Population Characteristics, Maturation and Spawning of the Blue Swimmer Crab Portunus pelagicus in Eastern Mediterranean Sea, Egypt

MATERIALS AND METHODS

Sample collection: Males, ovigerous and non-ovigerous females of Portunus pelagicus were monthly collected from the fishing centers of the eastern Harbor of the Egyptian Mediterranean Coast of Alexandria city. It lies between longitudes 29°53'-29°54' 40" E and latitudes 31°12'-31°13'N” during the period from March, 2015 to February, 2016.

Data analysis
Sex determination: Crab samples were brought to the invertebrate laboratory of the National Institute of Oceanography and Fisheries (NIOF), Alexandria, Egypt. Males of P. pelagicus have an inverted ‘T’ shaped abdomen. Three categories of females were defined: Triangular shaped abdomen (V-shape) for immature female crabs, globular shaped abdomen (U-shape) for adult “non-ovigerous females” and the third category is the ovigerous females (egg-bearing females) as described by Josileen²¹.

Morphological measurements: Morphometric measurements of P. pelagicus were assessed including: Body weight (BW), Carapace width (CW), Carapace length (CL), Gonad weight. Vernier calipers with 0.05 mm accuracy were used for the length measurements, while the weights of the crab samples and their gonads were measured using a digital balance to the nearest (0.01 g). Linear and power regression analysis were used to investigate the relationship between (CW), (CL) and (BW).

Gonad examination: Monthly samples of male and female P. pelagicus were dissected in order to morphologically determine their gonadal development stages according to the shape and the color of the gonads. Also, the maturation stages of fresh gonads were determined before fixation. Small portions of the anterior, middle and posterior gonad lobes were fixed in Davidson solution for 24 h. Then, the gonad was transferred into the 70% alcohol solution before proceeding with tissue processing. The tissue was dehydrated in ascending ethanol concentrations from 70-100%, transferred to cedar oil and embedded in paraffin. The tissue was sectioned to 5 μm using a rotary microtome, then the sections were mounted on clean glass slides, then stained with hematoxylin-eosin. Thereafter, stained sections were examined with Olympus CX31 light microscope.

Determination of size at first sexual maturity: The size at which P. pelagicus was considered sexually matured was investigated by using linear regression by setting a relationship between the ratio of matured gonad of males and non-ovigerous females for each sequential 10 mm CW, to determine the size at which 50% of the crabs would have become mature according to Hartnoll²².

Determination of gonadosomatic index (GSI): Monthly GSI was calculated according to the following equation:

Data are represented in mean and standard deviation, one-way ANOVA analysis (p<0.005) was used to test the differences of GSI between males and non-ovigerous females with time.

RESULTS

Population characteristics: A total of 445 samples of Portunus pelagicus were collected during the study period from the eastern Harbor fishery centers of the Egyptian Mediterranean Coast off Alexandria city. The crab sample was composed of 132 males, 184 non-ovigerous females and 129 ovigerous females. The weight and length characteristics of P. pelagicus are represented in Table 1.

Width/length-weight relationships: Linear and power regression analysis were used to investigate the relationship between CW, CL and BW. The values and constants of regression equations are given in Table 1.

The results of regression analysis showed that the increase in carapace width is always followed by an increase in the carapace length and body weight, so CW is positively correlated with CL and BW. In addition, the exponential values (b) of the length-weight relationships of males followed the cube law (CW-BW, b = 3.0970, CL-BW, b = 3.0300) indicating thereby nearly an isometric pattern of growth, whereas, the corresponding values of females (CW-BW, b = 2.9124, CL-BW, b = 2.8293) suggested marked departure from isometric pattern of growth.

Macroscopic and histological description of gonad development
Gonadal development stages of female P. pelagicus: The present observations showed that female gonadal development of the blue swimmer crab was classified into five stages: (I) Immature, (II) Early maturing, (III) Maturing, (IV) Ripe and (V) spent.

Macroscopic description:

Table 1:	Population measurements of male, non-ovigerous and ovigerous females of Portunus pelagicus in the eastern Harbor, Alexandria and population regression analysis of male, female of Portunus pelagicus
Numbers in parentheses refer to the average±SD. CW: Carapace width, CL: Carapace length, BW: Body weight, b: Exponential value, r: Correlation coefficient, R2: Coefficient of determination, *: p<0.005

Fig. 1(a-e):	Photomicrographs of light microscope showing the ovary in Portunus pelagicus Stage (I, II, III, IV and V), (a) Immature stage, (b) Early maturing stage, (c) Maturing stage, (d) Ripe stage and (e) Spent stage. Oc I: Oocyte I, Oc II: Oocyte II, Oc III: Oocyte III, Oc IV: Oocyte IV, Og: Oogonia, RO: Resorbing oocyte

Histological description: It is worth mentioning that the general ovarian structure and histology of P. pelagicus was similar to those of other brachyurans. During these five development stages, germ cells can be also divided into five growth development stages based on cellular diameters, shape and histological staining appearance. They were oogonia (Og) and four sequential types of oocytes (from Oc I to Oc IV). Histological description for gonadal development of female P. pelagicus is presented in Fig. 1a-e.

Gonadal development stages of male P. pelagicus: The progress of the male maturation was assorted to three stages according to the external morphology of testes and vas deferens: Immature (I), maturing (II), mature (III).

Macroscopic description:

Histological description: It was interesting to note that testis showed almost no histological difference in the development stages. The histological differences were shown in the vas deferens structure. Descriptions of each gonadal development stage are presented in Fig. 2a-g.

Size at 1st sexual maturity: The smallest recorded matured male and female crab during the period of study were at 88.6 and 72.5 mm (CW), respectively. On the other hand, by using the regression equation method, the calculated minimum biological sizes for mature male and female were found at 107.6 and 96.1 mm (CW), respectively (Fig. 3).

Gonadosomatic index (GSI): The mean values of monthly GSI of all males and females of P. pelagicus differ significantly (p<0.05) in all the study months (Fig. 4).

Distribution of different maturity stages and spawning activity
According to months: In the present study, for non-ovigerous females, the immature individuals were observed throughout the year with high percentages in August (56%) as shown in Fig. 5a. On the other hand, ripe stage occurred throughout the year except for the period from October -December. The high percentages of ripe individuals were in April and May, indicating that ripe females were expected to spawn with the starting of the warm months. While the highest percentage of occurrence of the spent individuals “which had accomplished breeding” was in October and November.

According to size: Ripe individuals of non-ovigerous females in the studied population started to appear from the size of 65 up to 135 mm (CW). On the other hand, the presence of immature females was represented by small size crab specimens with carapace width starting from 64.6-77.0 mm (CW) (Fig. 5b).

Fig. 2(a-g):

Photomicrographs of light microscope showing the vas deferens of P. pelagicus (Stage I, II and III): (a, b) Anterior vas deferens (AVD), Median vas deferens (MVD) and posterior vas deferens (PVD) were completely devoid of spermatophores in immature males, Sp: Spermatozoa, (c), d) AVD at maturing stage showing its packing with spermatophores (Sph), while MVD and PVD showing the entire lacking of spermatophores, (e) AVD and MVD at mature stage were full of spermatophores while PVD (f, g) May be empty or filled with spermatophores

Monthly ovigerous females distribution: The ovigerous females were available from March-October, with a maximum abundance in April and August. Figure 5c described the monthly proportion of ovigerous females of P. pelagicus. In general, during the study period, the highest values of female GSI were found to be during the period of April-May and August-September. On the other hand, the occurrence of non-ovigerous females that had mature and ripe ovaries was found throughout the period from March-September with high abundance percentage from April-July.

Fig. 3:	Size at 1st sexual maturity of male and female Portunus pelagicus using regression methods

Fig. 4:	Monthly mean values of Gonadosomatic index (GSI) of males and non-ovigerous females of Portunus pelagicus

Complementary to this, ovigerous females showed also a prolonged occurrence during the warm months from March to October as in Fig. 5c, with a maximum abundance in April and August. This tends to suggest the presence of two peaks of spawning activity, in April-May and in August-September. Hence, this indicated that ovigerous females may spawn more than one time during the spawning months.

DISCUSSION

The present study showed that the males of Portunus pelagicus are heavier than females, which is in conformity with Abdul-Sahib²³ and Murni et al.¹². Where Josileen⁸ mentioned that P. pelagicus females were marginally heavier than males up to a certain carapace size (120-125 mm) and thereafter; males became heavier than females. This could be due to the decline in the female growth during the period of gonad maturation rather than male¹².

A significant positive linear relationship with high (R²) values (0.9643 and 0.9224) was observed between CW and CL for males and females, respectively. This is in agreement with Josileen⁸, Abdul-Sahib²³ and Sukumaran and Neelakantan²⁴, for P. pelagicus.

Fig. 5(a-c):	Proportion of gonad maturity stages of non-ovigerous females of P. pelagicus (a) According to months, (b) According to size (mm) and (c) Monthly occurrence of ovigerous females of P. pelagicus

The exponential values (b) of the length-weight correlations showed that males of P. pelagicus only followed an isometric pattern of growth, which disagreed with the findings of Josileen⁸ and Sukumaran and Neelakantan²⁴ for the same species as males and females showed a marked departure from isometric pattern in the Mandapam and the Karnataka coasts of India.

In the current study, the ovarian development of P. pelagicus was comprised of five stages, reflected externally and histologically, which closely agrees with Ravi et al.¹⁰. At the present study, the oocyte diameter increased as long as the ovarian maturity stage developed, leading to an increase in the size of the ovary as shown in the increased gonadosomatic index and the ovarian external observation. This agrees with the results of Islam et al.²⁵ of the mud crab Scylla paramamosain.

Furthermore, the current results showed that (Oc I and Oc II) had basophilic cytoplasm because of its affinity for hematoxylin. Beninger et al.²⁶ pointed out that basophilic oocytes are typically linked with previtellogenesis and devoid of yolk in most decapods. In addition, it is apparent from the current observations that Oc III in the early maturing stage had a low eosinophilic cytoplasm, while the cytoplasm of the vitellogenic oocytes (Oc III-IV) was stained evenly with eosin, taking a pink color, because of the abundant presence of yolk granules. The clear shift from basophilic to eosinophilic cytoplasm in the present study is supported by the observations of King²⁷ in Litopenaeus setiferus and Vasudevappa²⁸, where it was recorded only at the late vitellogenic oocyte stage of Metapenaeus monoceros.

On the other hand, for male P. pelagicus, the current findings figured out that, the histological structure of the vas deferens in immature stage and maturing stage were in agreement with the findings of De Lestang et al.³ on P. pelagicus from the west coast of Australia. It was found also in the present results, PVD may or may not contain spermatophores in mature males. De Lestang et al.³ recorded that, spermatophores were absent in PVD of mature males of P. pelagicus.

The size at 1st sexual maturity differs with latitude and locations²⁹. Wardiatno and Fahrudin¹¹ found that the mean size of males and females of P. pelagicus from Indonesia reached the first sexual maturity at 103mm and 98 mm (CW) respectively. Sumpton et al.³⁰ mentioned that the smallest measured sexually mature female and male P. pelagicus were at 73.0 and 83.0 mm, respectively in the Australian waters. The differences observed between the minimum biological sizes of P. pelagicus can be explained by the effect of the difference in the site of sampling or fishing ground¹¹.

During the present study, ovigerous females occurred in all months (except from November-February) indicating that P. pelagicus breeds almost throughout the year except in winter months. As in temperate regions, reproduction process is restricted to the warm months³¹. While Batoy et al.³² reported that the blue swimmer crab breeds throughout the year in tropical regions. The spawning activity of females was pronounced with maximum values of GSI during April-May. While the abundance of ovigerous females during the period of study was recorded particularly from March-October suggesting prolonged breeding in this species. This observation agrees with the findings of Sukumaran and Neelakantan³³ on the blue swimmer crab from the southwest coast of India.

It is worth mentioning that the reproductive pattern analysis of P. pelagicus was performed by integration of the information concerning gonad maturation, GSI and the occurrence percentages of ovigerous females. Two peaks of spawning activity were detected in P. pelagicus, in April-May and in August-September. Hence, it is reasonable to say that ovigerous females may spawn more than one time during the spawning season. Wardiatno et al.¹¹ also recorded a similar pattern of spawning frequency for P. pelagicus from Indonesia. Further research is urgently needed on fecundity, embryonic development and the redevelopment of female ovaries to estimate the reproductive output of P. pelagicus during the spawning season.

CONCLUSION

The size at which P. pelagicus reached sexual maturity was at 107.6 and 96.1 mm carapace width (CW) for males and females, respectively. The size is proposed to be the minimum legal size for the crab fishery sustainable use. According to patterns in gonad maturation and GSI as well as the occurrence of matured and ovigerous females, two peaks were detected in spawning season, in April-May and in August-September. These findings could be used to support the implementation of policy for blue swimming crab’s sustainable fishery in the Egyptian waters.

SIGNIFICANCE STATEMENT

Despite Portunus pelagicus is quite abundant and of high commercial value, relatively little is known about its biology from the Egyptian waters. This study presents the results of a study conducted to characterize the maturation stages of male and female P. pelagicus through external morphological and histological examination during gonad development, with determination of gonadosomatic index (GSI), size at first sexual maturity and spawning population from Alexandria coastal waters of Egypt. That will inform the gaps which need to be addressed with regard to crab production and aquaculture in Egypt and could be used to support the implementation of policy for blue swimming crab’s sustainable fishery in the Egyptian waters.

ACKNOWLEDGMENT

The authors are grateful to Dr. Amal Ragae Khafage, Ph.D., Lecturer in the laboratory of Invertebrate Aquaculture, the National Institute of Oceanography and Fisheries (NIOF), for her great experience and constant help to conduct all the histology work of this research.