Vol. 4 (1)
Contributions to the Study of East Pacific Crustaceans
2006
Recent advances in the production and use of Tisbe monozota Bowman, 1962 (Copepoda:Harpacticoida: Tisbidae) in high-density cultures, and maintained under control conditions
Ana Carmela Puello-Cruz1, Eloy Yen-Ortega1, Blanca González-Rodríguez1, Gabriela VelascoBlanco1, Mario Nieves-Soto2 and Bruno Gómez-Gil1 1
Centro de Investigación en Alimentación y Desarrollo, A.C. (CIAD), Unidad Mazatlán en Acuicultura y Manejo Ambiental, A. P. 711, C.P. 82010, Mazatlán, Sinaloa, México. E-mail:
[email protected] 2 Universidad Autónoma de Sinaloa, Facultad de Ciencias del Mar, Ap. Postal 610 C.P. 82000 Mazatlán, Sinaloa, México.
ABSTRACT.- The Mexican aquaculture industry should consider producing large-scale live food alternatives because the successful rearing of many commercially important species depends on phytoplankton and zooplankton. In September of 2000, the tropical harpacticoid copepod Tisbe monozota Bowman, 1962 was placed under laboratory controlled culture conditions in order to evaluate its production rate. Throughout this study, the species showed good tolerance to handling and survival rates improved. The optimal mixed microalgae density was obtained with 320 cells µL-1 and in combination with a substratum made up of nylon twine inside PVC, copepods production was improved. The most favorable proliferation was achieved when cultures were maintained at 27° C. No significant difference in total number of copepods in either of the photoperiod treatments tested was observed. The renewal of filtered seawater (5 µm filter pore) in the depuration experiments decreased bacteria levels significantly. These results suggest that T. monozota readily adapt to specific culture conditions. However, further studies are recommended to substantiate the significance of these findings at large-scale for aquaculture industry. Keywords: Copepod culture, Tisbe monozota, Harpacticoidea, live food, substratum. Palabras clave: Cultivo de copépodos, Tisbe monozota, Harpacticoidea, alimento vivo, substrato.
Introduction Comprehending and producing new alternative cultures of live food are fundamental in the development of aquaculture. As reported by Nanton et al. (1998) and Shields et al. (1999), copepods have been successfully used as live food in larviculture and the harpacticoid species thrive in high density cultures. Literature refers to Tisbe as an opportunistic feeding organism consuming a wide variety of food such as vegetables, different microalgae and macroalgae species, detritus, bacteria, artificial diets, yeast, rice, wheat and soybean (Rieper 1982, Stottrup et
al. 1997, Williams et al. 1999, Payne et al. 2000, Puello-Cruz et al. 2004). When fed on planktonic microalgae, survival and production rates improved for Tisbe monozota Bowman, 1962 and other benthic species such as Tisbe carolinensis Volkmann-Rocco, 1972 and Tisbe battagliai Volkmann-Rocco, 1972 rather than when they were fed other diets (Lee et al. 1985, Sibly et al. 2000, Puello et al. 2004). Food quantity and quality are considered among the most important factors in copepod production, development and economic viability. Nutritional deficiency caused by an inadequate dietary source can impair growth, survival and reproduction. Light is also 13
Puello-Cruz, A.C., E. Yen-Ortega, B. González-Rodríguez, G. Velasco-Blanco, M. Nieves-Soto & B. Gómez-Gil
considered a predominant abiotic factor which affects both growth rates and survival (Miliou 1993, Stottrup 2000). Whether species of the genus Tisbe thrive and mass produce or not depends in part on the size of their habitat. Harpacticoid copepods compelled to shallow waters prefer to inhabit regions protected by thick vegetation. Phytal substrates are habitats that provide shelter from predators, an abundant supply of food and ample space for the copepods to settle on. In the case of benthic species, a lack of settlement space in the habitat is considered a limiting factor, even though these organisms can tolerate high population densities (Miliou 1992, Williams et al. 1999, Stottrup 2000). The use of live feed in shellfish mariculture applications always carries the risk of different bacteria entering the food chain. It is advisable that the use of any antimicrobials should be reduced if resistance formation is to be avoided. The Vibrio genus is important to the aquaculture industry because of its many pathogenic strains which take advantage of poor organism’s nutrition and alterations in abiotic factors resulting in serious production losses. This could impact a diverse range of economically important cultured species by reducing their growth and survival rates (Aguirre et al. 2000, West et al. 1986). According to Gómez et al. (2004), T. monozota is distributed between Virginia and Biscayne Keys, Florida, USA and on the coastal zone of Sinaloa, NW Mexico. Many marine benthic harpacticoid copepods have been successfully reared under controlled laboratory conditions, but large-scale batch cultures have not yet been reported (Sun et al. 1995). At the beginning of September 2000, staff at CIAD-Mazatlán started to carry out experiments on ways to improve the culture techniques for T. monozota in order to optimize their commercial potential. The species acclimatized well to handling and subsequently, when specific laboratory rearing conditions were applied, the number of copepods increased. Preliminary tests show that the optimum alternative diet for copepods consists of a mixture of three microalgae species (Tetraselmis suecica (Kylin, 1935), Chaetoceros muelleri Lemmerman, 1898 and Isochrysis galbana Parke, 1938) when administered at different ratios (3:1:1). However, in order for this species to reach its full 14
culture potential, further large-scale studies need to be conducted. This work describes the optimal temperature, food density and photoperiod for T. monozota culture and how an increase in available settlement area enhances production rate. A depuration procedure for reducing bacteria level is described, thus averting or eliminating antibiotic usage. Tisbe monozota can be considered as a potential alternative live food in larviculture when this species is reared under optimum culture conditions. Materials and Methods A batch of T. monozota was maintained under continuous culture conditions (27 ± 1º C, 35 ‰ salinity, 12 h :12 h light:dark photoperiod) at the Nutrition and Larviculture Laboratory in the Centro de Investigación en Alimentación y Desarrollo, Mazatlán, Mexico (CIAD-Mazatlán). An unknown amount of organisms were placed in a 300 L tank and fed once a day on a standard diet of mixed microalgae consisting of T. suecica, C. muelleri and I. galbana (3:1:1 ratio) at a cell -1 density of 320 µL . Eighty percent of the culture seawater was renewed once a week with filtered seawater (5 µm filter pore). The microalgae used in all the experiments were obtained from controlled cultures available at the Microalgae Laboratory of CIAD-Mazatlán. Cell density was measured using a haemocytometer (Brightline 0.1 mm deep). In order to test the effect of mixed microalgae densities on copepod production, five ovigerous females from the copepod maintenance tank were placed in glass flasks containing 20 ml of 5 µm filtered seawater and kept under laboratory controlled culture conditions (27 ± 1º C, 35 ‰ salinity, 12:12 h light/dark photoperiod). Seven different cell density ratios (20, 40, 80, 160, 320, 640 and 1289 µl-1) of mixed microalgae were tested as food. All treatments were run with five replicates. Eighty percent of the seawater was renewed daily, prior to the animals being fed. After a culture period of 15 days, the number of organisms were counted separately or in groups of ovigerous females (OF), and adults and copepodites (AC). Nauplii were excluded from the counts. To test the effect of different temperatures on copepod production, five ovigerous females from
Recent advances in the production and use of Tisbe monozota Bowman, 1962 in high-density cultures, under control conditions
the original maintenance tank were placed in glass flasks containing 20 mL of 5 µm filtered seawater and kept under controlled culture conditions (35 ‰ and 12 h :12 h light:dark). Four different temperatures (22, 27, 32 and 37º C) were tested by placing the flasks in water bath containers. All treatments were run with five replicates. Standard mixed microalgae diet was administered once a day. Eighty percent of the seawater was renewed with freshly filtered seawater daily. After a culture period of 15 days, the number of organisms were counted separately or in groups of ovigerous females (OF), and adults and copepodites (AC). Nauplii were excluded from the counts. To evaluate the effect of different photoperiods on copepod production, five ovigerous females from the original maintenance tank were placed in glass flasks containing 20 mL of 5 µm filtered seawater and kept under laboratory control culture conditions (35 ‰ and 27º C). Two different photoperiods were tested: 24 h light and 12:12 h light/dark. Both treatments were run with five replicates. Eighty percent of the seawater was renewed daily before the organisms were fed with the standard mixed microalgae diet. After a culture period of 15 days, the number of organisms were counted separately or in groups of ovigerous females (OF), and adults and copepodites (AC). Nauplii were excluded from the counts. To evaluate the effect of different diets and diet density on copepod production, two brands of commercially available diets (Lansy ZM and Cenzone) and Spirulina powder at five different densities (0.2, 0.1, 0.05, 0.025 and 0.0125 mg mL-l) were used. The standard mixed microalgae diet was also included. Five ovigerous females from the original maintenance tank were placed in glass flasks containing 20 mL of 5 µm filtered seawater and kept under controlled culture conditions (35 ‰, 27º C and 12 h : 12 h light : dark photoperiod). Each flask was fed on each treatment in five replicates (see Table 2). Eighty percent of the seawater was renewed daily, prior to the animals being fed. After a culture period of 15 days, the number of organisms were counted separately or in groups of ovigerous females (OF), adults and copepodites (AC) and nauplii (N). Ten ovigerous females of each treatment and replica were randomly selected and the amount of
eggs per female were counted using a Fisher Scientific Compound Microscope. Copepod production was assessed using two diets combined with two different substrata in order to obtain four different treatments: 1) PVC tubes with live diet, 2) PVC tubes with artificial diet, 3) rubber mats with live diet, and 4) rubber mats with artificial diet. The live diet consisted of the standard mixed microalgae diet and the artificial was Lansy ZM at density of 0.1 mg mL-1. One of the substrata was constructed by using nylon twine (cut up into 250 strands, each measuring 40 cm, then tied to another piece of the same twine of approximately 1.30 cm). Once assembled, each structure was placed inside Polyvinyl Chloride (PVC) tubes. The second substrata consisted of rubber floor mats, rolled up in order to simulate the shape and size of the first substrata. Both the PVC tubes and the rubber mats were of the same dimension (circumference of 7 cm by 25 cm long) and weight (93.74 ± 1 g). Thirty ovigerous females from the original maintenance tank were placed in plastic containers filled with 7 L of 5 µm filtered seawater and kept under controlled culture conditions (35 ‰, 27 ± 1º C and 12 h: 12 h light : dark photoperiod). All treatments were run with three replicates. The containers measured 27 cm long, 17.7 cm wide, and 18 cm height, and they were stacked half a meter apart in three separate rows of four columns. Eighty percent of the seawater was renewed daily, prior to the animals being fed. After 30 days the number of ovigerous females (OF) and eggs per ovigerous female (EF) were counted. The adults, copepodites and nauplii (Ud) were combined and counted. To asses the effect of the depuration process of bacterial levels on T. monozota, batches of copepods were taken from the copepod maintenance tank and kept under controlled conditions (same culture conditions as copepod maintenance) for a period of 10 days and fed with standard mixed microalgae diet once a day. Water was not renewed during the first five days of the experiment but after this time, eighty percent of the water in the culture container was exchanged every day to renew water quality and bacterial analyses were carried out on a daily basis. Initially, the copepods were rinsed in sterilized water and homogenized in a sterile saline solution (2.5% NaCl) with a hand tissue homogenizer. The 15
Puello-Cruz, A.C., E. Yen-Ortega, B. González-Rodríguez, G. Velasco-Blanco, M. Nieves-Soto & B. Gómez-Gil
45.11), 640 cells µL-1 (249.20 ± 81.68), 80 cells µL-1 (163.00 ± 10.12), 40 cells µL-1 (126.80 ± 13.31), 1280 cells µL-1 (123.40 ± 42.00) and 20 cells µL-1 (49.20 ± 6.22), respectively (Fig. 1). The best results for ovigerous females (38.00 ± 6.28 OF) was achieved with a stocking density of 160 cells µL-1 (Fig. 1). This result, however, hardly differed between the stocking densities of 320 cells µL-1 (32.20 ± 8.61 OF) and 640 cells µL-1 (29.80 ± 8.41 OF). On the other hand, poor results were obtained with the following densities: 20 cells µL-1 (4.60 ± 1.52 OF), 40 cells µL-1 (8 ± 3.08 OF), 80 cells µL-1 (10.20 ± 3.11 OF) and 1280 cells µL-1 (12.40 ± 3.36 OF), and no significant difference was found between any of these treatments (Fig. 1).
supernatant was diluted again in the saline solution and 0.1 mL was spread-plated on to marine agar medium (MA, Difco, Detroit, Michigan, USA) and on to thiosulphate citrate bile sucrose agar medium (TCBS, Difco, Detroit, Michigan, USA). The MA is a selective medium used for determining the amount of heterotroph colony forming-units (CFU) and TCBS is used to determine the amount of Vibrio CFU. The organisms were placed in an incubator at a temperature of 30º C for 24 h and later the CFU’s were counted. Results obtained from all the experiments were evaluated using the Statistica 4.10 (StatSoft Inc.) and the Sigma Stat 3.0 (SPSS Science, Chicago, IL) software packages. Data were analyzed by two-way ANOVA after equality and normality of variances were defined. Significant differences were observed at the p
