. The succession of developmental stages for each treatment is represented in Fig. 2. The duration of zoea I stage was longer in treatment B0 (Fig. 3; ANOVA: F = 124.126, P = 0.000) and zoea II stage
Fig. 1. Survival curves for the different feeding treatments: P B0 treatment; - - - B1 treatment;
B2 treatment;
B3 treatment and
B4 treatment.
F. Faleiro, L. Narciso / Aquaculture 295 (2009) 205–208
207
Fig. 3. Duration of larval stages for the different feeding treatments.
0.627, P = 0.601; ΔTL — ANOVA: F = 0.760, P = 0.474; ΔCL — ANOVA: F = 0.865, P = 0.428). Megalops presented an average TL of 2.8 ± 0.1 mm (X ± SE) and an average CL of 1.0 ± 0.1 mm (X ± SE). 4. Discussion
Fig. 2. Succession of developmental stages for the different feeding treatments (I — zoea I; II — zoea II; III + IV — zoea III and IV; M — megalop).
was longer in treatment B1 (ANOVA: F = 5.077, P = 0.003). No differences were found in time to reach megalop between treatments (ANOVA: F = 2.576, P = 0.067) neither in molt synchronism (ANOVA: F = 3.866, P = 0.100). In what concerns megalop size, no differences were found between treatments (TL — ANOVA: F = 2.185, P = 0.601; CL — ANOVA: F =
Artemia nauplii proved to be an inadequate feed to Upogebia pusilla larvae, except for the zoea IV stage. Larvae fed on Brachionus until zoea III or zoea IV had a higher chance of survival. In fact, almost no megalops were obtained when larvae were fed Artemia until zoea III or IV and only a few number reached the megalop stage when fed Artemia until zoea II. Indeed, the switch from Brachionus to Artemia nauplii had critical consequences for larvae metamorphosis. Once zoea I, zoea II and zoea III started eating Artemia, molt synchronism decreased and mortality increased during the metamorphosis to the next stage. The importance of rotifers during the early larval stages is mainly related to predator–prey size. Although crustacean larvae can capture and break the prey with their feeding appendages, prey size should not be disregarded in the feed selection. Capture and manipulation of over large preys can be more demanding to larvae and make difficult prey ingestion, affecting feeding efficiency. However, according to Agh and Sorgeloos (2005), as long as the prey size does not interfere with the ingestion mechanism of the predator, the use of larger prey (with a higher individual energy content) will be beneficial since the predator will spend less energy in taking up a smaller number of larger prey to fulfill its energetic requirements. Although data suggest that the prey size has an important effect on the feeding efficiency of Upogebia pusilla larvae, many other factors may explain the results obtained. Besides size, Brachionus and Artemia nauplii differ in several other visual aspects, such as form, color, optical reflectivity, motion and behavior. For instance, compared to Artemia, rotifers are a less conspicuous prey but their slow swimming
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velocity makes them easier to catch. Moreover, Artemia nauplii are positively phototropic organisms that tend to be unevenly distributed in the water column, making it difficult for the larvae to capture it. Besides prey ingestibility, differences in Brachionus and Artemia nauplii digestibility and nutritional quality may also be important. Fish and crustacean larvae have a poor digestive capacity, probably due to an insufficient enzymatic activity (Hammer et al., 2000; Kolkovski, 2001), which can be improved by exogenous enzymes from preys (Kolkovski, 2001). In carnivorous larvae, prey digestibility is particularly important because they have a short gut retention time, particularly during the early larval stages, making the rapid digestion so critical (D'Abramo, 2002). The digestive capacity of a species varies with the prey and is probably related with their natural diet. Since Artemia is not a common prey in natural conditions, it is expected that many species have a poorer capacity to digest Artemia nauplii. Nutritional quality of prey such as lipids and essential fatty acids is also extremely important for larval development (Sargent et al., 2002). The nutritional value of Brachionus and Artemia as food sources for marine larvae rely mainly on the content of EPA (20:5n-3) and even more importantly of DHA (22:6n-3) (Agh and Sorgeloos, 2005), a fatty acid essential to the neural function development (Bell et al., 1995). In addition to n-3 HUFA, some attention has also been paid in recent years to n-6 HUFA, especially to ARA (20:4n-6), an important precursor of eicosanoids (Sargent et al., 1995). Although the nutritional value of both rotifers and Artemia nauplii can be enhanced through enrichment with essential fatty acids, the HUFA metabolism in these two organisms is quite different. Unlike Artemia, rotifers are not selective for HUFA catabolism and high levels of DHA and ARA can be easily incorporated, without the risk of preferential catabolism of DHA as in Artemia (Dhert et al., 1993). Although early larval stages of Upogebia pusilla need a smaller prey like rotifers, Artemia nauplii showed to be an adequate feed to zoea IV larvae. An adequate predator–prey size proportion seems to be achieved at this developmental stage. Considering that zoea IV larvae can be successfully reared either with Brachionus or Artemia nauplii, the advantages and disadvantages of the production of these live feeds should be considered before choosing one of the prey species. Although temporal and spatial fluctuations on the availability and nutritional quality of Artemia cysts occur, they are a convenient source of live feed and hatching conditions involve reduced infrastructure, manpower and labor requirements. In contrast, rotifers require more complicated culture facilities (including microalgae production) and production can be frequently affected by crashes in rotifer populations. Moreover, considering that most production costs are associated with live feed production (Treece and Davis, 2000; Waning, 2002), the higher costs of rotifer production should not be disregarded. This study demonstrated that rotifers are essential for larvae survival and for the development of early larval stages in Upogebia
pusilla culture, but also that rotifers can be successfully replaced by Artemia nauplii in the zoea IV stage. There is however a scope for further experimentation on the benefits of rotifers and Artemia nauplii enrichment and on the potential of alternative prey such as copepods to improve Upogebia pusilla larvae culture. Acknowledgments The authors would like to thank Fundação para a Ciência e a Tecnologia (scholarship SFRH/BD/28943/2006) and the Portuguese Government for providing financial support. References Agh, N., Sorgeloos, P., 2005. Handbook of Protocols and Guidelines for Culture and Enrichment of Live Food for Use in Larviculture. Artemia & Aquatic Animals Research Center, Urmia University, Urmia. 60 pp. Bell, M.V., Batty, R., Navarro, J.C., Sargent, J.R., Dick, J.R., 1995. Dietary deficiency of docosahexaenoic acid impairs vision at low light intensities in juvenile herring (Clupea harengus L.). Lipids 30, 443–449. D'Abramo, L.R., 2002. Challenges in developing successful formulated feed for culture of larval fish and crustaceans. In: Cruz-Suárez, L.E., Ricque-Marie, D., Tapia-Salazar, M., Gaxiola-Cortés, M.G., Simoes, N. (Eds.), Avances en Nutrición Acuícola VI. Memorias del VI Simposium Internacional de Nutrición Acuícola, 3–6 September 2002, Cancún, Quintana Roo, México. Dhert, P., Sorgeloos, P., Devresse, B., 1993. Contributions towards a specific DHA enrichment in the live food Brachionus plicatilis and Artemia sp. In: Reinertsen, H., Dahle, L.A., Jorgensen, L., Tvinnereim, K. (Eds.), Fish Farming Technology. Balkema, Rotterdam, Netherlands, pp. 109–115. dos Santos, A., Paula, J., 2003. Redescription of the larval stages of Upogebia pusilla (Petagna, 1792) (Thalassinidea, Upogebiidae) from laboratory-reared material. Invertebr. Reprod. Dev. 43 (1), 83–90. Hammer, H.S., Bishop, C.D., Watts, S.A., 2000. Activities of three digestive enzymes during development in the crayfish Procambarus clarkii (Decapoda). J. Crust. Biol. 20 (4), 614–620. Kolkovski, S., 2001. Digestive enzymes in fish larvae and juveniles — implications and applications to formulated diets. Aquaculture 200, 181–200. Ruscoe, I., Williams, G., Shelley, C., 2004. Limiting the use of rotifers of the first zoeal stages in mud crab (Scylla serrata Forskaal) larval rearing. Aquaculture 231, 517–527. Sargent, J.R., Bell, J.G., Bell, M.V., Henderson, R.J., Tocher, D.R., 1995. Requirement criteria for essential fatty acids. J. Appl. Ichthyol. 11, 183–198. Sargent, J.R., Tocher, D.R., Bell, J.G., 2002. The lipids. In: Halver, J.E., Hardy, D.M. (Eds.), Fish Nutrition. Elsevier Science, USA, pp. 181–257. Treece, G.D., Davis, D.A., 2000. Culture of small zooplankters for the feeding of larval fish. Southern Regional Aquaculture Center Publication No. 701, USA, p. 8. Van Stappen, G., 1996. Artemia. In: Lavens, P., Sorgeloos, P. (Eds.), Manual on the Production and Use of Live Food for Aquaculture. Fisheries Technical Paper n° 361. Food and Agriculture Organization, pp. 107–136. Villalta, M., Estévez, A., 2005. Culture of Senegal sole larvae without the need for rotifers. Aquac. Int. 13 (5), 469–478. Waning, K.M., 2002. Two Bioeconomic Studies on Haddock Culture: Live Feed and Juvenile Production. M.Sc. thesis, University of Maine, USA, 78 pp. Zhang, D., Lin, J., Creswell, R., 1998. Effects of food and temperature on survival and development in the peppermint shrimp Lysmata wurdemanni. J. World Aquac. Soc. 29 (4), 471–476.