**5.2 Protein and carbohydrate requirement**

*Invertebrates - Ecophysiology and Management*

**Figure 7.**

*the second and following holdings.*

Rocky habitat and adhering to the substrates are problems. Limpet *C. sandwicensis* attach to the washing rocks in the wild. They cling to the culture tank with their muscular foot. It indicates that physical damaged may happen while removing them off the tank's wall. Similar observation has been made in abalone; they often succumb to wound suffered during removal off the substrates. Abalone blood has no clotting ability, and relatively minor cut can cause death due to loss of hemolymph [32]. Eventually, we developed plastic tank liners that were our breakthrough for transferring animals from one tank to another. Our study was the first to reveal that the Hawaiian limpet *C. sandwicensis* was healthy and fed well in the experimental

*A circular holding biofilm tank without plastic liner, and three aquaria with plastic sheer liner above, used for* 

We [18] began our studies in this area with several preliminary tests on biofilm

Nutrient requirement was our next step to develop the commercial feed available for limpet, and the authors assumed that the nutrient requirement of limpet and abalone is the same as they are marine gastropod [18]. For abalone, a series number of researches had been done, and the optimum nutrient requirement as protein, carbohydrate, and lipid was focused. However, the results still varied among researchers. For example, the protein requirements of abalone found by previous studies [33, 34] were higher than those reported in the previous studies [35–37]. Poor growth was found for abalone when the animal was fed with formulated diet containing amino acid profile that does not match the animals' tissue [38]. Moreover, other studies [39, 40] found that a significant lower growth rates when abalone fed with dried kelp *Ecklonia maxima* and *Laminaria*. Therefore, these studies raised the hypothesis that the growth rate of abalone is related to the degree

of the amino acid profile of feed and the amino acid profile of tissue.

because *C. sandwicensis* ate biofilm well which should be close to their natural diet. We also tested several dry diets, gelatin, and agar diets. We discovered that several were preferred and some were not. Several chemical attractants were tested including betaine, gamma aminobutyric acid (GABA), and dimethyl propiothetin (DMPT), but these did not enhance feeding. Among the feeds tested in a preliminary way, fish meal and soybean meal as well as feeds incorporating biofilm were preferred. Eventually, we found that *Porphyra* preparations could replace biofilm as

aquaria without intermittent water sprayed or dump tanks.

**5. Feed development and nutrient requirement**

**5.1 Development of formulated feed**

a feeding stimulant in formulated feed.

**94**

Based on the results of previous study [18], further studied on the determination of protein and carbohydrate requirement for Hawaiian limpet *C. sandwicensis* [41].

Experimental animals. Adult *C. sandwicensis* limpets (shell length above 3.0 cm) were collected from a remote area in Oahu, Hawaii, used for this study. After collection they were immediately placed into a 14 L ice plastic insulation box with plastic liner and then transported to the laboratory at the University of Hawaii in Monoa. The limpet was held in a plastic aquaria 150 L with water flow for a week; during this period, the animal were fed with the experimental diet and the commercial algae *Porphyra tenera* or *yezoensis*, known as Nori (Nishimoto Trading Co. Ltd., Korea).

Experimental diets. Formulations of dietary protein and carbohydrate levels are shown in **Table 5**. The first trial was done for dietary protein level, following by dietary carbohydrate. For carbohydrate trial, four different dietary carbohydrate levels of 18, 27, 32, and 37% were tested. The amino acid profiles of *C. sandwicensis* tissue and of the dietary protein in trial 1 were analyzed at the Aquatic Feed and Nutrition Laboratory, Oceanic Institute, Hawaii, USA, according to the described method [42]. The results are presented as A/E ratio (**Table 6**). Most of the essential amino acids of diets were identical and/or close to the amino acid profile of *C.* 


*1 This is commercial seasoned seaweed known as nori or the red algae Porphyra tenera or yezoensis. Nishimoto Trading Co. Ltd., Korea.*

*2 Corn oil and menhaden oil (1:1; v/v).*

*3 Commercial vitamin mix (NRC 1981) was kindly provided from Dr. Warren Dominy (Oceanic Institute).*

#### **Table 5.**

*Composition of formulated diet (% dry matter).*


#### **Table 6.** *The A/E ratio [(each EAA/total EAA) × 1000] amino acids of dietary protein and animal tissue.*

*sandwicensis* tissue except for Arg and Thr which were lower in the experimental diets compared to the tissue.

The process of feed preparation for extrusion of all diets was based on the methods described by the previous study [18, 41]. In brief, fish meal, soybean meal, and krill meal were mixed thoroughly with other ingredients. Wheat flour was used as starch, and diatomaceous earth was used as filter to balance in the diets. Wheat flour and alginate were gelatinized in boiling water (about 25% of total dried weight basis) before being mixed with other ingredients. Other ingredients were then mixed thoroughly with the gelatinized solution; thereafter the mixed (paste) was heated in boiled water bath again for about 2 min. The paste was shaped into sheets about 1.0 mm thickness and then cut into 1.2 cm2 /pieces and dried naturally in laboratory conditions for about 1–2 h. The pieces were then sealed in a plastic sample bag and stored at −4°C until use.

Each limpet was randomly placed into its own colander of 20 cm diameter (**Figure 8**). The colanders were placed in aquaria (150 L) with a recycled water flow rate (15 L min<sup>−</sup><sup>1</sup> ). Nice limpets were used for diet, and the experiment was run for 90 days.

The growth of animals in weight (g) and shell length (cm) was measured monthly. The growth was expressed in terms of specific growth rate (SGR), weight gain, and shell length increasing. The shell length was measured with an electronic digital caliper (0.01 μm), and the weight was determined with an electronic scale (0.01 g error) for every 4 weeks:

SGR = {(lnWf − lnWi)/T} × 100, where Wf is the final weight, Wi is the initial weight, and T is the total day of the experiment.

The result showed that the growth response of *C. sandwicensis* in terms of weight gain (%) of animals in dietary protein trial 2 was fitted into quadratic models (**Figure 9**). The best fit for the estimation of optimal protein level could be described as Y = −0.0003x<sup>2</sup> + 0.234x − 21.8 (R2 = 0.96). The trend of growth showed that maximum weight gain appeared to be about 35% dietary protein.

The response of *C. sandwicensis* in weight gain to dietary carbohydrate levels was then fitted to quadratic models (**Figure 10**). It shows that the weight gains of *C. sandwicensis* progressively increased and reached their maximum value at a carbohydrate level of about 27%, which could probably be described as Y = −0.0012x2 + 0.64x −56.7 with the correlation value of R<sup>2</sup> = 0.91.

**97**

**Figure 10.**

*Reproductive Biology, Seed Production, and Culture of the Hawaiian Limpet* Cellana*…*

*Experimental colander with an* C. sandwicensis *on it; a small square is a piece of feed.*

*Relationship between weight gain and dietary protein level of trial 2 for* C. sandwicensis *for 60 days.*

*Relationship between weight gain and dietary carbohydrate level for* C. sandwicensis*.*

*DOI: http://dx.doi.org/10.5772/intechopen.87128*

**Figure 8.**

**Figure 9.**

*Reproductive Biology, Seed Production, and Culture of the Hawaiian Limpet* Cellana*… DOI: http://dx.doi.org/10.5772/intechopen.87128*

**Figure 8.** *Experimental colander with an* C. sandwicensis *on it; a small square is a piece of feed.*

**Figure 9.**

*Invertebrates - Ecophysiology and Management*

**Essential AA**

diets compared to the tissue.

*sandwicensis* tissue except for Arg and Thr which were lower in the experimental

*The A/E ratio [(each EAA/total EAA) × 1000] amino acids of dietary protein and animal tissue.*

The process of feed preparation for extrusion of all diets was based on the methods described by the previous study [18, 41]. In brief, fish meal, soybean meal, and krill meal were mixed thoroughly with other ingredients. Wheat flour was used as starch, and diatomaceous earth was used as filter to balance in the diets. Wheat flour and alginate were gelatinized in boiling water (about 25% of total dried weight basis) before being mixed with other ingredients. Other ingredients were then mixed thoroughly with the gelatinized solution; thereafter the mixed (paste) was heated in boiled water bath again for about 2 min. The paste was shaped into

in laboratory conditions for about 1–2 h. The pieces were then sealed in a plastic

Each limpet was randomly placed into its own colander of 20 cm diameter (**Figure 8**). The colanders were placed in aquaria (150 L) with a recycled water flow

The growth of animals in weight (g) and shell length (cm) was measured monthly. The growth was expressed in terms of specific growth rate (SGR), weight gain, and shell length increasing. The shell length was measured with an electronic digital caliper (0.01 μm), and the weight was determined with an electronic scale

SGR = {(lnWf − lnWi)/T} × 100, where Wf is the final weight, Wi is the initial

The result showed that the growth response of *C. sandwicensis* in terms of weight gain (%) of animals in dietary protein trial 2 was fitted into quadratic models (**Figure 9**). The best fit for the estimation of optimal protein level could

+ 0.234x − 21.8 (R2

showed that maximum weight gain appeared to be about 35% dietary protein. The response of *C. sandwicensis* in weight gain to dietary carbohydrate levels was then fitted to quadratic models (**Figure 10**). It shows that the weight gains of *C. sandwicensis* progressively increased and reached their maximum value at a carbohydrate level of about 27%, which could probably be described as

+ 0.64x −56.7 with the correlation value of R<sup>2</sup>

). Nice limpets were used for diet, and the experiment was run for

**Preliminary protein trial (trial 1) Second protein trial Tissue 270 320 370 420 470 210 300 350 500**

Arg 224 123 118 129 125 139 208 198 190 173 His 33.8 39.5 37.4 41.5 39.9 44.6 29.1 29.4 29.8 32.3 Ile 81.4 88.7 89.5 87.7 88.6 99.5 80.4 80.9 81.2 80.8 Leu 146 158 157 155 156 177 149 148 147 142 Lys 69.2 159 170 149 160 178 110 112 114 116 Met/Cys 68.3 74.1 75.1 77.6 77.7 87.0 51.2 53.5 55.4 57.4 Phe/Tyr 123 158 161 159 160 179 187 194 200 219 Thr 136 97.9 92.0 97.8 93.4 98.0 78.9 79.1 79.3 78.4 Val 117 103 99.9 103 99.8 111 107 105 104 100

/pieces and dried naturally

= 0.96). The trend of growth

= 0.91.

sheets about 1.0 mm thickness and then cut into 1.2 cm2

sample bag and stored at −4°C until use.

(0.01 g error) for every 4 weeks:

be described as Y = −0.0003x<sup>2</sup>

weight, and T is the total day of the experiment.

rate (15 L min<sup>−</sup><sup>1</sup>

90 days.

**Table 6.**

**96**

Y = −0.0012x2

*Relationship between weight gain and dietary protein level of trial 2 for* C. sandwicensis *for 60 days.*

**Figure 10.** *Relationship between weight gain and dietary carbohydrate level for* C. sandwicensis*.*

### **5.3 Energy requirement**

Recent study found that limpet *C. sandwicensis* required no specific effect on dietary protein to energy (PE) ratio when the animal was offered with diet containing various PE ratios ranging from 87.2 to 102.9 mg/kcal [43]. There was no significant effect on growth performance of limpet among the diets, but a PE ratio of 87.2 mg/kcal produced the best tissue growth.
