ABSTRACT

Pinctada maxima is an oyster that produces highly valuable silver and gold South Sea pearls (Strack 2006; Southgate 2007)� Juvenile of P. maxima usually grows faster than the adult (Chellam 1978)� At least 18 months are needed to achieve sufficient size of oysters for pearl nuclei insertion�

CONTENTS

Introduction ������������������������������������������������������������������������������������������������������������������������������������ 131 Materials and Methods ������������������������������������������������������������������������������������������������������������������ 132

Material ������������������������������������������������������������������������������������������������������������������������������������� 132 Method �������������������������������������������������������������������������������������������������������������������������������������� 132

Experimental Design ������������������������������������������������������������������������������������������������������������ 132 Time and Location ��������������������������������������������������������������������������������������������������������������� 132 Low-Voltage Electricity Method Installation ����������������������������������������������������������������������� 133 Juvenile Stocking ����������������������������������������������������������������������������������������������������������������� 133 Shell Length Measurements ������������������������������������������������������������������������������������������������� 134 Weight Measurement ������������������������������������������������������������������������������������������������������������ 134 Growth Calculation �������������������������������������������������������������������������������������������������������������� 134 Survival Rate ������������������������������������������������������������������������������������������������������������������������ 135 Aquatic Environment Parameters ���������������������������������������������������������������������������������������� 135 Data Processing and Statistical Analytics ���������������������������������������������������������������������������� 135

Results �������������������������������������������������������������������������������������������������������������������������������������������� 135 Discussion �������������������������������������������������������������������������������������������������������������������������������������� 137 Conclusions ������������������������������������������������������������������������������������������������������������������������������������ 138 Acknowledgments �������������������������������������������������������������������������������������������������������������������������� 138 References �������������������������������������������������������������������������������������������������������������������������������������� 138

Efforts were taken to accelerate high survival and growth of P. maxima juveniles to achieve shorter production time of nuclei-insertion-ready oysters�

As in most mollusks, P. maxima grows with increased shell size and body weight� Shell growth is controlled by the mantle organ, which is located between the inner shell and outer epithelium of inner organs or visceral mass� Outer epithelial cells will produce calcium carbonate crystals (CaCO3) in the form of calcite and aragonite (Bubel 1984; Weiner et al� 1984; Simkiss and Wilbur 1989; Lowenstam and Weiner 1989)� These cells also form organic protein called conchiolin as the adhesive for lime crystals (Weiner 1984; Suzuki et�al� 2004)� The combination of calcite, aragonite, and conchiolin forms shells that protect the mollusk’s soft bodies (Bubel 1984)�

The composition of coral exoskeleton is similar to P. maxima shells containing calcium carbonate (CaCO3)� Coral growth can be accelerated by running a low-voltage electrical current through negative and positive terminals inside the seawater, using the low-voltage Biorock method (Dwija 2003; Arifin et al� Chapter 6)� The Biorock method, also known as mineral accretion, was developed to be the latest generation of artificial reefs method that is very safe and most effective� Bivalves show rapid growth when they are located near Biorock artificial reef� The purpose of this experiment is to determine the potential benefits of low-voltage electricity on the early growth stage of the pearl oyster P. maxima (Jameson)�

MATERIALS AND METHODS

Material

Pearl oyster juveniles of P. maxima, aged 6 and 10 months, were used� Juveniles of the same age came from the same spawning group at the hatchery department of PT� Cendana Indo Pearl in Penyabangan, Buleleng, Bali� Juveniles were selected based on the length of the shell by passing through a 4 cm hole of a plastic bucket lid for 6 months old and a 6 cm hole lid for 10 months old�

The materials used for installing low-voltage electricity were 6 V, 11 A wet battery as power supply, 1 mm diameter iron wire as cathode, titanium alloy as anode, 10 mm diameter cable, epoxy glue (made from resin and hardener), and 5 cm rubber tube to protect the anode and cable connection�

The tools used in this research were caliper, electric scales, stove oven, light bulb, incinerator (furnace), water sampling, GFF filter paper, thermometers, hand refractometer, pH meter, Erlenmeyer bottle, voltage meter, sounder, pocket net, multitester, and battery charger�

Method

Experimental Design

The method used in this experiment was a 3 × 2 factorial design with four replications� Each replication consists of 10 pearl oysters� There were two treatment factors: electrical treatment and age treatment� Electrical treatments consist of without electricity, electricity without accretion, and electricity with accretion (accretion/cement already exists in one-month cathode)� Age treatments consist of 6-and 10-month-old juveniles�

Time and Location

The experiment was conducted in August-October 2006 at PT� Cendana Indo Pearls, Buleleng, Bali, with pearl oysters in panels hanging from long horizontal surface lines� Experiments were carried out by hanging the PN24 rearing panels at 3 m depth from a raft/guarding house located about 500 m from the coastline�

Low-Voltage Electricity Method Installation

The low-voltage electrical treatment consists of four main components: the voltage source/voltage converter, cathode at the negative terminal, anode at the positive terminal, and cable� The cathode is a double-twisted steel wire inserted into the oyster rearing panel to have a direct contact with the juvenile oyster shells� The top edge, middle, and the lower end of double panels were connected by cathode twisted to the cable�

The anode (titanium alloy) was connected to the cable with a strong, electrically insulated seal resistant to water/air� The connection was enclosed in a rubber hose filled with resin and hardener, so water/air could not penetrate� The cathode was connected to the negative terminal, whereas the anode was connected to the positive terminal from the batteries, which had been wired in parallel and placed in a box to protect them from waves and protect the cable connections�

A total of four panels with cathodes were attached to the raft and electrified for one month until the cathode was covered by an accretion of CaCO3� Two wet batteries were used as power supply each day, which last for four hours/day� In the case of electricity with Biorock, the oysters were attached to substrate that had been electrified and built up a layer of minerals� In the case of electricity without Biorock, the oysters were attached to fresh metal substrate on which no minerals had yet grown� Because the mineral growth acts to some degree as an insulator, the electricity without Biorock should have received more electrical current than the electricity with Biorock�

After one month, all panels were hung as shown in Figure 11�1�

Juvenile Stocking

Oysters are selected based on the age and size by passing the oysters through a hole of a plastic bucket lid with diameter of 4 cm for 6-month-old juveniles and 6 cm for 10-month-old juveniles� The shells of the oysters were cleaned from fouling organisms, the excess water was absorbed using cloth or tissue paper, and the oysters were weighed and labeled� Next, the oysters were placed on the rearing panels with a density of 10 juveniles per panel and acclimatized two times� First acclimatization was performed in a tub of running water that contains eight panels and was placed in the room for one night� Second acclimatization was held on the next day at the juvenile rearing location in the sea without low electrical voltage for 24 hours�

Shell Length Measurements

Length measurements were performed at months zero, one, and two using a 0�01 mm accuracy caliper� Juvenile shell length was measured as shown in Figure 11�2�

Weight Measurement

The shells of the oysters were cleaned from fouling organisms, excess water on the oyster was absorbed using cloth or tissue paper, and then the oysters were weighed and labeled�

Growth Calculation

Absolute growth (Moyle and Cech 1982) in shell length was determined using the following equation from the study by Effendie (1997):

∆L L Lt= − 0

where ΔL: Absolute growth in the average individual length (mm) Lt: The average length at time t (mm) L0: The average length at the beginning of the experiment (mm)

The absolute growth for wet weight was determined using the following equation from the study by Effendie (1997):

∆W W Wt= − 0

where ΔW: Absolute growth in the average individual weight (g) Wt: The average individual weight at the end of the experiment (g) W0: The average individual weight at the beginning of the experiment (g)

Survival Rate

Juvenile mortality counts were made every two weeks and at the end of the research� Survival rate was calculated using the following equation (Effendie 1997):

S N Nt= ( ) ×/ %0 001 where

S: Survival rate (%) Nt: Number of living organisms at time t N0: Number of living organisms at the beginning of the experiment

Aquatic Environment Parameters

The observed environment parameters measured every two weeks were water temperature, water current, water clarity (Secchi disk depth), pH, and salinity�

Data Processing and Statistical Analytics

The mean of survival and growth were tested for homogeneity level using the Levene statistic, and continued using analysis of variance (ANOVA) and least significance difference (LSD)� All tests were performed by software SPSS 13�

RESULTS

Table 11�1 shows the results of ANOVA of final survival rates, and the experimental results of final survival rate can be found in Table 11�2�

Table 11�2 showed that the final survival rate of P. maxima juveniles reared by several electricity methods ranged between 67�5% and 90%� The final survival rate of P. maxima juveniles reared with electricity without accretion (90�0%) and electricity with accretion (87�5%) were significantly higher than the juvenile survival rate reared without electricity (67�5%)�

Figure 11�3 shows the juvenile survival rates observed during the experiment; there was mortality in each treatment, except for six-month-old juveniles reared by electricity without accretion�

The  lowest survival were 10-month-old juveniles reared without electricity� Solid lines refer to 6-month-old oysters, and dashed lines refer to 10-month-old oysters�

Table 11�3 shows that juveniles aged 6 and 10 months showed different growth to the electricity treatment� Juvenile aged six months generally had highest shell length on electricity without accretion treatment� Next were the juveniles reared without electricity, and the last were the juveniles reared with electricity and accretion� The highest wet weight was found on electricity without accretion, followed by without electricity, and last electricity with accretion, although they were not significantly different�

In contrast with 10-month-old juveniles, electricity with accretion treatment had the highest length increment� Juveniles reared without electricity and juveniles reared with electricity with accretion have similar values� Electricity with accretion also had the highest weight growth� The absolute wet weight and shell growth had the same trend, with electricity without accretion highest, followed by without electricity, and last, electricity with accretion�

Environmental quality parameters are shown in Table 11�4� The table shows that there were few parameters that were substandard for pearl oyster growth, namely low water-current velocity and water clarity, although, in general, the water quality was acceptable for pearl oyster maintenance� High suspended material density (plankton, sediment, organic matter, etc�) indicated that there was enough food for P� maxima juveniles�

DISCUSSION

Survival was influenced by environment, food, and diseases (Nasr 1982, Pass 1987, Taylor 1999)� Environmental data showed that water current velocity was below desired values� This could lead to reduced food supplies (plankton) and also feces accumulation that are consumed by the P� maxima juveniles (Wilson 1987 cit� Taylor 1999)� Crabs were common predators of oyster juveniles, and P. maxima uses its strong shell for self-defense against predators� Shells are strong not only because they are formed by hard aragonite crystals (CaCO3) but also because of the macromolecular biological matrix that functions to bond the aragonite crystals (Addadi and Weiner 1997)�

The Biorock low-voltage method works by influencing the environment and by increasing the pH, CaCO3 contents, and electron flow within several millimeters of the cathode (Global Coral Reef Alliance 2007)� Cathode electrochemical processes produce electrons� Electron flow influences the electron transport chain to produce Adenosine triphosphate (ATP) biochemical energy in juveniles (Goreau, Chapter 19)� This energy may be used by juveniles to compensate for the consequences of the byssus thread cut used by pearl oyster culture and for lack of food, and could aid formation of the macromolecular shell biological matrix and CaCO3 formation and increase the power of shell closure as self-defense mechanism by P� maxima juveniles against crab predation�

Oyster death can be caused by fragility of juvenile shells, making them susceptible to predatory attack by crabs� Shell fragility can be caused by low seawater current velocity causing accumulated organics and reduced food (plankton), and also frequent byssus cuts that will result in less energy to form the shell (Wilson 1987 cit� Taylor 1999, Mariani et al 2002)�

Absolute weight was not significantly different when varied low-voltage methods were applied to different aged juveniles� Although the highest absolute weight (Alagarswami et al 1989, Pouvreau 2000, Taylor 1999) was produced by electricity without accretion in two months of experiment, it is possible that if the experiment time was extended with a longer time of electricity supply, then the experimental result might be significantly different� Similar experiments should be done with longer rearing time (six months minimum) using longer electricity supply, with 12 hours during daylight� Experiments using a broader range of ages, including adult oyster, juvenile, spat, and larvae, should be done to understand the appropriate age of each oyster phase to apply low-voltage method and optimize the practical management benefits of the method�

These results might indicate that electricity without accretion has higher electron availability to stimulate ATP production� Electricity without accretion can increase the absolute length increase of six-month-old P� maxima juveniles� The absolute weight growth also had significant increases higher than other treatments� Two volts voltage without accretion had higher growth than electricity with accretion� Higher voltages produced higher electron currents and electrochemical reactions, and probably influenced the electron chain transport to produce ATP� These conditions are appropriate for six-month-old juveniles, which have greater proportional growth rates than larger oysters� Higher pH in seawater around the cathode helped juveniles to produce shell (calcification)�

Electricity with accretion can increase absolute elongation of P� maxima juveniles aged 10 months� Accretion that occurred on the cathode in the electricity with accretion treatment may act as an electrical insulator and reduce the current� No measurements were made of electrical currents or pH change on mantle performance in producing shell, but 1�1-1�3 V electricity did precipitate CaCO3� Minerals precipitated on accretion included aragonite (CaCO3) and brucite (Mg(OH)2), where the proportion depends on the voltage and accretion age� Higher voltage and younger accretion age give more brucite than aragonite (Hilbertz 1992)� Gonad development in 10-month-old juveniles may influence energy needs (Pouvreau 2000), so that mineral accretion formed during one month was more appropriate for juvenile P� maxima growth, and elevated pH in seawater near the cathode should increase juvenile shell calcification� Lower voltage causes lower electron currents, affecting electron transport chains that produce ATP� Energy produced from ATP is prioritized to gonad development and production of biological matrix macromolecule used for shell formation� Higher temperatures and higher salinity also favor faster accretion (Bachtiar 2003)�

CONCLUSIONS

The low-voltage method is very useful for early growth of P. maxima, increasing survival and growth� The highest growth was achieved by 6-month-old P. maxima juveniles reared with electricity without accretion and 10-month-old P. maxima juveniles reared with electricity with accretion method�

ACKNOWLEDGMENTS

Gratitude to Cendana Indo Pearls Company (Atlas South Sea Pearls Ltd�) along with the staffs and employers for the grants and provision of research tools and equipment, also to Tom Goreau from The Global Coral Reef Alliance for inspiration and discussion�

REFERENCES

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