Salinity tolerance of embryos of obscure puffer Takifugu obscurus

Transcription

1 Aquaculture 253 (2006) Salinity tolerance of embryos of obscure puffer Takifugu obscurus Zhou Yang a,b,c,, Yafen Chen a a Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, 73 East Beijing Road, Nanjing , People's Republic of China b Jiangsu Key Laboratory of Bioresource Technology, School of Biological Sciences, Nanjing Normal University, 122 Ninghai Road, Nanjing , People's Republic of China c Graduate School, Chinese Academy of Sciences, Beijing , People's Republic of China Received 21 April 2005; received in revised form 6 August 2005; accepted 11 August 2005 Abstract Salinity tolerance of embryos of obscure puffer Takifugu obscurus was investigated at salinities of 0, 4, 8, 12, 16, 20, 24, 28, and 32 parts per thousand (ppt), respectively. Survival rates of embryos at 0, 4 and 8 ppt salinities were higher than 90% throughout the experiment, and there were no significant differences between 0 and 8 ppt (P > 0.05), whereas most embryos died gradually at salinity of 12 ppt or higher after four days. Total hatch rates at 0, 4, and 8 ppt were 95.0%, 95.0%, and 91.0%, respectively. A few embryos hatched at salinities of 12, 16, 20, and 28 ppt; however, all larvae were deformed and died in 24 h post-hatch. Only at salinity of 8.0 ppt or lower, the embryos hatched into viable larvae. Hatching period at 0, 4, and 8 were 171, 182, and 182 h, respectively. Within the tolerable salinity range (0 8 ppt), there were no significant differences in total hatch rates, viability of 24 h post-hatch larvae, and rate of embryonic development (P > 0.05). We concluded that embryos of obscure puffer could not tolerate salinities of 12 ppt and higher, but could well adapt to salinities below 8 ppt. Thus, it is viable to incubate embryos of obscure puffer under lower salinity conditions Elsevier B.V. All rights reserved. Keywords: Obscure puffer; Takifugu obscurus; Salinity tolerance; Embryos; Survival rate; Incubation period 1. Introduction Puffer fishes of the order Tetradontidae exist in anadromous and non-anadromous seawater resident forms. Some economically important species have been well studied for potential aquaculture (Fujita and Abe, 1992; Chuda et al., 1997; Matsuyama et al., 1997; Duncan et al., 2003; Komar et al., 2004; Yang and Yang, 2004; Yang and Chen, 2003, 2004, 2005; Chen, 2005). Corresponding author. Jiangsu Key Laboratory of Bioresource Technology, School of Biological Sciences, Nanjing Normal University, 122 Ninghai Road, Nanjing , People's Republic of China. Tel.: ; fax: address: yangzhouff@vip.sina.com (Z. Yang). Obscure puffer Takifugu obscurus is an anadromous species (for detail about this fish see Yang and Chen, 2005 and references therein) whose intensive culture in freshwater has been developed rapidly in China in recent years owing to its large body size and highquality meat (Yang and Chen, 2004). Aquaculture production for obscure puffer has increased to 2600 t per year since 2002 (Yang et al., 2002) and plays an important role in meeting the rising demand. However, considerable amount of freshwater is consumed during the culture process from embryos to adults. This hampers further development of obscure puffer production, especially in the areas where freshwater resources are limited. If obscure puffer could be cultured in brackish water, a lot of freshwater would be saved as /$ - see front matter 2005 Elsevier B.V. All rights reserved. doi: /j.aquaculture

2 394 Z. Yang, Y. Chen / Aquaculture 253 (2006) extensive saline swamps in the coast of China have great potential for brackish water aquaculture. In the natural environment, obscure puffer confronts with drastic changes, especially salinity changes, in the external medium during their spawning movement from marine to freshwaters. Therefore, obscure puffer adult is expected to have better ability to regulate osmolality to adapt to various salinities. However, no information is available on the salinity tolerance in its early life stages. It is thus important to understand the salinity tolerance of obscure puffer in embryonic stages in both theory and application. This study aimed at investigating the salinity tolerance of obscure puffer embryos and also gaining an insight into the possibility of aquaculture of this species in brackish water. 2. Materials and methods 2.1. Fertilized eggs collection Artificially fertilized eggs of obscure puffer were obtained from a single pair of parents by induced spawning of the cultured broodstock by injections of [D- Ala 6 -Pro 9 -Net]-luteinising hormone releasing hormone analogue (Yang and Chen, 2003). When embryos were at the stage of middle blastula, dead and physically damaged embryos were removed using a wide-mouthed pipette and only normally developed embryos were used for the experiment Salinity treatments To obtain the required salinities, different proportions of the commercially available sea salt was mixed with fresh water. Salinities used for incubation were 0 (freshwater control), 4, 8, 12, 16, 20, 24, 28, and 32 ppt. There were three replicates for each treatment. Incubation units consisted of 100-ml glass beakers filled with 50 ml fresh or saline water. Fifty embryos were placed into each beaker. All salinity gradients were adjusted gradually at a rate of 1 ppt h 1 from a salinity level to next level within 4 h. Eggs were incubated statically in the beakers under natural light and photoperiod. Fifty percent of the water in each beaker was replaced with fresh medium daily. During the incubation period, water temperature fluctuated naturally between 17 and 20 C, dissolved oxygen mg l 1, and ph Data collection Live embryos were spherical and transparent. Thus, the embryonic developmental stages could be identified using a dissecting microscope. Since dead embryos became white and opaque, they were distinguished easily from live embryos by the naked eye. For all treatments, dead embryos were removed and counted each day, until all the larvae hatched or all the embryos died. Meanwhile, three embryos were sampled every day from each replicate using a wide-mouthed pipette and were observed quickly under a dissecting microscope for determination of the developmental stages. Sampled eggs were returned to their respective incubation units after the brief examination. Survival rate was calculated as the percentage of survivors during incubation period. Total hatch rate was calculated as the percentage of stocked embryos that hatched, regardless of viability. Newly hatched larvae were removed and held in separate containers for 24 h to observe their development and post-hatch viability. After 24 h, deformed and moribund larvae were counted. Viability of 24 h post-hatch larvae was calculated as the percentage of total hatch that were alive and normally developed at 24 h post-hatch. To determine the effect of salinity on the rate of embryonic development, we used incubation period as an index. Incubation period is defined as the time interval from egg activation until 50% of the fertilized eggs have hatched (Kamler, 2002). The percentages of eggs that have hatched were monitored every few hours and the time of these observations was recorded once hatching began. As only a few embryos (much lower than 50%) hatched in the higher salinities, incubation period was unusable for these salinities. Thus, we also used the time to the hatch of the first egg as an index to compare the rate of embryonic development at different salinities Statistical analysis All data (survival rate, total hatch rate, viability of 24 h post-hatch larvae, incubation period) were presented as mean ± standard error of mean (SEM). The influence of salinities on those indices was analyzed by two-way analysis of variance (ANOVA). Duncan's multiple range test (Duncan, 1955) was used to estimate the differences between treatment means. Statistical significance was set at α = Results 3.1. Survival rates of embryos during incubation period Survival rates of embryos in 0, 4.0 and 8.0 ppt salinities were always higher than 90% from the beginning to the end of the experiment, and there were no significant differences among 0 to 8 ppt (P>0.05).

3 Z. Yang, Y. Chen / Aquaculture 253 (2006) Survival rates in 12 and 16 ppt salinities remained high before day 4, but decreased sharply by day 5. At the salinities above 20.0 ppt, survival rates decreased more quickly than those at lower salinities. All embryos died on day 4 at the 32 ppt salinity (Fig. 1). Although the embryos at the salinities above 12 ppt remained live for several days, most of them seemed to cease development or developed slowly, and became opaque and dark gradually and died finally Total hatch rates Total hatch rates at 0, 4, 8, 12, 16, 20, 28 ppt were 95.0±7.07%, 95.0±3.96%, 91.0±4.24%, 4.0±2.83%, 4.0±2.65%, 2.0 ±1.73%, and 2.0±1.00%, respectively. No embryo hatched at 24 and 32 ppt. In addition, although a few embryos hatched at higher salinities, all larvae were abnormal or moribund. Hatching success was significantly lower (P < 0.05) at higher salinities from 12 to 32 ppt than at lower salinities (0 8 ppt). However, statistical analysis showed that total hatch rates among salinities of 0, 4, and 8 ppt were not significantly different (P > 0.05). Similarly, total hatch rates among salinities of 12, 16, 20, and 28 ppt were not significantly different (P > 0.05) Viability of 24 h post-hatch larvae Viability of 24 h post-hatch larvae at 0, 4, and 8 ppt were 98.0 ± 1.82%, 96.7 ± 2.55%, and 97.3 ± 3.05%, respectively. However, no larvae survived after 24 Survival rate (%) Time (Days) 0ppt 4ppt 8ppt 12ppt 16ppt 20ppt 24ppt 28ppt 32ppt Fig. 1. Survival rates of embryos incubated at different salinities. h post-hatch at 12 ppt and higher salinities. Viability of 24 h post-hatch larvae was significantly lower (P < 0.05) at high salinities (12 32 ppt) than at low salinities (0 8 ppt), while there was no significant difference (P > 0.05) among low salinities Hatching periods Hatching periods at 0, 4.0, and 8.0 were ±7.78, ± 16.27, and ± h, respectively, and there was no significant difference among them (P>0.05). The time to the hatch of the first egg at low salinities (0 8 ppt) were h, whereas those at high salinities (12 32 ppt) were h. Rates of embryonic development appeared to decrease with increasing salinities. 4. Discussion The results showed that hatching of obscure puffer eggs occurred within a wide salinity range of 0 to 20 ppt, no embryo hatched in 24 and 32 ppt. However, it is very harmful to embryos when salinity increased to 12 ppt or higher. Most embryos died gradually at high salinities after several days and only a few eggs survived. Although some embryos hatched into larvae at salinity of 12 ppt and higher, all larvae were deformed and died in 24 h post-hatch. Only at salinity of 8 ppt or lower, the embryos hatched into viable larvae. Within the tolerable salinity range (0 8 ppt), there were no significant differences in total hatch rate, viability of 24 h posthatch larvae, and rate of embryonic development (P>0.05). Based on these findings, we concluded that embryos of obscure puffer could not tolerate salinity of 12 ppt and higher but could well adapt to lower salinity brackish water with salinity up to 8.0 ppt. Thus, it is viable to incubate embryos of obscure puffer under lower salinity conditions. A number of studies on the effects of salinity on embryonic development and survival of embryos have been conducted for a variety of fish species. In general, many freshwater fish embryos can only tolerate low salinity, such as catfish Clarias gariepinus (Gbulubo and Erondu, 1998) and Heterobranchus longifilis (Fashina-Bombata and Busari, 2003), silver carp Hypophthalmichthys molitrix (Gao, 1965), whereas embryos of some marine and euryhalineous fish can tolerate a wide range of salinity change, such as black sea bass Centropristis striata (Berlinsky et al., 2004), European sea bass (Conides and Glamuzina, 2001), red sea bream Pagrus major (Mihelakakis and Yoshimatsu, 1998), greenback flounder Rhombosolea tapirina (Hart

4 396 Z. Yang, Y. Chen / Aquaculture 253 (2006) and Purser, 1995), and rabbitfish Siganus guttatus (Young and Dueñas, 1993). Embryonic development in an anadromous fish, Atlantic salmon Salmo salar, occurred at salinity up to 3 ppt, but at higher salinity (5 ppt) development was arrested (Li et al., 1989). In the present study, no negative influence was observed on embryos of obscure puffer when the salinity was not higher than 8 ppt. This indicated that embryos of obscure puffer can tolerate higher salinity than those of Atlantic salmon, although both are anadromous fishes. Therefore, based on the findings from this and other studies cited above, influences of salinity on fish embryos are species-specific. Within the tolerable salinity range (0 8 ppt), no negative effect was observed on the rate of embryonic development of obscure puffer embryos. This is similar to the majority of other fishes (reviewed by Kamler, 2002). However, rate of embryonic development of obscure puffer is retarded at high salinities. This effect may be caused by the excessive expenses of energy by the embryos on regulating osmolality, which negatively affected the developmental rate. Kinne and Kinne (1962) also demonstrated the retarding effect on embryonic development of a cyprinodont fish at high salinity and explained it as the result of reduced oxygen availability, which decreases with increasing salinity and retards ontogeny. In the present study, to obtain the practical information, temperature was allowed to fluctuate naturally during incubation. However, as the combined effect of salinity and temperature on the rate of embryonic development has been demonstrated in some other fishes (Kinne and Kinne, 1962; Mihelakakis and Yoshimatsu, 1998), and salinity tolerance of embryos of obscure puffer is likely different under various temperatures, further studies, therefore, are needed to clarify the combined effect of salinity and temperature on developmental rate and the effect of temperature on salinity tolerance in embryos of obscure puffer. In summary, our results showed that the embryos of obscure puffer can tolerate salinity up to 8.0 ppt. Within this salinity range, no significant difference was observed in total hatch rate, viability of 24 h post-hatch larvae, and rate of embryonic development. It is critical to incubate embryos of obscure puffer under low salinity conditions to ensure successful embryo development and hatching. It is promising to culture obscure puffer in brackish water from embryonic incubation. Acknowledgements We appreciate Xinke Zhu, Xi Chen and Jie Hua for their technical assistance, and constructive comments from three anonymous reviewers. Our sincere thanks are also due to Dr. Binhe Gu for valuable suggestions and polishing the English. This investigation was partially funded by Science Foundation for Young Teachers of NJNU (2001XQ22) and Three-Projects of Aquaculture in Jiangsu Province of China (PJ ). References Berlinsky, D.L., Taylor, J.C., Howell, R.A., Bradley, T.M., Smith, T.I.J., The effects of temperature and salinity on early life stages of black sea bass Centropristis striata. J. World Aquac. Soc. 35, Chen, Y.F., Induced ovulation and embryonic development of ocellated puffer, Takifugu ocellatus. J. Appl. Ichthyol. 21, Chuda, H., Matsuyama, M., Ikeda, Y., Matsuura, S., Development of the maturation- and ovulation induced method in cultured tiger puffer Takifugu rubripes by hormonal treatments. 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