As mentioned previously, the pilot pearl culture farm was established in 1984 at the AES in Mymensingh. In July 1985, the station was transferred to FRI (now called FARS). Mymensingh is located 120 km north of the capital city of Dhaka and can be reached by road or train in two hours. It is situated west of the Bangladesh Agriculture University (BAU). The station is primarily engaged in freshwater aquaculture research and it has numerous ponds of various sizes, hatchery tank facilities, an office building, employees quarters and also a complete scientific staff.
The ponds measure 15 × 60 m (900 m2). They are supplied with water through pipes that are connected to a canal that runs perpendicular to each pond. The ponds can be drained with water through the concrete monk gates. To hold the water in a pond, the monk gate was fitted with wooden flushboards set on two grooves. To prevent seepage/leakage of water, sawdust was placed in between the two sets of wooden flashboards. The average depth of the water in each pond was approximately 0.6 m during dry months and 1 m during rainy months. The pond soil is of clay and will remain hard even when submerged under water for days. Diagram 1 shows the culture pond.
At the start of the project, three ponds were initially used for culturing operated and unoperated mussels. Pond nos. 60 and 61 were for operated mussels and pond no. 59 was a stock (unoperated mussel) pond. Later in the project, four more ponds were added. Use of the ponds was as follows:
Pond no. 59 - for mussels newly operated on
Pond no. 60 and 61 - for mussels already operated on
Pond nos. 58, 59, 93 and 94 - stock ponds (newly collected mussels)
The FARS building was newly renovated with the assistance of the Danish Government. A room at the southeastern side was provided with furniture and utilized as a laboratory/office for the project. There pearl culture specialists work together with the government counterparts on the surgical operation of mussels to produce pearls.
There are two genera of freshwater pearl mussels found in Bangladesh, the Lamellidens and Perreysia. From the survey undertaken by the author of the various districts of Bangladesh in 1982, two species of Lamellidens were identified, the Lamellidens marginalis (Lamarck) and L. jenkinsianus var. obesus (Hanley and Theobald) and five species of Perreysia, the Perreysia daccaensis (Preston) P. wynegungaensis (Lea), P. (Radiatula) pachysoma (Benson). P. favidens var. assamensis (Preston) and P. favidens var. deltae (Benson). The mussels inhabit lakes, rivers, dams, ponds and pools.
Of the above species, the Lamellidens marginalis was selected for culturing for two reasons: first, due to its size, mature ones ranging from 7 to 10 cm (measured from anterior end to posterior end), it is suitable for operating on. Second, it is common in most rivers and lakes and is therefore available when needed. The Perreysia daccaensis have a golden colour inner layer shell, capable of producing pink pearls, but being smaller in size (usually less than 6 cm) will be more difficult to operate on.
1 All figures referred to in the text appear at the end of Appendix 1
For pearl culture, special instruments are needed in order that the nuclei and graft tissues can be inserted in the gonad of mussels. These instruments were similar to those used in Japan. From the previous project (TCP/BGD/2308), five sets of instruments were fabricated locally and turned over to the Government at the termination of the project. The instruments were still usable. The five sets were sufficient in training Government officers but for the scheduled training of rural farmers, it necessitated the fabrication of ten sets of instruments.
The following is a description of each instrument needed for pearl culture (Figure 2):
Shell Opener - Made of stainless steel. When its handles are pressed, the end opens. This was used to pry open the shells of mussels and prevent them from closing prior to the introduction of nuclei and grafts.
Graft cutter/knife - Made of flat stainless steel, and used in the preparation of graft tissues. The pointed end can easily be inserted in between the shells of mussels to cut the adductor muscles (both the anterior and posterior) to open the mussel. It was then used to cut the two mantles of the mussel which are cut into 2 to 4 squares.
Incision knife - Made of stainless steel with a plastic handle. It has a rounded flat sharp tip which was used to make slits on the gonad through which the nucleus and graft tissue were inserted.
Nucleus carrier/lifter - Similar to the incision knife except that the tip is cup-shaped. It is used to carry or lift a piece of nucleus/ irritant for insertion into the gonad. In doing so, the tip should first be wet with water. Due to surface tension (a physical property of water) this will enable the instrument to lift a nucleus.
Graft carrier/lifter - Also similar to the incision knife and nucleus lifter, except that the tip was pointed. By pricking it into a piece of mantle tissue, it can be lifted and inserted inside the gonad of a mussel.
Spatula with a hook - Made of stainless steel, with the flat end used to lift the gills of a mussel in order to have a clear view of the body prior to operation. The hook was used to hold the foot when making an incision during operation.
Mussel holder - A wooden block (10 × 10 × 18 cm) with a large ordinary paper clip fitted on one end, which holds the mussel to be operated on.
Pincher - An ordinary pincher which is used in the preparation of graft tissues, such as in picking up the cut/detached mantle from the mussel.
Graft cutting board - A wooden block (3 × 10 × 15 cm) used as a board when cutting the mantle to desired size and shape.
Wooden peg - Made of wood (usually cut from an ordinary pencil) 7 mm in diameter and 3.5 cm long. It is placed in between the shells of the mussel, after they have been opened by the shell opener, to prevent the shells from closing during the operation.
Additional equipment - Other items were needed during operations such as basins, trays, rubber sponges, dishes and bowls.
With the use of nuclei or irritants, pearls can be produced in shorter culture periods. The best material for nuclei would be made of shells as they have the same chemical composition as pearls. Shell nuclei are available in Japan with sizes ranging from 3 to 10 mm; but there are constraints in importing these items. Manufacturing them locally is also not advisable due to the non-availability of both the equipment for processing and the thick shells as raw material.
For use in the project, a substitute material was procured and used as nuclei. Round beads, made of ceramic, were ordered from a ceramic factory in Dhaka. Ceramic will be a good substitute material for pearl culture, due to its hardness and because it has almost the same weight as pearl. Sizes of the ceramic beads range from 2 to 4 mm in diameter, but they were not uniform in shape and size as they were made by hand.
For culturing pearls, mussels are needed. The freshwater mussels, Lamellidens marginalis, are present in some ponds of FARS. At pond no. 59, the mussel stock were those from a previous TCP project (TCP/BGD/2308). They have also reproduced at pond no. 83. As the stock of mussels at the FARS ponds was not sufficient for use in the project, it was necessary to gather them from areas outside the station, such as the university pond and from the low lying areas, of lakes, ponds and streams of the town of Trisal (18 km south of the station). These collection sites were already identified during the previous TCP project. For the gathering of mussels, an FAO vehicle was used.
Gathering the mussels was easy as they were usually in knee-deep water. While wading, a gatherer steps on them and their presence on the bottom can be felt, as they are only partly embedded in the sand or mud. The gatherer then just picks the mussels up from the bottom and accumulates them in a basin floated on the water. There was a problem in gathering mussels during the rainy season as the river, lakes or ponds were flooded, which necessitated diving for them. During the winter season when the water temperature goes down, the gatherers do not feel like wading in the cold water. The best season for gathering is after the rainy season when the water starts to recede, until early winter (September to November) and after winter until the start of the rainy season (February to May).
The following is a list of mussels collected monthly:
| September | 1 917 |
| October | 1 600 |
| December | 5 410 |
| January | 900 |
| March | 4 000 |
| TOTAL | 13 827 mussels |
The gathering was not continuously undertaken as the stock of mussels in the pond of the station was sufficient to serve the needs of the trainees. There was also a plan to propagate the mussels in ponds such as is done in ponds nos. 92, 93 and 94; however, this plan was abandoned when it was found that research on aquaculture would be utilizing the ponds.
The gonad, the reproductive organ of the mussel, is the best place to culture round pearls. It is the only organ that can contain a nucleus of sufficient size. The technique has been perfected in Japan, using their local oyster, the Akoya Gai, Pinctada matensii. To produce a round pearl, a round nucleus together with a graft tissue was inserted inside the gonad. The graft tissue is a piece of mantle taken from another mussel. When grafted or inserted inside the gonad, the graft tissue merges with the wall of the gonad and draws nutrition from there to stay alive. It then goes through a series of cell divisions and encloses the nucleus, thereby developing into a pearl sack. As the mantle produces the shell of the mussel, the small piece of mantle inserted inside the gonad will do the same. The pearl sack will then secrete the shell substance, calcium carbonate (or pearl substance) and coat the nucleus. Continuous deposits of shell substance will result in a cultured pearl.
In some cases, if the graft tissue is expelled by the mussel, there will be no pearl coating on the nucleus even though the mussel will be cultured for a long period of time. The nucleus will remain as it was. But in the case where the nucleus was expelled and the graft tissue remains, seed pearl will form. The pearl will be small as it was not started from a larger-sized nucleus.
The activity for pearl culture was concentrated on the above technique involving the gonad for growing pearls. The procedures undertaken were as follows:
Preparation of the Laboratory
The laboratory was first prepared by setting up the table with the necessary operating instruments, trays and pans. Mature-sized mussels were gathered from the stock pond and brought to the laboratory, where they were arranged in trays with water, ready for the operation.
Preparation of graft tissues
The mussel to be used as graft should be properly selected. It should be young and healthy. The two mantles of one mussel, after being cut into small pieces, will be sufficient for use in operating on 10 to 15 mussels.
To prepare the graft tissues, the mussel was opened to get at the mantles. This was done by slipping the pointed end of the knife between the shells of the mussels and cutting the two adductor muscles (anterior and posterior). With the shells widely opened, the two mantles were cut and separated from the body of the mussel and placed on a wooden board. The slimy fluid on the mantle was wiped off with a wet synthetic sponge. They were cut into long strips by cutting or eliminating the outer margin of the mantles which produces the prismatic layer of the shells. They were then cut into 2 to 4 mm sections and used as graft tissues.
Insertion of nucleus and graft tissues (Figure 3)
The mussels to be operated on were on trays in water. The end of the shell opener was inserted into a gaping mussel and the handle pressed to open the shells approximately 7 mm wide. A piece of wooden peg was placed between the shell valves and the shell opener removed. With the use of a spatula, the right gills were lifted up and the left gills down, in order to have a clear view of the visceral mass. If the body was thin or the gonad full of eggs (or sperm), the mussel was rejected. Weak specimens will usually not survive the operation, while in those with full gonads, the graft tissue or nucleus may flow out when the gonadal fluid oozes during the operation.
After a healthy mussel was selected, it was placed on a holder. With the spatula and hook in the left hand, the foot of the mussel was held in position so that the body would not move and for easier manipulation. Using the incision knife held in the right hand, a cut was made near the base of the foot, then pushed toward the gonad to cut its wall. A piece of graft tissue was then picked up with the graft carrier and inserted into the gonad through the slit. With the use of a nucleus carrier, a piece of nucleus was picked up and inserted into the gonad, to be in contact with the graft tissue. Another incision was made for the introduction of a second nucleus and graft tissue. The operation was then completed, the mussel taken out of the holder, the wooden peg removed and the mussel placed on a tray in water. The same procedure was done on the succeeding mussels. Each operated mussel was used on the following mussels. Each operated mussel was properly marked by etching a letter and number on the outer shell in order to identify the person who operated on the mussel and the month of operation, (i.e. the mussels operated on by Mrs Momtaz Begum in the month of April were marked M4 - M for Montaz and 4 for April). After operating on a sufficient quantity in a day, the counterparts returned the mussels to the pond for recuperation.
Care after the operation.
The mussels operated on were placed in a wooden cage (Figure 4). The cage measured 4 m2 by 0.3 m high, with netting on the sides and set on the bottom of the pond. As the mussels moved on the bottom of the pond seeking a suitable place to burrow, the cage prevented them from spreading around the pond. It was also easier to locate them for inspection, two weeks after the operation. During inspection, the mussels were collected from the cage and the dead shells (mortality due to the operation) were separated from surviving mussels. They were properly counted and recorded and the live mussels released in the pond for culturing.
Causes of mortalities
In order to minimize mortalities of mussels operated on, various trials were conducted and the results given to the Government counterparts. Following are some of the causes:
The mussel will die whenever a vital organ is damaged during the operation. The organ may be the intestine, stomach, liver or kidney. It was emphasized to the counterparts to avoid hitting or damaging any organ. On the tests completed by the pearl culture specialist, it was found that inserting the nucleus in the gonad, near the base of the foot, would result in a lower mortality rate as no vital organ was liable to be damaged.
Forcing open the mussel shells during the operation can damage the adductor muscles resulting in mortality. Mussels should therefore be opened gently.
Opening the shells of the mussels too wide can damage the adductor muscles and result in mortality. It was easier to operate on a mussel with a widely opened shell, but this will result in a higher mortality rate. The tests conducted in January 1986, showed negligible mortality to mussels on which a 7.5 mm dia wooden peg was used, as compared with those on which a 9.5 dia mm peg was used.
Weak or thin mussels should not be operated on as they will most likely not survive the operation and will eventually die. Instead, healthy ones should be selected.
As experienced in foreign countries, mortality was higher when using larger-sized nuclei than when smaller-sized nuclei were used.
Causes of expulsion of graft tissues and nuclei
The freshwater mussel crawls on the bottom of the pond by using its protruding foot. In doing this, the body is compressed somewhat and the nucleus that was inserted in the gonad may be accidently expelled. It may be emitted through the wound opening made during the operation, or the skin may give way, thus spitting out the nucleus. The expulsion rate of the nucleus, in the operations of the Government trainees, was found to be considerably high, prompting the pearl culture specialist to conduct tests to minimize the rate. From September 1985 to March 1986 the pearl culture specialist did not operate on mussels for the purpose of growing pearls in the gonad, instead his operations were for seed pearls on the mantles. After a series of tests were conducted, the expulsion rate was minimized when the nuclei were inserted deep inside, almost to the other side of the gonad (Figure 5). It was therefore necessary that the nuclei and graft tissues be inserted far enough inside the gonad. Another cause of expulsion of the nuclei occurs when mussels with full gonads are operated on. When making a slit in the gonad for insertion of the nucleus, it was observed that the gonadal fluid oozed out. Because of this, nucleus and graft tissue may be carried out immediately after they are inserted. It was not recommended to operate on mussels with full gonads. A higher expulsion rate will be attained when using larger-sized nuclei than when using smaller-sized ones, as experienced by the pearl culture specialist.
Pearls can be grown on the mantle of the mussels. This method has been practised in China and Japan using their freshwater mussels (Anodonta sp./Cristaria plicata). Only pieces of graft tissues were inserted between the layers of the mantle without any nucleus/irritant. The graft tissue easily attached to the mantle; if a nucleus/irritant is inserted, it can easily be expelled by the mussel.
In China and Japan, the freshwater mussels are large in size, mature ones measuring 10 to 15 cm (from anterior end to posterior end) and so numerous pearls can be cultured in each mussel. On each mantle five to ten graft tissues can be inserted allowing 10 to 20 pearls to be cultured on the two mantles of each mussel. These pearls are called seed pearls, having no irritant/nucleus as core and are commonly termed rice pearls. The shapes can be controlled by the shape of the graft tissues. A rounded-cut graft tissue will result in round-flat pearl (like a button) while a rectangularshaped graft tissue will result in long-shaped pearl (like rice).
Trial cultivation of pearls on the mantle
The pearl culture specialist has cultured seed pearls on the mantle of the local mussels using the following procedure: first, the graft tissues were prepared (in the same way as when used in the operation on the gonad). The mussel to be operated on was opened slightly with a 7 mm gap between the shells. A wooden peg was placed between the shells to prevent them from closing and the mussel was then placed on the holder. Using a spatula, the gills were raised to allow for a clearer view of the lower mantle lobe. Using the incision knife, slits were made on the mantle. The mantles are usually bulging due to the presence of a watery fluid so, after it was slit, the fluid oozed out. Using a graft carrier, a piece of graft tissue was then inserted inside the mantle through the slit and the succeeding slits. As the local mussels were smaller in size, two to three graft tissues were inserted on each mantle, although it was possible to insert up to five graft tissues on one mantle. On the two mantle lobes (left and right) a total of four to six graft tissues were inserted. Use of larger-sized graft tissues is preferable as the pearl will develop faster (the culture period will be shorter), although it is difficult to insert larger graft tissues and the rejection rate of the tissues is higher. After three weeks time, the slits were found to be completely healed and the graft tissues merged with the mantle. On inspection after three months, seed pearls were found to be developing on the mussels that were opened.
This process is quite difficult as it requires very steady hands. Also it should be executed quickly, as the mantle tends to contract after making the slits causing insertion to be difficult. The process was demonstrated by the pearl culture specialist to the Government trainees but they did not practise it because it is difficult. The pearl culture specialist was the only one who operated on the mussels for seed pearls (Table 2).
The production of blister pearls, pearls grown attached to the shell of mussels, was not given much attention as the size of the local mussels were small as compared with the freshwater mussels in China or Japan. It may not be practical to produce good quality half-round blister pearls from a small mussel.
For demonstration purposes, the process of operating on blisters was shown to the counterparts using mussels. Cross-shaped and crescent moon-shaped irritants were cut out of plastic material. They were then slipped through the inside of the mussels, between the shell and the mantle. After this process, the mussels were returned to the pond for culturing.
After ten months of culture the mussels were harvested, although they were not yet ready, in order to determine the result of the operations. The irritants were found to be coated with shell layer but still not sufficiently thick. It may take two years to develop a thick layer of pearl.
The principle behind the blister operation was that the irritant inserted will be coated when the mussel makes another shell layer. As the irritant was placed between the mantle and the shell of the mussel, it will be covered with the mantle secretion of calcium carbonate, following the contour of the irritant. Continuous deposits, layer after layer of shell substance will result in blisters the quality of which will be determined by the thickness of the coating. The shape of the blister pearl depends on the shape of the irritant and so it is possible to make a half-round, triangular, square, initial letters, numbers or any other configuration. In China, blister images of Buddha were produced, and in our case cross-shaped and crescent moon-shaped blisters were used. The cross-shaped blister was kept in a bottle preserved in formalein and displayed in the laboratory of FARS.
The total number of mussels operated on from September 1985 to July 1986 was as follows: (Tables 1 to 5 show the monthly list of mussels operated on by individual)
| Name | Quantity |
| R. Pagcatipunan | 1 070 pieces |
| Momtaz Begum | 1 693 |
| N. Nahar Begum | 1 456 |
| Jahirul Islam | 613 |
| Others | 29 |
| Trainees (29 Jun-3 Jul/86) | 1 057 |
| Trainees (27–31 Jul/86) | 819 |
| TOTAL | 6 737 pieces |
The Government trainees could have operated on more mussels but as they have other activities in fish culture research, they were not able to concentrate on pearl culture.
As of 31 July, the outstanding inventory of mussels operated on at the farm (excluding those of the rural farmer trainees) was as follows:
| Name | Quantity |
| R. Pagcatipunan | 701 |
| N. Nahar Begum | 835 |
| Momtaz Begum | 1 049 |
| Jahirul Islam | 413 |
| Others | 6 |
| TOTAL | 3 004 |
Of the above list, the mortalities incurred due to operations during culture and the trial openings were deducted. Most of the mussels were cultured at pond no. 61 but later transferred to pond nos. 59 and 60. Pond no. 61 will be used for fish/duck culture. Toward the end of the TCP project, only three ponds were being used: no. 58 for unoperated mussels and nos. 59 and 60 for operated mussels. During the daily operation on mussels or whenever they were operated on, the pearl culture specialist took note of the condition of the gonad and the body of the mussels. As observed during the period of operation from September 1985 to July 1986, the condition was observed to be as follows:
| Months | Condition of gonad/body |
| September-October | Spent, recovering, late spawning |
| October-December | Spent, recovering, maturing |
| December-February | Weak/thin, maturing |
| February-April | Maturing, spawning |
| April-July | Spawning, spent, recovering, weak |
It is also common to observe some mussels at maturity stages throughout the year. Temperature and rainfall could have a great influence on the life of mussels. The above record is important to pearl culture in scheduling the operations. From late December to early February it is not advisable to operate on the mussels as they are weak or thin and due to the very low temperature (less than 20°C), the mussels were found to recover slowly. They can be operated on the rest of the year, but attention should be given to choosing only the healthy ones and rejecting those with matured gonads, spawning and thin/weak mussels.
Both mussels operated on and unoperated are being cultured in ponds. As the pearl culture period is two to three years, it is necessary that the ponds be maintained in suitable condition (the culture period of two to three years is a calculation as there is no past experience in pearl culture in Bangladesh).
The temperature of the water was taken and plankton samples were collected for volumetric analysis. Whenever the plankton content was low, which can visually be determined (aside from volumetric analysis) by the transparency of water, chemical fertilizers were supplied to the pond.
The culture ponds were inspected every day for any unusual occurrence. The monk gates were inspected for any leakages/seepages on the flash-boards and stopped if any was detected. The growth of submerged and floating vegetation was prevented by removing them and drying them on the dikes.
In October 1985, Euglena bloom occurred at pond no. 59. The entire surface of the pond was red and as they consumed a lot of oxygen especially during the night, the cultured mussels might have been affected. It was decided then to check the Euglena bloom by draining the surface water. As the flagellates were accumulated at the surface layer, overflowing the water by lifting the upper flashboards of the monk gate, eliminated most of them. To compensate for the loss of water in the pond, new water was pumped in.
During the dry months the water level went down due to evaporation. It was necessary to maintain a high water level especially during not weather conditions, to allow for a greater difference between the surface and bottom water temperatures. It was noted that when the water temperatur reached 31°C, the body of the mussels became weak and thin.
Three Government officers designated as counterparts were trained in pearl culture. They were Mrs Nurun Nahar Begum, Mrs Momtaz Begum, both Scientific Officers and Mr Jahirul Islam, Research Assistant. Of the three, only Mrs Momtaz Begum was trained in the previous TCP project. This was conducted as on-the-job training. Activities included the gathering of mussels, the caring of mussels before and after the operations, maintenance of ponds, and the recording of the mussel stock. A large part was devoted to the surgical operation of mussels. They have operated on mussels regularly in order to perfect the technique. Pointers were given in order to minimize both the mortality rate of mussels operated on and the expulsion of nuclei inserted.
The counterparts were not able to concentrate full-time on pearl culture as they had other assignments in fish culture research. This was the reason for the small number of mussels they have operated on. Tables 3, 4 and 5 as appended, show the number of mussels each counterpart/ trainee operated on each month for the project period.
The programme to send two counterparts on a study tour to a foreign country with a developed pearl culture industry was not achieved due to the non-availability of a host country that was willing to offer the use of their facilities.
As part of the programme of the project, two groups of rural farmers, composed mostly of women, were given training in pearl culture. The plan of the training is shown as Appendix 2. The first training was initially scheduled for 15–19 June 1986 but was postponed to a later date, from 29 June to 3 July 1986 and was attended by 16 participants. The second training was held on 27–29 July 1986 and was attended by 15 participants. See Tables 6 and 7 for the names of the trainees and the quantity of mussels operated on by each participant.
The training was planned to concentrate on the practical application of the technique of pearl culture. Most of the time was spent in the laboratory where the operation of mussels was undertaken. Brief lectures on the biology of mussels and technique of pearl culture were given by assigned lecturers, slides on pearl culture were shown and field visits were made to the culture ponds and natural grounds.
The mussels operated on by the participants were inspected after two weeks. Judging on the mortality incurred during the operation (although this is not conclusive), seven participants from the first group and ten from the second group, showed promising results in their operations. Although the mortalities were quite high this can be attributed to the fact that this was the participants' first opportunity to operate. Those that incurred a high mortality rate could attribute it to unsteady hands while operating.
The trainees showed their eagerness to learn pearl culture in the five days of training. They were able to acquire the technique on how to produce pearls. One constraint here is that they have no instruments which they may use in their own villages. As an immediate solution to this problem, it was suggested that whenever the trainees intend to operate on mussels, they are welcome to bring the mussels to the FARS laboratory, operate on them and then bring these mussels home for culturing in their own ponds.
