Gonadic conditioning of the calafia mother-of-pearl oyster, Pinctada mazatlanica (Hanley, 1856), under two temperature regimes

Ž .Aquaculture 195 2001 103–119www.elsevier.nlrlocateraqua-online

Gonadic conditioning of the calafia mother-of-pearlž /oyster,Pinctada mazatlanica Hanley, 1856 ,

under two temperature regimes

Pedro Saucedo, Carmen Rodrıguez-Jaramillo,´ ´Cynthia Aldana-Aviles, Pablo Monsalvo-Spencer,´Teodoro Reynoso-Granados, Humberto Villarreal,

Mario Monteforte)

Centro de InÕestigaciones Biologicas del Noroeste, S.C., Mar Bermejo 195, Colonia Playa Palo de Santa Rita,´La Paz, Baja California Sur, Mexico

Received 23 September 1999; received in revised form 15 February 2000; accepted 17 October 2000

Abstract

Gonadic conditioning of pearl oysterPinctada mazatlanica was studied under two watertemperature regimes using cultured specimens. Before starting conditioning, specimens wereinduced to spawn with thermal shock. Two different regimes were evaluated, controlled tempera-

Ž . Ž .tures CT and gradually increasing temperatures IT . In both trials, specimens were fed dailywith Isochrysis galbana and Chaetoceros gracilis in a proportion of 7:3. Gonad and musclesamples were excised from the oysters every 15 days and processed for histological analysis.Condition Index and Muscle Condition Index were calculated. Similarly, oocyte frequency andoocyte diameter measurements were made. ANOVA analyses were used to test significantdifferences in the frequency and diameter of postvitellogenic oocytes, and values of both conditionindexes between temperature regimes, and over time. The IT showed more suitable conditions forgonad conditioning than CT, although the time needed for reaching maturity was higher in theformer regime. For CT, a temperature of 248C was the best for conditioning, since there were notraces of atretic oocytes, partially spawned specimens, and there were less previtellogenic oocytesand more postvitellogenic oocytes filling the follicles. At 208C, no positive results were noticed.Condition indexes appear to be reliable indicators of gonad activity during conditioning in bothtrials. The ANOVA analysis detected significant differences in almost all the statistical treatments

) Corresponding author. Tel.:q52-112-53633; fax:q52-112-54715.Ž .E-mail address: [email protected] M. Monteforte .

0044-8486r01r$ - see front matterq2001 Elsevier Science B.V. All rights reserved.Ž .PII: S0044-8486 00 00552-4

( )P. Saucedo et al.rAquaculture 195 2001 103–119104

run for oocyte frequency, oocyte diameter and condition indexes according to temperature regimeand over time.q2001 Elsevier Science B.V. All rights reserved.

Keywords: Pearl oysters; Gonadic conditioning; Reproduction; Temperature;Pinctada mazatlanica

1. Introduction

Pearl culture is today one of the most important aquaculture industries in the world,with a lucrative, well-established market whose value is between US$3 and 5 billion per

Ž .year Fassler, 1995 . At present, this industry is supported mainly by the cultivation ofŽ .only five marine species of pearl oysters Pteriidae :Pinctada martensi, P. fucata, P.

margaritifera, P. maxima, and Pteria penguin. Several Indopacific countries carry ontheir commercial exploitation mainly through wild spat collection andror by strictly

Žcontrolled extraction of adults from natural beds, such as in Australia Rose and Baker,.1994 .

However, in the last two decades, hatchery production has been considered as animportant alternate strategy to wild spat collection for pearl farming. Important advanceshave been made for practically all the species that are now used in commercial pearl

Žproduction Tanaka and Kumeta, 1981; Rose and Baker, 1994; Southgate and Beer,.1997 . India, for example, has developed a successful technology for artificial larval

Ž .rearing of the pearl oysterP. fucata Alagarswami et al., 1983a,b, 1989 , and hatcheryŽ .production Dharmaraj et al., 1991; Victor et al., 1995 . For other countries, however,

intensive cultivation for commercial pearl production is just starting, e.g. forP.Ž . Ž . Žmazatlanica Hanley, 1856 andPte. sterna Gould, 1851 of Mexico Mazon, 1988;´ ´

.McAnally and Valenzuela, 1990; Araya-Nunez et al., 1991, 1995 .˜In general, few reports describe the means by which pearl oysters are conditioned and

induced to maturation, and most are focused on studies of rearing larvae that wereobtained from already mature wild pearl oysters taken to the laboratory. This is becausemost of these species show the typical reproductive cycle found in the tropics, where itis common to find mature specimens throughout the year. This is not the case in Mexico´for P. mazatlanica and Pte. sterna. In the Gulf of California, mature specimens arefound only during well-defined seasons, summer for the former and winter for the latterŽ .Saucedo and Monteforte, 1997 .

Ž .In 1997, the Centro de Investigaciones Biologicas del Noroeste CIBNOR started´studies on the gonadic conditioning and larval rearing ofP. mazatlanica. The prelimi-nary results from these trials showed the need to control the main physical, chemical,and biological variables involved in the process of conditioning and maturity. This wasespecially to produce oocytes and spermatozoa of the best quality, which further willproduce viable larvae capable of completing their development. Water temperature anddiet are two of the most important variables that need to be controled. Our aim in thisstudy was to assess the effect of two water temperature regimes on the artificial

Ž .maturation of P. mazatlanica. Apart from the work of Mazon 1988 , whose results´were not completely satisfactory in terms of efficiency of gonad maturation andspawning, there is no other information on gonad conditioning for this species.

( )P. Saucedo et al.rAquaculture 195 2001 103–119 105

2. Materials and methods

2.1. Broodstock management

The rate and efficiency of gonad conditioning and maturity of adult pearl oysters wasŽ .tested at two temperature regimes, constant temperatures CT and gradually increasing

Ž .temperatures IT . For the former regime, three temperatures were selected; 208C, 248C,and 288C. These temperatures represent most of the annual range of variation observedfor organisms from the wild population. For IT, water was increased from 208C to 298C,28C every 15 days over 2 months.

In April 1997, 120 cultured specimens were collected and transferred to experimentalŽ .units 1200-l fiberglass tanks at the laboratory. Four experimental units were used, each

Ž .one corresponding to one temperature tested three for CT and one for IT . Eachexperimental unit held 30 oysters whose size ranged from 90 to 160 mm shell height.This size selection was chosen to increase the possibility of having both sexes present in

Žthe sample, considering the species is a protandrous hermaphrodite Saucedo and.Monteforte, 1997 . Before starting the experiment, specimens were induced to spawn by

Ž .thermal shock, following the methodology suggested by Loosanoff and Davis 1963 forbivalve mollusks. Only spawned specimens were used at the beginning of the experi-ment to assure that gonadic tissue was spent.

The seawater in each experimental unit was sterilized with UV and filtered with 10-and 5-mm Gaff bags. A titanium 1 1r2-HP water chiller was used for cooling andmaintaining seawater at the selected temperature. Similarly, a variable number of 250-Wwater heaters were used for heating and maintaining seawater at the desired temperature.

Oysters were fed daily withIsochrysis galbana var. tahitiana and Chaetocerosgracilis in a proportion of 7:3. Microalgae were supplied at a constant flow of 27.7mlrs using an open flow system. A sufficient airflow was provided to each unit tomaintain microalgae in suspension and oxygenate water. The daily routine also includedthe removal of feces by siphoning, both in the morning and in the afternoon, andmeasurements of temperature and salinity.

2.2. Histological analysis

During gonad conditioning, the ongoing process of gametogenesis was evaluated byhistological analysis. For that, gonad and abductor muscle were excised from eachoyster. Since pearl oysters do not have a gonad as a defined and functional organ, thegonad was considered to be the visceral mass in which gonadic tissue is intermingledŽ .Saucedo and Monteforte, 1997 . Sampling was done by collecting seven oysters fromeach unit every 15 days. For the histological examination of gonads, the scheme of five

Ž .stages, initially proposed by Tranter 1958a for pearl oysters, was followed with someŽ . Ž .modifications to the number of stages used. The stages are; 1 Inactive, 2 Gametogen-

Ž . Ž . Ž . Ž .esis either early, mid, or late , 3 Mature or Ripe, 4 Spawning partial or total , andŽ .5 Spent.


2.3. Oocyte examination

Parallel to the histological examination, the oocyte frequency for each temperatureregime was estimated in three different regions of the ovary by counting the total

Ž .number of oocytes appearing in a predetermined test area Grant and Tyler, 1983 . Thediameter range of oocytes was also determined in three different regions of an ovary.Only those oocytes sectioned through the nucleus were measured along their longestaxis. Thirty oocytes were measured per region. Both procedures were done using aneyepiece graticule.

Oocytes were classified according to their vitellogenic stage, following the criterionŽ .proposed by Gaulejac et al. 1995 as previtellogenic, vitellogenic, and postvitellogenic.

Previtellogenic oocytes are those immature, round oocytes that still are connected to thefollicular wall and lack yolk. Vitellogenic oocytes are those still immature oocytes foundconnected or free from the follicular wall, showing a peduncle shape. Postvitellogenicoocytes are those fully ripe, polygonal-shaped free oocytes.

( ) ( )2.4. Condition Index CI and Muscle Condition Index MCI

During tissue excision, morphometric measurements including the whole wet weightof the oyster with shell, wet weight without shell, wet weight of the visceral massŽ .where gonadic tissue gradually develops , and wet weight of the abductor muscle weretaken. These data were used to calculate two indexes.

Ž . Ž .1 Condition Index Sakai, 1979 :

CIsVMrW=100

where CI is the condition index, VM is the wet weight of the visceral mass, andW isŽ .the wet weight of the oyster without shell both in grams .

Ž . Ž2 Muscular Condition Index Marshall, 1979, modified by Saucedo et al. for this.experiment :

MCIsMWrW=100

where MCI is the muscular condition index, MW is the wet weight of the adductorŽ .muscle, andW is the wet weight of the oyster without shell both in grams . Because

Ž .muscle acts as an important site of glycogen storage Gabbott, 1975 , which is furthertransferred to the gonad to fuel gametogenesis, this index is a useful tool to understandthe relation between both organs, particularly the role of muscle as source of energy forthe gonad during gametogenic cycles.

Ž .The original proposal of Marshall 1979 to calculate MCI involved the measurementof the maximum diameter of the adductor muscle divided by the dorsoventral distance ofthe animal. Pectinid scallops have a round muscle, and measuring the maximumdiameter is easy for the calculation of this index. Pearl oysters, however, do not have around muscle. Instead, they have a large, curved, and irregularly muscle. Thus, themaximum diameter cannot be used as a reliable measurement to calculate MCI. Ourcriterion to solve this problem was the use of weight measurements in the formulainstead of linear measurements.


2.5. Statistical analysis

Considering the experiment was carried out with specimens of a native speciesclassified by the Mexican legislation as aAspecies under special protectionB, we wereunable to collect large quantities of broodstock from natural beds to be used as replicatesof each of the regimes tested in the experiment. Instead, we decided to use singleexperimental units for each regime without replication. Although this methodologicaloption was not the best for a proper statistical treatment, it allowed us to test more

Žexperimental variables e.g. two temperature regimes instead of only one, and three.experimental temperatures from the CT regime .

For the CT regime, a two-way ANOVA analysis was done to assess significantdifferences in the frequency and diameter of postvitellogenic oocytes between tempera-

Ž . Žtures factorT with three levels: 208C, 248C, and 288C , and over time factort with.four levels: 15, 30, 45, and 60 days of conditioning . Only postvitellogenic oocytes were

considered for the analysis because their frequency in time increases when the percent-age of previtellogenic andror vitellogenic oocytes decreases. For the IT regime, aone-way ANOVA was used for the oocyte frequency and diameter as only factort was

Ž .directly involved. Similarly, a two-way ANOVA for CT regime and a one-wayŽ .ANOVA for IT regime were used for significant differences in the value of both

condition indexes between temperatures and over time. When necessary, a Tukey testfor unequal mean comparisons was done to detect the levels where significant differ-ences were found. All analyses were made with STATISTICA 5.5 for Windows.

3. Results

( )3.1. Controlled Temperatures CT

3.1.1. Histological analysisA temperature of 208C seems to be inadequate for gonad conditioning and inducing

ripeness of adult oysters, either male or female. Although some gonad development wasobserved, only 24% of the total sample reached a late gametogenesis after conditioning,

Ž .and there was no evidence of ripe specimens Fig. 1 . In addition, a large number ofŽ .atretic oocytes were observed at this temperature Fig. 1D , suggesting the existence of

Ž .unsuitable conditions. The sex ratio was completely skewed to males 96.4% and onlyŽ .one female appeared during the whole experiment 3.6% . The femalermale sex ratio

was 0.04:1.The best conditions for inducing gonadic conditioning were found at 248C. At this

temperature, the highest proportion of fully ripe specimens was observed, with noevidence of atretic oocytes and a strong decrease in the percentage of specimenspartially spawned. Moreover, a large number of auxiliary cells were seen among

Ž .vitellogenic and postvitellogenic oocytes Fig. 1E . The sample was again skewed toŽ .males 71.4% and the femalermale sex ratio 0.4:1.


Fig. 1. Histological features of oocytes ofP. mazatlanica conditioned with controlled constant temperatures atŽ . Ž . Ž . Ž .208C A–D and 248C E–F . A Partially spent female in which residual oocytes ro that have lost nucleus

Ž . Ž .and undergo cytolysis are evident. B Immature connected vitellogenic oocytes vo of oysters that remainedŽ .on early and mid-gametogenesis. All are peduncle-shaped oocytes. C Inactive phase showing empty follicles

Ž . Ž . Ž . Ž .ef . D Atretic oocytes ao that have lost shape and tend to be lobular. E Immature vitellogenic oocytesŽ . Ž . Ž .vo surrounded by several auxiliary cells ac that help oocytes during its nutritional development. F

Ž .Postvitellogenic oocytes po that reached full maturity and have their typical polygonal shape. Scale bars25mm.

A temperature of 288C was also suitable for gonadic conditioning. No atretic oocytesand only few cases of partially spawned specimens were detected by histological

Ž .examination. The sex ratio was again skewed to males 89.3% . The femalermale sexratio was 0.12:1. At this temperature, however, oysters were found mostly at mid-game-

Ž .togenesis 41.6% , with only 8.3% reaching full maturity.


3.1.2. Oocyte examinationThe frequency of the three types of oocytes among temperature regimes and over

time is shown in Fig. 2. It is evident that there is a clear tendency for decreasingpercentage of previtellogenic oocytes and increasing postvitellogenic oocytes over timeat 248C and 288C, particularly at the lower temperature. The frequency of postvitel-

Žlogenic oocytes was significantly different between the three temperatures testedFs. Ž .23.09; Ps0.00001 , but not over timeFs1.852; Ps0.2169 . Here a significantly

lower percentage of postvitellogenic oocytes was found at 208C.The mean oocyte diameter in each type of oocyte at each temperature tested is

presented in Fig. 3. Previtellogenic oocytes were larger at 208C and smaller at 288C.Vitellogenic oocytes were largest at 288C and smallest at 248C. Finally, postvitellogenicoocytes were larger at 248C and smallest at 288C. However, there were no significant

Ž .differences in the mean diameter of oocytes between temperaturesFs3.52; Ps0.065 ,Ž .but differences were significant over timeFs19.63; Ps0.00033 . According to

Tukey’s test, previtellogenic oocytes remained significantly smaller than vitellogenicand postvitellogenic oocytes during the experiment.

Fig. 2. Temporal variation in the frequency of the three types of oocytes fromP. mazatlanica maintained withcontrolled temperatures of 208C, 248C, and 288C during gonadic conditioning. PREVsprevitellogenicoocytes; VITELs vitellogenic oocytes; POSTVspostvitellogenic oocytes.


Fig. 3. Mean diameter of the three types of oocytes fromP. mazatlanica maintained with controlledŽ .temperatures of 208C, 248C, and 288C during gonadic conditioning. Standard deviations"Std. Dev. ,

Ž .standard errors"Std. Err. , and means are shown.

3.1.3. Condition Index and Muscular Condition IndexŽ .The best yield for the CI was observed at 248C, and the worst at 288C Fig. 4 .

However, specimens never recovered the initial condition at which they started the

Fig. 4. Variation in the mean value of the condition index ofP. mazatlanica maintained with controlledtemperatures of 208C, 248C, and 288C during gonadic conditioning.


experiment, suggesting that gonadic conditioning was not completely successful. TheANOVA analysis detected significant differences in the values of the CI both between

Ž . Ž .temperaturesFs7.17; Ps0.0076 and over timeFs10.25; Ps0.0031 . Accord-ing to the Tukey test, the greatest difference in the value of the index was found at 288C,with this difference significant from day 45 of conditioning on.

The MCI behavior is presented in Fig. 5. In this case, the temperature of 288C offeredthe more suitable conditions to increase muscle activity and performance, whereas 248Cthe worst. The ANOVA test showed again significant differences in the values of the

Ž . ŽMCI between temperaturesFs9.87; Ps0.003 , but not over timeFs2.74; Ps.0.093 . The Tukey test found significant differences at 248C.

( )3.2. Gradually Increasing Temperatures IT

3.2.1. Histological examinationConditioning using increasing temperatures appears also to be an adequate strategy

for inducing gonad conditioning, because there were few cases of partial spawning andŽoocytes found at different developmental stages had their typical morphology whether

.connected or free , with no evidence of atretic oocytes. Moreover, during gonadconditioning all the sexual phases of female development were seen. However, the speedfor reaching maturity was slower using IT than with CT.

Gonad development reached advanced stages during conditioning, and the highestproportion of the sample was composed of fully ripe oysters at the end of the

Fig. 5. Variation in the mean values of the muscle condition index ofP. mazatlanica maintained withcontrolled temperatures of 208C, 248C, and 288C during gonadic conditioning.


Ž .experiment. The sample was again skewed to males 67.8% , with females at 28.6%,Ž .and an indeterminate specimen 3.6% . The femalermale sex ratio was 0.42:1.

3.2.2. Oocyte examinationOocyte frequency is shown in Fig. 6. All types of oocytes were present during the

experiment, and there is no clear pattern as to decrease previtellogenic oocytes andincrease vitellogenic and postvitellogenic oocytes over time. However, from day 30 to60 of conditioning, both a decrease in the frequency of previtellogenic oocytes and anincrease in the frequency of vitellogenic oocytes was observed. There were no signifi-

Žcant differences in the frequency of postvitellogenic oocytes over timeFs3.29;.Ps0.089 .

As conditioning proceeded, and temperature was gradually increased in the tank from228C to 29–308C, a large variation in oocyte diameter among each oocyte type was

Ž .observed Fig. 7 . At that temperature range variation, previtellogenic oocytes remainedconstant at 23–25mm until 45 days of conditioning, and dropped at day 60 whentemperature was 308C. In vitellogenic and postvitellogenic oocytes, although there is noclear pattern as to increasing oocyte diameter in time, it is evident that postvitellogenic

Ž .oocytes reached their maximum size 49mm diameter when temperature was also

Fig. 6. Temporal variation in the frequency of the three types of oocytes fromP. mazatlanica maintained withgradually increasing temperatures during gonadic conditioning. PREVsprevitellogenic oocytes; VITELsvitellogenic oocytes; POSTVspostvitellogenic oocytes.


Fig. 7. Temporal variation in the mean diameters of the three types of oocytes fromP. mazatlanica and itsŽ .relation to temperature changes during gonadic conditioning. Standard deviation"Std. Dev. , standard error

Ž ."Std. Err. , and means are shown.


Fig. 8. Variation in the mean values of the condition index and muscle condition index ofP. mazatlanicamaintained with gradually increasing temperatures during gonadic conditioning. CIsCondition Index; MCIsMuscle Condition Index.

highest at the end of conditioning. The ANOVA test found significant differences in theŽ .oocyte diameter over timeFs5.31; Ps0.0022 , where day 60 of conditioning

Ž .showed the greatest significant differences higher and lower in diameter for the threekind of oocytes.

3.2.3. Condition Index and Muscular Condition IndexFig. 8 shows the behavior of both indexes. Apparently, CI and MCI are reliable

indicators of gonad conditioning. As temperature was raised from 238C to 308C andgonad development proceeded normally, the values of CI and MCI showed the expectedinverse relation. There were significant differences in the values of both indexes over

Ž .time Fs24.86; Ps0.00001 for CI, andFs39.12; Ps0.000001 .

4. Discussion

In our work, two successful trials to stimulate gonad conditioning and maturity ofcultured specimens ofP. mazatlanica were made. The oysters were able to produce ripeoocytes and active spermatozoa after providing sufficient feeding and significant changesin water temperature. Other environmental variables were kept within narrow ranges,

Ž . Ž .such as microalgae concentration 75 000"5000 cellsrml and salinity 37"2‰ .However, none of the trials gave 100% gonad maturation and spawning, suggesting thepresence of other external factors affecting the overall process of maturation, of whichtemperature still seems to be the most important variable to be controlled. The test of


different temperature regimes allowed us to detect the presence of anomalies during theprocess of conditioning, such as the production of atretic oocytes and the high incidenceof specimens partially spawned found during the samplings. Both anomalies wereobserved only at 208C. This temperature clearly represents the lower limit of annualvariation recorded for Bahıa de La Paz. Apparently, the long period at which oysters´were maintained at a low temperature seems to be the cause of this.

Atretic oocytes are deformed cells, which further produce non-viable larvae incapableŽof completing the embryonic development and reach metamorphosis Gaulejac et al.,

.1995 . Thus, atreties and partial spawns indicate the oysters were subjected to unsuitableconditions at low temperatures, like 208C. This idea is supported by previous observa-

Ž . Ž .tions of Wada 1953 and Tranter 1958c . These authors suggested that anomalies ofthis kind were found in the pearl oystersP. maxima and P. margaritifera when exposedto some situation of stress, such as high temperatures or salinities, and high microalgaeconcentrations.

The quality and quantity of the diet supplied to oysters during its conditioningrepresents another important factor that affects gonadic development, gonad perfor-mance, and the type of oocytes produced at low temperatures. Although studies on diets

Ž .have been widely documented for bivalve mollusks Brown et al., 1989 , there are veryŽ .few data available for pearl oysters Teshima et al., 1987; Southgate et al., 1998 .

Today, diets for most bivalve mollusks are based mainly on some species of tropicalŽmicroalgae such asI. galbana var. tahitiana, Chaetoceros sp., Tetraselmis sp.,

.Nannochloris sp., PaÕloÕa sp., etc. , but microencapsulated lipids and yeast-based dietsŽ .have also been used with success Southgate and Beer, 1997; Southgate et al., 1998 . All

of them have important differences in their gross biochemical composition, especially inthe content of protein and total lipids, particularly cholesterol and highly unsaturated

Ž .fatty acids HUFA , and hence have different effects on the overall process of gonadŽ .maturation. This problem has been previously reported by Soudant et al. 1996a,b for

Pecten maximus. Unfortunately, no information regarding nutritional requirements ofpearl oysters based on such a biochemical and metabolic profile is available.

In our study, we used a diet composed of a mixture ofI. galbana andC. gracilis in aproportion of 7:3 in a continuous feeding system. Similar diets, and others includingTetraselmis sp. have also proved to be effective for adult conditioning and spawning of

Žadult Argopecten Õentricosus maintained under laboratory conditions Monsalvo-Spencer.et al., 1997; Racotta et al., 1998 . Other diets composed mainly ofI. galbana and other

microalgae in minor proportion have been successfully used for larval rearing of someŽ .species of pearl oysters, asP. fucata Alagarswami et al., 1983a,b ,P. margaritifera

Ž . Ž .Alagarswami et al., 1989 , andP. maxima Southgate et al., 1998 .Evidently, I. galbana is an important constituent of most diets used for artificial

maintenance of bivalve mollusks, including pearl oysters. However,P. mazatlanica andPte. sterna appear to have particular nutritional needs that are still unknown. Therefore,more research combining different species of microalgae with different nutritionalvalues, and artificial feeding strategies are needed to find an adequate or ideal diet.

During gonadic conditioning, the oysters behaved as protandrous specimens. Mostspecies of pearl oysters are protandrous hermaphrodites with a sex ratio reaching unity

Ž . Ž .at larger sizes, as forP. albina Tranter, 1958a,b ,P. margaritifera Tranter, 1958c ,P.


Ž . Ž .fucata Chellam, 1987; Behzadi et al., 1997 , andP. maxima Rose et al., 1991 . In aŽ . Ž .previous study, Garcıa-Domınguez et al. 1996 and Saucedo and Monteforte 1997´ ´

reportedP. mazatlanica also as a protandrous species. In this study, however, the strongskew to male sex seen at all temperatures was of particular interest, especially at 208C,where 27 of the 28 oysters sampled were male. Only large adult specimens, ranging 142

Ž .mm shell height"9.7 mm s , were used and a sex ratio near unity was expected. Thevalues of the sex ratios suggest a possible anomalous sex reversal from female to male.The stress generated by low temperatures maintained constant for long periods of time

Ž .may explain this phenomenon. Taylor 1999 , however, suggests that this kind of sexreversal is triggered in the pearl oysterP. maxima by high stocking densities at whichspecimens are maintained during the initial culture phase before transfer to the labora-tory.

In our study, although gonadic development for both temperature regimes did notreach advanced sexual stages at the frequency expected, and gonadic performance wasnot 100%, there was high reproductive activity detected at temperatures of 248C and288C for CT, and for all the cycle of IT. At 248C, for example, a large number ofauxiliary cells were seen among most vitellogenic and postvitellogenic oocytes. Thepresence and role of these cells in helping oocytes and spermatozoa during their

Ž .nutritional development has been previously reported by Gaulejac et al. 1995 for themarine bivalvePinna nobilis.

Under normal conditions, gonad development during oogenesis and spermatogenesisimplies a rapid increase in lipid and protein content in this organ, while spawning

Žimposes a decrease in these components and an increase in water content Barber and.Blake, 1991 . The occurrence of this biochemical process in our specimens was

supported by both condition indexes showing the expected inverse relationship duringthe whole conditioning process. At the beginning, low values of CI and high values ofMCI were measured. At this point, gonad development was poor or absent, and adductormuscle was growing as it acts as the most important site of glycogen storage and source

Ž .of energy Gabbott, 1975 . As gametogenesis proceeded, such energy was transferred togonad for its development, and thus, CI increased and MCI decreased.

More research about pearl oyster culture in the laboratory is needed. Although ourprevious results concerning collection of wild spat have been highly satisfactory, analternative to provide an abundant and constant input of healthy spat should also beavailable. The world pearl industry is relying more and more on hatcheries but thestudies on gonadic conditioning are few. Considering the reproductive characteristics of

Žour native pearl oyster population mature individuals are present only during limited.periods of the year , we believe that the correct management of gonadic conditioning is

one of the most important factors for sustaining a future development of pearl farms onthe Mexican Pacific coast. This control could ensure a constant availability of suitablejuveniles to be cultivated both for repopulation of natural beds and pearl production.

5. Conclusion

Both temperature regimes tested in this study to induce gonad conditioning andmaturity of adult specimens ofP. mazatlanica under laboratory conditions gave


satisfactory results in terms of the intense reproductive activity noticed since condition-ing started. However, an important differential effect of temperature in specific aspectsof gonad development was also detected. Thus, these results are preliminary and willserve to develop more detailed experiments of gonadic conditioning for the species. Ourobservations will also be useful to define the main needs for designing furtherexperiments about maturation, induction to spawning, and larval rearing for the species.Although temperature seems to be the most important variable to control in this kind ofexperiment, more detailed studies involving other variables are required. It is necessaryto test different management strategies of temperatures and diet components based onthe variation of biochemical profiles during gametogenesis. This research is in progressand the results will be available in the future.

Acknowledgements

This work is dedicated to the memory of Don Gaston Vives, pioneer of pearl culture´in the world. The study was done as part of an institutional project of CIBNOR on Pearl

Ž .Oyster Culture and Pearl Induction at Bahıa de La Paz Project AMB-7 . The study was´Ž .also granted by the Consejo Nacional de Ciencia y Tecnologıa CONACYT , Fondo´

Ž .Mexicano para la Conservacion de la Naturaleza FMCN , Consejo Nacional de la´Ž . Ž .Biodiversidad CONABIO , and Sistema de Investigacion del Mar de Cortes SIMAC .´ ´

The authors are grateful to MSc Horacio Bervera Leon and Tec. Juan Jose Ramırez´ ´ ´Ž .Rosas CIBNOR for the invaluable support provided during the SCUBA diving and the

Ž .collection of oysters. We also like to thank Dr. Ilie Racotta CIBNOR for his commentsŽ .made to the manuscript. Dr. Ellis Glazier CIBNOR also helped in the edition of the

English-language manuscript.

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