Aquaculture Research, 2014, 45, 1591–1599 doi:10.1111/are.12105 Mass selection for small-sized cysts in Artemia franciscana produced in Vinh Chau salt ponds, Vietnam Nguyen Thi Hong Van1,2, Nguyen Van Hoa2, Peter Bossier1, Patrick Sorgeloos1 & Gilbert Van Stappen1 1 Laboratory of Aquaculture & Artemia Reference Center, Faculty of Bioscience Engineering - Department of Animal Production, Ghent University, Ghent, Belgium 2 College of Aquaculture and Fisheries, Cantho University, Cantho, Vietnam Correspondence: N T H Van, Laboratory of Aquaculture & Artemia Reference Center, Ghent University, Rozier 44, 9000 Ghent, Belgium. E-mails: nthvan@ctu.edu.vn or Van.NguyenThiHong@Ugent.be Abstract Unidirectional mass truncation selection for smallsized cysts was carried out on brine shrimp (Artemia franciscana Kellogg 1906), Vinh Chau strain, Vietnam, through cyst sieving and then culturing the selected individuals (<5% of the population) under field and laboratory conditions over two successive generations (F1 and F2). The results showed that at both culture scales, the cyst size of the selected line was smaller (P < 0.05) compared with the control (non-selected) line, illustrating that there was a response to selection. The cyst size decreased about 3% per generation and the accumulated gain on the target trait was in the range 5.82–6.07% at the end of the selection process compared with the base population. The realized heritability estimation (h2) for cyst size was 0.23 0.06 and 0.74 0.19 in the field trials and 0.34 and 0.51 in the laboratory test for the F1 and F2 generation respectively. The variations found between the respective trials may be explained as the result of environmental interaction with the selection process, but the results show that a selection program for small-sized cysts in Artemia by mass selection is possible. Keywords: Artemia franciscana, mass selection, small-sized cysts, response, heritability Introduction The brine shrimp Artemia, together with rotifers, is the most widely used live prey in aquaculture © 2012 John Wiley & Sons Ltd (Leger, Bengtson, Simpson & Sorgeloos 1986; Lavens & Sorgeloos 2000; Conceicßao, Yufera, Makridis, Morais & Dinis 2010). The main advantage of Artemia is its ability to produce dormant cysts that are highly resistant to adverse environmental conditions and that can easily be stored for years. This is convenient for transporting and marketing, and eliminates the need to maintain live cultures, such as in rotifers Brachionus plicatilis (Lavens & Sorgeloos 2000). Artemia cyst diameter and the corresponding instar I nauplius length can largely differ, e.g. around 500–515 lm for the nauplii of parthenogenetic strains from China versus 400–418 lm for the San Francisco Bay (SFB) type Artemia franciscana Kellogg 1906 strain from Vinh Chau, Vietnam (Vanhaecke & Sorgeloos 1980). Cyst size is one of the parameters determining the price of the cyst product on the market. However, even the smallest Artemia instar I nauplii, such as in the Vinh Chau strain, are considerably bigger than the biggest rotifers (130–340 lm, average length 239 lm), which is a problem for ingestion by small-sized marine fish larvae (Baylon, Ma-Eugenie & Maningo 2004). Recently, a lot of efforts have been made to replace rotifers by Artemia nauplii at first feeding of shrimp, marine fish and crab. However, prey size limitations resulting in reduced larval survival are still the main challenge (Faleiro & Narciso 2009; Nhu, Dierckens, Nguyen, Tran & Sorgeloos 2009; Galley, Green, Watkins & Le Vay 2011). Consequently, there is a high potential for small-sized Artemia cysts on the world market. 1591 Artemia small-sized cyst selection N T H Van et al. Aquaculture Research, 2014, 45, 1591–1599 Scientists working on heritable traits in Artemia indicated that heritability values can be used to formulate a selective breeding program and to develop suitable lines with the specific aim to alter desirable traits in Artemia as live food for a specific group of aquatic animals (Browne, Moller, Forbes & Depledge 2002; Shirdhankar, Thomas & Barve 2004). Although selection is common in many other aquatic animals, such as crustaceans, shellfish, Tilapia, salmon and catfish (Rezk, Smitherman, Williams, Nichols, Kucuktas & Dunham 2003; Kenway, Macbeth, Salmon, McPhee, Benzie, Wilson & Knibb 2006; Li, He, Hu, Cai, Deng & Zhou 2006; Zheng, Zhang, Liu & Guo 2006; Rezk, Ponzoni, Khaw, Kamel, Dawood & John 2009), up to date very little work on Artemia selection has been reported. So far the genetic basis of cyst size as well as of selective breeding in Artemia cysts has remained unexplored. Initial work has been carried out by Shirdhankar and Thomas (2003) who realized substantial genetic gain from bi-directional selection for naupliar length of A. franciscana. However, this study was based on laboratory culture tests using a limited number of animals and did not consider large scale or field applications. The objective of our study was therefore to verify if the principle of unidirectional mass truncation selection, applied over successive generations, would result in a heritable smaller Artemia cyst size as compared with the original population. To assess the practical feasibility of the technique in view of mass production, the selection experiment was run both under pond conditions and laboratory conditions where more stable environmental conditions can be maintained. Material and methods Parental cyst material Artemia franciscana, Vinh Chau strain, Vietnam, was utilized as parental material in the laboratory and in two field selection experiments run in 2008 and 2010. Parental material in the first and second field tests originated from a cyst batch harvested inVinh Chau ponds in 2007 and 2008 respectively. The cysts used in the laboratory test were produced in a laboratory mass culture started with nauplii hatched from 2007 Vinh Chau pond cysts. 1592 Experimental set-up and selection procedure Based on the frequency distribution of the Vinh Chau cyst diameter, a cyst diameter below 210 lm, corresponding to about 5% of the population, was set as targeted selection truncation point. The parental cyst material (P) was sieved and hydrated for 1 h in 30 g Ll seawater over a nylon filter bag with mesh size 212 lm. The parental cysts were gently shaken on the mesh under a seawater flow until no cysts passed through the filter bag anymore. The fraction passing through the mesh was used as inoculation material Ps (Fig. 1). Nauplii hatched from Ps and non-sieved control cysts were each inoculated in three replicate ponds (in the field) or tanks (in the laboratory), producing F1s and F1c cysts respectively. F1s cysts were pooled and diapause was broken by oven drying to have sufficient inoculation material to produce a next generation after selection. To increase the selection pressure, as compared with the previous generation, the pooled F1s cysts were sieved over 200-lm mesh size targeting a truncation point of 1% based on the cyst size frequency distribution. The cysts passing through the mesh (F1ss) were hatched and inoculated in three replicate ponds and tanks to produce F2s cysts. In parallel, non-sieved pooled F1s cysts were used to produce the F2 control line (F2c) in three replicate ponds (Fig. 1). Laboratory test Production of parental generation Three gram of cysts from the 2007 Vinh Chau harvest were hatched according to standard procedures (Lavens & Sorgeloos 1996). The nauplii were stocked at 200 individuals L1 in a semiraceway 4 m3 polyethylene tank with brine water of salinity 80 g Ll collected from the salt ponds, and cultured for 35 days without water renewal. Faeces and other organic deposits were siphoned weekly. The culture was kept at room temperature (in the range 24–29°C) and fed twice a day with spray dried algae (Spirulina pacifica; Cyanotech-Kailua-Kona, HI, USA) combined with Lansy® PZ shrimp feed (INVE, Baasrode, Belgium) according to the feeding rate used by Hoa (2002). Cysts appearing on the water surface were collected daily using a small 200 lm scoop net, rinsed and stored in NaCl-saturated brine water for 2 weeks (P generation). To maximize diapause © 2012 John Wiley & Sons Ltd, Aquaculture Research, 45, 1591–1599 Artemia small-sized cyst selection N T H Van et al. Aquaculture Research, 2014, 45, 1591–1599 P (parental material) Selected line Ps (screening over 212 µm) Control line Inoculation in three replicate ponds or tanks and cultured to maturity F1s 1 F1s2 Inoculation in three replicate ponds or tanks and cultured to maturity F1s3 F1c1 F1c2 F1c3 F1s cysts pooled and dried Selected line F1ss (screening over 204 µm) Inoculation in three replicate ponds or tanks and cultured to maturity F2s1 F2s2 Control line Inoculation in three replicate ponds or tanks and cultured to maturity F2s3 F2c1 F2c2 F2c3 Figure 1 Schematic overview of selection experiments. deactivation, cysts were then rinsed with tap water and dried in an oven at 40°C during 7 h before being used for selection using the standard procedure described above. material available, the culture was run in 4 L plastic cones. F1 and F2 culture conditions Experimental field site Parental cysts were hatched according to standard procedures (Lavens & Sorgeloos 1996). The selected and control lines of the F1 generation were cultured in three replicate 50 L conical polyethylene tanks with pond water of 80 g Ll salinity and moderate aeration, using a stocking density of 400 nauplii L1. The feeding procedure was the same as used for the parental generation, but food quantity was adjusted every 3 days by examining the densities and population composition. Temperature was maintained at room conditions (24–29°C). Water exchange was carried out weekly after cyst collection. The culture lasted for 45 days and was ended before the ovoviviparously produced F2 generation approached maturation. Culture conditions for the oviparously produced F2 generation were the same as for F1. However, due to the smaller amounts of F1 cyst Two experimental runs were carried out during two dry seasons in the solar salt ponds of Vinh Chau, Soc Trang province, Mekong delta, Vietnam (106º05′–106º42′E and 9º22′–9º24′N; January– April 2008 and 2010). Field tests Pond management and data collection All experimental ponds used for production of F1 cysts had a surface area of 800 m² each (20 9 40 m), whereas those for F2 had 260 m² (13 9 20 m), as less inoculation material was available. In terms of production of green water, fertilization scheme, control of water level and salinity, the ponds were managed according to the standard culture procedures described by Anh, Hoa, Van Stappen and Sorgeloos (2009). Conditions in selected and control ponds were kept as identical as practically possible. The stocking density was 100 nauplii L1. © 2012 John Wiley & Sons Ltd, Aquaculture Research, 45, 1591–1599 1593 Artemia small-sized cyst selection N T H Van et al. Aquaculture Research, 2014, 45, 1591–1599 When the cysts started to appear on the pond surface (generally about 2 weeks after inoculation), they were daily collected by a scoop net, cleaned and stored in NaCl-saturated brine for further analysis and utilization. The culture period lasted around 3–4 weeks, and was terminated before the ovoviviparously produced nauplii became adult. Data collection and analysis Abiotic parameters Both in the laboratory and in the field tests temperature (mercury thermometer), salinity (temperature-corrected refractometer; Atago, Tokyo, Japan) and pH (WinLab® Data Line pH meter; Windaus, Clausthal-Zellerfeld, Germany) were monitored daily at 07:00 hours and 14:00 hours by direct measurement. The nutritional parameters, total ammonia nitrogen (TAN) and nitrite (NO2), were sampled every week and analysed in the laboratory using standard methods (Eaton, Clesceri & Greenberg 1995). S ¼selection differential; the difference in mean phenotype between the selected parents ðls Þ and the entire parental population ðlÞ : S ¼ ls l Genetic gain is the amount of increase in proportional phenotype that can be achieved through artificial genetic improvement programs. In this study, it is used to refer to the percentage reduction in cyst diameter between selected and nonselected lines after one generation, known as current genetic gain (GGc) or estimated response; it was calculated according to Zheng et al. (2006): GGC ¼ ððlselected lcontrol Þ=lcontrol Þ 100 where lselected ¼mean phenotype of the progeny in the selected line lcontrol ¼mean phenotype of the progeny in the non-selected line Cyst biometrical analysis Cysts produced both in ponds and tanks were sampled and measured, after that data from replicates were pooled for analysis of heritability. Cyst biometry of field samples (P, Ps, F1s, F1c, F1ss, F2s and F2c) was determined using Clemex® image analysis software at INVE Tech Company (INVE, Baasrode, Belgium). The cyst samples were first hydrated in sea water, after which they were put in vials followed by adding a few drops of lugol solution as staining agent and as prevention for possible hatching. The samples were then transferred into a sample holder using a micropipette and placed on a motorized stage with camera focusing from above. The individual cyst diameter (in lm) of at least 500 cysts per sample was determined, and the average value and standard deviation was calculated. Biometry of laboratory samples was determined manually by analysing at least 500 cysts per sample under a microscope equipped with a graduated ocular (Olympus SZ51, Tokyo, Japan). Calculation of heritability value and genetic gain The realized expressed as heritability h² (Lutz 2001) R ¼response; the difference in mean phenotype between the progeny generation ðl0 Þ and the previous generation ðlÞ : R ¼ l0 l 1594 is The realized response, or difference in mean phenotype between selected line and parental generation, expressed as percentage of the value of the parental generation, was calculated and reported as SR (size reduction). Statistical analysis Mean value and standard deviation of cyst diameter of the P, Ps, F1s, F1c, F1ss, F2s and F2c samples was determined using the Descriptive Statistics tool in Statistica version 7.0. One-way ANOVA and posthoc comparison (Fisher LSD test) was used to detect significance of differences at P = 0.05 between P, F1s, F1c, F2s and F2c samples. Results Abiotic culture conditions During the laboratory test, the salinity was kept stable at 80 g Ll; pH showed little variation (7.5 0.3) and temperature was 26.2 2.7°C. TAN and NO2 measurements were in the range 0.09–0.33 mg L1 and 0.25–0.60 mg L1, respectively, which is below the harmful levels for Artemia. In the field trials, the culture conditions were more unstable compared with the laboratory test. © 2012 John Wiley & Sons Ltd, Aquaculture Research, 45, 1591–1599 Artemia small-sized cyst selection N T H Van et al. Aquaculture Research, 2014, 45, 1591–1599 The salinity was initially maintained at the optimal level for Artemia culture (80–95 g Ll). However, heavy rain at the end of the dry season in the first run lead to a strong decline in salinity (with a final value below 50 g Ll). The temperature fluctuated in the wide ranging 23–40°C during daytime and pH was 7.8–8.4 (Table 1). Concerning to the nutritional value during the culture, TAN were recorded in the range 0.110– 0.980 and 0.028–0.950 mg L1; meanwhile NO2 0.001–0.071 and 0.008–0.376 mg L1, for F1 and F2 respectively. No obvious differences were found in these parameters between the selected and non-selected lines (Table 1). Cyst diameter The hatching percentage of all cyst samples in the control and selected lines used for inoculation and production of a subsequent generation was at least 89.4%. Ps cysts obtained after sieving accounted for 4.2 0.4% wet weight (mean value for laboratory and both field runs) of the non-sieved P material. During the second selection round, the cysts passing through the mesh (F1ss), accounted for 12.3 1.1% wet weight of the non-sieved F1s. As the P material used in the laboratory test and two field tests did not come from the same batch, different cyst diameter values were found. Consequently, different values were obtained as well for the respective Ps fractions in laboratory and field tests (Table 2). In general, the offspring (F1, F2) in the field and laboratory tests showed a tendency of decreasing cyst diameter over the successive generations for both control and selected lines. However, a significant difference (P < 0.05) with the parental generation (P) was found for the selected F1s in the laboratory test and in the second field run, whereas it was not significant for the control F1c in any of the three tests (Table 2). All F2 samples (both control and selected lines) were significantly smaller as compared with their respective P-values, but the F2s of all three tests (laboratory and two field runs) were significantly smaller (P < 0.05) than their corresponding control F2c and than the corresponding F1 samples of both control and selected lines (Table 2). Response and heritability In laboratory conditions, the cyst size reduction through selection was almost similar in each generation (7.4 lm in F1 and 7.0 lm in F2), whereas in field conditions, this response was found higher in F2 than in F1 (8.1–10.0 lm versus 3.3–4.3 lm respectively) because of the cumulative selection pressure over the two generations. The percentage reduction in cyst diameter (SR), obtained on average per generation, was very similar in both the laboratory and field experiment (3.09% and 2.87 1.47%). However, the estimated response (GGc) was smaller than the corresponding SR value, except in the second run of the field test. Nevertheless, values from both calculations were generally not dissimilar (Table 2). The heritability value (h²) in the laboratory test was lower in F1 than in F2 (Table 2). This was also observed in the field tests, although the range of variation between F1 and F2 heritability values was larger than in the laboratory test (0.19 and 0.27 in F1 versus 0.60 and 0.87 in F2 for the first Table 1 Abiotic factors in culture ponds during the field experiment (mean standard deviation of all observations throughout culture period) Temperature (°C) Treatment F1 (R1) F2 (R1) F1 (R2) F2 (R2) Selected Control Selected Control Selected Control Selected Control 1 Salinity (g L ) 93.6 94.2 63.4 60.8 81.2 81.0 92.7 94.3 6.2 4.9 14.8 15.2 5.9 6.6 6.4 5.5 07:00 hours 26.8 26.5 27.3 27.3 23.4 23.4 26.6 26.7 1.1 1.0 0.9 0.9 1.3 1.3 1.3 1.2 14:00 hours 36.3 36.4 31.5 31.3 31.9 31.9 34.7 34.7 1.7 1.8 2.6 2.7 2.2 2.2 2.3 2.3 TAN (mg L1) pH 8.2 8.3 7.8 7.8 8.2 8.2 8.1 8.4 0.3 0.3 0.1 .2 0.3 0.3 0.3 0.2 0.620 0.558 0.305 0.316 0.579 0.563 0.380 0.390 0.259 0.238 0.162 0.171 0.091 0.067 0.291 0.175 NO2 (mg L1) 0.006 0.004 0.017 0.015 0.012 0.010 0.082 0.114 0.004 0.002 0.011 0.009 0.005 0.006 0.046 0.049 R1, R2: first run and second run respectively. © 2012 John Wiley & Sons Ltd, Aquaculture Research, 45, 1591–1599 1595 1596 204.0 10.5 (1639) 1.69 1.51 0.27 215.5b 9.6 (752) F(R2) 206.5 12.7 (603) 219.2a 8.6 (1939) 218.7 10.3 (1755) 1.44 1.82 0.19 235.7a 10.5 (4082) 232.3ab 10.8 (3353) 214.1 11.5 (1150) Mean values sharing the same superscript in the same row are not significantly different at P = 0.05; the sieved cysts (Ps and F1ss) were not involved in the statistical analysis; F(R1), F(R2): first and second field run, respectively. For abbreviations of heritability parameters, see Material and methods. 5.26 4.64 0.87 2.05 3.49 0.60 2.79 3.05 229.8b 17.3 (943) 215.6 16.0 (700) Lab test F(R1) 237.2a 15.5 (654) 236.6a 12.3 (769) 218.8a 10.6 (563) 234.9a 17.2 (949) 0.34 3.12 2.17 216.1 17.0 (750) 222.8c 13.0 (521) 224.2c 11.4 (5576) 205.5c 10.6 (1068) 227.2b 13.4 (547) 230.0b 11.9 (3361) 216.9b 9.8 (1079) 0.51 GGC (%) SR (%) h2 F 2c F2s F1s Ps P F 1c h2 SR (%) GGC (%) F1ss Second selection First selection Table 2 Cyst diameter (mean standard deviation) and heritability parameters of cyst size selection experiments under field and laboratory culture conditions of P, F1 and F2 samples; number in parentheses is sample size Artemia small-sized cyst selection N T H Van et al. Aquaculture Research, 2014, 45, 1591–1599 and second field run, respectively, corresponding with average h² values of 0.23 0.06 in F1 and 0.74 0.19 in F2). Discussion Artemia is a crustacean characterized by its short generation time and high productivity; therefore, mass selection could be suitable for selecting desirable traits in this genus, such as small cyst size. The inbreeding rate is supposed to be low as the selected population still contains a very high number of individuals. In this study, selection experiments targeted the production of small-sized Artemia cysts by application of selection over two consecutive generations. The results indicated that the response to selection was similar in both F1 and F2, with a cyst size reduction in approximately 3% per generation. In the field trials, both estimated and realized response were markedly higher in F2 than in F1, with average values for both trials in the range of 3.7–4.1% and 0.02–1.7%, respectively, whereas this was not the case in the laboratory test. The variation in responses between laboratory and field condition as well as between generations could be attributed to different rearing conditions in the three tests. Although the conditions in the laboratory test were maintained stable, they were less controlled in the field tests. These conditions are supposed to interact differently with the genetic characteristics of the population and hence with the success of the selection process. This is illustrated by the difference between the estimated and realized response values over successive generations. The former is calculated based on populations (selected and control lines of the same generation) raised under more or less the same culture conditions, whereas this is not the case for the latter (parental and offspring generations). Another factor possibly accountable for the difference in response in field and laboratory tests is selection intensity, calculated as the selection differential S divided by the phenotypic standard deviation of the trait. According to Muir (2000), mass selection applied with higher selection intensity leads to a higher response to selection, but only over a limited number of successive generations. With the relatively big amount of cysts harvested from the pond culture, the selection differential in the field tests could be smaller than in the lab tests, where only tiny amounts of cysts © 2012 John Wiley & Sons Ltd, Aquaculture Research, 45, 1591–1599 Artemia small-sized cyst selection N T H Van et al. Aquaculture Research, 2014, 45, 1591–1599 were available for selection. The selection intensity in the field material varied little, from 1.16 (F1) to 1.18 (F2), whereas for the lab tests, these values were 1.39 at F1, but only 0.79 in F2. Nevertheless, the phenotypic gain (percentage decrease in cyst diameter) from both field and laboratory results were consistent in demonstrating that selection pressure played a very important role during selection. After short-term selection of two generations, the total response in size reduction (as compared with the P population) was very similar in field and laboratory conditions (5.82 1.44% and 6.07%, respectively, or 2.91–3.04% per generation) even though rearing conditions were different. This response was higher than what was found by Shirdhankar and Thomas (2003) with bidirectional mass selection of small- and big-sized A. franciscana nauplii from Great Salt Lake, USA; the response in the selection line for small nauplii was 7.65–9.31% size reduction over six generations, corresponding with 1.28–1.55% per generation, which is lower than the 2.91–3.04% in our results. However, our response values are rather low compared with body size values obtained for other aquatic animals for which responses in the range of 7–20% per generation have been documented (Rezk et al. 2003, 2009; Zheng, Zhang, Liu, Zhang & Guo 2004; Maluwa & Gjerde 2007; He, Guan, Yuan & Zhang 2008). In a selection program, realized heritability (h²) is considered the criterion to evaluate the effective transmission of a selected trait from parent to the next generation (Lutz 2001; Beaumont, Boudry & Hoare 2010). In our study, h² was of intermediate magnitude in F1 (0.34 and 0.23 0.06 in laboratory and field tests respectively) and high in F2 (0.51 and 0.74 0.19). The heritability of quantitative Artemia traits, although poorly documented compared with other aquatic animals, is highly variable, ranging from low (0.02) to high (0.95) depending on the observed traits (Browne, Sallee, Grosch, Segreti & Purser 1984; Browne et al. 2002; Shirdhankar et al. 2004; Briski, Van Stappen, Bossier & Sorgeloos 2008). The heritability values may be affected by the environmental conditions, which were different in our respective experiments. According to Browne et al. (2002), studying Artemia reproductive and life span traits, the environmental component contributes more to total phenotypic variation than the genetic one. This has been demonstrated for Artemia cyst size as well, which has been documented as negatively correlated with salinity and temperature of salt ponds during the reproduction phase (Guermazi, Habib & Lotfi 2009). Furthermore, the variation in heritability between generations and between laboratory and field conditions might also be related to parental genetic drift (Beaumont et al. 2010) or to the action of natural selection. If the targeted traits coincide with improved fitness, natural selection will work in synergy with the selection program; in the opposite case, it will counteract it (Gjedrem & Thodesen 2005). The action of natural selection, combined with phenotypic plasticity, may be an explanation of the increasing proportion of small cysts with a size less than 200 lm, amounting up to almost 30% of the population in the field tests and 14% in the laboratory test at the end of our selection (data not shown). In summary, our results from both laboratory and field trials demonstrated that there was a response to selection of small-sized cysts in Artemia. Realized heritability suggests that effective progress could be made through selection. 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