Use of abdomen looseness as an indicator of sexual maturity in male mud crab Scylla spp.
KEY WORDS: sexual maturity, abdomen looseness, mud crabs, Scylla
Determining sexual maturity in male portunid crabs is difficult and often ends up in the dissection of male specimens to assess their reproductive system development (Hartnoll 1969, Robertson & Kruger 1994, Ikhwanuddin et al. 2011). This is because unlike their female counterparts that show significant morphological changes in their abdomen shape and size (i.e., the abdomen of females becomes darkened and rounded) (Ikhwanuddin et al. 2011), males only exhibit changes in secondary sexual characters, such as increase in chela length and height once they attained sexual maturity (Hartnoll 1974). These secondary sexual characters are quantitative and are only useful in estimating sexual maturity morphometrically in a population as a whole (Ribeiro et al. 2013), but are not able to reveal the maturity status of each crab within the population.
The most used method to determine sexual maturity in male portunid crabs is the assessment of reproductive system development (Nascimento & Zara 2013, Soundarapandian et al. 2013b). This method evaluates sexual maturity in terms of physiological aspects (i.e., presence of spermatophores in the vas deferens as a sign of sexual maturity using the squash method) (Haley 1973) and is commonly termed as physiological maturity. Although the presence of spermatophores was used as physiological sexual maturity indicator in Scylla species (Robertson & Kruger 1994, Islam & Kurokura 2013), preliminary studies that were conducted in the present study showed contradicting results. In addition, determination of sexual maturity via assessment of reproductive system development is tedious, time consuming, and crabs need to be killed to assess the development of vas deferens. The presence of mating scars is an alternative to determine sexual maturity in males and is currently the only available maturity indicator to determine functional maturity in portunid crabs (Knuckey 1996). Functional maturity implies the capacity of the organism to mate effectively (Corgos & Freire 2006). Scars are formed on the sternum or first pair of walking legs of males as a result of repeated abrasion during precopulatory embrace when mating (Robertson & Kruger 1994). Nevertheless, this indicator in determining sexual maturity is inconsistent as mating scars are not always present in all mature male crabs (Knuckey 1996).
The abdomen segments of immature portunid crabs (e.g., blue swimmer crab Portunus pelagicus (de Lestang et al. 2003) and blue crab Callinectes ornatus (Nascimento & Zara 2013) are firmly attached to the thoracic sternum, whereas mature ones are able to detach their abdomen segments from thoracic sternum easily (Nascimento & Zara 2013). Significant changes in the looseness of abdomen segment in both male and female, and in the size and coloration of abdomen in females once they reach pubertal maturity were also observed in mud crab genus Scylla (H. Fazhan, Universiti Malaysia Terengganu, July 2012, personal observation). Thus, this criterion is explored further in the present study as a maturation indicator in male Scylla spp.
Mud crabs from the genus Scylla are commercially important crab species in Malaysia where three of the four mud crab species are found in Malaysian waters, that is, Scylla olivacea, Scylla tranquebarica, and Scylla paramamosain (Ikhwanuddin et al. 2011, Waiho et al. 2016a). In mud crabs, males are generally larger than females (Ikhwanuddin et al. 2011), and their detailed mating process have been described in a recent study (Waiho et al. 2015). Thus, the availability of basic reproductive knowledge, their economic importance, and high abundancy in local waters make Scylla spp. suitable candidates for the investigation of a new method to identify sexual maturity in males.
This study on the male mud crabs Scylla spp. (1) reevaluated the reliability of the presence of spermatophores in vas deferens (squash method) as indicator of physiological maturity, (2) explored the possibility of using the looseness of abdomen as functional maturity indicator in comparison with conventional functional maturity indicator (i.e., presence of mating scars) and their relationships with physiological maturity as indicated by vas deferens development, and (3) compared the body size of the three Scylla spp. to reach sexual maturity.
MATERIALS AND METHODS
Samples of the three species of the mud crabs Scylla were collected from Setiu Wetlands, Terengganu, Malaysia (5[degrees] 39' N 102[degrees] 43' E) using traditional crab pots from March 2013 to October 2013. Crabs were placed inside an ice box without water and were brought to marine hatchery of Institute of Tropical Aquaculture, Universiti Malaysia Terengganu, for sorting and examination. Species and sex identification were conducted based on the morphological keys provided by Keenan et al. (1998). Only intermolt males were used. The body size of crab in terms of carapace width (CW) was measured using standard vernier caliper to the nearest 0.01 mm.
Reevaluation of Physiological Maturity Indicator
The determination of physiological maturity using conventional method, that is, crabs dissected to check if spermatophores were present in vas deferens, was reevaluated. This was compared with the visual examination method where males with enlarged and swollen vas deferens that were milky white in color were classified as mature, whereas males with translucent and small vas deferens were considered as immature (Soundarapandian et al. 2013a, Waiho et al. 2016b). Before dissection, crabs were first anaesthetized in ice water for 5 min to immobilize them. The external morphology of the vas deferens in terms of color and overall size was recorded. The vas deferens (median vas deferens) were then placed on a glass slide, squashed with a coverslip, and observed under Nikon Eclipse 80i Advance Research Microscope for the presence of spermatophores. The size (diameter) of spermatophores was randomly selected, measured, and compared using Nikon Elements BR software version 4.1 (Nikon Instruments Inc).
Assessment of Abdomen Looseness as Functional Indicator
The functional maturity of the crabs was assessed by (1) the looseness of abdomen segments and (2) the presence of mating scars, whereas physiological maturity was determined by the status of vas deferens development as described in section Reevaluation of Physiological Maturity Indicator. The telson, located at the end of abdomen segments of all males, was examined by gentle flipping using dissecting needles or surgical syringes. Only minimal strength was applied during the examination. The determination of functional maturity was based on the looseness of the telson and the other parts of the abdomen segments. Males were classified as immature if only the telson can be flipped open when probed using a dissecting needle (Fig. 1 A), and mature if the entire abdomen segments are loose and can be opened easily with a dissecting needle (Fig. IB). The presence or absence of mating scars was determined by examining the first pair of walking legs of males (Fig. 1C) (Knuckey 1996).
All data analyses were conducted using IBM SPSS version 20 and Microsoft Excel 2013. Data were checked for normality using Shapiro-Wilk test and homogeneity of variance using Levene's test before proceeding with subsequent tests. Size of spermatophores in immature and mature males was compared using Welch's t test, an adaptation to the conventional Student's t test due to unequal variances. The size at sexual maturity ([CW.sub.50]) of crabs were estimated using logistic function fitted with least squares method with the equation: y = 1/(1 + [e.sup.r(CW - [CW.sub.50])]), where y is the proportion of mature crabs, r represents the slope, and [CW.sub.50] is the estimated CW when half of the population composes of mature crabs (Herrera et al. 2013). One-way analysis of variance (ANOVA) was used to determine the relationship between average CW and species. The statistical significance of [CW.sub.50] estimated between species and different maturity indicators was evaluated using two-way ANOVA. Tukey's honest significant difference (HSD) multiple comparison test was used to identify significant differences between treatments. All tests assumed significance level at P = 0.05.
A total of 479 male crabs made up of three species, that is, Scylla olivacea (n = 200), Scylla tranquebarica (n = 127), and Scylla paramamosain (n = 152) were examined. Average CW between three species differed significantly (ANOVA: F = 86.327, P < 0.001), with that of 5. olivacea ([[bar.x].sub.CW] = 82.51 [+ or -] 10.63) being significantly smaller (Tukey HSD: P < 0.001) than the CW of the other two species (P = 0.083) (S. tranquebarica'. [[bar.x].sub.CW] = 100.43 [+ or -] 14.89; S. paramamosain: [[bar.x].sub.CW] = 97.00 [+ or -] 15.13).
The reevaluation of using the presence of spermatophores as physiological maturity indicator involved 90 crabs (30 from each species) of various sizes. Morphologically, the vas deferens of mature males was easily recognizable, swollen, and whitish in color (Fig. ID, E), whereas that of immature males were translucent and barely visible to the naked eye (Fig. IF, G). When examined under the microscope, slides of vas deferens that were prepared using the squash method showed that spermatophores were present in vas deferens regardless of maturity status, although differences in spermatophore sizes were obvious (Fig. 2). The diameter of 700 spermatophores (n = 350 in immature males and n = 350 in mature males; maturity status was based on external morphology of vas deferens as described in Materials and Methods section under 'Reevaluation of Physiological Maturity Indicator') were randomly measured. The size of spermatophores was significantly larger in the vas deferens of mature males (Welch's t test: [F.sub.1,605.25] = 1,703.97, P < 0.001, Fig. 3). Spermatophores in vas deferens of immature males had an average diameter size of 49.89 [+ or -] 22.41 pm, ranging from 11.35 to 129.28 pm, whereas that of mature males ranged from 71.09 to 149.13 pm, with a mean value of 109.16 [+ or -] 14.82 pm. Thus, instead of the commonly used squash method that examines the size of spermatophores, the external morphology of vas deferens was used as physiological maturity indicator for subsequent experiments.
It was observed that there were three scenarios in which a male Scylla can be categorized as mature: (1) crab is mature in terms of all three maturity indicators tested (abdomen looseness, presence or absence of mating scars, and status of vas deferens development); (2) crab is mature only in terms of abdomen looseness and vas deferens development status; and (3) crab is mature only in terms of vas deferens development status (Fig. 4). This observation was consistent between all three Scylla species. This indicates that physiological maturity precedes functional maturity in mud crab genus Scylla. According to Figure 4, both Scylla tranquebarica and Scylla paramamosain reached physiological and functional maturities at the same CW size classes, 76-80 and 81-85 mm, respectively. In comparison, Scylla olivacea attained physiological and functional maturities at a smaller size, that is, 66-70 mm CW for both type of maturities.
No significant difference was found between [CW.sub.50] estimates based on three different maturity indicators (ANOVA: F = 1.152, P = 0.377, Fig. 5), but variation in [CW.sub.50] estimates occurred between species (F = 7.239, P < 0.05, Fig. 5). The [CW.sub.50] of Scylla olivacea was categorized as the smallest (Tukey HSD: P < 0.05), whereas Scylla tranquebarica and Scylla paramamosain were grouped together (P = 0.996).
This study reevaluated the common technique of determining physiological maturity in male mud crab genus Scylla, that is, the presence of spermatophores in vas deferens as maturity indicator. Physiological maturity as indicated by the presence of spermatophores via squash method was widely used in crustaceans (Aiken & Waddy 1980, Conan et al. 2001, Corgos & Freire 2006), including portunid crabs (Muino et al. 1999, Soundarapandian et al. 2013a, Vallina et al. 2014). Interestingly, results from the present study showed that spermatophores were present in the vas deferens of all Scylla specimens regardless of their size and species. This contradicts with the findings reported in two previous studies carried out on male mud crabs, that is, Scylla serrata by Robertson and Kruger (1994) and Scylla paramamosain by Islam and Kurokura (2013) in which they successfully determined the physiological maturity status of Scylla spp. based on the presence of spermatophores in the vas deferens (anterior vas deferens in S. serrata and median vas deferens in S. paramamosain). This may be due to the variation in the method used prior to the viewing of spermatophores under the microscope. Although the method used was not clearly described by Islam and Kurokura (2013), Robertson and Kruger (1994) reported that vas deferens of male crab was teased out in a drop of water on a glass slide prior to viewing under the microscope, whereas no water was added in the squash method used in the present study. Because of the low number and significantly smaller size of spermatophores in immature crabs, adding a drop of water presumably dispersed the available spermatophores within the vas deferens, thus reducing the chance of spotting them under the microscope with high magnification, even though they were present. Therefore, the use of the presence of spermatophores in vas deferens as physiological maturity indicator in male mud crab genus Scylla is inconclusive and until a more definite method is developed, visual examination of the external morphology of vas deferens (e.g., color and size) is already sufficient.
The conventional method that involved the monitoring of mating scars has proven to be not conclusive, whereas the examination of vas deferens status requires the culling of the studied crabs. In the present study, a simple, rapid and noninvasive method to accurately identify the sexual maturity status of male mud crab genus Scylla was tested. The maturity status of crabs as determined by abdomen looseness was confirmed by internal examination of vas deferens status. All crabs that were initially considered as mature by the means of abdomen looseness indeed turned out to be mature crabs with enlarged vas deferens of milky white coloration.
In all Scylla species, the estimates of [CW.sub.50] were not significantly different among all three maturity indicators. This clearly indicates that the looseness of abdomen as maturity indicator is as capable as vas deferens development status or mating scars observation in estimating [CW.sub.50]. The sensitivity of using looseness of abdomen in determining [CW.sub.50] was also proven in this study. The positive relationship between body size (CW) and sexual maturity was apparent. The smaller size class in reaching physiological and functional maturities in Scylla olivacea compared with Scylla tranqueburica and Scylla paramamosain could be attributed to their significantly smaller body size. On the other hand, males of S. tranqueburica and S. paramamosain (both with no significant difference in CW) attained physiological and functional maturities at the same size class. The size class in which S. tranqueburica and S. paramamosain attained physiological maturity is comparable to that of Scylla serrata from Natal, South Africa (smallest size class with mature crabs was 81-85 mm) (Robertson & Kruger 1994), although the smallest size class with mature S. serrata in terms of mating scars (115-119 mm) was larger compared with that of S. tranquebarica and .S', paramamosain in this study (86-90 mm). This indicates that S. serrata attains functional maturity at a larger size compared with S. tranquebarica and S. paramamosain, which is possible due to their comparably larger average body size.
The advantage of using looseness of abdomen to determine sexual maturity in male Scylla spp. is that this maturity indicator is far more superior in terms of accuracy compared with the method involving observation of mating scars and more time saving, and less effort is needed compared with using vas deferens development status. As shown in this study, the usage of mating scars observation to determine maturity in male Scylla spp. is inconclusive. There were instances where males were considered mature in both abdomen looseness and vas deferens status, but without the presence of mating scars. Such ambiguity in the usage of mating scars in determining sexual maturity was expected and was also observed in other portunid crabs (Knuckey 1996). Perhaps, some mature crabs do not develop any mating scars at all after mating, or that they lost their mating scars after subsequent molting.
In addition, determining sexual maturity by observing the development status of vas deferens ultimately requires the culling of crab prior inspection. The time and effort required are much higher compared with external examination of abdomen looseness. The feasibility of using abdomen looseness to accurately determine sexual maturity without having to kill the examined crab opens up new possibilities in fisheries and aquaculture sectors, such as stock monitoring activities can be carried out without affecting the local crab population, or the correct identification of crab maturity status for research and culture purposes.
It is however important to note that few cautions need to be taken when abdomen looseness is used for determining functional maturity in male Scylla spp. As observed in this study, this method can only be applied on live crabs, because once they are dead, their abdomen segments relaxed and can be opened easily, be it immature or mature crabs. It is also advisable that the strength used in assessing the looseness of abdomen segment should be kept to a minimum, as applying too much strength might force open the abdomen segment of immature crabs and cause injuries (i.e., the abdomen segment unable to close and return to its natural state).
The change in looseness of abdomen segment in Scylla spp. indirectly implies that they are functionally mature and are able to mate (Van Engel 1990, Nascimento & Zara 2013). During copulation, mature male Scylla spp. will flip open his abdomen segment and insert his gonopods into the gonopores of the female located underneath her abdomen segment for the transfer of sperms (Waiho et al. 2015). In our opinion, the inability of immature crabs to flip open their abdomen segments ensures that only mature males are involved in the copulation process and sperms are successfully transferred to females. This in turn guarantees that every copulated female has the sperms needed to fertilize the eggs once their ovaries matured, thus make sure that the time, energy, and effort spent during mating are not wasted. The close relationship in [CW.sub.50] estimated using abdomen looseness and vas deferens status might also indicate that functional maturity occurs simultaneously with physiological maturity in male Scylla spp. Further studies on the structural changes in abdomen segment of male Scylla spp. once they reach sexual maturity will aid in the validation of the proposed method. In addition, the applicability of this method in other crab species with different mating strategies needs to be tested to verify its universality.
In conclusion, the common method of using the presence of spermatophores to determine male physiological maturity is not reliable as spermatophores were found to be present in all squash slides of both mature and immature male crabs. In addition, the proposed method to determine functional maturity (via the looseness of the abdomen) is as reliable as conventional maturity-determining methods, with added advantages of being more accurate and simpler to conduct. The introduction of using abdomen looseness to determine sexual maturity will contribute greatly to ease in the conduct of research on reproductive ecology and in the stock assessment of Scylla spp. in the wild.
This research was financially supported by Malaysia Ministry of Higher Education under the Niche Research Grant Scheme (NRGS) (Vot. No. 53131). We gratefully thank Institute of Tropical Aquaculture, Universiti Malaysia Terengganu, for providing the needed infrastructure and laboratory space.
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KHOR WAIHO, (1) HANAFIAH FAZHAN, (1) JULIANA C. BAYLON, (2) WAN IBRAHIM WAN NORFAIZZA (1) AND MHD IKHWANUDDIN (1) *
(1) Institute of Tropical Aquaculture, Universiti Malaysia Terengganu, Mengabang Telipot, 21030, Kuala Terengganu, Terengganu, Malaysia; (2) Division of Biological Sciences, College of Arts and Sciences, University of the Philippines Visayas, 5023, Miagao, Iloilo, Philippines
* Corresponding author. E-mail: email@example.com
Please note: Some tables or figures were omitted from this article.