The Effects of Microalgae as Live Food for Brachionus plicatilis (Rotifer) in Intensive Culture System

Brachionus plicatilis is used to feed fish and crustacean larvae in the aquaculture industry. It is well established that the type of microalgae may influence rotifer production. This experiment was conducted to determine the effect of five different locally available microalgae species at Fisheries Research Institute (FRI), Kampung Pulau Sayak, Kedah, Malaysia on the instantaneous growth rate (μ) of rotifer. Nannochloris sp., Tetraselmis sp., Isochrysis sp., Chlorella sp., and Nannochloropsis sp. were used as feed at different algae densities (0.1, 0.3, 0.7 and 1.5 × 106 cells/ml) and culture volumes (20, 70 and 210 ml). At algae densities ranging from 0.3 to 1.5 × 106 cells/ml, an average μ value of more than 0.90 per day were recorded for all algae species. However, at density of 0.1 × 106 cells/ml, only Tetraselmis sp. resulted in the significantly highest μ value compared with others (p < 0.05). In terms of volume, smaller culture volume of Tetraselmis sp. (20 ml) showed significantly higher μ compared with higher volume (70 and 210 ml cultures).


INTRODUCTION
The rotifer Brachionus plicatilis Muller, 1786 is one of the major food sources for fish and crustacean larvae in the aquaculture industry (Hoff & Snell 2001;Sorgeloos & Laven 1996;Lubzens et al. 1997;Lubzens & Zmora 2003). The rotifer has the ability to tolerate a wide range of salinity besides having the shape, size, colour, slow mobility and rapidly reproduce to reach high density in a short time, which contribute to the utilisation as initial food for the larvae. Furthermore the body composition of B. plicatilis can be manipulated to suit the nutritional requirements of the fish and crustacean larvae feeding on them (Lubzens et al. 1989).
B. plicatilis reproduces asexually (parthenogenesis) under suitable condition by producing one or two large eggs of 80-100 × 110-130 µm every 4 to 6 hours and hatches to become an amictic female (Liao et al. 1993). In high density reproduction, in order to obtain mass production of rotifers, it is important to ensure that only asexual reproduction should prevail by avoiding factors that contribute to sexual reproductive phase (Liao et al. 1993;Theilacker & McMaster 1971). Factors such as high density of rotifers, food type, water temperature, salinity, light penetration, water quality and the genetic composition of rotifers can contribute to the types of reproduction of rotifers whether asexual or sexual reproduction (Hino & Hirano 1976, 1977Lubzens et al. 1980).
One of the crucial problems in the production of fish fry in hatcheries is insufficient live food for the early stages of fish larvae and until now no alternative food can replace the use of rotifers as the initial food for larvae (Hagiwara et al. 2001;Yoshimatsu & Hossain 2014). Production of nutritious rotifers depends on the production of microalgae used to feed them. Apart from the nutritional quality, rotifer's breeding rate is also strongly influenced by the species of microalgae given as food (Lubzens 1987;Lubzens et al. 2001).
Many species of microalgae have successfully been used as diet for rotifers such as Tetraselmis, Nannochloropsis, Chaetoceros, Rhodomonas and Isochrysis (Dhert et al. 2001;Hoff & Snell 2001;Treece & Davis 2000;Wikfors & Ohno 2001). Kostard et al. (1989a) reported higher growth rates when they were fed on Isochrysis galbana compared with Tetraselmis, Nannochloris, or mixture of Isochrysis and Tetraselmis, Isochrysis and Nannochloris, and Tetraselmis and Nannochloris. According to Sayegh et al. (2007), differences in strains of Isochrysis galbana used as feed resulted in some differences in the rotifer parameters such as growth rate, which was even greater than the differences of different diet species. Nevertheless, rotifers have different eating habits and varied structure and size of the corona, the mastax (specific pharynx) and also the mouth, which will determine the type of food that can be taken.
According to Yoshimatsu and Hossain (2014), various methods of microalgae cultures have been developed for successful production of rotifers, namely through batch-culture, semi-continuous culture and continuous culture system. To cater for the needs of live food for culturing fish larvae at high densities, rotifers can also be cultured under high density method. Nannochloropsis is the most common used as live food for rotifers because of its ease of culture and obtaining the yield expected with high density on time.
Mass scale production of rotifers comes with challenges of its own, such as drastic decrease in density due to the sudden collapse of rotifer cultures. In order to achieve high density and stability of rotifer cultures, microalgae play an important role as their food source. The Comparative study of the effects of microalgae Nannochloris sp., Tetraselmis sp., Isochryris sp., Chlorella sp. and Nannochloropsis sp. as live feed for rotifer on the growth rate of rotifer B. plicatilisis has yet to be determined in the confines of the Fisheries Research Institute (FRI). This study was aimed to examine the effectiveness of these species at different concentrations as live food and compared them with Nannochloropsis sp. because this algae is normally use in the hatchery. We assessed the effect of these differences by using a quantitative indicator (Instantanous growth rate of rotifer per day) and their relationship with the utilisation of different densities of food and volume of cultures.

MATERIALS AND METHODS
A female amictic Brachionus plicatilis with loricae length of 110-230 µm was isolated from existing stock culture in FRI Pulau Sayak, which has been maintained for over a year. Rotifers culture was carried out using natural seawater at 30±2 psu, and fed Nannochloropsis sp. as the main food. The seawater used for culture medium was obtained from the coastal areas of Kg. Pulau Sayak, Kedah. The seawater was sterilised (autoclaved) before it was used to culture the rotifer, as well as the algae. The study was carried out entirely under laboratory conditions, at FRI Kg. Pulau Sayak, Kedah.

Mass Culture of Brachionus plicatilis In Batch-culture System
One ml of stock culture, at density of 20 rotifers/ml was inoculated into a test-tube of 20 × 160 mm containing seawater at salinity 30±1 psu. The rotifers were fed Nannochloropsis sp. throughout the culture process at densities of 10-20 × 10 6 cells/ml, which is the recommended density for intensive rotifer culture (Lee and Tamaru 1993). The rotifer culture was maintained at room temperature of 25±1°C and illumination of 150 lux. This starter cultures were then transferred to a 500 ml Erlenmeyer flasks to increase the volume, and again were fed, Nannochloropsis sp. at a concentration of 10-20 × 10 6 cells per ml. The high density per ml of rotifer occurred in three days after inoculation. During this period there was no aeration given. Rotifers at exponential growth phase were then introduced into a 10 L aquarium filled with 5 L of seawater. Rotifers at this phase were cultured for three to four days before they were collected using a 30 µm mesh netting to be used in the experiments. The rotifers were always kept immersed in seawater during sieving.

Live food preparation
Pure cultures of local strains of Nannochloris sp., Tetraselmis sp., Isochrysis sp., Chlorella sp. and Nannochloropsis sp. were obtained from FRI Pulau Sayak, and were cultured in batch-culture system. The microalgae range from 1-2 µm for Nannochloris sp., 9-10 µm×12-14 µm for Tetraselmis sp., 4-8 µm for Isochrysis sp., 2-10 µm for Chlorella sp., and 4-6 µm, for Nannochloropsis sp. The algal cultures were maintained in exponential growth phase using natural seawater at 30 psu and were enriched with Conway medium. In order to produce the inocula of pure strain algal for a larger volume, inoculum transfers were started from an axenic culture stock in a small volume culture. Contamination by bacteria normally cannot be avoided at a large volume culture, therefore the success of microalgae production depends on the inoculation procedure. The flasks cultures were kept on illuminated shelf of 3000 lux at room temperature of 24°C. Algal densities were estimated three times using a haemacytometer to obtain the average density to be used at the beginning for each feeding trials. All aseptic techniques were followed at every steps of sub-cultures transferred.

Assessment of Live Feed for Brachionus plicatilis
The intensive cultures of Brachionus plicatilis were fed with Nannochloris sp., Tetrasemis sp., Isochrysis sp., Chlorella sp., and Nannochloropsis sp. separately and the instantaneous growth rate (μ) of the rotifers were determined. Prior to experiment, the rotifers were not fed for one day. Rotifers from the exponential growth phase were inoculated into a 50 ml Erlenmeyer flask filled with 20 ml of seawater at 30 psu. Assays were also performed at 70 and 210 ml volume cultures.
Each rotifer cultures were fed different microalgae species at densities of 0.1, 0.3, 0.7 and 1.5 × 10 6 cells/ml. The algae densities used in this experiment were based on recommendations by Hoff and Snell (2001) for optimum feeding rate in rotifer. Test containers were exposed to fluorescent light intensity of 150 lux to maintain microalgae survival but prevent algal reproduction. No additional aeration was given. In this experiment all treatments were conducted in triplicate.
Summary of experimental design to assess live feed for Brachionus plicatilis as follows: 1. Microalgae -Batch culture system 2. Isolated Amictic Rotifer -Batch culture system 3. High Density of Rotifer (exponential growth phase) 4. Assessment of Live feed for B. plicatilis a. Size of rotifer culture 20, 70 and 210 ml. b. Each trail was fed different microalgae species at densities of 0.1, 0.3, 0.7 and 1.5 × 10 6 cells/ml with triplicate c. After 24 h, Sedgewick rafter slide used for counting rotifers and their eggs

Rotifer Density Sampling
Triplicate of 1 ml sample from a homogenized rotifer culture were pipetted onto a sedgewick rafter slide using a micropipette. Counting of rotifers and their eggs were then conducted under a light microscope and their densities were determined. The number of rotifers and their eggs were summed up as the total number of rotifers. The instantanous growth rate (µ) was estimated after 24 h of treatments. The growth rate of rotifer (µ) is calculated as the slope of log e number of rotifers against time in days on the exponential growth phase.

Statistical Test
SPSS package statististics 20.0 was used to examine the effects of the treatments on the parameters measured. In all analyses, the level of significance were considered at α = 0.05.

RESULTS
The instantaneous growth rate of rotifers fed with Nannochloris sp. showed increased value with increasing food densities (Table 1). The highest values of instantanous growth rate were obtained at a density of 0.7 × 10 6 cells per ml (1.53 per day) and was decreased at density of 1.5 × 10 6 cells per ml, but showed no significant differences with density of 0.7 × 10 6 cells per ml (Table 1). At density of 0.1 × 10 6 cells per ml, the instantanous growth rate was only 0.10 per day. The increment in instantaneous growth rate of rotifers fed on Tetraselmis sp. occurred by increasing food densities but had no significant effects ( Table 1). The highest value of instantanous growth rate was occurred at density of 0.7 × 10 6 cells per ml (1.72 per day) and the mean was decreased at density of 1.5 × 10 6 cells per ml. At food density of 0.1 × 10 6 cells per ml, the mean value already exceeded the µ values for other spp. (Table 1). The instantaneous growth rate of rotifers per day fed on Isochrysis sp., increased with increasing food densities (Table 1). Even though the result showed a slightly lowered at density of 0.7 × 10 6 cells per ml and the highest values of instantanous growth rate occurred at density of 1.5 × 10 6 cells per ml (1.11 per day), however treatments at densities of 0.3 × 10 6 , 0.7 × 10 6 and 1.5 × 10 6 cells per ml indicated the mean values were not significant (Table 1). At density of 0.1 × 10 6 cells per ml was 0.50 per day. The instantaneous growth rate of rotifers per day fed on Chlorella sp. indicated increased value with increasing food densities (Table 1). At density of 0.1 × 10 6 cells per ml, the mean value obtained only 0.24 per day. At density of 0.7 × 10 6 cells per, showed significant increased in the mean value compared at density of 0.3 × 10 6 cells per ml (Table 1). At density of 1.5 × 10 6 per ml, showed the mean value was not significant compared to 0.7 × 10 6 cells per ml (Table 1).
The instantaneous growth rate of rotifers per day fed on Nannochloropsis sp. also increased with increasing food densities (Table 1). At density of 0.3 × 10 6 cells per ml, the mean value was achieved at 1.64 per day, but indicated that not significant to the densities of 0.7 × 10 6 and 1.5 × 10 6 cells per ml (Table 1). At density of 0.1 × 10 6 cells per ml, the mean was only 0.10 per day.
At the level of food density 0.1 × 10 6 cells per ml, among the five microalgae, Tetraselmis sp. resulted in significantly highest mean instantanous growth rates of rotifers (1.4 per day) ( Table 2). The second highest was Isochrysis sp. (0.5 per day) and was significantly then those fed on Nannochloris sp., Chlorella sp., and Nannochloropsis sp. (Table 2). Meanwhile, no differences were observed among Nannochloris sp., Chlorella sp., and Nannochloropsis sp. for the values 0.1, 0.24 and 0.10 per day respectively. Meanwhile, at density of about 0.3 × 10 6 cells per ml, all treatments recorded mean values higher per day. Nannochloropsis sp (1.64 per day) was higher than Isochrysis sp. and Chlorella sp., and Tetraselmis sp.
(1.54 per day) was significantly different to Chlorella sp. (Table 2).  Experiments were conducted in larger volume cultures of rotifers at 70 ml, at the same level of food density. Statistical tests showed that the means of instantaneous growth rate of the rotifer fed on different diet species were not significantly differed (Table 4). Meanwhile, in culture volume of 210 ml, the instantaneous growth rates were 1.39 and 1.48 per day for Tetraselmis sp. and Chlorella sp. respectively showed significant difference to Isochrysis sp. (Table 4). The effects of algal species were related to the densities of diet (two-way ANOVA; p < 0.01). The impacts of food densities (partial η 2 = 0.48) are stronger than food species (partial η 2 = 0.17; Table 5).

DISCUSSION
Previous studies showed that the production of rotifers were affected by their food (Lubzens 1987;Lubzens et al. 2001). Sarma and Rao (1987) reported, the growth in many rotifers is a function of type of food and food levels provided. This was due to many factors that could influence the suitability of microalgae species as live food such as size, shape, mobility, digestibility and nutrient composition. However this study showed similar instantaneous growth rate when the algal densities were at the abundance level (0.3 × 10 6 cells per ml). According to Yufera and Pascual (1980), the highest growth rate was recorded when using Tetraselmis suecica. Meanwhile Kostard et al. (1989a) reported highest growth rate when using Isochrysis galbana compared to others such as Tetraselmis sp., Nannochloris atomus or mixture of Isochrysis and Tetraselmis, Isochrysis and Nannochloris, and Tetraselmis and Nannochloris. Isochrysis sp. is a type of motile algae species, but with slow mobility compared to Tetraselmis. Although the use of Tetraselmis sp and Isochrysis sp. are ideal for rotifer, attaining high densitiy cultures are quite difficult compared with Nannochloris, Nannochloropsis and Chlorella. Previous studies also reported strong correlation between rotifers growth rate and food abundance (Edmonson 1965;Hotos 2002;Snell et al. 1983). Lee and Tamaru (1993), suggest that food densities at 10 to 20 × 10 6 cells per ml of Nannochloropsis oculata for intensive cultures of Brachionus plicatilis. In contrary, Hoff and Snell (2001), recommend a food density at only 0.1 × 10 6 cells per ml for optimal nutrition values.
This study indicated at each of the four levels of food densities tested (i.e. 0.1 × 10 6 , 0.3 × 10 6 , 0.7 × 10 6 and 1.5 × 10 6 cells per ml) for all species resulted in instantaneous growth rates were higher except at the lowest food density tested at 0.1 × 10 6 cells per ml for Nannochloris sp., Isochrysis sp., Chlorella sp. and Nannochloropsis sp.
At food density 0.1 × 10 6 cells per ml, the mean instantaneous growth rates were depend on the food species. Only Tetraselmis sp. recorded significantly higher instantaneous growth rate. This indicated that at low food densities, the types of food become a limiting factor. Feature of Tetraselmis sp. that able to move faster than Isochrysis sp. gave them advantage as preferred food for rotifers. This was followed by Isochrysis sp. with a mean value of the instantaneous growth rate at 0.50 per day and then by Chlorella sp., Nannochloris sp. and Nannochloropsis sp., which were non-motile species.
The mean instantaneous growth rates of rotifers increased significantly by increasing food densities from 0.1 × 10 6 cells per ml to 0.3 × 10 6 cells per ml, except Tetraselmis sp. Rotifers given food at density of 0.3 × 10 6 cells per ml showed mean instantaneous growth rates were higher for all algae species. Therefore it was assumed that in intensive rotifers culture, higher density culture can be achieved at this level of algal density. The mean instantaneous growth rates were also higher at 0.7 × 10 6 cells per ml and 1.5 × 10 6 cells per ml. This indicated that rotifers were getting enough foods. However, Tetraselmis sp. still remains the preferred food. This study generally showed that foods given at densities of 0.1 × 10 6 to 1.5 × 10 6 cells per ml contributed to higher instantaneous growth rates for rotifers. Snell et al. (1983) reported, B. plicatilis recorded a peak in growth rate using Chlorella sp. at density of 0.5 mg per ml followed by a decrease in instantaneous growth rate at a high density of algae. Hotos (2002) stated the ingestion rate of B. plicatilis increased with increasing density of algae given and decreased at density 620,000 per ml. Meanwhile, Kostard et al. (1989b), observed ingestion rate by rotifers were linearly increased with an increase in food density until a maximum level and then remained constant.
The results indicated, when all species of food given at the abundance conditions produced higher impacts on the mean instantaneous growth rates of rotifers. But Tetraselmis showed better growth than Chlorella, Nannochloropsis showed better growth than Isochrysis and Nannochloropsis showed better growth than Chlorella. Rotifers reared at 70 ml and 210 ml culture volumes also did not revealed any significant differences, except for Tetraselmis-Isochyris and Isochrysis-Chlorella in 210 ml culture. There was no significant differences because the mean instantaneous growth rates using Isochrysis sp. was lower. The characteristic of motile preys can be ignored when at high density of food is given to rotifers, thus mobility factor of the preys only significant at low food density.
Our study suggest that culturing of rotifers in intensive culture systems can be conducted at food densities between 0.3 × 10 6 cells per ml to 0.7 × 10 6 cells per ml except for Tetraselmis sp., which can be supplied at lower density. All algal species tested at 1.5 × 10 6 cells per ml showed declined pattern of rotifers growth rate, except for Chlorella sp..
Our study also revealed that culturing rotifers at high density were better when performed in small containers. Our result demonstrated that rotifers fed with Tetraselmis sp. at 0.1 × 10 6 cells per ml, showed the highest mean instantaneous growth rate when culture in 20 ml volume culture (1.40 per day), and became lower in the 70 ml (0.80 per day) and 210 ml (0.60 per day) volume cultures. According to Rimatulhana et al. (2006), the growth rate for the B. plicatilis size SS, which was cultured in 750 ml at 30 psu using Nannochloropsis sp. at 3 × 10 6 cells per ml was 0.787 per day. Yoshimatsu and Hossain (2014), reported in high density culture, the rotifers were exposed to a high pressure conditions such as from their own excreta, a situation that did not occur in their natural environment, and therefore will make the culture unstable.
Their study also showed the instantaneous growth rates of rotifers per day were higher when moving diets were used even at low food densities compared with the stationary food species that required higher densities for the same results. The use of Tetraselmis sp. and Isochrysis sp. at lower densities than the three other species produced a higher instantaneous growth rate of rotifers. Factor of mobility for Tetraselmis sp. and Isochrysis sp. were met with their predators become more frequent.
Statistical test revealed that the effects of food densities on the mean instantaneous growth rate values were varied between species (p < 0.01) and furthermore the food densities dominated over food species on rotifers production. According to Theilacker and McMaster (1971), high food densities are the most important parameters needed to ensure high production of rotifer.

CONCLUSION
Generally, the instantaneous growth rate of rotifers per day increased with increasing food densities. All food species assayed at density of 0.3 × 10 6 cells per ml, showed all the mean values of instantaneous growth rates were higher when the algal densities at the abundance level (0.3 × 10 6 cells per ml). The type of microalgae also can become a limiting factor which at food density of 0.1 × 10 6 cells per ml (recommended by Hoff and Snell 2001), showed only food species have the capability to move (motile food species) such as Tetraselmis sp. and Isochrysis sp. produced the high instantaneous growth rates. This study also shown that the smaller volume culture size has a better result for mass production of rotifers. stock cultures. The authors would like to thank Universiti Kebangsaan Malaysia and Universiti Teknologi MARA. We would also like to thank the Ministry of Higher Education Malaysia and the JSPS Core-to-Core Program, B. Asia-Africa Science Platforms. We thank the anonymous reviewers for their comments, which have helped improve the quality of this paper significantly.