**3. Seed production**

## **3.1 Maturation culture**

Two experiments were conducted to induce final maturation of limpet *C. sandwicensis* in the laboratory conditions. The first trial is formulated feed with the addition of arachidonic acid (ARA) into diet. The experimental diet is described and shown in the previous studies [12, 13]. In brief, limpet was fed with three diets including: control diet (without additional ARA), diet 2 containing 0.20% ARA, and diet 3 (0.33% ARA). Nice adult *C. sandwicensis* (3.07 ± 0.22 cm in shell length) species were fed with these diets for 90 days. Each limpet was randomly placed into its own colander of 20 cm diameter. The colanders were placed in aquaria (150 L) with a recycled water flow rate (15 L min<sup>−</sup><sup>1</sup> ). Seawater was exchanged weekly of about 30%. The experiment was conducted under ambient photoperiod and temperature ranging from 23 to 25°C. Salinity was maintained at 35. Prior to the beginning of the experiment, several limpets were randomly selected among the group and dissected to obtain initial GSI and gonad development status. During the experimental period, three animals were randomly examined monthly to assess maturation status as described in Section 2.3. At the end, their gonads were extracted and weighed to obtain the gonad's weight for the calculation of the GSI.

The result showed that gonad of limpet fed with diet containing ARA increased three times higher than the GSI of animals that fed with the control diet (**Table 3**). There was a significant difference (P < 0.05) in GSI of animal that fed with diet incorporated with ARA as compared to those fed with control diet. There was no significant difference in GSI of those limpets fed with both diets 0.2% ARA and 0.33% ARA with the same ARA/EPA ratio of 0.70.

In the following trial, the final maturation of limpet was induced by using OvaRH (Syndel Laboratories Ltd. Canada) which is a synthetic salmon GnRH analog (sGnRHa). The hormone was injected directly into the gonad of limpets. Twelve limpets (9.17 ± 3.17 g/ind.) were tagged and weighed. Each limpet received a total of five to seven injections, at 7-day intervals at dose of 250 ng/g body weight (BW). The control treatment was run without hormone injection. During the period, experimental limpets were held on biofilm aquaria with water movement by an aquarium biofilter pump (567 L per hour). The maturation of limpet was examined weekly by randomly selected and sacrificed two limpets in each treatment. Their gonads were collected for calculation of GSI, and a piece of gonad was immediately fixed in 10% formalin for histological examination. The experiment was conducted during the final maturation and spawning season.


*a,b The same letters in the row indicate no significant difference in eggs sizes, the empty grids indicate no egg was observed.*

**89**

**Figure 4.**

*Gonadosomatic growth of limpet* C. sandwicensis *by hormone induction.*

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

with three injections (**Figure 4**). The GSI of limpet *C. sandwicensis* increased rapidly from initial 12.0–28.3% after the third injection and reached 32.9% for the final maturation stage after the fourth injection. GSI of limpets in the control group

The results showed that the gonad of limpet was rapidly increased after 3 weeks

It is reported that the reproduction of aquatic animal is controlling by external and internal parameter factors such as photoperiods, food availability and hormone regulation. Therefore, our study focused on three factors including photoperiod, nutrient requirement as the ratio of highly unsaturated fatty acid arachidonic acid (ARA) and eicosapentaenoic acid (EPA), and gonadotropin-releasing hormone. In our hands photoperiod is important for the maturation of limpet. The effect of photoperiod may be seen more clearly in the following maturation trials when the experiment was run before and during the spawning season [11, 12]. This showed the role of environmental conditions in the regulation of the timing of the reproductive cycle of limpet. For example, the limpet *C. exarata* was found that the reproductive resting phase coincided with day length above 13 h [10], suggesting that a higher 13 h day length could inhibit gametogenesis of limpet. Photoperiod has also been reported to be influential on reproductive cycles of many marine

Final maturation of *C. sandwicensis* was successfully induced by the addition of arachidonic acid (ARA) into diet to obtain an appropriate ARA per eicosapentaenoic acid (EPA) ratio. Arachidonic acid serves as a precursor for the synthetic of prostaglandins which are functional for reproductive process [16]. Prostaglandins play a critical role during the ovulatory process in teleost fishes [17]. Our previous study found that *C. sandwicensis* preferred to feed on benthic diatoms in the wild [18]. Several benthic diatoms such as *Nitzschia*, *Amphora*, and *Navicula* were predominant in the stomach content of *C. sandwicensis*, and literature studies found that these diatoms contained high level of ARA and EPA [19–21]. Our review found that an ARA/EPA ratio of about 0.70 was found in several benthic diatoms such as *Nitzschia* and *Chaetoceros* suggesting that this would be a good starting point for experimental diet on adult limpet *C.* 

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

invertebrates [14, 15].

remained the same until finishing the experiment.

#### **Table 3.**

*Gonadal somatic index and egg size of limpet fed different dietary ARA for 95 days.*

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

The results showed that the gonad of limpet was rapidly increased after 3 weeks with three injections (**Figure 4**). The GSI of limpet *C. sandwicensis* increased rapidly from initial 12.0–28.3% after the third injection and reached 32.9% for the final maturation stage after the fourth injection. GSI of limpets in the control group remained the same until finishing the experiment.

It is reported that the reproduction of aquatic animal is controlling by external and internal parameter factors such as photoperiods, food availability and hormone regulation. Therefore, our study focused on three factors including photoperiod, nutrient requirement as the ratio of highly unsaturated fatty acid arachidonic acid (ARA) and eicosapentaenoic acid (EPA), and gonadotropin-releasing hormone. In our hands photoperiod is important for the maturation of limpet. The effect of photoperiod may be seen more clearly in the following maturation trials when the experiment was run before and during the spawning season [11, 12]. This showed the role of environmental conditions in the regulation of the timing of the reproductive cycle of limpet. For example, the limpet *C. exarata* was found that the reproductive resting phase coincided with day length above 13 h [10], suggesting that a higher 13 h day length could inhibit gametogenesis of limpet. Photoperiod has also been reported to be influential on reproductive cycles of many marine invertebrates [14, 15].

Final maturation of *C. sandwicensis* was successfully induced by the addition of arachidonic acid (ARA) into diet to obtain an appropriate ARA per eicosapentaenoic acid (EPA) ratio. Arachidonic acid serves as a precursor for the synthetic of prostaglandins which are functional for reproductive process [16]. Prostaglandins play a critical role during the ovulatory process in teleost fishes [17]. Our previous study found that *C. sandwicensis* preferred to feed on benthic diatoms in the wild [18]. Several benthic diatoms such as *Nitzschia*, *Amphora*, and *Navicula* were predominant in the stomach content of *C. sandwicensis*, and literature studies found that these diatoms contained high level of ARA and EPA [19–21]. Our review found that an ARA/EPA ratio of about 0.70 was found in several benthic diatoms such as *Nitzschia* and *Chaetoceros* suggesting that this would be a good starting point for experimental diet on adult limpet *C.* 

**Figure 4.** *Gonadosomatic growth of limpet* C. sandwicensis *by hormone induction.*

*Invertebrates - Ecophysiology and Management*

with a recycled water flow rate (15 L min<sup>−</sup><sup>1</sup>

0.33% ARA with the same ARA/EPA ratio of 0.70.

during the final maturation and spawning season.

Two experiments were conducted to induce final maturation of limpet *C. sandwicensis* in the laboratory conditions. The first trial is formulated feed with the addition of arachidonic acid (ARA) into diet. The experimental diet is described and shown in the previous studies [12, 13]. In brief, limpet was fed with three diets including: control diet (without additional ARA), diet 2 containing 0.20% ARA, and diet 3 (0.33% ARA). Nice adult *C. sandwicensis* (3.07 ± 0.22 cm in shell length) species were fed with these diets for 90 days. Each limpet was randomly placed into its own colander of 20 cm diameter. The colanders were placed in aquaria (150 L)

of about 30%. The experiment was conducted under ambient photoperiod and temperature ranging from 23 to 25°C. Salinity was maintained at 35. Prior to the beginning of the experiment, several limpets were randomly selected among the group and dissected to obtain initial GSI and gonad development status. During the experimental period, three animals were randomly examined monthly to assess maturation status as described in Section 2.3. At the end, their gonads were extracted and weighed to obtain the gonad's weight for the calculation of the GSI. The result showed that gonad of limpet fed with diet containing ARA increased three times higher than the GSI of animals that fed with the control diet (**Table 3**). There was a significant difference (P < 0.05) in GSI of animal that fed with diet incorporated with ARA as compared to those fed with control diet. There was no significant difference in GSI of those limpets fed with both diets 0.2% ARA and

In the following trial, the final maturation of limpet was induced by using OvaRH (Syndel Laboratories Ltd. Canada) which is a synthetic salmon GnRH analog (sGnRHa). The hormone was injected directly into the gonad of limpets. Twelve limpets (9.17 ± 3.17 g/ind.) were tagged and weighed. Each limpet received a total of five to seven injections, at 7-day intervals at dose of 250 ng/g body weight (BW). The control treatment was run without hormone injection. During the period, experimental limpets were held on biofilm aquaria with water movement by an aquarium biofilter pump (567 L per hour). The maturation of limpet was examined weekly by randomly selected and sacrificed two limpets in each treatment. Their gonads were collected for calculation of GSI, and a piece of gonad was immediately fixed in 10% formalin for histological examination. The experiment was conducted

**Day Parameter Control 0.2% ARA 0.33% ARA** Initial GSI (%) 3.10 ± 2.48 3.10 ± 2.48 3.10 ± 2.48 45 GSI (%) 5.94 ± 5.65 11.0 ± 6.82 8.13 ± 0.52

75 GSI (%) 6.11 24.5 23.7

95 GSI (%) 4.21 ± 0.82a 10.8 ± 4.47<sup>b</sup> 15.5 ± 5.47<sup>b</sup> *a,b The same letters in the row indicate no significant difference in eggs sizes, the empty grids indicate no egg was* 

*Gonadal somatic index and egg size of limpet fed different dietary ARA for 95 days.*

Egg size (μm) — 118 ± 9.71a 121 ± 9.42a

Egg size (μm) — 123 ± 4.23a 121 ± 5.93a

). Seawater was exchanged weekly

**3. Seed production**

**3.1 Maturation culture**

**88**

*observed.*

**Table 3.**

*sandwicensis*. Thus, experimental trial on different ARA to EPA ratios of 0.70 was conducted; as a result, *C. sandwicensis* reached final maturation [12]. This result provided significant data on the effect of ARA/EPA on maturation of limpet and gastropods as well.

GnRH-like peptides that existed in the central nervous system and peripheral chemosensory organ of sea hare *Aplysia* were detectable by antisera against mGnRH [22]. These GnRH-like peptides controlled egg laying of *Aplysia*. For abalone, studies had demonstrated the existence of GnRH-like peptides in the neural ganglia and ovary of abalone [23, 24], and the existence of GnRH-like peptide in the neural ganglia was determined by using immunohistochemistry and reverse-phase highperformance liquid chromatography [24, 25].

The Hawaiian limpet *C. sandwicensis* were also induced to final maturation using salmon GnRH analog (sGnRHa) at dose of 250 ng/g BW. The sGnRHa stimulated gonad development and final maturation in limpet in 5 weeks when they injected at 7-day intervals at low concentration 250 ng/g BW [13]. The GSI increased significantly from the third week of injection and developed rapidly and reached to the maximum level after 4 weeks of injection as compared to the control, which did not show gonadal development (**Figure 4**). This shows that GnRH also involved in regulating reproductive development in limpet. Similar finding was also reported in abalone; the adult abalone was induced to final maturation in 5 weeks by weekly injection of these GnRHs at low dose (250 ng/g BW) and induced spawning at higher dose of 1000 ng/g BW [23]. The existence of GnRH-like peptides in the neural ganglia and ovary of the abalone [23, 24] and the existence of GnRH-like peptide in the neural ganglia were determined by using immunohistochemistry and reverse-phase high-performance liquid chromatography [24, 25]. GnRH-like peptides that existed in the central nervous system and peripheral chemosensory organ of sea hare *Aplysia* were detectable by antisera against mGnRH [22]. These GnRH-like peptides controlled egg laying of *Aplysia*. The mammalian GnRH analog was known to stimulate maturation and induced spawning in abalone [23]. The responses of molluskan to environmental cues are controlled by hormones, and the principal sources of hormones within molluscan nervous system are neurosecretory cells [26]. Our results suggest that diatom blooms may be the environment cues. GnRH could stimulate reproductive process by acting directly on the gonad in limpet. Both limpet and abalone are marine gastropod species. This process would be also facilitated by the reproductive photoperiod, and/or the right photoperiod would stimulate the increased secretion of luteinizing hormone and follicle-stimulating hormone that enhances the reproductive process in limpet *C. sandwicensis*.

#### **3.2 Spawning induction**

Two different spawning methods were conducted to examine the optimal method of spawning for the Hawaiian limpet. The first method was conducted using hydrogen peroxide. Hydrogen peroxide is a traditional method used for spawning induction in abalone. **Figure 5** shows the addition of H2O2 to seawater is believed to produce hydroperoxy free radicals (HOO<sup>−</sup>) and peroxy radicals (OO<sup>−</sup>); these radicals of activated oxygen suitable for the cyclooxygenase catalyzed addition of prostaglandin [27–29].

Experimental animal. Limpet broodstock (>3.0 cm in shell length) were collected at the shoreline from a remote area on Oahu island. They were held on biofilm aquaria for 2 days before use for the experiment. Sexually matured broodstock were selected as described in Section 2.3. Eight matured limpets

**91**

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

(approximate sex ratio, male:female = 1:1) were selected from the holding tank then placed into a spawning container (3 L) with fresh clean seawater for each trial. The spawning container was gently aerated, and pH in the spawning container was first adjusted to pH 9–9.5 by 1 M of Tris-base for about an hour. Thereafter, stock 6% of H2O2 was slowly added to spawning container to obtain the desired concentration. The broodstock were exposed to five different concentrations of H2O2. These are control (without H2O2), 0.6 × 10<sup>−</sup><sup>2</sup>

45 min depending on the response of animals. Then the spawning activities were observed. The results show that the highest number of spawners was induced at

These results highlighted the nonspecific toxic effect of the chemical. Similarly, at this level all animals were dead eventually, but this level induced 10–15% spawning [10]. However, we concluded that this method may not be used as a practical method and not recommended for spawning induction in limpet. This was due to a nonspecific effect, and the broodstock eventually died within a week after being exposed to H2O2. The second method with GnRH at dose of 1000 ng/g BW may be considered as the most practical induction spawning method for limpet because

This could probably be due to the instability of H2O2. The H2O2 was fresher, and we ordered before use. No mortality occurred in the 24 h after spawning at

trial at this concentration, all animals died within a week after exposure to H2O2. We used this level in further spawning the trials. The limpet may have released gametes because they thought they were dying. This is a well-known phenomenon among fruit trees that are sometimes even sprayed with herbicide to get them to fruit. Under the microscope we found that a high percentage of immature eggs with different sizes, these eggs were not successfully fertilized. This concluded that

Induction of spawning by using sGnRHa is an applicable technique and was the most practical method. There were no mortalities after injection of sGnRHa, and 100% animals survived after spawning. However, it is noticed that spawning induc-

Different larval development of *C. sandwicensis* is shown in **Table 4**. There were 18 distinct stages of larval development of *C. sandwicensis* in this study. Spawned eggs were 111 ± 5.64 μm (**Figure 6a**). The first polar body appeared in about 30–45 min after spawning indicating fertilized eggs (**Figure 6b**). The two-cell

% and no mortality occurred in the 24 h after spawning. Most of animals

%, and 1.80 × 10<sup>−</sup><sup>2</sup>

*Mechanism of hydrogen peroxide in spawning induction of mollusk species.*

% and 1.80 × 10<sup>−</sup><sup>2</sup>

there were no mortalities occurring after spawning.

hydrogen peroxide is not a practical method.

**3.3 Embryonic and larval development**

%. This led to the thought that 0.6 × 10<sup>−</sup><sup>2</sup>

tion of limpet by GnRH is effective only on ripe *C. sandwicensis*.

%,

%. The exposing time ranged from 5 to

% in the 24 h after exposure to these levels.

% may be safe, but in the last

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

1.20%, 1.49 × 10<sup>−</sup><sup>2</sup>

died at 1.49 × 10<sup>−</sup><sup>2</sup>

0.6 × 10<sup>−</sup><sup>2</sup>

**Figure 5.**

0.6 × 10<sup>−</sup><sup>2</sup>

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

**Figure 5.** *Mechanism of hydrogen peroxide in spawning induction of mollusk species.*

(approximate sex ratio, male:female = 1:1) were selected from the holding tank then placed into a spawning container (3 L) with fresh clean seawater for each trial. The spawning container was gently aerated, and pH in the spawning container was first adjusted to pH 9–9.5 by 1 M of Tris-base for about an hour. Thereafter, stock 6% of H2O2 was slowly added to spawning container to obtain the desired concentration. The broodstock were exposed to five different concentrations of H2O2. These are control (without H2O2), 0.6 × 10<sup>−</sup><sup>2</sup> %, 1.20%, 1.49 × 10<sup>−</sup><sup>2</sup> %, and 1.80 × 10<sup>−</sup><sup>2</sup> %. The exposing time ranged from 5 to 45 min depending on the response of animals. Then the spawning activities were observed. The results show that the highest number of spawners was induced at 0.6 × 10<sup>−</sup><sup>2</sup> % and no mortality occurred in the 24 h after spawning. Most of animals died at 1.49 × 10<sup>−</sup><sup>2</sup> % and 1.80 × 10<sup>−</sup><sup>2</sup> % in the 24 h after exposure to these levels. These results highlighted the nonspecific toxic effect of the chemical. Similarly, at this level all animals were dead eventually, but this level induced 10–15% spawning [10]. However, we concluded that this method may not be used as a practical method and not recommended for spawning induction in limpet. This was due to a nonspecific effect, and the broodstock eventually died within a week after being exposed to H2O2. The second method with GnRH at dose of 1000 ng/g BW may be considered as the most practical induction spawning method for limpet because there were no mortalities occurring after spawning.

This could probably be due to the instability of H2O2. The H2O2 was fresher, and we ordered before use. No mortality occurred in the 24 h after spawning at 0.6 × 10<sup>−</sup><sup>2</sup> %. This led to the thought that 0.6 × 10<sup>−</sup><sup>2</sup> % may be safe, but in the last trial at this concentration, all animals died within a week after exposure to H2O2. We used this level in further spawning the trials. The limpet may have released gametes because they thought they were dying. This is a well-known phenomenon among fruit trees that are sometimes even sprayed with herbicide to get them to fruit. Under the microscope we found that a high percentage of immature eggs with different sizes, these eggs were not successfully fertilized. This concluded that hydrogen peroxide is not a practical method.

Induction of spawning by using sGnRHa is an applicable technique and was the most practical method. There were no mortalities after injection of sGnRHa, and 100% animals survived after spawning. However, it is noticed that spawning induction of limpet by GnRH is effective only on ripe *C. sandwicensis*.

#### **3.3 Embryonic and larval development**

Different larval development of *C. sandwicensis* is shown in **Table 4**. There were 18 distinct stages of larval development of *C. sandwicensis* in this study. Spawned eggs were 111 ± 5.64 μm (**Figure 6a**). The first polar body appeared in about 30–45 min after spawning indicating fertilized eggs (**Figure 6b**). The two-cell

*Invertebrates - Ecophysiology and Management*

performance liquid chromatography [24, 25].

gastropods as well.

*sandwicensis*. Thus, experimental trial on different ARA to EPA ratios of 0.70 was conducted; as a result, *C. sandwicensis* reached final maturation [12]. This result provided significant data on the effect of ARA/EPA on maturation of limpet and

GnRH-like peptides that existed in the central nervous system and peripheral chemosensory organ of sea hare *Aplysia* were detectable by antisera against mGnRH [22]. These GnRH-like peptides controlled egg laying of *Aplysia*. For abalone, studies had demonstrated the existence of GnRH-like peptides in the neural ganglia and ovary of abalone [23, 24], and the existence of GnRH-like peptide in the neural ganglia was determined by using immunohistochemistry and reverse-phase high-

The Hawaiian limpet *C. sandwicensis* were also induced to final maturation using salmon GnRH analog (sGnRHa) at dose of 250 ng/g BW. The sGnRHa stimulated gonad development and final maturation in limpet in 5 weeks when they injected at 7-day intervals at low concentration 250 ng/g BW [13]. The GSI increased significantly from the third week of injection and developed rapidly and reached to the maximum level after 4 weeks of injection as compared to the control, which did not show gonadal development (**Figure 4**). This shows that GnRH also involved in regulating reproductive development in limpet. Similar finding was also reported in abalone; the adult abalone was induced to final maturation in 5 weeks by weekly injection of these GnRHs at low dose (250 ng/g BW) and induced spawning at higher dose of 1000 ng/g BW [23]. The existence of GnRH-like peptides in the neural ganglia and ovary of the abalone [23, 24] and the existence of GnRH-like peptide in the neural ganglia were determined by using immunohistochemistry and reverse-phase high-performance liquid chromatography [24, 25]. GnRH-like peptides that existed in the central nervous system and peripheral chemosensory organ of sea hare *Aplysia* were detectable by antisera against mGnRH [22]. These GnRH-like peptides controlled egg laying of *Aplysia*. The mammalian GnRH analog was known to stimulate maturation and induced spawning in abalone [23]. The responses of molluskan to environmental cues are controlled by hormones, and the principal sources of hormones within molluscan nervous system are neurosecretory cells [26]. Our results suggest that diatom blooms may be the environment cues. GnRH could stimulate reproductive process by acting directly on the gonad in limpet. Both limpet and abalone are marine gastropod species. This process would be also facilitated by the reproductive photoperiod, and/or the right photoperiod would stimulate the increased secretion of luteinizing hormone and follicle-stimulating hormone that enhances the reproductive process in

Two different spawning methods were conducted to examine the optimal method of spawning for the Hawaiian limpet. The first method was conducted using hydrogen peroxide. Hydrogen peroxide is a traditional method used for spawning induction in abalone. **Figure 5** shows the addition of H2O2 to seawater is believed to produce hydroperoxy free radicals (HOO<sup>−</sup>) and peroxy radicals (OO<sup>−</sup>); these radicals of activated oxygen suitable for the cyclooxygenase catalyzed addi-

Experimental animal. Limpet broodstock (>3.0 cm in shell length) were collected at the shoreline from a remote area on Oahu island. They were held on biofilm aquaria for 2 days before use for the experiment. Sexually matured broodstock were selected as described in Section 2.3. Eight matured limpets

**90**

limpet *C. sandwicensis*.

**3.2 Spawning induction**

tion of prostaglandin [27–29].


#### **Table 4.**

*Embryonic and larval development of limpet* C. sandwicensis *at (22°C).*

stage (stage 3) was found within 2 h after spawning (**Figure 6b**). About 10 h post-fertilization, protrochophore stage with cilia appeared (**Figure 6b**). Larvae started hatching out at 12–14 h. The length and width of free swimming larvae were 85.5 ± 9.5 μm and 79.6 ± 7.9 μm, respectively. Larvae continued to develop velum from cilia, and apical region became flat for shell formation in about 18–20 h after spawning (**Figure 6o** and **p**).

#### **3.4 Larval rearing**

Several studies [10, 11] on settlement of *C. sandwicensis* larvae on different combinations of diatom and pelagic algae were conducted. The results showed that mixture of diatom *Amphora* and pelagic *Palova* induced the highest survival rate (21.7 ± 7.07%) of settled larvae. Diatom *Nitzschia* seemed not to be preferred by *C. sandwicensis* larvae because the observation noticed that high mortalities occurred from 4 to 6 days. Pelagic algal *Palova* may be preferred over *Isochrysis*. Among the surviving larvae, all of them settled after 3 days and fed on diatoms. On the other hand, different plate substrates reported to be affected on larval settlement of gastropod species, such as abalone of roughened plexiglass, and corrugated plastic sheet, and the rubberized canvas seemed to be preferred for settlement over fibrocement board. The results of our study were higher than previous study which was attempting to induce the settlement of Hawaiian limpet *C. sandwicensis* larvae on different substrata [10]. She found that mylar plastic and plexiglass induced a significantly higher larval settlement compared to glass, smooth and rough basalt

**93**

*sandwicensis*.

**4. Culture system**

solves this problem (**Figure 7**).

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

rocks, coral skeleton, and textured and untextured plastic. However, the settlement rates were very low ranging from 1.58 to 7.73%. It could probably be due to inappro-

*Embryonic development stage of Hawaiian limpet* C. sandwicensis*. (a) Spawned egg and stage 1 spermatozoids; (b) stages 2 and 3, discharge of polar body and first cleavage (2 cells), and stage 4, second cleavage (4 cells); (c) stage 5, third cleavage (8 cells); (d) stages 6–7 morula and blastula; (e) stage 8, gastrula; (f and g) stage 9, appearance of cilia forming the prototrochal; (h) stage 10, trochophore larvae ready for hatch out; (k) stage 11, trochophore larvae free swimming; (m) stage 12, trochophore free swimming larvae with extend cilia; (n) stage 13, complete girdle, cilia develop and apical; (o) stage 14, early larvae shell formation; (p) stages 15 and 16, veliger larvae exhibiting flat apical from larval shell and complete developed velum and* 

*cilia; (q) stage 17, appearance of eye spot; (r) stage 18, appearance of muscle attached.*

The role of benthic diatoms *Navicula*, *Amphora*, *Nitzschia*, and others were reported to best diatom species induced the settlement and metamorphosis of abalone larvae [19, 30, 31]. The effects of different benthic diatoms grown on different plate substrates on metamorphosis of the tropical abalone *Haliotis asinina* were reported by [31]. They found that mixture of diatoms induced significantly higher metamorphosis rate of abalone larvae than other group including *Amphora*, *Amphora* + *Nitzschia*, and *Nitzschia* with any plate substrate. This suggested that mixture of benthic diatoms is better than single once. Another study also found that a mixture of benthic diatoms consisting of *Navicula* and *Amphora* produced a significantly higher growth and survival rates for abalone larvae *H. discus hannai* than monocultures benthic diatoms [19]. The report showed that the monocultures of benthic diatoms produced a poor growth and did not support survival for more than 2 weeks especially *Nitzschia*. The authors also stated this could be due to the difference in nutritional value of these benthic diatoms. In particular, the EPA value in *Navicula* and *Amphora* was reported to be higher than the value in *Nitzschia* [19]. These results support our study that mixture of diatom and pelagic algae induced better survival rate of Hawaiian limpet and mixture of diatom *Amphora* and pelagic *Palova* would be recommended for future use of larval rearing of the *C.* 

There is a lack of study on aquaculture system for limpet species as well as the Hawaiian limpet *C. sandwicensis*. We have attempted to raise the limpet *C. sandwicensis* in system with water flow through, but transfer mortality is a challenge because the animals cling tightly to the cultured tank walls. It was hard to get them off the wall without injury. Later we found that putting plastic sheets as tank liners

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

priate benthic diatoms.

**Figure 6.**

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

#### **Figure 6.**

*Invertebrates - Ecophysiology and Management*

**Sequent stage**

stage (stage 3) was found within 2 h after spawning (**Figure 6b**). About 10 h post-fertilization, protrochophore stage with cilia appeared (**Figure 6b**). Larvae started hatching out at 12–14 h. The length and width of free swimming larvae were 85.5 ± 9.5 μm and 79.6 ± 7.9 μm, respectively. Larvae continued to develop velum from cilia, and apical region became flat for shell formation in about 18–20 h after

*Embryonic and larval development of limpet* C. sandwicensis *at (22°C).*

16 Exhibiting flat apical from larval shell and complete developed velum and

cilia

17 Eye spot 20.00–21.30 18 Completed muscle formation 21.30–24.0

**Embryo, larval development stage Time (h)**

18.00–20.00

 Fertilization 0.00 Discharge of first polar 0.30–0.45 First cleavage (2 cells) 1.00–1.30 Second cleavage (4 cells) 2.00–2.30 Third cleavage (8 cells) 3.00–3.30 Morula 3.30–4.00 Blastula 4.00–4.30 Gastrula 4.30–5.00 Appearance of cilia forming prototrochal 8.00–10.00 Trochophore ready to hatch out 10.30–11.30 Trochophore free swimming larvae 12.00–14.00 Continue extended cilia 13.30–14.30 Completion of girdle and cilia develop 14.30–16.00 Larval shell formation 14.30–16.00 Advance larvae shell formation 16.30–18.00

Several studies [10, 11] on settlement of *C. sandwicensis* larvae on different combinations of diatom and pelagic algae were conducted. The results showed that mixture of diatom *Amphora* and pelagic *Palova* induced the highest survival rate (21.7 ± 7.07%) of settled larvae. Diatom *Nitzschia* seemed not to be preferred by *C. sandwicensis* larvae because the observation noticed that high mortalities occurred from 4 to 6 days. Pelagic algal *Palova* may be preferred over *Isochrysis*. Among the surviving larvae, all of them settled after 3 days and fed on diatoms. On the other hand, different plate substrates reported to be affected on larval settlement of gastropod species, such as abalone of roughened plexiglass, and corrugated plastic sheet, and the rubberized canvas seemed to be preferred for settlement over fibrocement board. The results of our study were higher than previous study which was attempting to induce the settlement of Hawaiian limpet *C. sandwicensis* larvae on different substrata [10]. She found that mylar plastic and plexiglass induced a significantly higher larval settlement compared to glass, smooth and rough basalt

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spawning (**Figure 6o** and **p**).

**3.4 Larval rearing**

**Table 4.**

*Embryonic development stage of Hawaiian limpet* C. sandwicensis*. (a) Spawned egg and stage 1 spermatozoids; (b) stages 2 and 3, discharge of polar body and first cleavage (2 cells), and stage 4, second cleavage (4 cells); (c) stage 5, third cleavage (8 cells); (d) stages 6–7 morula and blastula; (e) stage 8, gastrula; (f and g) stage 9, appearance of cilia forming the prototrochal; (h) stage 10, trochophore larvae ready for hatch out; (k) stage 11, trochophore larvae free swimming; (m) stage 12, trochophore free swimming larvae with extend cilia; (n) stage 13, complete girdle, cilia develop and apical; (o) stage 14, early larvae shell formation; (p) stages 15 and 16, veliger larvae exhibiting flat apical from larval shell and complete developed velum and cilia; (q) stage 17, appearance of eye spot; (r) stage 18, appearance of muscle attached.*

rocks, coral skeleton, and textured and untextured plastic. However, the settlement rates were very low ranging from 1.58 to 7.73%. It could probably be due to inappropriate benthic diatoms.

The role of benthic diatoms *Navicula*, *Amphora*, *Nitzschia*, and others were reported to best diatom species induced the settlement and metamorphosis of abalone larvae [19, 30, 31]. The effects of different benthic diatoms grown on different plate substrates on metamorphosis of the tropical abalone *Haliotis asinina* were reported by [31]. They found that mixture of diatoms induced significantly higher metamorphosis rate of abalone larvae than other group including *Amphora*, *Amphora* + *Nitzschia*, and *Nitzschia* with any plate substrate. This suggested that mixture of benthic diatoms is better than single once. Another study also found that a mixture of benthic diatoms consisting of *Navicula* and *Amphora* produced a significantly higher growth and survival rates for abalone larvae *H. discus hannai* than monocultures benthic diatoms [19]. The report showed that the monocultures of benthic diatoms produced a poor growth and did not support survival for more than 2 weeks especially *Nitzschia*. The authors also stated this could be due to the difference in nutritional value of these benthic diatoms. In particular, the EPA value in *Navicula* and *Amphora* was reported to be higher than the value in *Nitzschia* [19]. These results support our study that mixture of diatom and pelagic algae induced better survival rate of Hawaiian limpet and mixture of diatom *Amphora* and pelagic *Palova* would be recommended for future use of larval rearing of the *C. sandwicensis*.

#### **4. Culture system**

There is a lack of study on aquaculture system for limpet species as well as the Hawaiian limpet *C. sandwicensis*. We have attempted to raise the limpet *C. sandwicensis* in system with water flow through, but transfer mortality is a challenge because the animals cling tightly to the cultured tank walls. It was hard to get them off the wall without injury. Later we found that putting plastic sheets as tank liners solves this problem (**Figure 7**).

**Figure 7.**

*A circular holding biofilm tank without plastic liner, and three aquaria with plastic sheer liner above, used for 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 aquaria without intermittent water sprayed or dump tanks.
