The effects of algal diets on population growth and egg hatching success of the tropical calanoid copepod, Acartia sinjiensis

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1 Available online at Aquaculture 273 (2007) The effects of algal diets on population growth and egg hatching success of the tropical calanoid copepod, Acartia sinjiensis Michael Milione, Chaoshu Zeng Tropical Crustacean Aquaculture Research Group School of Marine and Tropical Biology, James Cook University, Townsville, Queensland 4811, Australia Received 10 June 2007; received in revised form 16 July 2007; accepted 17 July 2007 Abstract As natural diets of fish larvae, a number of calanoid copepod species are being investigated for use as live prey in aquaculture hatcheries. One of these, the tropical calanoid copepod, Acartia sinjiensis, has good potential as a live feed for tropical reef fish larvae. However, the rearing techniques for A. sinjiensis require further development to improve productivity. This study was carried out to investigate the population growth and egg hatching success of A. sinjiensis when fed a range of mono-and binary algal diets, including algae in the form of frozen paste. For the population growth experiment, the final A. sinjiensis population, including eggs, nauplii, copepodites and adults, was determined after feeding eight algal diets (two frozen algae, four live monoalgal and two live binary algal diets) for 8 days at temperature 28±1 C; salinity 34±1 psu and photoperiod 12 L:12 D. Five replicates, with an initial 12 adult A. sinjiensis per replicate, were set up for each treatment. In a separate experiment, effects of diets on egg hatching success were examined after 48 h incubation of eggs produced by A. sinjiensis fed the same eight diets. The results showed that diet significantly affected both population growth and hatching success of A. sinjiensis. Of the diets tested, the binary algal diets were more successful than monoalgal diets, while the frozen algae had little dietary value. The highest population growth was recorded on A. sinjiensis fed a binary diet of Tetraselmis chuii and the Tahitian strain of Isochrysis sp. (T- ISO) (final population: 1091 ± 80), which was significantly higher (P b 0.001) than all other diets tested except for the other binary diet of Nannochloropsis sp. and T-ISO (final population: 897±123). Diet also had a significant effect (Pb0.001) on egg hatching rate, though the highest hatch rate was recorded with eggs produced by A. sinjiensis fed binary diet Nannochloropsis sp. and T-ISO (88.1±2.1%), this was not significantly different from that of eggs produced by A. sinjiensis fed either T-ISO alone (88.0±1.7%) or the binary algal diet of T. chuii and T-ISO (76.4±7.1%). The results of this study suggest that among the diets tested, a combination of live T. chuii and T-ISO was the best for the culture of A. sinjiensis Elsevier B.V. All rights reserved. Keywords: Microalgal diets; Tropical copepod; Acartia sinjiensis; Population growth; Egg hatching rates 1. Introduction Corresponding author. Tel.: ; fax: address: Chaoshu.Zeng@jcu.edu.au (C. Zeng). Copepods are the natural prey items for most fish larvae in the wild (Stottrup, 2000). Their nutritional value to fish larvae is known to be superior to the rotifer Branchionus spp. and brine shrimp Artemia spp, the two /$ - see front matter 2007 Elsevier B.V. All rights reserved. doi: /j.aquaculture

2 M. Milione, C. Zeng / Aquaculture 273 (2007) main live prey currently widely used in aquaculture hatcheries (Stottrup, 2000; Lee, 2003). More importantly, copepods are considered essential prey items that promote good survival for early larvae of some fish species with small mouth-gape sizes, including many high value reef fish species, such as tropical groupers Epinephelus sp. (Knuckey et al., 2000; McKinnon et al., 2003; Toledo et al., 2005), snapper Pagrus sp. (Payne, 2000) and Lutjanus sp. (Doi et al., 1997; Ogle et al., 2005; Phelps et al., 2005; Su et al., 2005). However, in comparison to rotifers and Artemia, the major drawback of copepods as live prey for larvae culture is their low productivity in mass culture. While selecting microalgal diets that could support maximum copepod production as well as supply appropriate nutrients to the cultured copepods is crucial (Kleppel et al., 2005; Lee et al., 2006), labour requirements and costs involved in algal culture are also important considerations. Ideally, algal diets used as copepod feed should be species that are commonly available, relatively simple to culture, not prone to culture crash and at the same time, support maximum fecundity and development of the copepod (Knuckey et al., 2005; Lee et al., 2006). Among microalgae species commonly used as a diet for the culture of copepods, Rhodomonas sp. is often reported as being effective as a sole diet or as part of a mixed algal diet for Acartia species, including A. sinjiensis (Stottrup and Jensen, 1990; McKinnon et al., 2003; Knuckey et al., 2005; Morehead et al., 2005; Holste and Peck, 2006; Peck and Holste, 2006). However, reliance upon Rhodomonas can be problematic because it is relatively unstable in culture and is prone to suddenly dying off (Knuckey et al., 2005). Therefore, finding alternative microalgae species that are more reliable to culture and are equally successful as diets for A. sinjiensis could represent a significant improvement in feeding protocols when culturing this species. In addition, the use of concentrated frozen algae paste may also provide another option for simplifying culture protocols for A. sinjiensis. Algae paste has become more popular in hatcheries recently because it provides off the shelf convenience and it can be used as a back-up food source in the case of algal culture crashes, which are not unusual occurrences in hatcheries. Although frozen algae have generally been successful as an alternative to live algae for the mass culture of rotifers (Lubzens et al., 2001), this option appears not to have been tested for copepods. Acartia sinjiensis is a planktonic calanoid copepod indigenous to the coastal and estuary waters of tropical North Queensland, Australia (Uye, 1985, McKinnon et al., 2003). This species is amenable for use in aquaculture hatcheries as live food for tropical fish larvae, as it occurs naturally in tropical waters and is likely to be their natural prey. The copepod species is also adapted to sudden shifts in temperature and salinity, which make it suitable for the rearing larvae of tropical fish species that occur in both estuarine and oceanic environments (McKinnon et al., 2003). Moreover, the nauplii of A. sinjiensis are small enough to be efficiently ingested by fish with small mouth-gape sizes at the time of first feeding (Doi et al., 1997, Knuckey et al., 2000). However, despite two recent investigations on the effects of algae diets of Chaetoceros muelleri, Dunaliella sp., Rhodomonas sp., Heterocapsa niei, Tetraselmis chuii, T-ISO and Pavlova salina on its egg production (McKinnon et al., 2003) and algae diets of Cryptomonad sp., Rhodomonas sp., Heterocapsa niei, Tetraselmis sp., T-ISO and Pavlova salina on nauplii development (Knuckey et al., 2005), further information is needed to optimize the culture conditions for A. sinjiensis. To date, there have been no studies on how diet may affect the population growth and egg hatching success of this species. Furthermore, comprehensive studies that include algal species that are relatively simple and reliable to culture but have not yet been tested for A. sinjiensis are likely to provide valuable information that could lead to substantial improvement in the productivity of A. sinjiensis mass culture. Consequently, this study was conducted to investigate the effects of different algal diets, including frozen algae and species/specie combinations that have not been tested previously, on the population growth and egg hatching success of A. sinjiensis. With the relative ease of the production of the algae being taken into consideration when selecting algae species, and with a view to establishing an alternative to the use of Rhodomonas, the aim of this research was to identify an optimal algal diet that would maximise the productivity of A. sinjiensis culture. 2. Materials and methods 2.1. Algal culture Except for the green algae Nannochloropsis sp. which was mass cultured in 2000 L outdoor tanks on a commercial fertilizer (AQUASOL, Yates Ltd, New South Wales, Australia), all other live microalgae used for the diet experiments were cultured indoors in 20 L polycarbonate carboys, and were inoculated from starter cultures supplied by the Commonwealth Scientific and Industrial Research Organization (CSIRO) Microalgae Supply Service, Hobart, Tasmania, Australia. The strain codes of these microalgae were: Rhodomonas maculata (CS-85), the Tahitian strain of Isochrysis sp. (T-ISO) (CS-177) and Tetraselmis chuii (CS-26). The indoor-grown algae were inoculated into 1 μmfiltered and UV irradiated seawater of salinity 30 psu and

3 658 M. Milione, C. Zeng / Aquaculture 273 (2007) vigorously aerated. Nutrients used for all species were f/2 concentration (Guillard and Ryther, 1962), except for Rhodomonas, wheref concentration was used. Cultures were maintained at 25 C, on a 12 h:12 h light:dark cycle. Two commercial frozen algae pastes (instant algae) were also used, which are frozen paste of Tetraselmis sp. and a paste of mixed Nannochloropsis sp. and Tetraselmis sp., both were produced by Reed Mariculture Inc., California, USA Acartia sinjiensis stock culture A. sinjiensis used in current experiments was initially obtained from a culture kept at the Northern Fisheries Centre (NFC), Queensland Department of Primary Industries, Cairns, Queensland, Australia. The strain was originally isolated from Townsville, Queensland, Australia in 1996 and has since been kept at NFC. Upon their arrival, the copepods were kept in 20 L carboys filled with 1 μm-filtered and UV-irradiated seawater with gentle aeration. Salinity was 34±1 psu, and the culture temperature was 27±1 C. Light intensity was 1050 lux (MC- 88 light meter) on a photoperiod of 12 h:12 h light:dark cycle. Seventy to eighty percent of the culture water was exchanged daily using a siphon with a 38 μm mesh attached to the end to prevent the removal of the copepods. The copepods were fed daily with a mixture of the microalgae Isochrysis sp. (T-ISO), Rhodomonas maculata, Tetraselmis chuii and Nannochloropsis sp., at a biomass ratio of T-ISO : Rhodomonas : Tetraselmis : Nannochloropsis=1:1:1:1. The carboys were completely drained every 10 days to remove the build up of detritus while the copepods were retained on a 150 μm sieve. The carboys were then cleaned and sterilised with chlorine before the cultures were restarted again Experimental design and setup The study consisted of two experiments, the first experiment examined population growth while the second compared the egg hatching success of A. sinjiensis in relation to diets they were offered. Both experiments were carried out using 500 ml conical bottles that were placed under similar culture conditions as described for the stock cultures (i.e. gentle aeration, 1050 lux light intensity, 12 h:12 h light:dark cycle, salinity 34 ± 1 psu, temperature 28±1 C). For both experiments, there were eight diet treatments with five replicates for each treatment. The eight diet treatments include two frozen microalgal diets: i.e. 1) a mixture of Nannochloropsis sp. and Tetraselmis sp. dead cells from defrosted frozen paste (Nan & Tet F) and 2) Tetraselmis sp. defrosted from frozen paste (Tet F); four live monoalgal diets: i.e. 3) Rhodomonas maculata (Rho); 4) T-ISO (T-is); 5) Tetraselmis chuii (Tet); 6) Nannochloropsis sp. (Nan); and two live binary algal diets: 7) Nannochloropsis sp. and T-ISO (Nan & T-is) and 8) Tetraselmis chuii and T-ISO (Tet & T-is). Concentrations of algal cultures were determined daily using a haemocytometer to count the number of cells per ml under a microscope (except for frozen algae where cell density was stipulated on the commercial packaging). For all treatments and throughout the experiments, feeding ration of all algae in cells/ml were based on their equivalent to optimal feeding levels of ash free dry weight (AFDW) ml 1 as recommended by Knuckey et al. (2005). The only exception was Nannochloropsis sp., whose required amount in cells/ml was estimated based on the cell size compared to the other species. For binary algal diets, the equal proportion of two algal species based on their AFDW was used. As the AFDW of different algae species varies (Knuckey et al., 2005), the feeding rations of the algal diets used in this experiment were as follows: Nan & Tet F: 100,000 cells/ml (approximately 95,000 cell/ml Nannochloropsis and 5000 cell/ml Tetraselmis respectively based on cell counts, as the Nannochloropsis to Tetraselmis ratio of the product was not specified by the manufacturer); Tet F: 6300 cell/ml; Rho: 20,000 cell/ml; T-is: 80,000 cell/ml; Tet: 6250 cell/ml; Nan: 100,000 cell/ml; and for the binary diets: Nan & T-is: 50,000 and 40,000 cell/ml respectively; Tet & T-is: 3150 and 40,000 cell/ml respectively. At the beginning of the population growth experiment, 12 adult A. sinjiensis were stocked into each replicate culture bottle. Adult copepods were separated from the stock culture by draining culture water through a 180 μm sieve. The copepods caught on the mesh were then immediately placed in a petri dish with a small amount of seawater. Individuals were randomly captured using a fine-tipped pipette and transferred to the experimental bottles. During the following 8 days, the copepods were fed the designated diets daily, and approximately 50% culture water was exchanged daily using a siphon with a 38 μm mesh attached to the end to prevent removal of the copepods. After 8 days, all the contents of each replicate bottle were drained through a 38 μm sieve and the total number of eggs, nauplii, copepodites and adults of A. sinjiensis retained were fixed in 10% formalin solution. The counting of A. sinjiensis eggs, nauplii, copepodites and adults were made using a Sedwick Rafter counter and a high-powered microscope (Leica CME). For the egg hatching experiment, the same procedure as per population growth was followed except that the replicate bottles were stocked only 6 adults initially. After allowing a four-day period for egg production, the contents of each bottle were drained onto a 38 μm sieve, which were then rinsed and placed in fresh water for 2.5 min to kill all post-egg-stage copepods (Knuckey et al., 2005). The eggs were then returned to seawater and the number of eggs per replicate was counted under a highpowered microscope before being placed in 2 ml of seawater in sealed specimen containers for incubation under identical conditions (12 h:12 h light:dark cycle, 28±1 C and salinity 34± 1 ppt). Based upon an pilot trial, a incubation period of 48 h was selected. After 48 h, the number of unhatched eggs per replicate was counted and the hatching rate (%) was subsequently calculated Statistical analysis Data for both population growth and egg hatching experiment were analysed using one-way ANOVA. The percentage hatching rate data were arcsine transformed prior to analysis. When significant differences (P b 0.05) were found, Tukey's multiple comparisons test was used to determine

4 M. Milione, C. Zeng / Aquaculture 273 (2007) specific differences among treatments. All statistical analyses were conducted using SPSS program, version 11. Data are presented as mean ± standard error (SE). 3. Results 3.1. Population growth Fig. 1 shows the mean total number of copepod population, including eggs, nauplii, copepodites and adults, for each diet treatment after 8 days of culture. The results showed that diet had a significant effect on the population growth of A. sinjiensis (df=7,f=36.1, Pb0.001). Mean population growth of A. sinjiensis after being fed a designated diet was highest for the two live binary diets, with a final population of 1091±80 for Tet & T-is treatment and 896±123 for Nan & T-is treatment (Fig. 1). Statistical analysis revealed that while the final A. sinjiensis populations fed these two binary diets were not significantly different from each other, population growth of the Tet & T-is treatment was significantly higher than all the other diet treatments (Pb 0.05) while the Nan & T-is treatment were only significantly different from four other treatments, i.e. the two frozen diets, Nan & Tet F, Tet F and two monoaglal diets, Nan and Tet. The frozen diets produced the lowest population number at the end of the experiment: 3±1 for Nan & Tet F and 26±9 for Tet F. The final population numbers of both treatments were significantly lower (Pb 0.05) than all the other treatments except for the live monoaglal treatment of Nan, which also had a significantly lower population number (Pb 0.05) than three other live monoalgal treatments. Of the live monoalgal diets, mean final population for T-is treatment (723±84) was higher than both Rho (592±59) and Tet treatments (413±85) though they were not statistically significant (Fig. 1). Fig. 2 shows a breakdown of different life-cycle stages of the A. sinjiensis population fed various algal diets for 8 days. It showed that there were substantial differences in the numbers of the four life-stage categories, i.e. eggs, nauplii, copepodites and adults in different diet treatments. Most notably, the number of nauplii was strikingly higher for the Rho diet treatment. Also noteworthy was that the three most productive diets, i.e. Tet & T- is, Nan & T-is, and T-is, differed from the other diets in that they all had substantially higher numbers of both nauplii and copepodites relative to eggs and adults. These three diets also had markedly higher numbers of adults when compared to the rest of diet treatments (Fig. 2) Egg hatching rate Among eight diet treatments, the data from three of them, i.e. Nan & Tet F, Tet F and Nan, were excluded because the numbers of eggs produced by A. sinjiensis fed these diets were too few (0-4) and therefore did not constitute reliable data. For the five remaining diets, there were significant differences in egg hatching rates over 48 h incubation period (df=4,f=18.9, Pb 0.001). The mean percentage egg hatching success was not significantly different among three of the diet treatments, i.e. the binary diet Nan & T-is (88.1±2.1%), the monoalgal diet T-is (88.0±1.7%) and the other binary diet Tet & T-is (76.4±7.1%) (PN0.05). However, the other two monoalgal diet treatments, Rho and Tet, had significantly lower hatching rates (Pb 0.01) although the difference between them was not significant (Fig. 3). 4. Discussion Previous research comparing the effects of different microalgae diets on the productivity of calanoid copepods Fig. 1. Mean total population (including eggs, nauplii, copepodites and adults) (± SE) of Acartia sinjiensis cultured on eight different algal diets for 8 days at temperature 28±1 C, salinity 34±1 ppt and photoperiod 12 L:12 D. Nan: Nannochloropsis sp.; Tet: Tetraselmis chuii; Rho: Rhodomonas maculata; T-is: Tahitian strain of Isochrysis; F: frozen algae. Different letters on the top of histogram bars indicate significant differences (Pb0.05).

5 660 M. Milione, C. Zeng / Aquaculture 273 (2007) Fig. 2. Mean number of four life-stages (eggs, nauplii (N1 N6), copepodites (C1 C6) and adults) within the population of A. sinjiensis cultured for 8 days on eight different algal diets at temperature 28±1 C, salinity 34±1 ppt and photoperiod 12 L:12 D. Nan: Nannochloropsis sp.; Tet: Tetraselmis chuii; Rho: Rhodomonas maculata; T-is: Tahitian strain of Isochrysis; F: frozen algae. targeted for aquaculture use has focused mainly on egg production rates (Stottrup and Jensen 1990; Kleppel et al., 1998; McKinnon et al., 2003; Morehead et al., 2005), although there are a few studies on hatching success (Lacoste et al., 2001; Peck and Holste, 2006; Holste and Peck, 2006), and nauplii growth and development (Payne and Rippingale, 2000; Knuckey et al., 2005; Lee et al., 2006). The findings of these studies have shown clear Fig. 3. The mean 48 h hatching rates (%) of Acartia sinjiensis eggs produced by adults fed five different microalgae diets. Eggs were incubated under identical condition of 28±1 C, 34±1 ppt and photoperiod 12 L:12 D. Nan: Nannochloropsis sp.; Tet: Tetraselmis chuii; Rho: Rhodomonas maculata; T-is: Tahitian strain of Isochrysis. Different letters on the top of histogram bars indicate significant differences (Pb0.05).

6 M. Milione, C. Zeng / Aquaculture 273 (2007) Table 1 Nutritional composition of four major microalgal species used as diets for the culture of Acartia sinjiensis in present study Reference Linolenic acid 18:3n-3 (% total fatty acids) EPA 20:5n-3 (% total fatty acids) DHA 22:6n-3 (% total fatty acids) Total fatty acids (pg/ cell) Lipid (% dry weight) Carbohydrate(% dry weight) Protein (% dry weight) Species Size (μm) Trace Trace (Volkman et al. 1989; Muller-Fuega et al. 2003) Nannochloropsis sp. Tetraselmis sp Trace (Volkman et al. 1989; Fernandez-Reiriz et al. 1989; Renaud et al. 1999; Muller-Fuega et al. 2003) T-ISO (Volkman et al. 1989; Fernandez-Reiriz et al. 1989; Renaud et al. 1999; Muller-Fuega et al. 2003) Rhodomonas sp (Fernandez-Reiriz et al. 1989; Renaud et al. 1999; Muller-Fuega et al. 2003; Tremblay et al. 2007) effects of diets on those parameters related to the productivity of copepods. Egg production is one of the principal factors determining population growth in calanoid copepods, and it has been linked to the quality of diet (Uye, 1981; Castro-Longoria, 2003; Ianora, 2005), in particular the EPA (eicosapentaenoic acid) to DHA (docosahexaenoic acid) ratio of the diet (Stottrup and Jensen, 1990; Kleppel et al., 2005; Lee et al., 2006). EPA and DHA are highly unsaturated fatty acids (HUFAs), which are essential nutrients for most cultured fish and crustaceans as they are required for normal growth, development of the nervous system, and cell membrane functions (Koven et al., 2001; Anderson and De Silva, 2003). Aside from egg production, copepod productivity may also be linked to other factors, such as the hatching rate of eggs and subsequent survival and development of the nauplii and copepodites, as well as life expectancy and sex ratio of the adults (Knuckey et al., 2005). Therefore, evaluating the effects of diets on overall copepod population dynamics after a certain period of culture rather than focusing on a single stage of the copepod life cycle is likely to provide more complete information for the purposes of aquaculture. Analysis of population growth is probably more pertinent to the ultimate goal of improving productivity of copepod culture for hatcheries because it provides a summary of the dietary effects on a range of inter-related parameters, including egg production, egg hatching rates, nauplii and copepodite development time and survival. Of the many algal species that have been used in aquaculture, Rhodomonas and T-ISO have been conspicuously successful as a food for rearing copepod species (Stottrup et al., 1986; Lacoste et al., 2001; Rippingale and Payne, 2001; Lee et al., 2006). T-ISO contains a high concentration of DHA and medium to high amounts of 18:4n-3 and 18:5n-3 (Table 1; Whyte, 1987; Coutteau, 1996; Van-Thinh et al., 1999), the fatty acids commonly associated with promoting high growth rates in aquatic larvae (Nell and O'Connor, 1991; Renaud et al., 1999). The other algae species Rhodomonas sp. is often favoured in copepod culture because it contains substantial levels of both EPA and DHA (Table 1). The algal species has been successfully used as a sole diet or as part of a mixed diet for the culture of a number of Acartia species, including A. tonsa Dana (Stottrup et al., 1986; Peck and Holste, 2006), A. tranteri (Morehead et al., 2005), Baltic strain A. tonsa (Holste and Peck, 2006) and A. sinjiensis (McKinnon et al., 2003; Knuckey et al., 2005). McKinnon et al. (2003) found that Rhodomonas supported high egg production rates of A. sinjiensis (up to 33 eggs/female/ day) while Knuckey et al. (2005) found Rhodomonas supported higher development rates of A. sinjiensis

7 662 M. Milione, C. Zeng / Aquaculture 273 (2007) nauplii than a binary diet of Tetraselmis and T-ISO. However, evaluating the relative effectiveness of a given algal diet in supporting overall copepod productivity is complex, as there are many factors that may affect the final outcomes. Studies have shown that an algal diet may perform well for one particular parameter of copepod productivity but not for another one. For example, while McKinnon et al. (2003) found that egg production of A. sinjiensis was lower on a T-ISO diet when compared to Tetraselmis or Pavlova, Knuckey et al. (2005) reported that T-ISO supported better nauplii development of A. sinjiensis than either Tetraselmis or Pavlova. The results of this study showed that algal diets can significantly affect both the population growth and egg hatching success of A. sinjiensis and it confirms that both binary diets used supported the best results overall: A. sinjiensis fed binary diet of Tetraselmis & T-ISO produced the highest mean population growth, in 8 days, reaching a final mean population of 1091 from an initial 12 adults. The other binary diet of Nannochloropsis & T-ISO also produced the second highest population growth with a final population at 896. Meanwhile, the highest egg hatching rate (88.1%) was obtained from the binary diet of Nannochloropsis &T- ISO while the other binary diet of Tetraselmis & T-ISO produced the third highest hatching rate (76.4%). Statistic analysis showed that both population growth and egg hatching success of A. sinjiensis fed these two binary diets were not significantly different. A number of studies have confirmed that copepods fed binary algal diets often exhibit higher fecundity compared to those fed monoalgal diets (Stottrup, 2000; Rippingale and Payne, 2001; Knuckey et al., 2005; Morehead et al., 2005; Lee et al.,2006), and binary algal diets are thought to provide a better balance of nutrients than a single algal diet (Coutteau, 1996; Southgate, 2003). The algae Isochrysis, for instance, contains high DHA but it has low levels of EPA, while Nannochloropsis contain high levels of EPA, but it has virtually no DHA (Table 1). In fact, in a study on the effects of algae diets on the survival, development and egg production of calanoid copepod Gladioferens imparipes, Payne and Rippingale (2000) suggested that a combined diet of Isochrysis and Nannochloropsis oculata is likely to compensate each other on their essential fatty acid profile to achieve a balanced EPA to DHA ratio and thereby boost G. imparipes productivity. Similarly, Knuckey et al. (2005) showed that a binary diet of Isochrysis and Tetraselmis produced better results for nauplii development of A. sinjiensis than fed either Isochrysis or Tetraselmis alone, and was almost as effective as Rhodomonas. The monoalgal diets used in this study, Nannochloropsis, T-ISO, Tetraselmis and Rhodomonas all produced substantially lower population growth and hatching success than the binary diets, the only exception was that T-ISO produced a hatching rate (88.0%) that was comparable to the two binary diets. Although Rhodomonas has been shown to be the best among algal diets tested for egg production (McKinnon et al., 2003) and nauplii development rates (Knuckey et al., 2005) of A. sinjiensis, the results of this study indicate that the binary diet of Tetraselmis & T-ISO supports significantly better population growth as well as egg hatching success than Rhodomonas. Given that algal diets have been shown to affect various parameters of copepod production, these results are not surprising, nor do they necessarily contradict those of previous studies. Actually, the breakdown of A. sinjiensis population fed Rhodomonas showed it had substantially high number of nauplii compared to other diets, which may imply that Rhodomonas supports good egg production and nauplii survival and development, but was less effective for the copepodites and adults. Population growth results were lowest for the two frozen diets of Nan & Tet F (3±1) and Tet F (26±9), and for the live algae diet Nan (42±7). The population growth results of these three diets were significantly lower than all other diet treatments. These diets also produced very low egg numbers in egg hatching experiment and had to be excluded for data analysis. The green algae Nannochloropsis sp. has been found to perform poorly in other studies as a monoalgal diet for calanoid copepods. Payne and Rippingale (2000) found that Nannochloropsis oculata supported very low nauplii development for G. imparipes as compared to T-ISO and Morehead et al. (2005) also found that N. oculata did not support population growth in Acartia tranteri. The ineffectiveness of N. oculata as a monoalgal diet may relate to its lack of DHA and other HUFAs despite its high EPA content. Alternatively, Payne et al. (2000) suggested that the poor results associated with Nannochloropsis may be due to the toughness and indigestibility of its cell wall, which is also perceived as a major problem in Artemia culture (Dhont and Lavens, 1996). The final population counts of A. sinjiensis fed on both frozen algal diets of Nan & Tet F and Tet F were very low. While the poor result for Nan & Tet F may be explained by the fact that the Tetraselmis to Nannochlorposis ratio was very low in the binary frozen diet supplied by the commercial supplier, the striking contrast in the results between A. sinjiensis fed frozen and live Tetraselmis is more difficult to explain, as frozen diets have been successfully used in aquaculture for the culture of a range of filter feeding organisms, such as rotifers (Brown, 2003). However, as selective filter feeders (Mauchline,

8 M. Milione, C. Zeng / Aquaculture 273 (2007) ; Stottrup, 2000), A. sinjiensis may preferentially select live motile algae cells as food items and may therefore have avoided feeding on the dead cells. It is also possible that the dead algae cells did not remain suspended as well as live algae in the water column and higher levels of aeration may help prevent deposition of the algal cells at the bottom. In addition, the potential significant differences in the availability of bioactive chemicals, such as enzymes, from live and dead algae could also be a reason. Obviously, further research is required to clarify this issue. In conclusion, based upon the results of this study, it is recommended that for the culture of A. sinjiensis, an AFDW based equal ration of binary algal diet consisting of Tetraselmis chuii and T-ISO is used, which equals to a density of 3150 cells/ml of Tetraselmis chuii and 40,000 cells/ml T-ISO respectively due to their respective AFDW values. 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