**4.1 Importance of temperature during incubation**

Fish are affected by many intrinsic and extrinsic factors. These factors can affect developmental controls resulting in phenotype alterations (Johnston *et al*.,1996; Adriaens &

Fig. 12. Percent of rotifers and copepods found in the snook larvae stomach contents from four diets.

Fig. 13. Percent of larvae with food in the digestive system from the three diets (SS type rotifers, 100 and 150µm micro-diets).

Fig. 12. Percent of rotifers and copepods found in the snook larvae stomach contents from

Rotifers 100µm 150µm

75/25 50/50 25/75 **Feeding treatment**

Fig. 13. Percent of larvae with food in the digestive system from the three diets (SS type

1234567

**Days from 2 DAH**

rotifers, 100 and 150µm micro-diets).

0 5 10

**Percentage of larvae with food** 

35 40

four diets.

0.00

5.00

10.00

15.00

20.00

**% of fish with both preys**

25.00

30.00

35.00

Verraes, 2002). Among the abiotic factors, temperature has the most significant effect on development and growth (Blaxter, 1992; Kamler, 1992a; Hochachka & Somero, 2001), influencing developmental timing and formation and function of key tissues and structures (Kamler, 1992b; Fuiman *et al*., 1998; Koumoundouros *et al*., 1999) and the synchronization of these continuous developmental paths (Kovac, 2002). Temperature can also have a direct effect on physiology through its effects on enzyme reaction rates (Hochachka and Somero 2001).

Hatching rate of common snook varied with temperature in this study. Eggs incubated at 28ºC showed the best hatching rates; however, no growth trials were conducted. These results confirm Rideout *et al*., (2004) findings of a direct influence of temperature during the incubation period on larval growth, which also has a direct effect on hatch rates (Pepin *et al*., 1997). It has also been argued that survival may be proportional to larval size, since larval mortality rates have been shown to be inversely proportional to growth rates (Pepin, 1991).

## **4.2 The effect of stocking density on larval survival**

Egg stocking density is a key factor in larviculture; without an optimal stocking density, overall survival can be affected. The common snook stocking density experiments results showed the lower stocking density (100 eggs/l) to be the one with the higher survival. This finding agrees with Hernandez-Cruz *et al*., (1999), who obtained low survival in red porgy when eggs were stocked in high densities. On the other hand, Tagawa *et al*., (1997) found higher survival at higher rearing densities in Japanese flounder and this finding was confirmed in three other marine teleosts (Kaji *et al*., 1999). Tagawa *et al*., (2004). The higher survival at high densities was attributed to substances (proteins) secreted by larvae that were beneficial for their survival. Although more experiments need to be done on common snook density, the results clearly showed low survival at high densities, which might be due to the a high level of competition for prey or over-crowding the environment and reduced water quality conditions.

### **4.3 Influence of 'green water' on larval survival**

Food availability is a key factor during first feeding and the consumption rate is dependent on availability. Additionally, the developmental stage of the individual affects the consumption rate (Houde and Schekter, 1980; Kentouri, 1985). Based on the previous findings, this study examined survival of larvae in green water and in clear systems. Final results showed a significant difference in survival between the two treatments, where larvae grown in a green water (*N. occulata* had ingested the algae and had higher survival than those reared in clear water. This result agrees with previously published data (Papandroulakis *et al.,* 2002; Divanach *et al*., 1998; Oie *et al*., 1997; Holmejford *et al*., 1993). Also, it has been reported in cod (Van der Meeren, 1991) and halibut (Reitan, *et al.,* 1991). This may support the idea that microalgae are used as a direct food source at the start of feeding confirming the important role that phytoplankton has during the early stages of several species. Another explanation for the increased survival in green water is the role of phytoplankton in stabilization and improvement of the rearing medium and its direct (Moffatt, 1981; Reitan *et al*., 1993; Van der Meeren, 1991) or indirect nutritional effect (Tamaru *et al*., 1993). Phytoplankton has been reported as being a protective agent, antagonistic towards pathogenic bacteria (Kennedy et al., 1998; Støttrup *et al*., 1995). Skjermo & Vadstein (1999) noticed that microflora in rearing tanks of *Hippoglossus hippoglossus* were more stable in the presence of phytoplankton, increasing the total bacteria population by about 45%. The same authors noticed also that bacteria in larval gut were similar to the ones in rearing water, being mostly species with low growth rate. In addition, Nicolas *et al*., (1989) showed that stomach microflora affects survival during early stages of marine larvae. Other studies have hypothesized that green water produces a background effect that allows the fish to better locate its prey (Marliave, 1994). This effect has been documented to improved the larval rearing in a number of fish species, including dolphinfish *Coryphaena hippurus* (Ostrowski, 1989), yellow perch *Perca flavescens* (Hinshaw, 1985), walleye *Stizostedion vitreum* (Corraza & Nickum, 1981; Colesante, 1989), white bass *Morone chryops* (Denson & Smith, 1996), grouper *Epinephelus suillus* (Duray *et al*., 1996), Dover sole *Solea solea* (Dendrinos *et al*. 1984), barramundi *Lates calcarifer* (Pearce 1991); and red porgy *Pagurs pagrus* (Rotllant *et al*. 2003).

#### **4.4 Importance of rotifer density in common snook larviculture**

Prey density is an important factor in successful larval rearing (Fushimi, 1983). Low density can cause larval starvation or nutritional deficiencies leading to high mortalities. High densities can deteriorate water quality and lead to system fouling, decreased oxygen levels, and increased ammonia levels. Finding the right prey density is crucial to avoid these problems and also to reduce costs associated with live food production.

During the rotifer density experiments, both 15 and 30 rotifers/ml treatment resulted in high survival. Although 30 rotifers/ml had the highest larval survival, the difference between the two treatments was not significant; therefore, the higher cost to produce 30 rot/ml treatment indicates that 15 rot/ml is the optimal density for the 2 and 6 L tank systems. Recent attempts to calculate optimal prey density for cod larvae (*Gadus morhua*) found that survival reaches a maximum level and then begins to decrease if prey densities are further increased (Puvanendran and Brown, 1999). The decrease in larval survival when higher prey densities are used may be a result of poor water quality due to the release of metabolites by the prey (Houde, 1975) or it may be related to a reduction in the ability of the larvae to capture prey; what Laurel et al. (2001) term a ''confusion effect''. Optimal rotifers densities differ between species, for instance the black sea bream needs 1-3 rotifers/ml (Kafuku & Ikenoue, 1983), and the red sea bream needs between 3-10 rotifers/ml (Fushimi, 1983). In the case of common snook, results showed that 5 rotifers/ml was inadequate to meet their food demand. Further experiments need to be carried out to find out the optimal density, this time focusing between 10 to 20 rotifers per ml.

#### **4.5 Alternative prey for common snook larvae**

The suitable size of prey for fish larvae varies with larval mouth size (Shirota, 1970), and fish larvae select larger prey size as they grow (Ivlev, 1961). Although many researchers have reported larval rearing trials with marine fishes, only a few studies have been conducted to compare the appropriate rotifer size among fish species and among different growth stages (Oozeki et al., 1992, in Hagiwara *et al*., 2001). Four strains of rotifers and one copepod species were tested to find the optimal prey type for common snook larvae; prey that will suit the physical needs as the larvae develops and grows in size.

Previous work done on snook larval culture used L type rotifers and had little success. The experiments run with the L type rotifers in these experimental trials, showed extremely low

more stable in the presence of phytoplankton, increasing the total bacteria population by about 45%. The same authors noticed also that bacteria in larval gut were similar to the ones in rearing water, being mostly species with low growth rate. In addition, Nicolas *et al*., (1989) showed that stomach microflora affects survival during early stages of marine larvae. Other studies have hypothesized that green water produces a background effect that allows the fish to better locate its prey (Marliave, 1994). This effect has been documented to improved the larval rearing in a number of fish species, including dolphinfish *Coryphaena hippurus* (Ostrowski, 1989), yellow perch *Perca flavescens* (Hinshaw, 1985), walleye *Stizostedion vitreum* (Corraza & Nickum, 1981; Colesante, 1989), white bass *Morone chryops* (Denson & Smith, 1996), grouper *Epinephelus suillus* (Duray *et al*., 1996), Dover sole *Solea solea* (Dendrinos *et al*. 1984), barramundi *Lates calcarifer* (Pearce 1991); and red porgy *Pagurs* 

Prey density is an important factor in successful larval rearing (Fushimi, 1983). Low density can cause larval starvation or nutritional deficiencies leading to high mortalities. High densities can deteriorate water quality and lead to system fouling, decreased oxygen levels, and increased ammonia levels. Finding the right prey density is crucial to avoid these

During the rotifer density experiments, both 15 and 30 rotifers/ml treatment resulted in high survival. Although 30 rotifers/ml had the highest larval survival, the difference between the two treatments was not significant; therefore, the higher cost to produce 30 rot/ml treatment indicates that 15 rot/ml is the optimal density for the 2 and 6 L tank systems. Recent attempts to calculate optimal prey density for cod larvae (*Gadus morhua*) found that survival reaches a maximum level and then begins to decrease if prey densities are further increased (Puvanendran and Brown, 1999). The decrease in larval survival when higher prey densities are used may be a result of poor water quality due to the release of metabolites by the prey (Houde, 1975) or it may be related to a reduction in the ability of the larvae to capture prey; what Laurel et al. (2001) term a ''confusion effect''. Optimal rotifers densities differ between species, for instance the black sea bream needs 1-3 rotifers/ml (Kafuku & Ikenoue, 1983), and the red sea bream needs between 3-10 rotifers/ml (Fushimi, 1983). In the case of common snook, results showed that 5 rotifers/ml was inadequate to meet their food demand. Further experiments need to be carried out to find out the optimal

The suitable size of prey for fish larvae varies with larval mouth size (Shirota, 1970), and fish larvae select larger prey size as they grow (Ivlev, 1961). Although many researchers have reported larval rearing trials with marine fishes, only a few studies have been conducted to compare the appropriate rotifer size among fish species and among different growth stages (Oozeki et al., 1992, in Hagiwara *et al*., 2001). Four strains of rotifers and one copepod species were tested to find the optimal prey type for common snook larvae; prey that will

Previous work done on snook larval culture used L type rotifers and had little success. The experiments run with the L type rotifers in these experimental trials, showed extremely low

**4.4 Importance of rotifer density in common snook larviculture** 

problems and also to reduce costs associated with live food production.

density, this time focusing between 10 to 20 rotifers per ml.

suit the physical needs as the larvae develops and grows in size.

**4.5 Alternative prey for common snook larvae** 

*pagrus* (Rotllant *et al*. 2003).

survival and DHA analysis of larvae showed a steep decrease in DHA from 1 to 6 DAH. DHA values in 6 DAH larvae were below 1% of total lipid concentration (Yanes-Roca et al. 2009). In both experimental and production systems mass mortality regularly occurred between 5-6 DAH (Yanes-Roca et al. 2009) and 75-85% of all stocked tanks did not have live larvae after 6 DAH. These results along with the finding that only 5% of the larvae fed L type rotifers had food in their stomachs lead to the conclusion that the snook larvae were dying of starvation. This is likely due to the L type rotifer prey size, which was larger than the snook larvae mouth gape. Common snook larval rearing was successful when the larvae were fed copepods grown naturally in outdoor ponds (Lau & Shafland 1982), confirming the prey size hypothesis. Experimental trials comparing survival and growth with several strains of rotifers and copepods (*Acartia tonsa*) also showed an increase in snook larval survival. These findings agree with Doi *et al.,* (1997a,b) and Toledo *et al.,* (1997) found nauplii of copepods to be effective when fed to red spotted grouper, *Epinephelus coioides* and with Støttrup *et al.,* (1997) who found an increase in larval survival when rotifer feeding was supplemented with *Tisbe* sp copepod.
