4. Discussion
4.1. Pearl quality The overall quality of this pearl crop was low. This small percentage of gem quality
Ž .
pearls is similar to that reported by Cabral 1990 in P. margaritifera and by Scoones Ž
. Ž
. 1990 in P. maxima. Furthermore, pearl quality Fig. 4 had a normal distribution with
the mode around the score of 19. Ideally, the distribution needs to be skewed towards the maximum score of 30 so that a larger proportion of pearls are in the upper,
gem-quality range. The lack of effect of the treatments on the qualitative aspects of pearl quality suggests the need for reevaluation of what factors might be influencing pearl
quality.
4.2. Pearl sac formation As the mantle tissue implanted into the operated oysters grew to form a pearl sac
Ž .
around 76 of the 78 beads recovered Group I, 6 weeks , it is unlikely that any significant host vs. non-host tissue reaction had occurred. If implanted tissue is to be
Ž .
rejected, this rejection occurs within 27 to 40 days, as demonstrated by Wada 1989 using freshwater mussels.
Ž .
In pearl sac formation in P. imbricata, Aoki 1966 found the mean heights of periostracal, prismatic and nacreous epitheliums to be 55.8 21.4, 8.1 9.0 and
3.4 2.1 mm, respectively. The pearl sac epithelium thins progressively as it develops. Ž
. Kawakami 1952 also working with P. imbricata, reported that the nacreous layer
began to be secreted about 40 days after transplantation when the epithelial cells of the Ž
. pearl sac became flat. Aoki 1966 stated that it is difficult to distinguish pearl sac
epithelium which is producing a nacreous layer from that which is producing a prismatic layer on the basis of morphological features of these cells. In contrast, epithelial cells
Ž .
producing periostracum s conchiolin are quite distinct since they are extremely tall, cylindrical and ciliated and contain numerous acidophilic small granules in the cyto-
plasm. It seems that more than 60, but not all, of cultured pearls in P. imbricata have Ž
. Ž
. an organic layer of periostracum Tsujii, 1968 . Furthermore, Wada 1957 noted that
periostracum does not always cover the whole surface of the bead. As the epithelium of Ž
. the pearl sac is very sensitive to both internal and external stimuli Tsujii, 1968 , one
Ž . wonders what factor s control whether these cells secrete an organic layer of perios-
tracum. In this study of P. margaritifera, where 78 of beads were wholly or partially brown
in colour 42 days post-operation, it seems that at least these beads had received a layer of periostracum. This does not preclude that all beads received a periostracal layer: it
may be obscured by further layers in the other beads. The total or partial silverrgrey colour of 26 of the beads suggests that these were at the prismatic or nacreous stages.
However, the mean epithelial heights of the pearl sacs, 7.7 to 12.9 mm, suggest that the pearl sacs were at about the prismatic stage; that is, if the data for the pearl sac
Ž .
epithelium of P. imbricata Aoki, 1966 are an appropriate basis for assessment of stage. None of the specimens of P. margaritifera had a pearl sac epithelium that had
thinned to the mean height of nacreous epithelium in P. imbricata. There is thus some conflict between the observed colour of a substantial proportion of beads in P.
margaritifera and the presumed stage of the pearl sac. If colour is a reliable indicator of the onset of the nacreous layer, then the age of these beads, 42 day post-operation, is
Ž .
comparable to the 40 days observed by Kawakami 1952 for P. imbricata. If epithelium height, based on P. imbricata, is a better indicator of pearl sac development, then P.
margaritifera develops more slowly than the former species. Ž
. The ‘tails’ attached to many of the beads Table 3 occurred in the ‘dead’ space
above the shell bead. This ‘dead’ space was the persisting passageway made by the pearl knife and along which the shell bead was directed until it reached its final resting
Ž .
position adjacent to the mantle graft. Aoki 1961 observed that to produce superior pearls, the operation should be made without leaving a space between the bead and
adjacent tissues. It would appear that this ‘dead’ space adjacent to the shell bead is ideal for the accumulation of haemolymph, bacteria and inflammatory cells. There would
have been bacteria on the operating instruments, bead and mantle graft, as the pearl surgery technique used here was the usual industry technique, which was not aseptic.
The pearl sac grew around this accumulation of dead inflammatory cells which had accumulated in the tail, as well as around the bead, producing a calcified ‘tail’ attached
Ž .
to the bead Fig. 3C . Similar haemocyte accumulations were reported by Scoones Ž
. 1996 in 38 of 59 pearl sacs examined by him in P. maxima pearl oysters. He
concluded that the cells were a response to infection or tissue damage and that they contributed to the formation of low-value baroque pearls.
The significantly lower number of ‘tails’ on beads from oysters on which adhesive Ž
. was used Table 3 suggests that adhesive closure of the incision helped to reduce or
eliminate the ‘dead’ space adjacent to the shell bead. This was followed by a significant reduction in the mean length of harvested pearls and the mean proportion of lengthrdi-
ameter from oysters treated with adhesive compared to those without this treatment. It appears that reducing the development of ‘tails’ during early pearl formation continues
into the subsequent pattern of nacre deposition, so that the pearls have significantly lower lengthrdiameter ratios.
4.3. Antiseptic use The lack of any significant effect on post-operation mortality, bead rejection or pearl
Ž .
‘tail’ formation from the antiseptic treatment of the incision site Tables 3 and 4 accords with observations of the likely sources of bacteria in the bead and mantle graft
Ž .
site. Norton unpubl. data found that the densities of bacteria on the gonad and mantle tissues of healthy pearl oysters were very low. Hence there is likely to be little
introduction of bacteria into the incision from the tissue surface. The bacteria in a wound will come from the unsterile shell bead, surgical instruments, cutting boards, cloths and
piece of mantle tissue. Histological studies revealed the presence of large numbers of inflammatory cells in the bead and mantle graft site of the majority of oysters. They
clearly demonstrate that post-operative bacterial infections are very common. These infections are associated with cultured pearls that are at least initially malformed with
‘tails’, or with no formation of the pearl sac. Thus, there is considerable value in seeking
to lower the frequency of these infections. Similar conclusions on the role of bacterial Ž
. infections were made by Scoones 1996 when working with P. maxima pearl sacs.
4.4. Oyster mortality Ž
. The high mortality rates in relaxed oysters Tables 3 and 4 contrast with previous
Ž work in the laboratory on small numbers of pearl oysters P. albina and P. margari-
. Ž .
Ž tifera
Norton et al., 1996 and in the field on large numbers of farmed pearl oysters P. . Ž
. maxima
Mills et al., 1997 . Oyster survival was very high in both cases, e.g. with losses of 1 or less. Several factors may have contributed to the high mortality with
relaxant treatment. Immersion of each oyster in the relaxant bath for 15 min should not Ž
. have been a factor since Norton et al. 1996 immersed oysters for up to 60 min without
mortalities. However, these latter oysters were not subjected to multiple stresses, including forced opening, haemolymph haemorrhaging and prolonged emersion. The
oysters in this current study were held in the air for periods of 30 min to 1 h before and after the bead insertion operation. Water drained off or evaporated from the delicate
oyster tissues during the period of emersion and some drying may have occurred.
The large loss of haemolymph during the pearl operation is also of concern. As much as one-third of the circulating haemolymph volume can be lost in this way from
Ž .
non-relaxed P. margaritifera Lintilhac, 1987 . This volume may be greater from Ž
. relaxed oysters with collapsed mantles. Harley and Harley 1973 performed surgical
operations on the gastropod mollusc, Aplysia californica, and found that if there was a Ž
. weight loss of more than 15 during surgery mainly due to loss of haemolymph , a
significant proportion of the animals died. Further support for added stress arising from use of relaxant is the finding that the
pearls from this treatment were significantly smaller than those from untreated oysters Ž
. Table 5 . Pearl diameter and weight are related to bead size and to thickness of nacre on
the bead. It is conceivable that significantly smaller beads were inserted into the oysters that were treated with relaxant and the final size effect is related to bead size. However,
this seems unlikely. There was no significant difference between treatments in bead size and there was nothing to suggest any reason for such a bias during the course of the
bead insertion program. The most likely explanation is that the nacre layer is thinner in pearls from relaxant-treated oysters. This suggests that the process of nacre secretion
was delayed or inhibited, probably in the period after the stress of the bead insertion procedure.
Ž .
The increased mortality of oysters receiving the adhesive Tables 3 and 4 may be related to the extent of inflammation. The adhesive causes inflammation of the tissues to
Ž .
which it adheres Norton, unpubl. data . If a large quantity of adhesive is applied, the resulting large area of inflammation may lead to death.
4.5. Bead rejection Ž
. The higher bead rejection rate associated with the adhesive treatment Table 3 is
contrary to the expectation that adhesive closure of the incision may promote bead retention. It again probably relates to the adhesive’s inflammatory effect on in-contact
tissues, allowing the bead to escape. The use of a smaller quantity of adhesive may help to reduce these losses or another method needs to be developed to close the incision,
e.g., sutures. However, suturing is potentially unacceptable in terms of the time required, Ž
especially when used at the current incision site for bead insertion Norton, unpubl. .
data . For this reason, suturing in combination with a more accessible incision site for bead insertion e.g., the gonad surface, is being investigated. Such a site also has the
Ž .
advantage of reducing tissue damage and haemorrhage see above .
5. Conclusions
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