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Chapter one Introduction
1.1 Background
The mollusk fishery has significant contribution in the aquaculture consumption of Bangladeshi coastal tribal communities. Small scale fishermen living near the coast collect bivalve and univalve for domestic consumption as well as for economic purpose. In many part of the Asia, bivalves have been recognized as a valuable food source collected from wild or as a byproducts from culture operations, and still bivalve resources are widely exploited from the capture fisheries. In Bangladesh consumers of Perna viridis is confined to the tribal people living along the coastal region and they collect them directly by hand picking from the wild. This wild exploitation can be resulted into reduced the population stock in their natural habitat.
Like other Asian countries Bangladesh can develop commercial shellfish farming in small scale to attain the viability and meet the local demand. In recent years competition for fisheries resources has increased particularly in Cox’s Bazar region due to resource conflict and increasing demand for food production and decrease in traditional finfish fisheries, development of shellfish farming practices can create a new horizon to support this situation. Green mussel provides the highest conversion of primary producers (phytoplankton) to human food as a low-priced source of animal protein, and culture of mussels in the water column can boost the seafood production several folds (Pillai et al., 2000).
Among the exploited bivalve resources of Bangladesh green mussel (Perna viridis), oyster (Crassostrea sp.) and clam (Meretrix meritrix) are economically important species. They have a number of consumers where green mussel is most abundant and widely distributed particularly in Cox’s Bazar. Shahabuddin et al., 2010 reported the presence of Green mussel along the coast of Cox’s Bazar, Bangladesh and abundance was highest in Moheskhali comparing with Cox’s Bazar Sadar upazila and Teknaf.
Study on abundance, distribution and population dynamics of Perna viridis was previously done by many authors (Kamal and Khan., 1998; Shahabuddin et al., 2010;
Amin et al., 2005) but reproductive biology study for the purpose of revealing breeding cycle of this species in the Moheskhali Channel have not been investigated
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yet. Study on reproductive parameters is essential to develop management strategies and give initial information on reproductive season for which will be beneficial in mussel culture. Development of green mussel (Perna viridis) culture system can be a good initiative to use the concept of blue economy and it will be an ideal species for mariculture which is already established in many Asian countries.
Gametogenic cycle in mussel occurs annually or several times in a year (Vakily, 1989) depending on the condition of water quality, particularly temperature gradient and salinity. It consists of several steps from activation of gonad, through gametogenesis to the gamete release and the subsequent reduction of the gonad (Seed, 1969). Gametogenesis is the process of formation of reproductive cells or gametes (oocyte or egg, spermatozoa). Oogenesis is the process of egg formation and production of spermatozoa is known as spermatogenesis (Dorange and Le Pennec, 1989). Several oocyte and spermatocyte stages can be present. Acini are the sacs of connective tissue containing oocytes and follicle cells and sometimes it is mistakenly termed as follicles (Beninger and Le Pennec, 1991). Follicles are made of an oocyte surrounded by layers of acini cells (Tyler and Sumpter, 1996). In temperate regions, several mussel species starts gametogenesis in winter fueled by the reserve of protein, lipid and carbohydrate accumulation during the summer time and autumn (Seed, 1969; Seed and Suchanek, 1992). Spawning followed by a resting and storage period before winter gametogenesis starts again (Buchanan, 2001). To obtain reproductive cycle the most reliable and detailed information can be found from the histological preparation of gonad (Seed and Suchanek, 1992). In mussel gametogenesis occurs mainly in the mantle tissue but genital tissue can be found in all body tissue (Seed and Suchanek, 1992). During non-breeding season it is nearly impossible to distinguish male and female as they are spent, even from histological preparation (Seed and Suchanek, 1992). Histological sectioning can recognize various stages in reproductive cycle (Buchanan, 2001; King et al., 1989). The general reproductive condition of the population can then be assessed by calculating a mean gonadal index (GI). Condition of the mussel is directly related to gametogenic cycle in its lifetime depending on the habitat they live. Gametogenic cycle can also be identified using different condition indices and gonad index. Among those, gonadosomatic index is widely used to identify fish reproductive cycle but this is a new concept for bivalve population.
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Meat content, condition and biochemical composition of bivalve vary seasonally depending on the latitude. These are strongly related to water temperature, food availability and the gametogenic cycle of the species (Baird 1958, 1966; Lutz 1980;
Zandee et al., 1980; Smaal and Stralen, 1990).Condition index is using as the indicator of seasonal tissue storage cycle which ultimately measure the fatness and marketability of the species. Together with biochemical composition, it can particularly monitor gametogenic activity as condition indices changes in different life stage.
1.2 Research objectives
The specific objectives of the study were as follows-
1. Determining the ratio of female and male individual.
2. Finding a pattern of their reproductive cycle with seasonal variation from the collected wild stock of P. viridis population from Moheshkhali Channel.
1.3 Research questions
When does Perna viridis gonad development start?
How many cycles occur in a year?
How do water quality parameters influence gametogenic cycle and condition indices along with seasonal variation?
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Chapter two Review of literature
2.1 Importance of green mussel in Bangladesh
The coastal area of Bangladesh is suitable for mollusks habitats as sandy and rocky ground, mangrove and coral reefs exists (Shahabuddin et al., 2010). It is also suitable for development of shellfish culture though vast majority of population do not consider shellfish favorable for eating. Mollusks species in the Bay of Bengal can be an important part of our economy. Among the shellfish species green mussel (Perna viridis) have potentiality to be exported and being an economically important species (Shahabuddin et al., 2010). Kamal and khan, 1998 said that Cultivation and export of Caulerpa and mollusks like Green Mussel (Perna viridis), Crassostrea sp. and Mretrix meretrix could enrich the country's economy immensely. He also said, shellfish flesh can be exported to foreign countries along with shrimp and crabs and technologies can be developed for culturing shellfish in coastal area to ensure the conservation of shellfish biodiversity in nature as well as keeping harmony with the future fast growing industry which will provide future employment opportunities, alternative protein to 0.2 million tribal people, earn foreign currencies and open a new arena in coastal aquaculture of Bangladesh.
2.2 General features of Green mussel:
Perna viridis species is widely distributed in the Indo-Pacific region, extending from Japan to New Guinea and from the Persian Gulf to South Pacific Islands (Siddall, 1980). External feature of the green mussel includes wavy posterior end of the paleal line and the large kidney-shaped anisomyarian muscle. Periostracum can be vivid green to dark brownish-green near the outer edge and olive-green near the attachment point. According to FAO Species fact sheet, the interior of the shell valves is shiny and pale bluish green. They forms dense populations up to 35000 individuals per square metre area. On a variety of structures including vessels, wharves, mariculture equipment, buoys and other hard substrata. This species is an efficient filter feeder, feeding on small zooplankton, phytoplankton and other suspended fine organic material. Sexes are separate and fertilization is external in this species. Spawning generally occurs twice a year between early spring and late autumn (Rajagopal et al., 1998), however, in the Philippines and Thailand spawning occurs year round. Sexual
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maturity occurs at 15-30 mm shell length. Life spans is about 3 years. Found in estuarine habitats with salinities ranging from 18-33 ppt and temperatures from 11-32
°C (Segnini de Bravo et al., 1998).
2.3 Gametogenic cycle
According to NIMPIS (2002) Sexes in this Perna viridis are separate and fertilization is external. Fertilized eggs develop into larvae and remain in the water column for two weeks before settling as juveniles. They reach sexual maturity within 2-3 months age.
Growth rates and reproduction are influenced by environmental factors such as temperature, food availability and water movement (Rajagopal et al., 1998). In mussels, the gametogenic process occurs mainly in the mantle tissue, but reproductive tissue can also be found in the visceral mass and mesosoma (Bayne et al., 1978; Lowe et al., 1982; Walter, 1982). The genital tissue practically invades all body tissues, filling the mantle lobes, mesosoma, the outer surfaces of the digestive gland, and the floor of the pericardium (Seed and Suchanek, 1992). Only the dorsolateral walls of the pericardium, foot, gills and muscles remain free from genital tissue (Seed, 1969).
Gametogenesis proceeds at the same rate in both the mantle and mesosoma (Walter, 1982). As gonadal development advances, a progressive increase in the ramification and size of the acinus occurs throughout the body mass (Grizel, 2003). The gonadal tissues color varies in both males and females and intensifies until maturation (Walter, 1982). During this time it is possible to sex individuals by the color and texture, female tends to be orange-red, while male being creamy white. Male acinus tend is round, even in size and regularly distributed, female acinus vary in size, smooth in texture, and less granular in appearance than the male. During the non-breeding season it is impossible to sex individual mussels when they are completely spent, even from histological preparation (Seed and Suchanek, 1992).
2.4 Water quality parameters affecting gametogenic cycle
The onset of reproductive cycle of mussels are believed to be controlled by an interaction between environmental and endogenous factors (Richard 2013; Gosling 2003; Rajgopal et al., 1991). The temperature influence the course of oocyte development, the number of differentiating oocytes and the onset of spawning in bivalves (Gosling 2003; Rajgopal et al., 1991; Urian 2009). Green mussels in the Indo-Pacific region experience an average annual water temperature range between 12
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and 32 °c (Rajagopal et al., 1998) with an optimal range between 26-32 °c (Power et al., 2004; Sreedevi et al., 2014). No considerable mortality was observed up to 30 °C, 21% mortality was observed at 32 °C and further increase of temperature from 36 °C lead to the 100% mortality. Moderate or suitable current speeds within the range of 0.1 – 0.3 m ⁄ sec have been reported to be good for mussel growth and reproduction (Lovatelli 1988).Slow water movement usually results in slow growth of the mussels and also promotes the settling of organic and inorganic particulate on organisms.
(Aypa 1990) observed a water current of 0.17–0.25 m ⁄ sec during flood tide and 0.25–0.35 m ⁄ sec at ebb tide should be observed. According to Lovatelli (1988), a site having a disc reading of less than 25 cm should be considered unsuitable for mussel culture. Green mussel is reported to tolerate a high range of salinity. (Sivalingam 1977) observed that the species has 50% survival salinity tolerance at 24 ppt and 80 ppt for a period of 2 weeks in a laboratory experiment. Tropical green mussel occurs typically in estuarine or coastal water that is rich in plankton has high salinity (27 ppt to 33 ppt) and warm temperature (26°C to 32°C) (Hickman 1989). Studies done by (Rajagopal et al. 1998) show the green mussel can grow in water salinity ranging from 5.2 ppt to 39.8 ppt. Water temperature also affects the growth of green mussel.
Sivalingam (1977) demonstrated the green mussel has 50% survival temperature tolerance from 10°C – 35°C under experimental testing. . It was reported that the optimal temperature for green mussel ranges from 26°C to 32°C (Hickman 1989), 27°C to 30°C (Aypa 1990), 25.3°C to 34.6°C (Rajagopal et al. 1998).
2.5 Condition index
Condition index (CI) a measurement of the meat content relatively to total size of the organism. The concept was developed primarily for the examination of oyster populations (Hawkins and Rowell, 1987). Grave (1912) was the first to use CI to evaluate the quality of oyster meat. In 1922, Havinga, B., was the first to describe an index based on substituting dry meat weight for meat volume (Hawkins and Rowell, 1987). Yap et al. (2002) have suggested that the CI of P. viridis can be used as a physiological indicator of metal pollution in the coastal waters.
There are a number of indices used in the eco physiological studies with meaningful physiological or biochemical variables, indicating of the metabolic state of the bivalve (Lucas and Beninger, 1985). If the CI is reduced, then the amount of tissue relative to
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its shell size or volume has decreased (Lawrence and Scott, 1982). The CI when used to indicate growth, defined as the ratio of the soft tissues dry weight over shell weight, appears to be a good indicator of growth rate (Yap et al., 2002; Thebault et al., 2005).
Better living conditions for the mussels would result in a two folds increase in CI from their initial settling natural grounds in Hong Kong (Lee, 1985a). Spawning or gametogenesis requires energy, and various species have different way to obtain energy (Gosling, 2003). Some species use the recently ingested energy others use the stored energy in various organs (Gabbott, 1976; Bayne, 1976). A close relationship has been reported between the gametogenic cycle, CI and the storage consumption cycle of reserves particularly glycogen and meat quality (Gabbott, 1976). When CI is used together with approximate of biochemical composition of the bivalve, it is probably the most practical and simplest method of monitoring gametogenic activity (Okumus and Stirling, 1998). Therefore the use of CI as an index to measure reproductive output is a common practice in the literature (Villalba, 1995; Orban et al., 2002; Pampanin et al., 2005). The CI depicts a clear seasonal trend in its values, showing higher values in spring and summer in Mytilus gallaprovincialis in Italy (Pampanin et al., 2005). The growth of gonadic tissues increases the body mass, and consequently the values of CI results are higher. Low values of CI correspond to emission of gametes. Therefore, a drop in CI of bivalves indicates a spawning.
This previous established study from literature review helped to interpret result and summarize the findings.
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Chapter three Methodology
In this section, detail description of procedures and materials are described how the experiment was carried out.
3.1 Sample collection and transportation:
Monthly samples were collected randomly once in every month from bottom settled wild P. viridis from November 2017 to October 2018 from the Choufaldandi Bridge, Maheshkhali Channel, Cox’s bazar, located at, 21°30′20′′ N and 91°59′19′′ E (Figure 1). Total twenty live samples were collected per month, which were immediately stored in the ice box with ratio of 1:2 samples and ice, transported to the oceanography laboratory, CVASU. (Sampling schedule has been given in table 1).
Sampling was done during the low tide in every case.
Figure 1: Sampling site
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Table 1: Sampling schedule
Date 2/11/17 1/12/17 5.1.18 2/2/18 2/3/18 2/4/18 3/5/18 2/6/18 8/7/18 3/8/18 6/9/18 11/10/18 Time 11:30
am
12:00 pm
11:20 am
10:45 am
11.00 Am
12:30 pm
11:40 am
10:30 Am
10:45 am
11:30 am
11:00 am
12:30 pm
3.2 Morphometric data collection:
Sampled twenty mussels were used to determine the individual total dry weight from February 2018 to October 2018. Mussels were initially cleaned from all the encrusting organisms and their byssus were removed. Biometric measurements such as length (maximum length along the anterior-posterior axis), height (maximum length along the dorsal-ventral axis), and width (maximum length through both valves) were measured individually using a Vernier caliper to the Vernier constant of 0.01. The total weight of each individual was recorded after the inter-valval (or mantle) fluid was drained.
They were than dissected and the wet soft tissue weight and the shell weight were recorded with electronic weight meter (AS 220.R2, Radwag, Poland).
Figure 2: Draining out inter-valval fluid 3.3 Length-Weight Relationship:
The relationship of shell length (SL) to total weight (TW), meat weight (MW) and shell weight (SW) were calculated according to the allometric equation (Le Cren, 1951):
W= a* Lb
Since weight is a power function of length, the logarithm is taken so that the exponential relationship can be expressed by a linear equation. This equation can be expressed in its linearized form:
log W = log a + b log L
This equation is related to regression equation: 𝐘 = 𝐚 + 𝐛𝐗
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W is the shell weight (total weight, total unshelled meat, shell weight L is the shell length whilst ‘a’ is the intercept (initial growth coefficient) and ‘b’ is slope (relative growth rate of variables).
3.4 Condition index (CI):
Dry tissue weight was measured after drying the sample tissue at 105 ̊ C for 12 hours in hot air oven and was cooled in desiccator.CI was calculated following Yep et al.
(2003).
CI (g/cm³) =𝒕𝒐𝒕𝒂𝒍 𝒔𝒐𝒇𝒕 𝒕𝒊𝒔𝒔𝒖𝒆 𝒅𝒓𝒚 𝒘𝒆𝒊𝒈𝒉𝒕 (𝒈) 𝒔𝒉𝒆𝒍𝒍 𝒗𝒐𝒍𝒖𝒎𝒆 (𝒄𝒎³) ×1000
Figure 3: Morphometric data Collection 3.5 Determination of meat yield:
In the calculation of meat yield, preferences was taken into consideration along the consumers of Bangladesh. Meat yield was calculated as considering the edible portion includes all the muscle part and organs such as gonad, digestive gland, gill and others.
Meat yield calculation was carried out throughout the year in each sampling for identifying seasonal variation. The equation which was followed is given below- Meat yield = [meat weight (gm) /total weight (gm)] ×100
Differences between meat yields of different groups were determined by one-way ANOVA using Tukey’s multiple comparison test.
3.6 Gonadosomatic Index (GSI):
After length, weight and width data collection the mantle of mussel was opened by knife to collect soft tissue and blotted. After removing the unwanted dirt, digestive parts and intestine, the gonad of the mussel was collected carefully by using forceps.
GSI is the ratio of samples gonad weight to body weight, which is particularly helpful to identify the spawning seasons. To determine the spawning period gonadosomatic index (GSI) plays a major role as there is a cyclic change in gonad weight in relation
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to total body weight. The GSI was calculated by month-wise and sex-wise using the equation by Vladykov (1956).
GSI= Weight of gonad
Total weight of mussel ×100
(a) (b) (c)
Fig. 4: (a) Green Mussel Gonad, (b) Gravid Female Mussel and (c) Matured Male P. viridis
3.7 Histological process
The histological processes involved several steps as described below. A small part of the gonad from the mesosoma and the mantle lobe from each dissected mussel was sub-sampled by cutting a transverse section midway along the anteroposterior axis.
The gonads were fixed in Bouiuns fixative for 24 hours and then post-fixed in 70%
alcohol prior to dehydration after washing in 50% alcohol for 2-3 hours. All procedures were followed manually following the procedure described in the table 1.
Table 2: Protocol of histological procedure of green mussel gonad
STEPS CHEMICALS TIME (Hours)
A. Fixation Bouins Fixative 24
B. Dehydration I. ethanol 50% 2-3
II. Ethanol 70% 2-3
III. Ethanol 80% 2-3
IV. Ethanol 90% 2-3
V. Ethanol 95% 2-3
VI. Ethanol 100% 2-3
VII. Ethanol 100% 2-3
C. Clearing 1. Alcohol (50%) +Xylene (50%)
2hours or overnight
2 Xylene 2 hours
3 Xylene 2 hours or overnight
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D. Infiltration a) Parafin + xylene 2 hours or overnight
b) Paraffin 2 to 4
c) Paraffin 2 to 4
E. Embedding
The gonadal tissues were then embedded in paraffin blocks. After embedding the tissues, the paraffin blocks were trimmed to facilitate accurate sectioning.
F. Trimming:
Trimming is a process in which the undesirable wax layers of the embedded blocks are trimmed by knife to obtain suitable blocks. It helps for easy sectioning. After proper embedding, trimming was done to trim undesirable wax layers.
G. Sectioning
The blocks were then sectioned at 5 to 7 μm thickness using microtome. The sections were then mounted on slides and dried overnight in an incubator at around 40 ̊C.
Before staining, the sections were dewaxed through immersing them into different bathes of xylene, alcohol, and water.
H. Staining and mounting
Staining was performed using Hematoxylin (Harris Hematoxylin) and Eosin through standard methods (Bancroft and Stevens, 1996). Upon reaching the desired staining
levels, the slides were mounted permanently with D. P. X. mounting media.
Fig. 5: Tissue Embedding, Tissue sectioning, Sectioned tissue in microtome and Staining
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Table3: The staining schedule Sl.
No.
Solutions Time Process
1 Xylene 10 minutes Clearing
2 Xylene 10 minutes
3 Xylene 10 minutes
4 100% alcohol 5 minutes Rehydration
5 100% alcohol 5 minutes
6 90% alcohol 3 minutes
7 80% alcohol 3 minutes
8 70% alcohol 3 minutes
9 50% ethyl alcohol 2 minutes Staining
10 Distilled water 15 dips
11 Haematoxylene 3 minutes
12 Wash in tap water 15 minutes
13 50% ethyl alcohol 10-15 dips
14 95% ethyl alcohol 30 seconds
15 Eosin Y 1 minute
16 95% ethyl alcohol 2 minutes Dehydration
17 100% ethyl alcohol 1 minute
18 100% ethyl alcohol 3 minutes
19 100% ethyl alcohol 1 minute
20 Xylene 20 minutes Clearing
21 Xylene 20 minutes
22 Drying at room temperature Overnight Drying
I. Microscopic observation:
The mounted slides were observed under a microscope, which was connected to computer with Digital camera. By the help of this mechanism several photographs were taken at different magnifications.
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3.8 Ranking:
The numerical ranks were valued following Buchanan (2001) and these rankings were used during GI determination:
Table 4: Ranking criteria for determining gonad index
Stage Name Ranking
1 Resting 1
2-5 Development (A,B,C,D) 2
6 Mature 3
7-9 Spawning (A, B, C) 2
10 Spent 1
3.9 Gonadal index
The maturity stage of each sample was determined by screening the histological slides following Grizel (2003) and the gonad index (GI) was calculated following Buchanan (2001).
GI = Number in each stage ×Numerical ranking of that stage Number of animals in the samples
3.10 Data Analysis
Microsoft excel 2016, Past 3.25 and SPSS software were used for data analysis and graphical representation. % gonad development stages was calculated by Microsoft excel on the other hand chi square test was used for sex ratio and graph representing proportion of sex, GSI, MY and CI (dry) data were analysed with SPSS and Past. Principal Component analysis was done and illustrated by using past software.
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Chapter Four Result
4.1 Length- weight Relationship
Total 143 samples were used to evaluate the relationship of different shell dimension (Total length, shell width and shell height) and weight (Total weight, Soft tissue weight) which were collected from the Moheskhali Channel during the study period.
Simple linear regression were used to identify the morphometric relationships and represented in figure 6. The “b” values obtained from the regression analysis were evaluated whether it is significantly different to 3.0. Highest correlation values were observed in the SL-TW (r2 = 0.9033) and TL-STW (r2 = 0.713) relationships (table 5).
Figure 6: Simple linear regression between length and weight dimensions of P.
viridis
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The value of correlation coefficient (r) was close to 1 considering total length as growth indicator but shell width and shell height in contrast of wet tissue weight was far from one (01). Coefficient of determination was also higher in case of shell length- total weight regression. The coefficient of determination was very low in SH-STW, SW-STW and SW-TW. Overall, results of the statistical analysis showed a significant relationships (P <0.01) between paired variables. This suggests that there was an increase in the both total weight (TW) and soft tissue weight (STW) of the species as its shell length (SL), shell width (SW) and shell height (SH) increases over time.
Table 5: Observation of relationship between different lengths dimensions and weight dimensions
Variables R r2 Regression
Co-efficient (b)
Growt h test
Growth pattern
shell length –Total weight 0.950 0.903 2.296 b<3 Allometric Total length -wet tissue weight 0.845 0.714 2.1266 b<3 Allometric Shell height – Total length 0.878 0.771 2.567 b<3 Allometric Shell height – wet tissue weight 0.752 0.565 2.2899 b<3 Allometric Shell width- Total weight 0.817 0.667 1.8846 b<3 Allometric Shell width - wet tissue weight 0.706 0.499 1.6984 b<3 Allometric
4.2 Sex Ratio
Among 242 slides 67 were male, 62 were female, and 113 were undifferentiated. The sex ratio was not significantly different from 1:1 (χ2, P > 0.05). No hermaphrodites were observed during the research. Male contributes 27.69% and 25.62% were identified as female based on their histological analysis (Figure 7). Overall sex ratio of male and female was found to be 1:0.93 but considering the monthly ratio in some months male was dominant and in others female dominance was established ( Table 6). Male dominancy was observed in November’17, March, April and July of 2018;
female dominancy was seen in February and October 2018.
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Figure 7: Proportion of male, female and undifferentiated individuals of Perna viridis collected from Moheskhali Channel
Table 6: A review of the number of male and female and their sex ratio of Perna viridis
4.3 Developmental stages of gonad
The stages represented by the gonads in this study were classified as follows:
4.3.1 Resting
Inactive, no traces of sexuality were in the mantle. Gonad comprised mostly of storage cells. This stage included virgin animals where the reproductive system was rudimentary, and those animals which had completed spawning (Figure 8).
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Fig. 8. Resting or immature stage, no traces of gonad (mag. ×40) (n= neucleus) 4.3.2 Development A
Gametogenesis had started; acinus visible but no mature gametes apparent.(Figure 9;FD(a), MD(a))
4.3.3 Development B
Acini were larger and occupied a large part of the mantle. Ripe gametes first appeared in the centre of the acinus and were mainly small, with numerous oocytes. Larger spaces were apparent in female acini with much connective tissue between each. The spermatogonia and spermatocytes in the male were more abundant, and spermatids were found. Mature oocytes occupied about 30 % of each acini while the other 70%
was occupied by early stages of gametogenesis.
4.3.4 Development C
At this stage the acini had increased in size and there was evidence of rapid gametogenesis with mature oocytes occupying approximately 50% of each acini and the other 50% revealing early stages of gametogenesis.
4.3.5 Development D
The acini were fully ripe and maximum proliferation of genital tissue had been almost attained. The acini contained approximately 70% mature gametes, the number of spermatozoa had increased, and their tails were directed toward the lumen.
n
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4.3.6 Mature
Acini were fully mature and ripe. It differs from development D only in the greater reduction of the number of small oocytes in the germinal epithelium and the disappearance of round mature ova of the female (Figure 10; Fm), and a great reduction in the numbers of spermatogonia and spermatocytes in the male which can be visible as narrow band. Ova were compacted into polygonal configuration, whilst in the males the acinus was distended with morphologically ripe spermatozoa (Figure 10; Mm).
4.3.7 Spawning A
Emissions of gametes began. Acini still relatively full of ripe gametes (Figure 10;
FS(a)). This was obvious from the general reduction in density of spermatozoa where the laminae appearance was lost (Figure 10; MS(a)), and the rounding off of the remaining ova as the pressure within the acini was reduced following partial emission.
4.3.8 Spawning B
Release of more gametes started. Acini were half empty as in development C stage (Figure 10; FS(b)). However, less early stages of gametogenesis were present in this
Figure 9: Development stages of male and female
(F= Female; D= development;a=acinus, aw=acinus wall, yv= young vitellogenic oocyte, lv= late vitellogenic oocyte, Previtellogenic oocyte, ; mo= mature oocyte, n= nucleus, M= Male; D= Development ; s=spermatozoa)
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stage relatively which was the results of some re-development at the margin of the acinus. Acini in male became elongated and the boundaries were not easily distinguished. Evacuating ducts were evident from the reduction of spermatozoa as a result of spawning (Figure 10; MS(b)).
4.3.9 Spawning C
In this study acini were 70% empty at this stage. The lamellae of sperm showed a reformation in the acinus (Figure 10; FS(c) and MS(c)).
4.3.10 Spent
Acinus collapsing was started. Oocytes were reabsorbed and sperms were broken down. Only residual gametes remained which may be undergoing cytolysis. It was same as resting stage (Figure 11).
Figure 11: Histological view of spent gonad stages in female (F) and male (M)
Perna viridis
Figure 10: Mature and Spawning stages of male and female P. viridis
(F= Female; m= Mature; mo= mature oocyte, n= nucleus,S= Spawning; M=
Male; m= Mature; s= spermatozoa) )
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4.4 Seasonal variation in gametogenesis
Seasonal variation in gametogenesis was evaluated considering three parameter- monthly gonad development stages, gonad index based on ranking of histological analysis and gonadosomatic index (GSI). The description is given below-
4.4.1 Monthly gonad development stages
Active gametogenesis of Perna viridis in Moheskhali Channel was observed annually though a minor gametogenesis was also present after a clear inactive or resting stage.
During the initial stage of study, on November’17, mussels were in their development stages for 100% case (Fig. 19). Higher percentage of development C and D (late development stages) in male than female indicated that male starts gametogenesis process before female.
Development A and B was considered as the early development stages and development C and D as late development stages. In the following month most of the gametes were in late development stage and mature gonads was spotted first for both male (30%) and female (10%). In January 2018 early spawning stages of gonad was observed in male and female. During February mature stage was more abundant in female than male whereas early and late spawning was abundant in male. Both male and female were in their late spawning season throughout March and April 2018.
Undifferentiated gonad also was evident from April 2018 indicating resting phase before redevelopment. Presence of spent gonad was evident for both male and female though female spent their gametes before. In male highest percentage of development, mature and spawning gametes were noted during NOV’17 (100%), FEB’18 (43%) and APR’18 (45%). Female showed highest development, maturation and spawning in NOV’17 (100%), FEB’18 (23%) and MAR’18. May’18 and August’18 had 100%
undifferentiated gamete so graphical representation avoided. Only 10% gonad development was noted in June and July of 2018 indicating a minor development.
Gametogenesis of male and female was significant (t- test, p<0.05). Percentage of different gametogenic stage is presented in Figure 12 and 13.
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Figure 12: Percentage of gonad development stage of male and female Perna viridis ( Dev = Development, Spaw = Spawning)
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Figure 13: Percentage of gonad development stage of male and female Perna viridis from (In May and August 2018 male and female were 100% resting phase occurred; Dev = development, Spaw = Spawning).
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4.4.2 Gonad index
Based on gonad development stage ranking of Perna viridis in Moheskhali Channel indicated major active gametogenesis occurred only once in a year and a minor spawning was also present. The ranking value was observed more than 1.5 during the first months of study (Nov’17 to March’17). An increasing trend of CI in both male and female indicating gonad growth and maturation and lasted till January for male and February for female. After January a decreasing line was interpreted as spawning started and spawning completion was noted during May 2018 (Figure 14).
Figure 14: Monthly mean gonad index of male and female
A minor gonad development was observed in the month of June and July 2018 .GI value started to increase again in October, 2018. Nonparametric Kruskal Wali’s test showed significant differences (p<0.05) in GI of male and female and monthly variation was also significant (t-test, p<0.05).
4.4.3 Gonadosomatic index
Significant monthly variation was found from one way ANOVA (p<0.05). Random effect of components of variance, homogeneity of medians, and variance of means was also significant (p<0.05). Welch F test in the case of unequal variances also defined significant difference (F=63.55, df= 17.34, p=1.757E-11). Tukey pairwise comparison of male and female GSI was not significantly different. Test for equal means (p=0.37), Levene’s test for homogeneity of variance from mean (p= 0.99) and median (p= 0.94) was also not significantly different.
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Figure 15: Monthly variation of GSI (%); B) variation of GSI in male and female Overall study showed that Perna viridis population of Moheskhali Channel underwent one major peak breeding cycle in spring and a minor cycle in the fall throughout the study period. The gametogenic cycle based on monthly gonad development, gonad index and GSI value the following cycle can be interpreted.
Figure 16:
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4.4 Egg diameter:
Only mature egg was measured to identify the egg diameter. Mussel egg were not fully round rather polygonal in shape so few lines was drawn and average value was counted. Egg diameter varied from 0.09 to 0.29 micrometer. The mean egg diameter was found to be 0.22 micrometer.
Figure 17: Egg diameter measurement 4.5 Water Quality Parameters
In total 15 water quality parameters were observed throughout the study period. No monthly significant differences regarding nitrate, nitrite and ammonium were not significantly different. The detail of ANOVA test are presented in appendix 4. From PCA analysis of 15 variables showed that only PC1 and PC2 covers almost 99% cases of variation (Table 7).
Table 7: Eigenvalue and % variance of Principal components
PC Eigenvalue % variance
1 15544.6 80.72
2 3607.38 18.732
3 59.3927 0.30842
4 39.845 0.20691
5 3.14503 0.016332
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Loading variables graph of PC1 and PC2 we observed that turbidity, alkalinity, salinity and transparency were the main water quality parameters which had high seasonal variation. PCA plot of 15 water quality variables also depicted that October’17, November’17, December’17 and January’18 was in 3rd quadrant which were basically consist of early gonad development, late gonad development and maturation occurring months. February’18, March’18 and April’18 was the months when spawning phase was observed in the gametogenic cycle and these months were placed in 4th quadrant. So biplot distribution from figure represented the monthly variation of water quality parameters were related to the gametogenic development cycle.
Figure 18: PCA graph (x axis- Component 1, Y axis– Component 2) based on 15 water quality variables of Moheskhali Channel
Loading variables of PC1 and PC2 showed the positive and negative indent of the biplot which were given in the table 5. From the loading variables of PC1 showed that water temperature, turbidity, current speed, transparency influenced the component
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(value closely near to 1 and -1). On the other hand, alkalinity, ammonia, water depth, salinity influenced PC2.
Table 8: Loading variables for principal component 1 and 2
4.6 Seasonal variation in meat yield
Highest mean meat yield (MY) was observed in the month of June 2018 (mean32.48±
SE 1.7%) and lowest mean value was found in April 2018 (mean 22.839±SE0.79 %).
Grand mean MY was 27.23. Meat yield of undifferentiated or resting stage was highest (28.45±0.40) followed by male (27.82± 0.63) and female (25.788± .702). LSD multiple comparison of meat yield showed male, female and resting stage had significant differences in mean value. Two way ANOVA test of meat yield showed significant monthly differences (p<0.05) as well as significant difference between sex (p<0.05).
Loading variables PC 1 PC 2
Water Depth (m) 0.019 0.67
Temperature ( ͦC) 0.75 -0.03
Transparency (cm) -0.86 0.15
Turbidity (NTU) 0.99 -0.04
Current Speed (m/s) 0.90 0.09
pH -0.22 -0.39
DO (mg/L) -0.23 -0.26
Salinity (ppt) 0.11 0.46
Alkalinity (mg/L) 0.17 0.98
Nitrite (ppm) -0.14 0.038
Nitrate (ppm) -0.001 -0.002
Phosphate (ppm) -0.1 -0.23
Ammonia (ppm) 0.43 0.52
Ammonium (ppm) -0.17 0.29
Chlorophyll-a(µg/L) -0.106 -0.24
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Figure 19: Monthly meat yield of Perna viridis from Moheskhali Channel (data represented as mean, standard deviation and standard error)
Figure 20: Monthly meat yield of Perna viridis from Moheskhali Channel (data represented as mean, standard deviation and standard error)
4.7 Seasonal variation in Condition Index:
Highest mean value was observed in June 2018(21.813± 2.699 g/cm³) and lowest value of condition index was noted in March 2018. One way ANOVA test of condition index showed significant differences in between groups (F=4.242, df= 8, p=0.0001) and within groups (F=164, df=164, p=0.0001). Significant differences in unequal variance was also found by Welch F test (F=3.396, df= 65.37, p=0). Mean dry condition index of male (20.322±1.349) was higher than female (17.683±1.527 g/cm³) but resting stage possess highest value (21.70±.624g/cm³).
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Figure 21: Monthly dry condition index of Perna viridis from Moheskali Channel (data represented as mean, standard deviation and standard error)
Figure 22: Dry condition index of Perna viridis from Moheskhali Channel (data represented as mean, standard deviation and standard error) between genders
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Chapter Five Discussion
This chapter is made up with the interpretations and descriptions of findings of research work. Explanations about the results and the associated problems are also included. Discussion of gametogenic cycle and relationship of condition indices with reproduction is discussed here comparing with previous study.
5.1 Length - weight relationship
Results of the morphometric analysis revealed that shell dimensions such as length, width and height have a strong positive correlation with total weight and soft tissue weight. The highest value of r and r² in shell length and weight dimensions indicates that shell length was the ideal weight growth estimator (Table 5). Pauly (1980) also found similar r² value (0.968). Results showed values of the parameter “b” in all cases were significantly different to the isometric value and as the value was observed lower than 3 (allometric growth pattern) which reflect weight gain of Perna viridis in Moheskhali Channel was in negative way. The values obtained showed that there was a proportionate increase in the total weight and soft tissue weight of green muscle samples with increases in shell length, width and height of the species. In this study,
“b” values for TL-TW, TL-STW, SH-TW, SH-STW, SW-TW, and SW-STW relationships were 2.29, 2.13, 2.567, 2.30, 1.88 and 1.70 respectively. A similar length weight relationship pattern was observed by Aban et al., 2107. Compared to the studies conducted by Rao et al. (1975) and Qasim et al. (1977), Sundaram et al.
(2011) and Narasimham (1980) on the length-total weight relationships of Perna viridis in Goa, Versova creek and Kakinada Bay, respectively, value of “b” in this present study is lower. The “b” values obtained by these authors varied from 2.42 to 2.86.
5.2 Sex ratio and gametogenic cycle
In this research no hermaphrodite was reported from the 243 specimen slides. Many researchers in the region and as far as in the Caribbean, and the USA have not documented any hermaphrodite individuals of P. viridis population in their localities (Agard et al., 1992; Bigatti et al., 2005; Huang et al., 1985; Rao et al., 1975; Rylander et al., 1996; Urbano et al., 2005; Walter, 1982;). The high percentage reported by Anon (1960) could have resulted from the misidentification of the gender especially
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that he relayed on visual gender identification in his study. Lee (1988) has encountered 3 hermaphrodite individuals in his study. Since P. viridis was classified both as dioecious and hermaphrodite in different regions and different habitats, the actual identification is still debatable. No evidence of hermaphroditism during the study conclude that the species is diocious. Having some low reported cases of hermaphroditism make it difficult to draw conclusion as to what triggers this sexuality phenomenon in P. viridis.
The overall sex ratio in this study was 1: 1 except in January and February with significant female dominance while in October, March the male have shown some significant dominance over the females. Male dominance was observed during the early development stages and in late spawning seasons which indicated male gonad development starts before female but male releases their gamete slowly. This occurs due to attaining success in reproduction as P. viridis is a broadcast spawner. Anon (1960) have reported a male dominance in sex ratio in the months of February to April which coincided with the early phase of the gametogenetic cycle. However, Anon (1960) based his identification on the coloration of the intermantle and mesosomal gonads which is believed to be unreliable for some period of time of the gametogenic cycle (Mc Ferland, 2017; Brousseau, 1982; Lee, 1988). A second peak was noted during the months of June and July. Sivalingam (1977) have reported a similar observation for a population of P. viridis at Penang, Malaysia though frequency of gonad development was much higher. In the second peak only 10%
individual was gone through active gametogenesis and the size of the individual with active gonad was 5.2 to 5.5cm. Considering the growth rate this species attained sexual maturity within 6 to 7 months age. So it can be said that individuals who attained sexual maturity during second peak was born in the very early major peak spawning season. P. viridis in India was also reported to have considerable variation in spawning from year to year; some authors have reported a single spawning event (Narasimham, 1980; Mohan and Kalyani, 1989) while other have reported more than one event (Rajagopal et al., 1998).
Clear spent phase was observed in the months of April and May. A true resting phase in the month of May and August though resting period longed from May to September. In many studies no resting phase was observed as the phenomenon of yearly two even three peak spawning season was recorded.
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5.3 Water quality parameters
Gametogenic cycle is directly related to the environmental condition as species have special requirements on environmental parameters for their gamete development and reproduction. In the study we conducted PCA analysis to identify the major factors causing monthly fluctuation. Development of gametes started October which was the late autumn. Considering temperature gradient of Bangladesh temperature starts to reduce this time triggering the initiation of gonad development; continuous reducing temperature influenced the maturation of gonad. Spawning started when the temperature was lowest and all the individual spawned before starting the summer time when temperature exceeds 30°c Reproduction of P. viridis is directly related to temperature, salinity turbidity and plankton abundance (Sivalingam, 1977).According to Rajagopal (1991), the abundance and growth of mussel larvae in the coastal waters is linked to sufficient food availability, which enhances the chance of larval development and successful settlement. It has been therefore hypothesized that under adverse hydrological conditions, e.g. high temperature, the ecophysiological strategy of P. perna may be that a portion of the population is able to spawn over extended time (Newell et al. 1982).
The resting phases observed in the study was during the monsoon period. Heavy rainfall reduce the salinity even below 15ppt and turbidity increased as mouth of Moheskhali Channel opens in the BOB and carry freshwater runoff with sediment particles. This created an unfavorable condition for the reproduction and resulted in resting stages. But places where water is less turbulent and have constant salinity and temperature showed multiple spawning throughout the year in many studies.
5.4 Condition Indices:
Condition index, meat yield etc. condition indices are also influenced by different physicochemical conditions and related to the reproductive season. In this study meat yield was observed throughout the study period but condition index was recorded in 9 months. Monthly significant variation was observed for both the parameters but significant differences between male and female was evident only in case of Meat yield. This happened may be because not collecting the dry CI data from November 2017 to January 2018 as these month showed significantly active gametogenesis.
Average mean CI and Meat yield both showed highest mean value during resting
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period. It can be explained that energy was stored during the resting phase for further gametogenic activity. Mean CI value reduced when gonad development started and for both MY and dry CI, gradual rise in the mean level was observed until maturation and lowest value was found in the month of May when all the gametes were released.
The range of CI values for P. viridis in this study was relatively within the range reported in the literature (Table 9).
Table 9: List of condition index from previous study Species Ranges
(g⁄cm³)
Location Reference
P.viridis 10.15 - 20.92 Malaysia Yap et al. (2003) P.viridis 12.06 - 16.15 Muar, Malaysia Barwani et al.(2007) P.viridis 15.18 - 19.41 Pasir Panjang, Malaysia Barwani et al.(2007) P.viridis 21.06 - 27.18 Sebatu, Malaysia Barwani et al.(2007)
Barnes and Barnes (1977) emphasised that under tropical marine conditions, factors such as temperature and food availability were maintained at near constant levels, and therefore, seasonal breeding cycles were often superimposed.
The drop in CI in May 2018 and again in October 2018 due to release of gametes through spawning (Lutz, 1980; Vakily, 1989; Seed and Suchanek, 1992). This is also supported by the salinity data. This phenomenon was reported to naturally induce the spawning in P. viridis (Choo, 1983; Cheong and Lee, 1984).Walter (1982) reported that spawning of P. viridis in the Philippines had no obvious relationship with temperature fluctuations. The use of CI in reproduction evolves since reproductive cycle of marine bivalves is regulated by many exogenous and endogenous factors (Giese, 1959; Bayne, 1976). The effects of those factors are apparent on the condition of the bivalve which in turn can give an indication of the reproduction status of the bivalve (Orban et al., 2002). The most important environmental stimuli influencing bivalve reproduction are water temperature (Sastry, 1979) and food availability (Newell, 2002) which was also documented to influence the condition of the bivalves (Lutz, 1980; Hickman and Illingworth, 1980; Okumus and Stirling, 1998).
The present study supports the use of CI as a potential indicator of reproductive cycles of P. viridis. Site selection of areas free from tourist activities and pollution sources would help in sustaining the culture industry and provide a suitable culture product.
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Chapter six Conclusion
Green mussel (Perna viridis) at Moheskhali channel had shown a yearly major gametogenic cycle starting from October till April and a minor peak in the month of June and July. A true resting phase also observed in May and August. The overall sex ratio of male and female was 1:1 with male dominance in early development stages and female dominance during mature stages. Gonadal index results show that spawning event in the spring not year round though a primary development is present in the monsoon. Gonadosomatic index of male is higher than female. Water quality parameters play a vital role in gonad development and maturation. Decreasing water temperature during the prewinter season triggers the gonad development and spawning occurs when temperature begin to rise. Before starting summer season when temperature reaches 30°C mussel spent all the gametes and a resting phase starts.
Relationship of length dimension and weight dimension showed positive correlation with allometric growth pattern where shell length will be the ideal indicator of growth for both total weight and soft tissue weight. Condition indices (Meat yield and dry condition index) was proved to be related to gametogenic cycle and physicochemical parameters. The present study supports the use of CI as a potential indicator of reproductive cycles of P. viridis. Site selection of areas free from tourist activities and heavy developments would help in sustaining the culture industry and provide a suitable culture product. This basic biology study related to reproduction will be the baseline information for aquaculture practitioners who will take steps in artificial breeding technology development and mussel culture establishment.
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Chapter seven
Recommendation and future perspectives
Through study on different biological aspect like feeding behavior, reproductive system, impact on ecology are the primary need not only in green mussel population of Moheskhali channel but also in other coastal mussel habitat. Culture of mollusks especially mussels and oysters considered as rising an urgent option of coastal aquaculture. Side by side an export market in the neighboring Southeast countries needs to be explored so that excess product can be sold. A large quantity of mussel and oysters’ meat is destroyed during the collection, which can be prevented through improving methods of exploitation, transportation and storage. The most important of all, a distinct local community in the southern region should be identified who consume mollusk to develop local market. Proper processing technology can also ensure quality supply in local restaurants. This study provide the baseline information regarding the development of this culture. Culture development is necessary to reduce stress on natural stock of green mussel and will ensure the conservation of shellfish biodiversity as well as keeping harmony with the future fast growing industry. Culture development will also provide economic support to the small scale fisher’s life by creating employment opportunity.
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