Determination of Gonad Development of Mangrove Clam Polymesoda expansa (Mousson 1849) by Histological Classification

MATERIALS AND METHODS

Study area and sampling procedure: The study area is situated in the mangrove site of Kelulit river (03°58.649’ N, 113°44.244’ E) Miri, Sarawak, Malaysia. Samples were collected monthly in random over a period of 14 months from October, 2010 to November, 2011. However, no samples were collected in February, April and August, 2011 due to over exploitation by the locals. Physico-chemical properties of adjacent estuarine water such as temperature, salinity, pH and dissolved oxygen were measured and recorded using WQC-24P Multi-parameter during sampling. Live clam were transferred to the laboratory for further processing.

Gonads collection and index values calculation: A small part of the gonad from the mesosoma and mantle lobe from each dissected clams were taken out and processed following histological techniques¹⁶.

Monthly Gonad Index (GI) was computed utilizing a numerical grading system to obtain a quantitative value that represents the reproductive activity. From the results of the histological studies, the sexes of the specimens were further confirmed with the initial visual identification. Later, macroscopic gonad stages of each sample were assigned and gonad index were calculated^17-19:

(1)

The stages of gonad developmental were classified as rest (indifferent), develop (immature), mature, spawn and spent ²⁰.

Histology: Histology was observed following histological techniques¹⁶.

Statistical analysis: Pearson correlation analysis was implied to find any correlation between GI and physico-chemical properties of estuarine water parameters including rainfall in Kelulit, Miri¹⁷.

RESULTS

Environmental parameters: Study showed that small differences among monthly measurement for salinity and DO for water quality (Table 1). However, temperature assessment indicated month of September, 2011 as the highest and the lowest recorded in January. May, 2011 has as the highest pH value and November, 2010 as the lowest. The data on monthly rainfall from October, 2010 to November, 2011 was collected from the Department of Meteorology of Malaysia (Fig. 1). Rainfall found fluctuated throughout the year with highest in January, 2011 and lowest in October, 2011.

Gonad development stages: Five stages of gonad development were identified in P. expansa both for males and females are resting, developing, mature, spawning and spent (Plate 1, 2). The colors of gonads were grayish in females whereas, whitish in males and no hermaphrodite found in P. expansa at Kelulit. Different gonad development stages for both sexes are summarized as in Table 2.

Fig. 1:	Rainfall data collected taken from Kelulit, Miri, Malaysia, during the sampling period from October, 2010 to November, 2011 (Source: Malaysia Meteorology Department)

Table 1:	Physico-chemical properties of estuarine water (Mean±SD) measurement from October, 2010 to November, 2011

Table 2:	Description and criteria for the gonadal stages of Polymesoda expansa

Plate 1(a-f):	Gonad development stages in females of P. expansa (a) Resting, (b-c) Developing, (d) Mature, (e) Spawning, (f) Spent

Reproductive cycle: Since no hermaphrodites found, the reproductive cycle on P. expansa were easier to determine. Active gametogenesis was recorded throughout the study period with all stages of development usually occurring simultaneously within the same sample population. Relative frequencies of the maturity stages for P. expansa at Kelulit are illustrated in Fig. 2.

Though gametogenesis started early of January and the clams developed gametes, some of the gonads were seen still in resting stage (Fig. 2). Active maturation was observed in the month of January with gametogenesis where gametes matured slowly and from March-October, mature gametes were found less than 25% until it became higher in November (Fig. 2). From March to November, the gonads were maturing and the spawning started. Gonads were observed to be spawned in September and continuously throughout the year it became less in percentage.

Spermatogenesis and oogenesis appeared to continue to some extent through the breeding season in parallel to spawning. From December until June, a small percentage of the gonads were spent, which was the follicle progressively occurring with fewer and fewer mature gametes.

Plate 2(a-f):	Gonad development stages in males of P. expansa (a) Resting, (b-c) Developing, (d) Mature, (e) Spawning, (f) Spent

Spawning occurred all year around. However, in January, when water temperature were at their lowest (Fig. 2), no spawning was recorded. Polymesoda expansa population at Kelulit, Sarawak was thus characterized by all year around spawning, although only in September spawning occurred showing maximum frequency.

Monthly gonad index: Gonad index results represented that spawning in P. expansa was year around except in December and January. Spawning between sexes was fairly synchronized in November and May (Fig. 3). Between January and July, spawning activity was occurred mainly in males. Kruskal-Wallis non-parametric test showed no significant differences at p>0.05 in Gonad Index (GI) between the males and females.

The GI showed a seasonal trend along the year, with high values related with mature individuals while the fall of values were due to spawning activity. The GI values for pooled of P. expansa were higher in November and March and were lower from December to January (Fig. 4). The GI values indicated that the reproduction was inactive from December to January and gametogenesis started in March and continued until October.

Table 3:	Pearson Correlation analysis of Gonad Index (GI) of P. expansa with different physico-chemical properties of estuarine water parameters in Kelulit, Miri
*Correlation is significant at the 0.05 level (2-tailed), r: Pearson correlation coefficient, N: number of sample, DO: Dissolved oxygen

Fig. 2:	Reproductive stages of P. expansa in Kelulit, Miri (n = 67)

Fig. 3:	Gonad Index (GI) values for both male and female of P. expansa

Fig. 4:	Gonad Index (GI) values for pooled of P. expansa

A Pearson correlation analysis test (Table 3) was applied between GI and the different physico-chemical properties of estuarine water parameters from Kelulit, Miri, however, no significant correlation (p>0.05) was found. The GI presented a positive correlation only in temperature and dissolved oxygen while other physico-chemical properties of estuarine water parameters showed negative correlation.

DISCUSSION

Environmental parameters: Water temperature showed considerable seasonal variation with extreme values of 27.7°C in January, 2011 and 31.6°C in September, 2011 when monthly temperature was recorded with the mean of 29.4±1.3°C while salinity was 2.79±3.15 psu. Temperature is the most important parameters, which influenced on bivalve reproduction regulation²¹. Besides that, changes in water salinity affect a wide variety of biochemical, physiologic processes and growth in marine bivalves^22,23.

Different temperature and sampling dates indicate synchronous gonad maturation in male and female gonad of bivalve ²⁴. This is due to the faster gonad maturation rate that was observed at 25-30°C rather than at 20°C which is possibly because of the higher metabolic rates found at higher temperature in this species. When the variation in temperature and the gonadosomatic index examined, water temperature at 24°C seems to be the lower threshold for gonad development²⁵. However, present study on reproductive cycle was found neither temperature nor salinity showed a clear relation with their reproductive cycle since spawning appear during the annual cycle.

Gonad developmental stages: The sexes were determined for each individual by the presence of egg and sperm in the tissue section under a microscope. Many schemes for observation of histological samples for classifying gonad developmental stages in mollusc can be found²⁶. From the microscopic observations of gonadal sections it was determined that P. expansa is a dioecious (separate sex) species.

Five stages of gonad maturation of P. expansa were clearly described. Resting stage represented minimum gonadal proliferation where gonad and interfollicular areas were fully covered by connective tissue²⁷. No oocytes, spermatocytes or later stages recognizable and sex undetermined²⁸. Gametogenesis started in developing stage and was characterized by a rapid thickening of the gonad and branches of the developing follicles²⁹. Follicle size increased, the first spermatocytes were observed in males and the first previtellogenic oocytes appeared on the follicular wall in female³⁰.

Mature stage was characterized by over 90% of the volume of the gonad being occupied by mature follicle with well-developed gametes for most individuals³¹. The inter follicles connecting tissue disappeared and follicles reached to maximum size²⁰. In male, acinus seemed full of spermatozoa, with their tails towards the acinus lumen while in female, this stage was at the end of vitellogenis process. Spawning in male, the lamellar arrangement of spermatozoa was disappearing and follicles were less densely packed³². Empty spaces were observed in the follicular lumen and a low density of free ripe oocytes with a broken germinal vesicle was observed in female³¹. Finally, complete destruction of the follicle walls and gonad volume fast covered by the connective tissue in male spent stage²⁰. Whereas, follicules were getting smaller, connective tissue re-formed and oocytes in some follicules absorbed.

Reproductive cycle: The reproductive strategy found in P. expansa was clearly observed as dioecious. The existence of dioecious within the family Corbiculidae was observed for P. erosa³³. The two sexes can be separated from very small sizes on 14 mm in P. solida³⁴. Annual gametogenic cycle was exhibited throughout the study period, which began in October to November with rapid proliferation of gametes especially in November to July.

Most of the marine bivalves in Malaysian coastal waters showed a reproductive pattern that is active all year around. Therefore, the extensive culture system is only practiced for bivalve molluscs on coastal mudflats³⁵. Continuous spawning activity all year around was reported on Perna viridis from Penang¹³. Similar observations were made for P. viridis at Quezon, where the mussels undergo gametogenesis throughout the year with no repetitive during two years of study³⁶.

Monthly gonad index: Numerical grading system from the sectioning of the gonads tissue was used to calculate the Gonad Index (GI)¹⁹. The objective of the analysis was to find the spawning period or seasonal variations of P. expansa. Continued gonad developmental and spawning throughout the year in P. expansa in the region was an expected trend. This was an acceptable trend since they occur in a tropical condition where they are subjected to stable and warm temperature all year around. However, the Pearson correlation analysis indicated that an environmental factor was not influencing the gonad growth.

Determination of first maturity length and spawning period are the basic requirements for protection and conservation of the species. Several methods might be used to study the gonad development and reproductive cycle; however, the most precise results were obtained through histological technique. This was because, other approaches such as metabolomics and genetic methods were not being able to determine an absolute gender in some gonad developmental stages³⁷.

CONCLUSION

Polyemesoda expansa from Kelulit, Miri was observed dioecious and no hermaphrodite individual was found. Through histological classification, five stages of gonad development had been carried out and clearly identified for both male and female which were resting, developing, mature, spawning and spent. Reproductive cycle of P. expansa showed gametogenesis that continuously spawned all the year round since they occur in tropical conditions where most of the physical and chemical parameters are fairly stable throughout the year. The GI was found not to be influenced by any of the physico-chemical parameters recorded in Kelulit, Miri, Malaysia. It was proven by the pearson correlation analysis that showed no significant correlation (p>0.05) between GI and physico-chemical parameters of estuarine water in the study area.

SIGNIFICANCE STATEMENTS

This study discover, characteristic of the gametogenic development in the P. expansa through one year observation. Polymesoda expansa is categorized as dioecious and able to spawn every month. This study will help the researcher to uncover the critical area especially in the artificial breeding and larvae rearing for P. expansa under laboratory condition that many researchers were not able to explore. Thus, new technique in artificial breeding and rearing may be discovered.

ACKNOWLEDGMENTS

Authors would like to thank Universiti Putra Malaysia for research grant (Research University Grant Sheme No. 05-01-11-1267RU, RUGS Project No. 9199876) to be possible for this study conducted. The authors would like to thank the deanery and staff from the Department of Animal Science and Fishery, Faculty of Agriculture and Food Sciences, Universiti Putra Malaysia, Bintulu Sarawak Campus and University Malaysia Terengganu for technical, logistic and facilities support, which made this study possible.