Gonadal cycle and spawning of date mussel Lithophaga lithophaga (L.) (Bivalvia: Mytilidae) in Egyptian water

Egyptian Journal of Aquatic Research 45 (2019) 293–299

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Gonadal cycle and spawning of date mussel Lithophaga lithophaga (L.) (Bivalvia: Mytilidae) in Egyptian water Amal R. Khafage a,⇑, Fatma A. Abdel Razek a, Somaya M. Taha a, Hamdy A. Omar a, Mahmoud A. Attallah b, Rabab S. El-Deeb a a b

National Institute of Oceanography and Fisheries, Egypt Marine Biology and Ichthyology Section, Zoology Dept., Al-Azhar University, Egypt

a r t i c l e

i n f o

Article history: Received 28 February 2019 Revised 8 April 2019 Accepted 8 April 2019 Available online 19 September 2019 Keywords: Lithophaga lithophaga Sex ratio Gonad development Spawning season Egypt

a b s t r a c t This study describes the gonads of Lithophaga lithophaga (Linnaeus, 1758) inhabiting the coast of Alexandria City, Egypt, on the Southeastern Mediterranean Sea. Investigated samples were collected from five sites situated between Sidi Gaber and Montaza. Seven hundred and forty six individuals were collected through one year from December 2017 to November 2018. Two hundred and thirty eight specimens with shell length of 46.83 ± 12.28 and total weight of 7.29 ± 5.74 were selected representing the monthly abundance and were used for histological analysis. The sex of L. lithophaga is dioecious with no sexual dimorphism and distinct by the gonad color. However, differentiation of sex was accurately defined histologically. The investigated animals showed a statistically unbalanced sex ratio. The male to female ratio was 3.5:1. However, the results of the present study showed that the ratio of males decreased as the shell length increased. The developmental stages of gonads were identified as five stages. The spawning activity occurred during July and August, while the spent stage was found mostly in September. Furthermore, the results showed that L. lithophaga has one annual gonadal cycle. There is a clear correlation between the maximum values of condition index and meat yield of these studied date mussels with their spawning activity during July–August, followed by a decrease in these values from October to the following June. This result can be a more validating method to identify the reproductive conditions of L. lithophaga’s population. Finally, each gonad developmental stage was described microscopically in details for both sexes. Ó 2019 Hosting by Elsevier B.V. on behalf of National Institute of Oceanography and Fisheries. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction Lithophaga lithophaga (L., 1758) is considered one of the popular boring bivalve in the Mediterranean and Eastern Atlantic. It appears in almost every subtidal calcareous rock (GalinouMitsoudi and Sinis, 1994). The conservation importance of L. lithophaga results from its gastronomic value and the correlated environmental problems caused by its harvesting (Zˇuljevic´ et al., 2018). The habitat damage caused by the exploitation of this species has influenced wide coastal areas along the Mediterranean and Adriatic seas. This has led up to large-scale destructive effects on associated benthic communities (Devescovi et al., 2005). It is well recognized that the date mussel has the slowest growth rate among bivalves (Galinou-Mitsoudi and Sinis, 1995;

Peer review under responsibility of National Institute of Oceanography and Fisheries. ⇑ Corresponding author. E-mail address: [email protected] (A.R. Khafage).

Fanelli et al., 1995). On the other hand, its colonization patterns of exploited surfaces have been rarely investigated. The genus Lithophaga has developed a habitat specialization in rock or in either live or dead corals (Scott, 1988). Moreover, it has a long association with reef-building corals as well as with fossil records (Kleemann, 1980). Various studies were done on the date mussel that deal with its biology, fecundity, and population dynamics. In addition, studies were done on the impact of its harvesting on the surrounding habitats as well as on its over-exploitation in areas such as in the Atlantic and Mediterranean Sea (Kefi et al., 2014). In Egypt, studies on the date mussel have been very limited to the variations in the environmental conditions, in particular with reference to marine pollution (El-Morshedy, 2015), as well as on its biometric variables and relative growth along Alexandria Coast (Taha et al., 2018). The present study is essential to predict the reconstitution of natural populations of these date mussels after harvesting and also gives details on their gonadal cycle and sex ratio in the Egyptian waters on the southeastern part of the Mediterranean Sea.

https://doi.org/10.1016/j.ejar.2019.04.001 1687-4285/Ó 2019 Hosting by Elsevier B.V. on behalf of National Institute of Oceanography and Fisheries. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Materials and methods

Statistical analysis

During the study period (2017–2018), five sites were selected on the coast of Alexandria, Egypt, in shallow areas of depth ranging from 3 to 6 m. These sites ranged between 31.16, 31.14 latitudes and 29.59, 29.57 longitudes, as shown in Fig. 1, and they covered about 12 km of Alexandria coastline with different distances from the beach ranging between 300 and 700 m. The average water temperature in the area during the collection period was 17.3 °C, 27.4 °C, 23.0 °C and 14.0 °C; and the average of water salinity was 38.7‰, 38.8‰, 38.6‰, and 38.2‰ in spring, summer, autumn, and winter, respectively. The method of collection of samples was as described by Taha et al. (2018). The collected L. lithophaga mussels were maintained in clean aquaria in fresh flowing sea water. In the laboratory, morphometric characteristics of the shell of each bivalve were measured along its maximum anterior-posterior axis to the nearest 0.5 mm, and total weight in grams was recorded. The condition index (CI) was estimated using the formula CI = dry meat weight (g)/dry shell weight (g)  100, according to Crosby and Gale (1990). While, meat yield (MY) was calculated according to Freeman (1974) as MY = wet meat weight (g)/total weight (g)  100. Sex was determined, in the adults, by gonad color (creamywhite for males, orange for females), but this was usually confusable. For histological preparations, a total of (238) samples was studied with 15 to 20 individuals monthly, however, they reached 32 individuals during January and July 2017. The gonads of the date mussels are embedded in the visceral mass. Portions of gonads from live specimens were fixed in Davidson solution, dehydrated using a graded ethanol series and embedded in paraffin wax. Then sections of 5m thick were stained with hematoxylin-eosin (Kim et al. 2006). Sex was clear-cut confirmed by histological examination using the light microscope (OPTIKA B-150) with magnification from X4 to X40. The histological sections were photographed using a mounted digital camera (TOUPCAM TM-UCMOS-3.1MP1/2, Model No.3.2). The sex ratio of males to females in the present study was determined. The stage of the gonads was categorized based on the dominant number in 10 randomly selected follicles from each sample, according to Gribben (2005) in razor clam, Zenatia acinaces. The studied gonad developmental stages were classified into five gametogenic stages according to the maturity state of each sex. These stages are resting, developing, ripe, spawning and spent. The developing stage has three substages, early developing (I, II) and late developing.

A chi-squared goodness of fit test (a = 0.05) was applied to examine the hypothesis that there was an equal representation of males and females of the L. lithophaga mussels in the population depending on the histologically examined samples (238 individuals). The bio-physiological indices changes were examined monthly and a comparison between the sexes was done by oneway ANOVA. All statistical analyses were done using SPSS v.15 and Excel 2013. Results Sex ratio The sex ratio (male to female) of 215 individuals, out of the 238 specimens of the date mussels used in the study, was 3.5:1 (n = 167:48). The remainder, twenty three individuals, were not sexually differentiated because they had inactive gonads (Table 1). The sex ratio analysis was based on categorizing the shell length with 10 mm intervals. This showed that the proportion of males was slightly decreased to be 2.7:1 (72.7:27.3) at shell size class 60–70 mm. The same ratio was observed at 70 mm and more than this size revealed a non-significant p-value, thus indicating that the proportion of males decreased with an increase in the shell length (Table 1), nevertheless, it differed statistically from the ratio 1:1 (X2 test for p = 0.05). Gonadal cycle The results of analysis of the monthly changes of gonadal development, reported in Table 2, showed that the gonadal cycle of L. lithophaga was divided into five stages: resting stage (November and December); developed stage, which is subdivided into early developed I (EDI), early developed II (ED II), and late developed (LD) (January to June); ripe and spawning stages (July and August); and finally spent stage (September and October). The monthly analysis of gonadal developmental stages in L. lithophaga showed that the proportion of ripe and spawning gonads were found to be the highest for both sexes from July to October (27% and 44%, respectively). Gametogenesis The gonadal tissue in bivalves occupies the region between the digestive diverticulum and the foot, which is obvious in

Fig. 1. Investigated sites on Alexandria Coast, Southeastern Mediterranean Sea.

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A.R. Khafage et al. / Egyptian Journal of Aquatic Research 45 (2019) 293–299 Table 1 Sex ratio with reference to the shell length of L. lithophaga. Size class

N

Undifferentiated (Resting)

Male

Female

Sex ratio (M:F) %

Chi square

P-value

20–29.9 30–30.9 40–40.9 50–50.9 60–60.9 70–80

18 50 90 46 23 11

4 7 5 6 1 0

11 34 71 27 16 8

3 9 14 13 6 3

3.7:1 3.8:1 5.1:1 2.1:1 2.7:1 2.7:1

(78.6:21.4) (79.1:20.9) (83.5:16.5) (67.5:32.5) (72.7:27.3) (72.7:27.3)

4.571 14.535 38.224 4.900 4.545 2.273

0.032509a 0.000138a 6.31E-10a 0.026857a 0.033006a 0.131668b

Total

238

23

167

48

3.5:1 (77.7:22.3)

65.865

4.83E 16a

* Superscript a indicates significant P-value, b indicates non-significant P-value.

Table 2 The monthly distribution of the different maturity stages for males and females of L. lithophaga during the period of study (December 2017–November 2018). Month

Dec 2017 Jan 2018 Feb Mar Apr May Jun Jul Aug Sep Oct Nov

Male

Total No.

ED I

ED II

LD

8 19 8 10 18 11 5 6 2

1 7 2 1 3 4 3 3

2 4

3 4

2 2

Ripe

Spawning 1

1

5 7 1 4 3

2 1 2 2

Total

L. lithophaga. It appears from the gametogenesis in bivalves is a very present study confirms this view, includes a number of developmental

2 1

1 1

1 1

Resting

Spent

3

Female ED I

12 30 10 12 21 15 13 21 5 3 12 13

7 2

167

23

available literature that complicated process. The since in L. lithophaga it stages starting very early

4 2

Total No. ED II

LD

Ripe

Spawning

Spent

4

4 4

4

5 1 2 1 6 2

before stages within mental

4 1

1 1 2

5 1

1 1 2 2

2 5 1 2

2 11 13 6 4 4 48

maturation (Table 3 and Figs. 2–4). Some of these are closely intersecting with each other. The gonad a single month may have different successive developstages.

Table 3 Gametogenic developmental stages of L. lithophaga (L.) for males and females: Stage Resting stage

Early developing I

Early developing II

Late developing

Ripe stage

The gonad possesses undifferentiated view; only germinal tissue is free from any gonadal activity. The tissue is present in the region between the digestive diverticulum and the muscular foot (Fig. 2A). Few to many small scattered narrow bands with collapsed wall appear in the germinal tissue, as for the prospective gonadal follicles, most of them are empty and few may contain undifferentiated cells. The animal is still in a quiescent state. In general, this stage represents the initial stage of gametogenesis (Fig. 2A and B). Male The testicular follicles are small in size, some follicles essentially have germ cell nests embedded in the follicle wall. Early immature spermatozoa are present in the lumen (Fig. 3A and 3A1) It is the actual initiation of gametogenesis process; the germinal tissue is reduced and replaced with the gonadal follicles that are increased in size and filled with male immature gametes (Fig. 3B). Several mature spermatids (male) are identified. The follicles become more heavily occupied with spermatocytes and spermatids by high magnification (Fig. 3C). Most follicles are full of spermatids, flagella of spermatozoa packed together in the lumen’s center. The follicle wall has a small layer of the early stages of spermatogenesis including spermatogonia, spermatocytes and spermatids. Greater number of densely stained mature spermatozoa in the lumen are found. The follicles increase greatly in size, and fuse with each other (Fig. 3D).

Spawning stage

The follicles are occupied with spermatozoids. So, individuals are ready for partial emission of male’s products. Males are characterized by a reduction in the number of spermatozoa (Fig. 3E).

Spent stage

Follicles begin to degenerate and their walls appear collapsed to discharge spermatozoa (Fig. 3F).

Female The beginning of differentiation of female germ cells in early follicle is marked by the appearance of mitotically dividing very early oogonia that have a rounded shape. They also contain large nucleus surrounded by a thick layer of cytoplasm (Fig. 4A). Follicles contain well distinguishable pyriform oocytes still embedded in the wall (Fig. 4B). Oocytes are identified. They gradually become mature and free-laying polygonal cells also appear with peduncle (Fig. 4C) Most oocytes begin to lose their peduncles and fall into the lumen of the follicles. Numerous oogonia and different developing oocytes attach to the wall. Oogonia of the mussel L. lithophaga have a rounded shape. They contain nucleus with a nucleolus surrounded by a thin layer of cytoplasm, and the volume of the nucleus exceeds the volume of the cytoplasm. The female follicles increase in size and fuse with each other. They contain essentially ripe ova with different stages of immature oocytes that are attached to the walls of the follicles. However, there are some mature oocytes that are free in the lumen (Fig. 4D). The majority of female gonads are occupied completely by spherical oocytes, where they lose their peduncles and leave the wall of the follicles to reach the central lumen. The germinal tissue is highly reduced in between the follicles (Fig. 4E). Follicles contain many atrestic oocytes beside very few numbers of oocytes (Fig. 4F).

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A

gt

B

B1

eg

ef

fl nb Fig. 2. Photomicrographs of transverse section of gonads of the date mussel L. lithophaga (Linnaeus, 1758): A – Resting Stage, B – shows the start of gametogenesis, B1 – Enlarged part shows some early gonadal follicle, ef: early follicle; eg: early gametes; fl: follicle lumen; gt: germinal tissue; nb: narrow bands.

Discussion The present results showed that the sex ratio was male-biased (3.5:1) and this was in accordance with Galinou-Mitsoudi and Sinis (1994) who found that the population of L. lithophaga collected from Evoikos Gulf in Greece has a sex ratio of 3.1:1 and 2.7:1 in favor to males. However in the northern and middle Adriatic, it was 1.8:1 (Valli et al., 1986) and 1.29:1 (Šimunovic´ et al., 1990), which differed significantly from the theoretical sex ratio 1:1 of bivalves (Mackie, 1985; Morton, 1991). Kefi et al. (2014) reported that the sex ratio in the Bay of Bizerte was found to be unbalanced in the date mussels’ population. The individuals up to 48 mm had a sex ratio of 2.03:1 with respect to males, and they reported that many individuals of that size class were still undifferentiated. This male-biased sex ratio of the date mussels was suggested to be due to the fact that the mature females’ shell sizes are larger than those of males. The studied species L. lithophaga is a dioecious bivalve, which is a common character in mussels, where both sexes could not be recognized even macroscopically in accurate manner. This was also discussed by Gosling (2003), who reported that the gonads in bivalves are paired but are usually so close together that the pair is difficult to detect. The present study showed that there is a wide variation in the color of the gonads; the creamy and orange color for both male and female respectively. The same conclusion was discussed for some mytilids, where the gonads are usually lighter in color in males and darker in females (Nascimento, 1968; Arrieche et al., 2002; Gosling, 2003). On the contrary, Gomes et al (2009) stated

that in Mytella guyanensis the gonads’ colors were darker in males and lighter in females. However, they added that due to the degree of overlap in color, only 50% of the cases were sexually differentiated in a conclusive manner by microscopic observation. This study proves that, the histological structure of the gonads of L. lithophaga is the same as that of most bivalves (Gosling, 2003). Many of the gonads of bivalves studied in the available literature showed that the date mussel’s gonad has a definite pattern. However, the monthly changes in the gonad structure were distinct. The present study of the date mussel L. lithophaga gave a detailed interpretation for all gonadal stages in both sexes. The gametogenesis in the studied species was divided into five stages (Table 3). The classification of these stages depended on the histological results, which revealed the resting, developed, ripe, spawning and spent stages. The developed stage was subdivided to early developed (I, II), and late developed substages. The resting stage consists of special undifferentiated tissue, which we referred to as ‘‘germinal tissue” but not connective tissue as usually was encountered in the available literature. Only Galinou-Mitsoudi and Sinis (1994) were the ones to refer to the previous stage. They determined its period without further interpretation either written or by illustration. Moreover, Kefi et al. (2014) described it as a distinct stage in which gonads are detectable, and sex can be determined beside some few shrunken follicles and some atretic oocytes. However, this description is completely different from our observations. The same stage in boring bivalve Barnea davidi was expressed by Jeon et al. (2012) as an inactive stage, so it appeared negligible. On the contrary, we considered this stage as a fertile soil for all the prospecting stages. In the developed stage,

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A

B



 A1

gt

D

C gt

f 

gt

 E

sz

F

sz 

Fig. 3. Photomicrographs of transverse section of male gonads of the mussel L. lithophaga (Linnaeus, 1758). A – Early developed I, A1 – Enlarged testicular follicle, B – Early developed II, C – Late developed, D – Ripe stage, E – Spawning stage, F – Spent stage. ftf: fused testicular follicles; gcn: germ cell nests; gt: germinal tissue; tf: testicular follicle; sz: spermatozoa.

where gametogenesis is degrading, the gonadal follicles appear carrying the gameteic products. This is why it was divided into 3 subdivisions in this study (Table 3). The first is the initiation of gametes activity; the second is the actual gameteic activity; and the third where densely arranged gametes are observed. This description is in accordance with Kefi et al. (2014). Galinou-Mitsoudi and Sinis (1994) reported that L. lithophaga reproduction started simultaneously for both sexes in July and the full ripe stage was observed at the same time for both sexes. Hence, the period of full maturity extended mainly from July to November. In the present study, it was observed that the first production of spermatozoa preceded that of mature oocytes. While statistically, the ripe gonads were found in July, and the spawned were perceived in August. Scott (1988) reported that the growth and reproduction of bivalves is a continual process, and is of great reproductive potential. Kefi et al. (2014) revealed the presence of a single reproductive cycle per year in Bizerte Bay for L. lithophaga. Spawning occurred at the end of August as well as in early September, and is correlated with water temperature. While in the Split area (middle Adriatic

Sea), Šimunovic´ et al. (1990) registered several spawning events from late June to mid-October for L. lithophaga mussel and this activity was reduced by autumn. In the present study, the peak of active spawning is during July, August and September, which correlates also with the warmest period of the year. The spawning activity of L. lithophaga occurs during July and August, then spent individuals appear in September-November, thus showing also one annual reproductive cycle during increased water temperature. Kautsky (1982) reported that the reproductive cycle consists of the period of the weight-change of the animal, the beginning of gametogenesis, spawning and the build-up of the following gonads. The results of Jeon et al. (2012) on Barnea davidi showed the reverse effect of condition index on gonadal development by a low correlation. From the present result, it is interpreted that the rise of water temperature is the essential factor for spawning especially for L. lithophaga. A seasonal reproductive pattern is characteristic of most bivalve molluscs that live in temperate latitudes. The timing of gametogenesis and spawning of individual species depends on

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nu

A

eog

B

gt epl

n og oc

oc

C

n

D

poc

nu roc gt

ovf E

moc n

ovf

moc F ao

nu L

ovf epl Fig. 4. Photomicrographs of transverse section of female gonads of the mussel L. lithophaga (Linnaeus, 1758). A – Early developed I, B – Early developed II, C – Late developed, D – Ripe stage, E – Spawning stage, F – Spent stage. ao: atrestic oocytes; eog: early oogonia; epl: epithelial layer; gt: germinal tissue; l: lumen; moc: mature oocytes; n: Nucleus; nu: nucleolus; oc: oocytes; Og: oogonia; ovf: ovarian follicles; poc: pedunculate oocytes; roc: relict oocytes.

the seasonal changes of environmental factors (Šimunovic´ et al., 1990). Since date mussels are distributed in European warm seas, their reproduction was expected to occur during summer. The histological examination of the date shell’s gonad sections (Table 3) and the seasonal changes of flesh weights, as described by Taha et al. (2018), showed one annual active cycle with several spawning events from late July to the end of August, at water temperature over 27 °C. While the spawning event in the case of Mytilus galloprovincialis, a close relative species of the present animal, extends all year round with two distinct spawning peaks in December and March (Hrs-Brenko, 1971) or only one in JanuaryFebruary (Da Ros et al., 1985) in the Northern Adriatic. In the present investigation, the proliferation of gametes in the date mussel coincides with the increase of water temperature in the spring season. In July, rapid gametogenic processes in L. lithophaga occur with the development of sex cells in July and early August, and continue to the end of August. Afterwards, the spent stage occur in September, October and early November at the beginning of the new resting period in the winter season. The parameters of allometric growth equations, exponent and intercept of date mussel during the study of Taha et al. (2018) in the same area, fluctuated with seasonal variations of environmental

factors and sexual cycle of the animal as similarly recorded by Wilbur and Owen (1964). Sea water temperature and salinity were not the only factors controlling the exponents of allometric equations for the present study. The differences between the equations are also affected by feeding conditions. There is a clear correlation between the maximum values of condition index (CI) and meat yield (MY) in the studied date mussels during July-August and then the decrease in their values from October to the following June. This increase in their values was correlated with the spawning cycle of this mussel and can be a more validating method to identify the reproductive conditions of L. lithophaga. The reproductive activity of L. lithophaga appears to continue throughout its whole life span (Galinou-Mitsoudi and Sinis, 1994). This is in accordance with the findings of the present results, as the oldest individuals (54 years) were found to have active gonads. Studies on the effect of the trophic factors on the reproductive activity of the ‘‘red list” endangered Egyptian population of L. lithophaga are important and recommended to be done in future research. The resulted information of the present study will be useful for proposing future management measures to protect such vital local wild stocks.

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Acknowledgements The authors wish to thank Mrs. Elzahrae M. Elmasry for her help and support throughout the course of this work. Special thanks for Dr. El-Geziry T. for his help in preparing the map of the investigated sites. We also would like to thank the anonymous reviewers for their suggestions and comments.

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