Morphometrics and Reproductive Characteristics of the Freshwater Crab Sartoriana spinigera from the Habitat of Ratargul Swamp Forest, Bangladesh: An Approach to Conservation

A total of 332 freshwater crab Sartoriana spinigera samples were collected for eight consecutive months from the Ratargul freshwater swamp forest, Bangladesh, to examine their general morphometrics and reproductive traits. The length-weight relationships of carapace weight with carapace length, width, and depth indicated negative allometric growth. The logarithmic relationship between carapace weight and carapace length, carapace weight and carapace width, and carapace weight and carapace depth exhibited a correlation value of 0.86, 0.79, and 0.56, respectively. Fulton's condition factor and allometric condition factor were found to be highest in March and lowest in October. The sex ratio was 1.59 : 1 for the male to female, which did not show a considerable divergence from the expected 1 : 1 ratio in the chi-square test (P < 0.05). In July, a maximum of mature individuals was collected and investigated. The fertilization was 1297 ± 629 ova, the ovary weight was 447.22 ± 359.39 mg, and the egg diameter was 375.15 ± 130.18 µm. Histological analysis showed that the male hepatopancreas was well organized and decomposition was reported in the female during the vitellogenic stages of the ovary. Spermatogonium cells were found in the male gonad, and cells from the female gonad were in the vitellogenic mature stage. In conclusion, the research will serve as a foundation for future research work on freshwater crab species as well as the conservation and maintenance of the ecological balance of this species in the Ratargul freshwater swamp forest.


Introduction
Freshwater swamps are distinct ecosystems with particular types of vegetation and abundant invertebrate assemblages [1].Swamp forests perform a variety of signifcant hydrological supporting functions, such as providing water storage space, controlling and protecting food peaks, maintaining subsurface water levels, recharging runof, absorbing pollutant degradation, and purifying the quality of water [2].Te Ratargul swamp forest is a distinct ecosystem in the northeast of Bangladesh with lowland vegetation, narrow depressions, and seasonal changes in water [3].As this distinct habitat has great potential values for foral and faunal biodiversity, and other intangibles, management, and resource investigations become very important [4].Freshwater crabs are one of the key ecologically signifcant invertebrate groups in tropical and subtropical water areas [5,6].Tey provide an excellent food source for many human cultures and are crucial to the food chain of the aquatic ecosystem.Crabs now contribute signifcantly to the fshery wealth of several countries [7].Te world's freshwater crab species are thought to serve as more than just a source of food; they are also used as fertilisers, food additives, and sources of many therapeutic elements [8].
However, little is known about the freshwater crab resources in Bangladesh, and very little research on freshwater crabs has been done so far.To form a complete inventory of species and a crab resource management program in Bangladesh, a survey is required on the range of crab resources from various regions of the country.
Te freshwater crab Sartoriana spinigera is a substantial deep brown crab that is found moving, burrowing, and even buried in the mud soil of the littoral zone of wetlands.Te environments of wetlands are the most advantageous for this crab's reproduction.S. spinigera was reported to be present in the Ratargul swamp forest by Islam et al. [9].Tis species ofers potential food value and has been reported to contain 30-59% of protein and 7-11% of lipid content [10].However, very little is known about its reproduction, morphometrics, and sexual maturity in Bangladesh.Terefore, the current research aimed to investigate their major morphometric dynamics, sexual maturity, sex ratio, and reproductive features in the Ratargul swamp forest, Bangladesh habitat.

Study Area, Sample Collection, and Morphometric
Measurement.Random mature samples of S. spinigera were collected from Ratargul swamp forest between March and October 2022 (Figure 1).It is located in the Gowain River, Fatehpur Union, Gowainghat upazila, Sylhet, Bangladesh, and receives its fows from the Gowain River system.Te forest is drowned under 6.09 to 9.10 m of water during the rainy season due to runof from India, while the water level remains at about 3.05 m deep for the remainder of the year [1].About 8-10 traditional bamboo traps with approximately 0.5 cm × 0.5 cm × 1.5 cm mesh sizes for adult freshwater crabs were loaded with the help of local fshermen for 8 hours in diferent parts of the swamps forest each month, and chopped small fsh were used as bait.No additional care has been taken to attract ovigerous females.Any bycatch other than crab was immediately discarded in the water.Te water quality parameters, i.e., temperature, dissolved oxygen, electric conductivity, total dissolved solids, and pH were measured on-site using a professional YSI digital multiprobe meter, Model 58, and depth was recorded for each sampling point.Just after collection, the animals were placed in large fbreglass tanks to be carried to the wet laboratory facility of Fish Biology and Genetics, Sylhet Agricultural University, Bangladesh.Major morphometric measurements, i.e., weight, carapace length, carapace width, and carapace depth, were taken using automated battery-powered vernier callipers and electric balance.Te length parameters were calculated based on the description of Ashkenazi et al. [11].Te depth has been measured by putting the crabs ventrally on vernier callipers to reach the maximum depth.Details of measurement are illustrated in Supplementary Figure S1.Te sex of the animals was confrmed by observing their ventral sides and the gonadal stage was confrmed by dissecting them.A part of the gonads and hepatopancreas of the mature sample was preserved in 10% neutral bufered formalin (NBF) to identify gonadal cells and liver changes in response to gonadal maturation.

Analysis of Length-Weight Parameters, Condition Indices,
Sex Ratio, GSI, and HPI.Te empirical relation of diferent length-weight parameters was constructed by the description of Cren 1951 [12], and a, b, and r 2 parameters were obtained by building logarithmic plots between the measurement of length and weight measurement [13].Condition factors were computed as allometric K A � W/L b [14], Fulton's K F � 100 × (W/ L 3 ), and relative K R � W/(aL b ) [12]; where W refers to the weight of the individual crab (g), L for the length of the carapace (mm), and a and b are the parameters obtained from the length-weight correlation.Te size at frst sexual maturity was calculated as log (L m ) � ⟶ 0.1189 + 0.9157 × log (L max ) [15].Te sex ratios were recorded in each monthly sample and compared with the chi-square test to determine the deviation from the 1 : 1 ratio.Te value of the gonadosomatic index (GSI) and hepatopancreatic index (HPI) was calculated as the percentage of the ratio between the weight of the respective organ and the body weight.

Fecundity, Gonadal Maturation, and Histology of the
Gonad and Hepatopancreas.Te mature and immature phases of both male and female gonads were identifed in each sampling month following the criteria of Silva et al. [16].Te female gonad is classifed into four consecutive stages, i.e., stage I-immature previtellogenic phase, stage II-ripening vitellogenic phase, stage III-mature complete vitellogenic phase, and stage IV-spawned; while the male gonad is distinguished as immature stage I, maturing stage II, and mature stage III.Only mature ovaries during July were included in the estimation of fertility.Initially, three subsamples of 0.25 to 0.30 g of each ovary were separated and then diluted with Gilson fuid to disintegrate the ovum.Finally, the number of ova in all subsamples was manually 2 Scientifca counted on a stereomicroscope.Hereafter, fertilization was computed using the description of Rasheed and Mustaquim [17].Standard histological procedure for crustaceans, followed as described by Santos et al. [18].Te method started with fxing the gonadal and liver samples in Bouin fxative.Ten they were cleaned with xylene and dehydrated in graded ethanol.Parafn-embedded blocks were sectioned and stained with hematoxylin and eosin dyes used to visualize cells under the Primo Star v3.0 microscope.Te a, b, and r 2 parameters for diferent length-weight data are demonstrated in Table 3.All the weight and associated length features were calculated to be highly correlated.Te growth types were negative allometries for all cases, as seen by the negative values.Te length at maturity was recorded as 25.43 mm carapace length, 36.34 mm carapace width and 19.44 mm carapace depth for female and 25.21 mm carapace length, 30.39 mm carapace width, and 16.51 mm carapace depth for male.Te b values were below 3 for all cases as well.Fulton's condition factor F k and the allometric condition factor K A followed the same pattern of variation throughout the sampling period, occupying a peak in March, while the relative condition factor K R represents several peaks that are opposite to the above condition factors (Figure 3).

Data
Monthly changes in the sex ratio of collected samples are shown in Table 4.A total of 204 male and 128 female crabs had been spotted during the study period.Te χ 2 test at P < 0.05 revealed that the male and female ratio of the crab samples collected varied signifcantly from the 1 : 1 ratio.A moderately male-dominant population had been estimated in most of the sampling periods.
Te PCA plots (Figure 4) showed a clear formation of TDS, SW, CW, CD, CL, K F , K R , pH group, and K A , DO, depth, temperature, and electric conductivity group.All variables showed moderately strong infuence with diferent monthly slots.Te PCA between morphometrics and water quality in diferent months is explained as 96.69% and 2.49% of the total variance.
Te water parameters showed a slight to moderate correlation with most morphometrics in March, July, June, August, October, and September.However, DO, depth, temperature, and EC were found to have a weak correlation with allometric condition factors in May, October, and September.

Hepatopancreatic Index (HPI) and Gonadosomatic Index (GSI). Female individuals exhibited the highest HPI during
March and the lowest during July (Figure 5).In contrast, the peak of GSI in females was reported in July and the lowest in March.On the other hand, the HPI values in male crabs showed numerous peaks in March, May, and September, with the lowest value in October.Te GSI value of males has also been reported to show several peaks between May and September.

Gonadal Maturation Phases, Histology, and Fecundity.
Diferent phases of mature and immature gonadal stages have been reported for the entire studied period (Figures 6(a) and 6(b)).Te highest mature ovaries occupying stage 4 and testes in stage 3 were spotted in June, July, and August.Crabs having matured or fnal stages of ovum were only used for fecundity estimation.Te fecundity of the female crabs collected during July was accounted as 1297 ± 629 ova with an average ovarian weight of 447 ± 359.39

Scientifca
Te mature female ovaries were characterized by the presence of vitellogenic and previtellogenic oocytes with well-pronounced nuclei, animal poles, and vegetal poles (Figure 7(b)).Again, the histology of the testes revealed the presence of mature spermatid cells (Figure 7(e)).Increased liver B cells and disintegration of lumen structure in mature stages of female hepatopancreases were recognizable (Figures 7(a) and 7(b)) in liver tissue, while there was no such sign in the male liver (Figure 7(d)).

Discussion
Te isometric value of b (>3) seems to be common in crustaceans.A b value of 2.931 in blue crab Callinectes sapidus [19], 2.71 in brown crab Charybdis callianassa [20], and 2.91 in Potamon algeriense [21] had already been documented.Te current research also demonstrated a value of b between 2.60 and 2.89 for diferent length-weight relationships.Again, several studies also well reported a strong correlation between the length and weight parameters of crustaceans.An r 2 value of 0.97 in mud crab Scylla olivacea [22], 0.906 in Callinectes sapidus [19], and 0.993 in Potamon algeriense [21] were noted as well.Te length at sexual maturity was reported to be between 27 and 32 mm for both sexes in the freshwater crab Potamon algeriense [21], and 108-130 mm for female brown crab Cancer pagurus [23,24].However, the size at maturity difers between species and is highly dependent on the methodology or model applied to the calculation [23,25].A value of r 2 0.81 has been documented for this species in East Kolkata Wetland, India, with a size range of 32-37 mm carapace length [26].Terefore, the fndings of current research follow similar trends in diferent length-weight parameters.
Te DO level above 5 mg L −1 and pH ranged from 5 to 10 deemed reasonable to favour the growth of the majority of aquatic macro-fauna [27,28].Te measurement of turbidity and water temperature in the current research was ideal for the growth of tropical aquatic organisms [27,29].A very similar form of water quality parameters has already been documented in the Ratargul Swamp Forest and other freshwater habitats in Bangladesh [1,30,31].Termal alterations are due to periodic fuctuations in temperature.Te highest turbidity in July was due to heavy rainfall and the mass siltation of anthropogenic runof.Additionally, the escalating trend of O 2 in the rainy season suggested better saturation associated with increased water circulation.Te water characteristics registered in this study were found to be within the optimal range for aquatic organisms' growth and reproduction [27,32].Nur Syafaat et al. [33] reported that the physiology of mud crab Scylla paramamosain is triggered in large part by water quality parameters which in turn results in changes in growth and morphometrics.Hossain et al. [1] recorded a moderate to strong correlation between diferent parameters of water quality and the growth of freshwater mussels.Te sheltering environment, water quality, and food availability are key parameters in determining the morphometric features of crabs [34].Te correlation between morphometrics and water quality characteristics in the current research also remained supportive of previous reports.
Male dominance in diferent crab populations is wellestablished in several studies [21,35].A female-to-male sex ratio distorted towards 1 : 0.3 had been observed in freshwater crab Trichodactylus fuviatilis [36].A distinctive population of males (94%) and females (74%) has already been reported among three sympatric Scylla species has already been reported [37].It is already been reported that male crabs have a higher tendency to disperse more in comparison to females [38].A moderately higher abundance of females in August, September, and October might be due to environmental factors.Elevated rainfall therefore increasing water depth in sampling sites makes the crabs dispersed more widely.Te current research aligned with the diferent reports of previous observations.Low fertilization seems very common in freshwater crabs.Abit et al. [39] reported fecundity ranged from 26 to 81 eggs, with an ovum diameter of 3.7 to 4.2 mm in Purple crab Isolapotamon bauense from Sarawak, Malaysia, while 340 to 800 ova were accounted for Potamon mesopotamicum from Iraq [40], and 533-1306 ova for Paratelphusa spinigera in India [41].Scientifca Terefore, S. spinigera could be treated as a slightly higherfecundity freshwater crustacean like Paratelphusa spinigera in India [41].
Te value of GSI seemed to increase during the onset of the breeding season among aquatic animals [25].Te value of GSI ranged from 3 to 5 for both sexes in the crab Sesarma intermedia [42].Higher GSI values were reported in Scylla olivacea before the breeding season [25].Te current research also showed a strong increase in the value of GSI in response to the breeding season.Te value of HPI in females tends to be inversely related to GSI, which is also reported in several aquatic animals [43].Tis is due to the release of vitellogenin from hepatic tissue to the ovary [44], which is also evident in the intense degeneration of the hepatic lumen in female crabs.Aaqillah-Amr et al. [45] stated that oocyte development was strongly related to GSI in S. serrata, and increased B cell and R cells were reported in the hepatopancreatic tubules as the ovarian development process progressed.Mekuleyi and Fakoya [46] also described hepatopancreas changes during the onset of advanced ovarian development in C. pallidus.However, no such trend was described in HPI of male crabs, and crabs are reported to have mature gonadal cells throughout the year [47].Te higher GSI value for both sexes reported in June and July suggesting breeding peak for this species at studied area.Also, water quality properties in this month favour the availability of more food and suitable parameters for breeding.
In conclusion, the length-weight relationship showed negative allometric growth and the sex ratio of S. spinigera did not show a considerable deviation in the study area.Te calculated length-weight relationship and diferent condition factors in the current research will be valuable data sources for the establishment of a monitoring and management system of these species in diferent freshwater ecosystems.Tis research may also help to conserve S. spinigera species as an unconventional fsheries resource by providing key data on their morphometrics and reproduction.
Analysis.Te data were managed in Ofce 365 Excel tools and analyzed in SPSS v26 (IBM Corporation, Armonk, USA).Te assumptions of normal distribution and homogeneity were tested by employing the Shapiro-Wilk test in SPSS.A one-way analysis of variance (ANOVA) was performed to compare the means and Tukey's HSD post hoc test was used to confrm signifcance at P < 0.05.Te correlation coefcient values were calculated from Pearson's correlation and the F test was employed to test the significance of the regression at P < 0.05.Te principal component analysis (PCA) between diferent morphometrics and water quality parameters has been constructed by using multivariate tools of software past version 4.06b and illustrated in scattered biplot.. Results3.1.Water Quality Properties.Te water temperature and total dissolved solids were declared to be very unstable throughout the study period, while the pH and dissolved O 2 showed moderate variation (Table1).Te maximum water temperature was obtained in August (29.53°C)while the lowest was in March(23.33°C).Te value of the TDS level reached its highest in June, while the lowest was during the prewinter seasons in October.Similarly, the pH reached its highest in April and lowest in March.Te electrical conductivity was maximum in September and lowest in May.males, and 17.16 ± 2.45 g, 27.66 ± 1.28 mm, 36.53 ± 1.85 mm, and 16.97 ± 0.96 for male in July (Table2).Again, regression analysis of diferent morphometric characteristics was found to have a strong r 2 value.Carapace weight and carapace length, carapace width, and carapace depth exhibited r 2 values of 0.86, 0.79, and 0.56, respectively (Figures2(c) and 2(e)).

Table 1 :
mg and an ovarian diameter of 375.15 ± 130.18 μm.Major water quality parameters at the study site.Te column with diferent superscripts indicates signifcant diferences at P < 0.05; values are means ± SD.
aTe column with diferent superscripts indicates signifcant diferences at P < 0.05; values are means ± SD.

Table 3 :
a, b, r 2 , and L m parameters of S. spinigera.

Table 4 :
Sex ratio of S. spinigera from the Ratargul freshwater swamp forest, Bangladesh.