**1. Introduction**

Limpets are marine gastropods. They distribute at different intertidal zones of most oceans, from the upper littoral to the shallow subtidal on the rocky coasts. They feed by grazing on macroalgae, benthic diatom growing on rocky substrate because they attach themselves to rocks, and/or any substratum using pedal mucus and a muscular "foot," which also enables them to go against dangerous wave action, desiccation, and predator.

*Cellana* genus is a marine gastropod mollusk in the family Nacellidae [1]. This genus distributes in the temperate and tropical Indo-Pacific Oceans, Hawaii,

Australia, and New Zealand. Species are also found around the coasts of Japan, the Red Sea, Madagascar, South Africa, and the subantarctic island. There are more than 58 species of this genus. Among of those, many of them are of high economic value and aquaculture, for example, the two species *Cellana talcosa* and *Cellana sandwicensis* are expensive in Hawaii.

In Hawaii, there are three main endemic Hawaiian limpet species, called "opihi," including black foot or makaiauli (*Cellana exarata*), yellow foot or ālinalina (*C. sandwicensis*), and the largest species, giant limpet or kōʻele (*C. talcosa*) [2]. Natural ecological distribution of Hawaiian limpet is different intertidal zones of habitation on rocky shores. *C. exarata* is commonly found at higher intertidal zones, and *C. sandwicensis* is at low intertidal zones and rarely exposed by tide, whereas *C. talcosa* distributes in deep water [3–6]. These species are herbivorous grazers that feed on benthic microalgae, diatoms that growing hard substrates such as rocky substrates, death coral reef, and so on. They use teeth in their radula to graze on the toughest crustose coralline algae [3]. They are considered as highvalue food market and high-potential candidate species for commercial aquaculture. High commercial catch reduced significantly from 150,000 pounds in the 1900s to about 10,000 pounds in 1978 [7]. The scarcity has boost up prices to about \$200/gallon with shell on [8].

In addition to important food sources, these Hawaiian limpet species are also culturally important in Hawaiian society. Many people (opihi pickers) were asked to collect these opihi for parties or family gathering with high prices. Besides that limpet's shell also continue to be used as tools for scraping skin off taro plant and sweet potato and grating coconut meat before eating [9] and as decorative elements in jewelry.

The success of any aquaculture species depends on seed production in captivity. Understanding the completion of the life cycle of limpet would make limpet aquaculture sustainable. The first priority is to understand some reproductive characteristics of the limpet species. This would also provide us with better knowledge for breeding limpet in the hatchery. In this chapter, we describe the current finding on reproductive biology, seed production, nutrient requirement, and culture techniques for the Hawaiian limpet *Cellana sandwicensis*.

## **2. Some reproductive characteristics of Hawaiian limpet** *C. sandwicensis*

Reproductive characteristics of Hawaiian limpet *C. sandwicensis* have been reported by several studies [10–12]. The main focused reproductive criteria were spawning season, gonad development stage, fecundity, and maturity size.

#### **2.1 Spawning season**

A total of 266 specimens (**Table 1**) were sampled for a 1-year cycle (November 2011–December 2012) in Hawaii Island, and gonadal somatic index (GSI) was determined. The GSI was calculated according to equation GSI = (GW/BW) × 100, where GW is gonad weight and BW is body weight or soft body tissue. Gonad development stage was also evaluated by using histological examination. The result showed that the highest average GSI of *C. sandwicensis* was noticed between November and January. This suggests that spawning seasons of *C. sandwicensis* may occur right after this period, whereas the lowest GSI was found from March to August, this could probably be the resting season of the species. Similarly, the same GSI pattern (**Figure 1**) of males and females *C. sandwicensis* suggested synchronized spawning of male and female *C. sandwicensis* in the wild [10].

**85**

**2.2 Gonad development stage**

*data values from n individuals are presented as mean ± SD.*

male gonad (**Figure 2F**).

**2.3 Sexual determination**

The maturity stages of gonad development of Hawaiian limpet *C. sandwicensis* were reported and classified in **Table 2** and **Figure 2**. Multiple development stages (**Figure 2A**) were observed in the same ovary of female during the final maturation season. **Figure 2D** showed resting stages (April to August), because oocytes were not of clear formation from the ovary cell wall. Similar observation was made for gonad of the male (**Figure 2E**). The testes were densely packed with spermatozoa which appeared as dark blue stained by hematoxylin. Sperm were less densely in the

*Average size and GSI of sampled opihi for the reproductive cycle study from November 2011 to December 2012;* 

Sexual determination of all limpet species is not known from external morphology. Render and maturation status of any limpet species could only be sexed after killing and dissecting. Our efforts were trying to examine ripeness of live animal

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

**Total weight (g)** **Body weight (g)**

13 4.26 ± 0.59 15.3 ± 5.49 5.93 ± 2.43 1.68 ± 0.92 26.8 ± 6.27

30 3.46 ± 0.51 7.67 ± 3.63 2.54 ± 1.39 0.31 ± 0.22 11.7 ± 5.22

17 3.55 ± 0.96 9.31 ± 5.74 2.97 ± 2.40 0.93 ± 1.06 22.9 ± 12.4

27 3.11 ± 0.31 4.83 ± 1.74 1.86 ± 0.66 0.17 ± 0.09 9.05 ± 3.65

16 2.86 ± 0.48 4.68 ± 2.62 1.64 ± 0.71 0.20 ± 0.09 12.0 ± 5.41

12 3.26 ± 0.58 5.20 ± 2.47 1.81 ± 1.03 0.15 ± 0.14 8.73 ± 5.31

12 3.27 ± 0.56 5.20 ± 2.47 2.90 ± 3.50 0.14 ± 0.13 8.10 ± 5.25

17 3.17 ± 0.16 5.88 ± 0.91 2.19 ± 0.55 0.17 ± 0.11 7.25 ± 3.69

23 3.25 ± 0.43 5.98 ± 1.67 2.21 ± 0.78 0.16 ± 0.11 7.19 ± 4.51

12 3.95 ± 0.50 9.49 ± 3.91 3.47 ± 1.50 0.30 ± 0.28 7.56 ± 5.37

20 3.43 ± 0.44 5.43 ± 2.19 1.89 ± 0.91 0.28 ± 0.29 11.5 ± 7.43

21 3.71 ± 0.92 7.07 ± 5.38 2.86 ± 2.01 0.65 ± 0.73 18.7 ± 8.77

27 3.96 ± 0.49 8.75 ± 3.21 3.73 ± 1.47 1.31 ± 0.73 31.8 ± 7.72

19 3.96 ± 0.83 10.2 ± 8.64 4.10 ± 3.77 1.52 ± 1.94 28.4 ± 3.75

**Gonad weight (g)** **GSI (%)**

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

**n Shell length (cm)**

**Date of sampling**

November 12, 2011

December 04, 2011

January 31, 2012

February 28, 2012

March 28, 2012

April 24, 2012

May 28, 2012

June 28, 2012

July 21, 2012

August 03, 2012

September 11, 2012

October 05, 2012

November 25, 2012

December 30, 2012

**Table 1.**


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

#### **Table 1.**

*Invertebrates - Ecophysiology and Management*

*sandwicensis* are expensive in Hawaii.

\$200/gallon with shell on [8].

niques for the Hawaiian limpet *Cellana sandwicensis*.

in jewelry.

**2.1 Spawning season**

Australia, and New Zealand. Species are also found around the coasts of Japan, the Red Sea, Madagascar, South Africa, and the subantarctic island. There are more than 58 species of this genus. Among of those, many of them are of high economic value and aquaculture, for example, the two species *Cellana talcosa* and *Cellana* 

In Hawaii, there are three main endemic Hawaiian limpet species, called "opihi,"

In addition to important food sources, these Hawaiian limpet species are also culturally important in Hawaiian society. Many people (opihi pickers) were asked to collect these opihi for parties or family gathering with high prices. Besides that limpet's shell also continue to be used as tools for scraping skin off taro plant and sweet potato and grating coconut meat before eating [9] and as decorative elements

The success of any aquaculture species depends on seed production in captivity. Understanding the completion of the life cycle of limpet would make limpet aquaculture sustainable. The first priority is to understand some reproductive characteristics of the limpet species. This would also provide us with better knowledge for breeding limpet in the hatchery. In this chapter, we describe the current finding on reproductive biology, seed production, nutrient requirement, and culture tech-

**2. Some reproductive characteristics of Hawaiian limpet** *C. sandwicensis*

Reproductive characteristics of Hawaiian limpet *C. sandwicensis* have been reported by several studies [10–12]. The main focused reproductive criteria were spawning season, gonad development stage, fecundity, and maturity size.

A total of 266 specimens (**Table 1**) were sampled for a 1-year cycle (November

2011–December 2012) in Hawaii Island, and gonadal somatic index (GSI) was determined. The GSI was calculated according to equation GSI = (GW/BW) × 100, where GW is gonad weight and BW is body weight or soft body tissue. Gonad development stage was also evaluated by using histological examination. The result showed that the highest average GSI of *C. sandwicensis* was noticed between November and January. This suggests that spawning seasons of *C. sandwicensis* may occur right after this period, whereas the lowest GSI was found from March to August, this could probably be the resting season of the species. Similarly, the same GSI pattern (**Figure 1**) of males and females *C. sandwicensis* suggested synchronized

spawning of male and female *C. sandwicensis* in the wild [10].

including black foot or makaiauli (*Cellana exarata*), yellow foot or ālinalina (*C. sandwicensis*), and the largest species, giant limpet or kōʻele (*C. talcosa*) [2]. Natural ecological distribution of Hawaiian limpet is different intertidal zones of habitation on rocky shores. *C. exarata* is commonly found at higher intertidal zones, and *C. sandwicensis* is at low intertidal zones and rarely exposed by tide, whereas *C. talcosa* distributes in deep water [3–6]. These species are herbivorous grazers that feed on benthic microalgae, diatoms that growing hard substrates such as rocky substrates, death coral reef, and so on. They use teeth in their radula to graze on the toughest crustose coralline algae [3]. They are considered as highvalue food market and high-potential candidate species for commercial aquaculture. High commercial catch reduced significantly from 150,000 pounds in the 1900s to about 10,000 pounds in 1978 [7]. The scarcity has boost up prices to about

**84**

*Average size and GSI of sampled opihi for the reproductive cycle study from November 2011 to December 2012; data values from n individuals are presented as mean ± SD.*

#### **2.2 Gonad development stage**

The maturity stages of gonad development of Hawaiian limpet *C. sandwicensis* were reported and classified in **Table 2** and **Figure 2**. Multiple development stages (**Figure 2A**) were observed in the same ovary of female during the final maturation season. **Figure 2D** showed resting stages (April to August), because oocytes were not of clear formation from the ovary cell wall. Similar observation was made for gonad of the male (**Figure 2E**). The testes were densely packed with spermatozoa which appeared as dark blue stained by hematoxylin. Sperm were less densely in the male gonad (**Figure 2F**).

#### **2.3 Sexual determination**

Sexual determination of all limpet species is not known from external morphology. Render and maturation status of any limpet species could only be sexed after killing and dissecting. Our efforts were trying to examine ripeness of live animal

#### **Figure 1.**

*Seasonal changes in GSI of males and females limpet* C. sandwicensis*.*


#### **Table 2.**

*Maturation stages of gonad of limpets [10–12].*

without killing them. We eventually found a way to assess the gonad, by placing them upside down on a table or putting them close to the edge of a substrate. When they try to attach to the substrate, they move their foot toward the substrate, and sometimes the gonads may be seen from the top of their head. Males were identified if the animals had milky white gonad near the edge of the shell, as shown by the arrow in **Figure 3A**. The gonad of the ripe female was dark brown or dark green in color (**Figure 3C**). It is noticed that this way, it can only be conducted when the animal reaches maturity stage or during the spawning season. Ultrasound was also an option method, but it's inconvenient and is not a practical way.

#### **2.4 Fecundity and maturity size**

Absolute fecundity (F) of mature female (n = 5) limpet *C. sandwicensis* is varied, and related maturity stage and body weight of animal found that total

**87**

77.3 (R<sup>2</sup>

**Figure 3.**

*with shell removal.*

**Figure 2.**

larger [2].

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

*Cross sections showing stages of limpet* C. sandwicensis *gonad development. (A) Most oocytes in early development stages in ovary of female, (B) oocytes deforming shape in the ovary, (C) ripe stage and some oocytes were still in late development stage, (D) resting development stages of female, (E) mature male gonad* 

*with dark blue stained by hematoxylin, and (F) spermatic in gonad of male.*

eggs counted ranged from 42.080 to 157.000 eggs per g body weight (BW). Fecundity was plotted against body weight with linearly correlated to body weight (*P* = 0.019), and the best is described by the equations F = 28.4 BW −

*Determination of sexually mature male and female limpet. (A) Live limpet before dissecting, the arrow shows a sign where mature male gonad could be identified; (B) soft body tissue was removed off the shell, the mature male gonad (milky white) took up all the space around the digestive gland (dark color). This supports the location of mature gonad where it was seen as pointed in picture A; (C) live female limpet before dissecting, the arrow points where mature female's gonad would be seen; (D) showing a dark brown mature female gonad* 

 = 0.96). The monthly recorded GSI data combined with histology analysis showed that the Hawaiian limpet *C. sandwicensis* would attain sexual maturity size about 1.5–2.0 cm in shell length. Other studies also found that *C. sandwicensis* attained reproductive stage at shell length of 2.3–2.5 cm or

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

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

#### **Figure 2.**

*Invertebrates - Ecophysiology and Management*

**Stage Description**

**Figure 1.**

5 Spawning and reabsorbing

*Maturation stages of gonad of limpets [10–12].*

**2.4 Fecundity and maturity size**

**Table 2.**

1 Resting stage The gonad is characterized by little or germinal epithelium, unclear

2 Early development Nucleus enlarged, oocyte diameter about 7–10 μm. The male gonad is

3 Late development The ovaries are swollen laterally and some oocytes in the final stages of

4 Ripe Ovaries are swollen with dark brown color. Oocytes diameter ranging

without killing them. We eventually found a way to assess the gonad, by placing them upside down on a table or putting them close to the edge of a substrate. When they try to attach to the substrate, they move their foot toward the substrate, and sometimes the gonads may be seen from the top of their head. Males were identified if the animals had milky white gonad near the edge of the shell, as shown by the arrow in **Figure 3A**. The gonad of the ripe female was dark brown or dark green in color (**Figure 3C**). It is noticed that this way, it can only be conducted when the animal reaches maturity stage or during the spawning season. Ultrasound was also

Absolute fecundity (F) of mature female (n = 5) limpet *C. sandwicensis* is varied, and related maturity stage and body weight of animal found that total

and/or dark blue stained by hematoxylin

less densities of mature oocytes relative to ripe gonads.

oocytes about 2 μm

of the testes lobes

to 100 μm

*Seasonal changes in GSI of males and females limpet* C. sandwicensis*.*

an option method, but it's inconvenient and is not a practical way.

distinguishable from ovary wall cells and also for spermatid, the initial

shaped like around tubule and a thick germinal epithelium lines the edges

vitellogenesis. Cytoplasm granular, the oocytes diameter ranging from 50

from 110 to 130 μm. The testis is dense with spermatozoa, milky white

Spawning testis contained about 80% mature sperm; the ovary contains

**86**

*Cross sections showing stages of limpet* C. sandwicensis *gonad development. (A) Most oocytes in early development stages in ovary of female, (B) oocytes deforming shape in the ovary, (C) ripe stage and some oocytes were still in late development stage, (D) resting development stages of female, (E) mature male gonad with dark blue stained by hematoxylin, and (F) spermatic in gonad of male.*

#### **Figure 3.**

*Determination of sexually mature male and female limpet. (A) Live limpet before dissecting, the arrow shows a sign where mature male gonad could be identified; (B) soft body tissue was removed off the shell, the mature male gonad (milky white) took up all the space around the digestive gland (dark color). This supports the location of mature gonad where it was seen as pointed in picture A; (C) live female limpet before dissecting, the arrow points where mature female's gonad would be seen; (D) showing a dark brown mature female gonad with shell removal.*

eggs counted ranged from 42.080 to 157.000 eggs per g body weight (BW). Fecundity was plotted against body weight with linearly correlated to body weight (*P* = 0.019), and the best is described by the equations F = 28.4 BW − 77.3 (R<sup>2</sup> = 0.96). The monthly recorded GSI data combined with histology analysis showed that the Hawaiian limpet *C. sandwicensis* would attain sexual maturity size about 1.5–2.0 cm in shell length. Other studies also found that *C. sandwicensis* attained reproductive stage at shell length of 2.3–2.5 cm or larger [2].
