ReseaRch PaPeR Journal of Agricultural and Marine Sciences Vol. 20 (2015): 40 – 46 Reveived 5 Aug. 2014 Accepted 19 Feb 2015 Culture conditions affect the nutritional value of the copepod Acartia tonsa Arne M. Malzahn1,2,*, Nicole Aberle-Malzahn2, Katherina Schoo1,3, Maarten Boersma2 1Department of Marine Science & Fisheries, College of Agricultural and Marine Sciences, Sultan Qaboos University, P.O. Box 34, PC:123 Al-Khod. Arne Malzahn ( ) email: arne.malzahn@gmail.com 2Alfred Wegener Institute for Polar and Marine Research, Ostkaje 1118, 27498 Helgoland, Germany 3Shannon Point Marine Center, Western Washington University, 1900 Shannon Point Road, Anacortes, WA 98221, USA. Introduction The global yield of the capture fisheries has been stagnating at around 85 million metric tonnes per year since the mid 1980ies. Since more or less the same time, aquaculture production is on an exponential rise, replacing the missing growth in capture fisheries production (Fig. 1). The vast majority of the 80 million tons of aquaculture production in 2010 was made up by seaweeds and cyprinid fishes (20 and 25 million tonnes respectively) (Fao). However, focussing on the value per unit the picture completely changes and next to the high value products such as abalone, shrimps and sturgeons with values between 20,000 and 50,000 US$ tonne-1 we find species such as groupers, soles and pompanos in the same price range. The main difference between the first and the latter group is that the first is relatively easy to culture and the price is based on slow growth and relatively low area based biomass production. The latter group of groupers, soles and jacks have in common that they are sought after food fish with dwindling stocks and that they are not easy to grow, reproduce and wean in captivity. One of the bottlenecks for instance in grouper culture is the larval weaning (Sugama et al. 2012), and they, like many other species, rely on live feeds (Lavens and Sorgeloos 1996; Lavens et al. 1994). This is mirrored by the still increasing number of publications on the use of live feeds in aquaculture (Fig. 2). The typical succes- sion of live feeds for very small, gape limited larvae is to start with ss-type or s-type Brachionus spp., followed by larger Brachionus spp. strains and eventually Artemia spp. larval stages (Lavens and Sorgeloos 1996; Lavens et al. 1994). Artemia and Brachionus species are not always the most suitable first feeds for marine fish larvae due to inappropriate size (Pepin and Penney 1997; Van Der Meeren 1991), their swimming behaviour, which makes them less susceptible to predation (Buskey et al. 1993; Von Herbing and Gallagher 2000), and they are suspect- ed of being of insufficient nutritional quality (Støttrup and Norsker 1997). Calanoid copepods as live feed have been shown to improve growth and survival in groupers (Doi et al. 1997), and several other species (Stottrup Acartia tonsa حاالت اإلستزراع وأتثريها على القيمة الغذائية للكواببودا آرين مالزان1ونيكول ابرييل مالزان2 وكاتريينا سكو1,3ومارتن بورمسا 2 Abstract. Live feed are still necessary for the rearing of larval stages of several fish species, especially marine ones. Compared to Artemia, copepods are of superior quality. This is based on a suite of traits like size, movement, and nu- tritional value. Copepods are for example usually high in protein and fatty acids. Essential fatty acid profiles reflect to a large degree the fatty acid supply, which provides the opportunity to manipulate fatty acid profiles of, amongst oth- ers, copepods. By manipulating nutrient supply of the algae Rhodomonas salina we were able to double essential fatty acid concentrations in naupliar and copepodit life stages of the copepod Acartia tonsa. However, this lead to growth depression rather than to increased growth rates in a series of consumer species, including larval fish. The reason for the growth depression is likely to be mineral deficiencies occurring along with the nutrient manipulation of the algae. Keywords: copepod, nutritional value, live feed, aquaculture, Acartia املســتخلص: تبقــى املغذيــات احليــة ضروريــة لرتبيــة الريقــات للعديــد مــن األمســاك وخصوصــا البحريــة منهــا. وتعتــر الكوبابــودا ذات جــودة عاليــة مقارنــة مــع األرتيميــا. ويســتند هــذا علــى جمموعــة مــن الصفــات مثــل احلجــم، واحلركــة، والقيمــة الغذائيــة. وحتتــوي الكوبابــودا علــى ســبيل املثــال علــى نســبة عاليــة مــن الروتــن واألمحــاض الدهنيــة. وتعكــس مالمــح األمحــاض الدهنيــة األساســية إىل حــد كبــري إمــدادات األمحــاض الدهنيــة، والــي تتيــح الفرصــة ملعاجلــة مالمــح األمحــاض الدهنيــة مــن بــن أمــور أخــرى. ومبعاجلــة التزويــد باملــواد الغذائيــة الطحلبيــة )رودومــاس ســلينا( متكنــا مــن مضاعفــة تركيــز األمحــاض الدهنيــة األساســية يف مراحــل حيــاة الكوبابــودا . إال أن ذلــك أدى إىل القصــور يف النمــو بــدل الزيــادة يف معــدالت النمــو يف سلســلة مــن األنــواع املســتهلكة مبــا يف ذلــك الريقــات. وقــد يكــون النقــص يف املعــادن ســببا مرجحــا لذلــك، نتيجــة ملعاجلــة املغذيــات اخلاصــة بالطحالــب. الكلمات املفتاحية: غراء الببتيدات، السيلوكسانات، اخلاصة بالطحالب. 41Research Article Malzahn, Aberle-Malzahn, Schoo, Boersma 2000), and larval grouper actively select for copepod nauplii over rotifers (Toledo et al. 2004). Similar selec- tivity patterns have been reported for a suite of marine fish larvae (Monteleone and Peterson 1986; Stoecker and Govoni 1984). Food quality can be expressed in many ways, such as through the concentration of polyunsaturated fatty acids (Paulsen et al. 2014; Paulsen et al. 2013), sterols (Lee 2001), amino acids (Awaiss et al. 1992) or the el- emental composition (Malzahn et al. 2007; Shao et al. 2008) of the food item. All of the latter play a major role in fish nutrition (Lavens et al. 1994), and it is crucial to find the food organisms which suits the demand best. It is very likely that marine copepods are a good match for the nutritional demands of larval fish as they are the main prey items in nature, hence an adaptation of the needs of larval fish to the main prey in supply can be assumed. Wild zooplankton has been successfully used for larval rearing (Otterlei et al. 1999) with growth rates superior compared to Artemia larvae. However, availability of natural zooplankton is a problem, as zoo- plankton size spectrum (Beaugrand et al. 2002; Greve et al. 2004), density (Beaugrand et al. 2003) and species composition (Beaugrand et al. 2002; Greve et al. 2004) are permanently changing. Even if the supply of natural zooplankton was reliable, the nutritional value of cope- pods varies with species (Gismervik 1997a; Gismervik 1997b), life stage (Villar-Argaiz et al. 2002; Villar-Argaiz and Sterner 2002) and season (Villar-Argaiz et al. 2002; Villar-Argaiz and Sterner 2002) and is hence again an unreliable source of live feeds. Consequently, in-house life feed production in the form of copepods seems to be a reliable way to fulfil the nutritional demand of early life stages of fish until they can be weaned on more easily accessible live feeds like Brachionus and Artemia or even formulated diets. This paper aims to sum up a series of experiments carried out to determine how copepod culture condi- tions can be manipulated to produce copepods of an ele- mental and biochemical composition of choice. Materials and methods We conducted a series of experiments in which co- pepods were fed on phytoplankton which in turn was grown in nutrient manipulated growth media. The data we show here are representative for the nutrient treat- ments and can easily be reproduced. The phytoplankter R. salina was reared either under nutrient replete condi- tions using the f/2 medium following Guillard and Ryter Figure 1. Global wild fish capture and aquaculture pro- duction in million tonnes, 1950–2010. Data source: FAO (Fao). Figure 2. Number of publications retrieved from Web of Science using the search words ‘aquaculture’ and ‘live feeds’ Table 1. Fatty acid concentrations and elemental ratios of phytoplankton grown on nutrient replete (f/2) and nutrient depleted (-P and –N) media. Measure f/2 +/- sd -N +/- sd -P +/- sd sum unsaturated FA (µg*µgC-1) 0.035 0.022 0.066 0.032 0.105 0.079 total FA (µg*µgC-1) 0.058 0.023 0.145 0.056 0.146 0.090 20:5n3 (EPA) (µg*µgC-1) 0.009 0.007 0.011 0.006 0.016 0.011 22:6n3 (DHA) (µg*µgC-1) 0.007 0.005 0.017 0.017 0.024 0.039 C:N (mol*mol-1) 7.59 0.66 10.14 2.65 8.03 1.72 C:P (mol*mol-1) 231.13 86.71 173.12 84.60 579.24 108.17 42 SQU Journal of Agricultural and Marine Sciences, 2015, Volume 19, Issue 1 Culture conditions affect the nutritional value of the copepod Acartia tonsa (1962) or under nitrogen or phosphorus limitation. The phosphorus limited treatment was realized by adding all f/2 ingredients but phosphorus to sterile filtered natural seawater. This means that the phytoplankton could only use the phosphorus which was available in the seawater at the moment of filtration. Adding all of the other mac- ro- and micronutrients assured a Liebig/Sprengel type phosphorus limitation (Sprengel 1839). Nitrogen limita- tion was realized by adding 20% of the N usually added to the f/2 medium. The addition of some nitrogen was necessary to produce enough N-limited phytoplankton to suit the experimental needs. In order to ensure con- stant algal quality, a new batch of algae was set up every day and cultured well into the stationary phase for the N and P limited cultures. F/2 algae were always harvested in the exponential growth phase (Malzahn et al. 2007; Malzahn and Boersma 2012; Malzahn et al. 2010). The calanoid copepod Acartia tonsa was then fed on these nutrient manipulated phytoplankton for a pre- defined period, after which the copepods were analysed for elemental and biochemical composition as well as their developmental rates. Carbon and nitrogen were analysed by means of an elemental analyser. Phospho- rus was analysed as orthophosphate after acidic oxida- tive hydrolysis with 5% H2SO4 (Grasshoff et al. 1999). Fatty acids were analysed as fatty acid methyl esters and gas chromatography (for details see Malzahn et al. 2007; Malzahn and Boersma 2012; Malzahn et al. 2010; Schoo et al. 2013a; Schoo et al. 2013b). Developmental rates were calculated by dividing the mean developmental stage by the age of the animals. Due to the isochronal growth of A. tonsa, which means that all developmental stages are equally long (Berggreen et al. 1988; Miller et al. 1977) under constant growth conditions, it was not necessary to apply a weighing factor for certain devel- opmental stages. Results and Discussion The different nutrient limitations the algae were subject- ed to resulted in significant differences in carbon-to-nu- trient ratios and fatty acid profiles. Phytoplankton sig- nificantly varied in their C:N and C:P ratios with respect to the nutrient treatment (Fig. 3, Fig. 4 and Table 1). The concentrations of the limiting elements were al- ways lower than the non-limiting elements, pointing on Figure 3. C:P ratios of R. salina grown on nutrient replete (f/2) and nutrient depleted (-P and –N) conditions and A. tonsa reared on these algae. Figure 4. C:N ratios of R. salina grown on nutrient replete (f/2) and nutrient depleted (-P and –N) conditions and A. tonsa reared on these algae. Table 2. Statistical information (one way Anova followed by Tukeys HSD test for unequal n) on various fatty acid measures (µg FA*µg carbon-1) from phytoplankton reared on nutrient replete (f/2) and nutrient depleted (-P and –N) conditions. Measure SS df F p f/2 vs -P f/2 vs -N -N vs -P Total Fatty acids Intercept 0.741 1 192.020 >0.05 < < n.s. Treatment 0.096 2 12.449 >0.05 Error 0.201 52 Unsaturated FA Intercept 0.258 1 101.979 >0.05 n.s. < n.s. Treatment 0.046 2 9.119 >0.05 Error 0.131 52 20:5n3 (EPA) Intercept 0.008 1 117.231 >0.05 n.s. < n.s. Treatment 0.001 2 4.047 >0.05 Error 0.003 52 22:6n3 (DHA) Intercept 0.013 1 22.242 >0.05 n.s. n.s. n.s. Treatment 0.003 2 2.283 >0.05 Error 0.031 52 43Research Article Malzahn, Aberle-Malzahn, Schoo, Boersma the non-homoeostatic nature of phytoplankton growth (Droop 1973; Droop 1974). The concentration of fatty acids also varied significantly with nutrient limitation, showing generally higher fatty acids concentrations when grown under nutrient limitation (Table 1 & Ta- ble 2). Similar, as well as opposing patterns, have been reported for other phytoplankton species (Reitan et al. 1997). The majority of variability in fatty acid concen- trations in general seems to be introduced by taxonomic group. However, the variance due to culture conditions can be substantial as well (Reitan et al. 1994). This spe- cies specific behaviour of altering fatty acid production with nutrient supply enables the keen aquaculturist to tailor single species cultures or even mixes of different phytoplankton species to suit the needs of live food and subsequently the larval fish. Considering fatty acids not as concentrations but as percentage of total fatty acids revealed a different picture (Table 3). Here we found fewer differences between the treatments, which indi- cate that fatty acids production might vary in quantity, but that the variation in relative proportions is less pro- nounced. When copepods were fed such manipulated phyto- plankton we found the same pattern of increased ele- mental ratios and fatty acid enriched copepods as when they were fed on phosphorus limited phytoplankton (Figure 3 & Figure 4, Table 4 & Table 5). Looking at the relative contribution of fatty acids to the total fatty acid pool we found no differences between the treatments (Table 6), which points to the conservative nature of the propagation of fatty acids from one trophic level to the next (reviewed in Dalsgaard et al. 2003). However, not only did the fatty acid concentrations change in cope- pods when fed on nutrient limited phytoplankton, but so did the elemental composition. We found increased C:P ratios in copepods reared on phosphorus limited phytoplankton in several experiments (Malzahn et al. 2007; Malzahn and Boersma 2012; Schoo et al. 2010; Schoo et al. 2012; Schoo et al. 2013a). Consumers have a dome-shaped growth response to food carbon to phos- phorus ratios, growth being carbon (energy) limited on the low C:P side and phosphorus limited on the high C:P side (Boersma and Elser 2006). High C:P ratios create problems because of the excess carbon which has to be taken up with every unit of phosphorus. The handling of the excess carbon seems to create costs high enough to significantly depress consumers’ growth. The phyto- plankton in our experiments showed superior fatty acid profiles and inferior C:P ratios under phosphorus lim- itation, the former known to accelerate growth (Dals- gaard et al. 2003; Engstrom-Ost et al. 2005; Izquierdo et al. 2000), the latter known to depress growth (Sterner Table 3. Statistical information (one way Anova followed by Tukeys HSD test for unequal n) on various fatty acid measures (expressed as %of total FA) from phytoplankton reared on nutrient replete (f/2) and nutrient depleted (-P and –N) conditions. Measure SS df F p f/2 vs -P f/2 vs -N -N vs -P % sum unsaturated FA Intercept 143652.115 1 541.516 >0.05 n.s. n.s. n.s. Treatment 1388.279 2 2.617 0.08 Error 13794.431 52 % 20:5n3 (EPA) Intercept 5270.416 1 243.938 >0.05 > > n.s. Treatment 269.813 2 6.244 >0.05 Error 1123.487 52 % 22:6n3 (DHA) Intercept 6571.439 1 61.112 >0.05 n.s. n.s. n.s. Treatment 62.834 2 0.292 0.75 Error 5591.643 52 Table 4. Fatty acid concentrations and elemental ratios of copepods grown on nutrient replete (f/2) and nutrient depleted (-P and –N) phytoplankton. Measure f/2 +/- sd -N +/- sd -P +/- sd sum unsaturated FA (µg*µgC-1) 0.077 0.024 0.119 0.053 0.136 0.058 total FA (µg*µgC-1) 0.013 0.006 0.024 0.011 0.036 0.016 20:5n3 (EPA) (µg*µgC-1) 0.002 0.003 0.003 0.004 0.006 0.003 22:6n3 (DHA) (µg*µgC-1) 0.003 0.003 0.013 0.012 0.013 0.008 C:N (mol*mol-1) 4.989 0.127 5.303 0.628 5.617 0.580 C:P (mol*mol-1) 180.912 39.585 186.018 65.281 280.917 96.898 44 SQU Journal of Agricultural and Marine Sciences, 2015, Volume 19, Issue 1 Culture conditions affect the nutritional value of the copepod Acartia tonsa 1993; Sterner et al. 1993; Sterner and Hessen 1994). The question arising from this is distinguishing which factor is more important for e.g. larval fish growth. The unique biochemical composition of the phytoplankter R. salina and the relatively strong conservation of fatty acids as well as C:P signals in the copepod A. tonsa allowed us to test this. In all of the above mentioned experiments high copepod C:P resulted in reduced growth rates in larval herring (Malzahn et al. 2007), gelatinous zoo- plankton (Schoo et al. 2010) and larval European lobster (Schoo et al. 2012; Schoo et al. 2013a). This finding leads to the conclusion that mineral requirements have to be fulfilled first before biochemical requirements like fatty acids can promote growth. Consequently, not one single measure should be the focus when optimizing live feeds for aquaculture, but a more holistic approach will lead to better results. References Awaiss, A., P. Kestemont, and J. C. Micha. 1992. Nutri- tional suitability of the rotifer, Brachionus calyciflo- rus Pallas for rearing freshwater fish larvae. Journal of Applied Ichthyology 8: 263-270. Beaugrand, G., K. M. 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