Morphology of the female reproductive system and seasonality in reproduction of the freshwater crab Arcithelphusa cochleariformis (Brachyura: Gecarcinucidae)

Abstract

This study details the female reproductive system morphology and seasonality in reproduction of the freshwater crab Arcithelphusa cochleariformis, widely distributed in the wetlands of Wayanad, India. Histo-morphological analyses reveal that the H-shaped female reproductive system displays the general eubrachyuran pattern. The seminal receptacle exhibits a novel combination of both heterotreme and thoracotreme patterns with ventrally attached oviduct and the presence of a mono-layered columnar secretory epithelium lining the dorsal and ventral regions. The simple type vagina, covered by a cuticle, follows the heterotreme pattern. The seminal receptacle and vagina undergo changes parallel to the annual mating and ovarian cycle. The seminal receptacle appears small, collapsed, enclosing very few secretions before mating in the proliferation phase and swollen, containing enormous spermatophores and seminal plasma after mating in the previtellogenic phase. The seminal receptacle has a withered appearance in the primary and secondary stages of vitellogenic phase and with decreased amounts of seminal plasma and several freely dispersed spermatozoa concentrated at the ventral region during the tertiary vitellogenic stage.

Keywords:
Brachyurans; ovary; oviduct; seminal receptacle; vagina

INTRODUCTION

The female reproductive system in brachyurans is composed of H-shaped paired ovaries, oviducts, seminal receptacles (SRs), vaginae and gonopores (Hartnoll, 1968). The ovarian lobes, situated under the carapace and over the hepatopancreas, are joined beneath the stomach and extend to the posterior region underneath the pericardium (Warner, 1977). The oviduct is a narrow tube between the ovary and the SR, which in turn links to the gonopore on the sixth thoracic sternite through the vagina (Sternberg and Cumberlidge, 2001). The oviduct either enters the SR dorsally, ventrally or at a position in between and accordingly is termed dorsal, ventral or intermediate-type SRs (Diesel, 1991; McLay and Lopez-Greco, 2011; Gonzalez-Pisani et al., 2012). In heterotremes, the dorsal-type SR is generally seen in soft-shelled mating crabs, belonging to the families Portunidae and Xanthidae, while the ventral-type is observed in hard-shelled mating species (Calappidae, Geryonidae, Leucosiidae, Parthenopidae, Parathelphusidae, Corystidae) and the intermediate-type is in some majoids (Diesel, 1991). During copulation, spermatozoa from males are directly transferred to the SRs of females, where they are stored and maintained for an extended period of time (Hartnoll, 1968; Guinot and Quenette, 2005). Mature ova released into the oviduct during ovulation are led to the SR, where they come in contact with the stored spermatozoa, initiating fertilization (Diesel, 1989; Sainte-Marie and Sainte-Marie, 1998). Generally, the SRs enclose spermatozoa from multiple males (Christy, 1987; Orensanz et al., 1995), resulting in the enhancement of fertilization success by sperm competition (Jensen and Bentzen, 2012) and cryptic female choice (Dennenmoser and Thiel, 2015).

Despite the extensive information available regarding the morphological, histological, histochemical, ultrastructural, and functional details of the female reproductive system in marine crabs (Hartnoll, 1968; Beninger et al., 1988; Jensen et al., 1996; Sainte-Marie and Sainte-Marie, 1998; Sal Moyano et al., 2010; Becker et al., 2011; de Souza et al., 2011; Vallina et al., 2014; Antunes et al., 2016; Farias et al., 2017; Kienbaum et al., 2018; Ocampo et al., 2017; Vehof et al., 2017), and estuarine/brackish water crabs (Lopez-Greco et al., 1999; Sant’Anna et al., 2007; de Souza and Silva, 2009; Castilho-Westphal et al., 2013), there are very few studies devoted to freshwater crabs. Those reported are restricted to the SR secretory activity (Anilkumar and Adiyodi, 1977) or ovarian development (Sharifian et al., 2015; Smija and Sudha Devi, 2015; Sun et al., 2018). This knowledge is uneven and inadequate, considering the worldwide freshwater brachyuran fauna which represents about 1568 species (Raj et al., 2021).

The relationship between the ovarian cycle and the activity of the female reproductive tract have been extensively studied in marine and estuarine brachyurans. Hinsch (1988) reported seasonal fluctuations in the reproductive tract and the relationship between reproductive patterns of male and female Geryon fenneri (currently Chaceon fenneri (Manning and Holthuis, 1984)). Synchronization of morphological and histological changes in the SR such as size, consistency, wall and luminal contents with different stages of ovarian growth were observed in the estuarine crab Neohelice granulata (Dana, 1851) (Lopez-Greco et al., 1999) and the marine crab Arenaeus cribrarius (Lamarck, 1818) (Zara et al., 2014). Ocampo et al. (2017) confirmed a mating event during adult stage V in the parasitic castrator pea crab Calyptraeotheres garthi (Fenucci, 1975) by evaluating histological and ultrastructural variations in the SR and vagina during different stages of the female reproductive cycle. However, thus far, very few studies examined the seasonal variation in histology of the female reproductive tract and ovary in freshwater crabs. One exception being Lan and Chun-yuan (1999), who noted cytochemical changes in the wall and luminal constituents of the SR during different phases of the vitellogenic cycle in the freshwater crab Sinopotamon yangtsekiense (currently Longpotamon yangtsekiense (Bott, 1967)).

The freshwater crab Arcithelphusa cochleariformisPati and Sudha Devi, 2015, belonging to the family Gecarcinucidae (Heterotremata) and presently known only from Wayanad district of Kerala, India, inhabits burrows adjacent to the water canals in the Areca palms (Areca catechu), rice plant (Oryza sativa) and banana (Musa spp.) plantations. So far, only two species were reported under this genus: A. cochleariformis and Arcithelphusa tumpikkai Pati, Sujila and Sudha Devi, 2019. The meat of both species is edible and is a major source of protein for the local native people. Despite its abundance and edible nature, it is poorly exploited in scientific research, except for the taxonomic description by Pati and Sudha Devi (2015) and seasonal studies on the male reproductive system by Sneha and Sudha Devi (2019). The present study provides a holistic description of the morphology and seasonal variations in histology of the ovary and the female reproductive tract. Histo-anatomical studies of the female reproductive system may provide valuable information on reproductive strategies, evolution and phylogeny of the species concerned (McLay and Lopez-Greco, 2011; McLay and Becker, 2015; Ewers-Saucedo et al., 2016) and help to develop appropriate management strategies for species of commercial potential (de Souza et al., 2011).

MATERIAL AND METHODS

A total of 240 adult females (20/month; body weight, (BW): 25.35 ± 3.70 g; carapace width, (CW): 3.05 ± 0.50 cm) were sampled over a period of one year (March 2019 to February 2020) from the paddy fields near Ondayangadi, Mananthavady, Wayanad (11.82°N 76.02°E). They were carried to the laboratory, housed in clean plastic containers (46 ( 20 cm; 4-5 per container) sufficiently filled (nearly 15%) with natural water (temperature 27 ± 1 °C; pH 7.2; total dissolved oxygen 6.3-6.8 mg/L). The wet weights of animals were registered with a standard electric balance (Shimadzu) to 0.001 g accuracy and the carapace widths were measured using a precision vernier caliper (to 0.01 mm). The correlation (Pearson and Spearman models) between CW and BW of the female A. cochleariformis was calculated.

Figure 1.
Carapace width-body weight relationship in female Arcithelphusa cochleariformis.

After anaesthesia in crushed ice for ten min, the ovaries, SRs and vaginae of females (n= 20/month) were dissected out under a Trinocular Stereo Zoom Microscope (Radical) and photographed with a Nikon Coolpix L340 camera. The macroscopic characteristics (color, size, and texture) and the wet weights of ovaries and SRs were also recorded. The color evaluation of the ovary was carried out based on previous studies in freshwater crabs (Sharifian et al., 2015; Swetha et al., 2015; Smija and Sudha Devi, 2015; Sun et al., 2018). Some tissues (one ovary, SR, and vagina/female/month) were then preserved in Bouin’s solution (picric acid/formaldehyde/acetic acid in the ratio 75:25:5) overnight at room temperature, dehydrated in a graded alcohol series (30-100% ethanol), infiltrated in xylene, and embedded in paraffin wax (melting point 54-56 °C). Using a MicroTec CUT4050 rotary microtome (Germany), 5-7 μm thick sections (both longitudinal and transverse) were prepared and double stained with hematoxylin and eosin as described by Junqueira and Junqueira (1983). Observations of the stained sections were made under a Nikon Eclipse Ni-U Phase Contrast Microscope; and micro-photography was performed on a Nikon Y-TV55 camera attached to the microscope. The images were processed with the Nikon NIS Elements Imaging Software.

The oogenic phases were ascertained by evaluating the color, mean ovarian index, mean oocyte diameter, prevalence of the oocyte stage and histological appearance of the ovaries (Minagawa et al., 1993; Smija and Sudha Devi, 2015). The mean ovarian index was calculated as the percentage of wet weight of the ovary (g) to the wet weight of the crab (g). From each dissected female, one half ovary was torn open and the diameters of 100 randomly chosen oocytes were measured with a calibrated ocular micrometer under a compound microscope (Olympus) and the mean oocyte diameter was calculated. Both freshly dissected ovaries and their histological sections were used for calculating the prevalence of oocytes. From dissected females, the percentage of each oocyte stage was calculated as the number of oocytes in that particular stage of development to the total number of oocytes counted (n = 100) in one ovarian half. This was calculated for 10 females/month and the average of each oocyte stage was taken for each ovarian phase. Longitudinal sections of ovaries were scanned for determining the prevalence of oocytes. One whole histological section/slide/ovarian half/female (n = 10/month) was scanned, not a pre-determined area. The percentage of each oocyte stage was calculated to the total number of oocytes/longitudinal section and the most dominant stage was determined. Prevalence, here referred to as the most predominant oocyte stage in a particular phase of development of the ovary, was determined from this data.

Seasonal changes in the wet weight of the SR, width of the connective tissue layer and epithelium and nature of the luminal contents of the SR and vagina were also recorded. The SR index was calculated as the percentage of wet weight of the SR (g) to the wet weight of the crab (g) (n = 20/month). Ten spermatophores/histological section/slide/female (n = 10) were randomly selected, and their width along the major axes measured.

The data were expressed as mean ± SD. The differences in mean ovarian index, mean oocyte diameter values, SR index, height of muscle/connective tissue layer and columnar epithelium of SR and vagina, and width of the lumen of the vagina between oogenic phases (n = 30/oogenic phase) were compared through one way analysis of variance (ANOVA) followed by Tukey's post-hoc multiple comparison tests (SPSS version 24.0). Before applying mean and ANOVA, the data were checked for normality and homogeneity by Kolmogorov-Smirnov and Levene’s tests, respectively. Significant difference was set at a p value < 0.05.

RESULTS

Morphology and histology of the female reproductive system

The bilaterally symmetrical, H-shaped female reproductive system of A. cochleariformis consists of paired ovaries, oviducts, SRs, vaginae and gonopores. The anterior and posterior lobes of the ovaries, placed amid the hypodermis of the carapace and the hepatopancreas, were linked posteriorly to the stomach by a fine transverse commissure. The posterior lobes were continued as short, white, translucent tubes, the oviducts. The oviduct on each side was joined ventrally to an expandable translucent, thick walled pouch, called the seminal receptacle (SR). Each SR extended into a straight, flattened, soft tube, the vagina which opened to the exterior by a small circular gonopore (Fig. 2).

Figure 2.
Female reproductive system of Arcithelphusa cochleariformis during various phases of oogenesis. A, Previtellogenesis phase (PRE, April); B, primary vitellogenesis phase (PRI, July); C, secondary vitellogenesis phase (SEC, February). Note the mature oocytes (white arrowhead) passing through the oviduct towards the seminal receptacle (SR). GP, gonopore; OD, oviduct; OV, ovary; SR, seminal receptacle; VG, vagina.

Histologically, the ovary, circumscribed by an epithelial layer, encompasses oocytes in six different maturity stages: oogonium, chromatin nucleolus stage (CN), perinucleolar oocyte (PN), early vitellogenic oocyte (EV), middle vitellogenic oocyte (MV), and late vitellogenic oocyte (LV). The comparatively small, oval oogonia (9.12 ± 2.30 μm) have large, spherical nuclei (7.07 ± 1.90 μm) with granular chromatin, distinct nuclear envelope, and a narrow rim of mildly basophilic cytoplasm with indistinct oolemma (Fig. 3 A ). The oval CN oocytes (189.57 ± 5.71 μm) enclosed moderately basophilic cytoplasm and large, centrally situated round nuclei (27.11 ± 11.56 μm) with a network of fibrillar chromatin and peripherally arranged highly basophilic nucleoli (2-6) (3.01 ± 0.01 μm). A few elliptical, basophilic follicle cells (5.87 ± 2.31 μm) were seen scattered around these oocytes (Fig. 3 B ). The CN oocytes further increase in cytoplasmic volume to become the round PN oocytes (324.44 ± 24.56 μm), characterized by the appearance of small, basophilic yolk granules (5.15 ± 1.12 μm) and lucent vesicles (12.14 ± 1.23 μm) in the peripheral ooplasm and a multinucleolated (2-3) (6.72 ± 2.10 μm) nucleus (57.54 ± 1.09 μm). Basophilic follicle cells were seen arranged loosely around these oocytes (Fig. 3 C ). The EV stage demonstrates a large number of dense, highly basophilic mature yolk granules (19.40 ± 1.23 μm) and lucent vesicles (16.22 ± 2.16 μm), and a nucleus (67.21 ± 3.45 μm) with enlarged nucleoli (2-3) (7.56 ± 1.10 μm) in the EV stage (489.76 ± 97.05 μm). By this time, the follicle cells have organized to form a complete layer around the oocytes (Fig. 3 D ). In the MV stage (840.98 ± 15.50 μm), numerous large lucent vesicles (25.31 ± 3.02 μm) and mature yolk granules (30.08 ± 0.15 μm) encroach on the shrunken, condensed nuclei (26.23 ± 3.11 μm). Many mature yolk granules were seen integrated into the eosinophilic yolk bodies (19.45 ± 5.61 μm) in the peripheral ooplasm. A ring of follicular epithelium produced by the follicle cells was observed around these oocytes (Fig. 3 E ). The LV oocytes (mature oocytes), largest of the oocytes (1453.12 ± 90.79 μm), were characterized by the absence of a visible nucleus and follicular epithelium. Their ooplasm was filled with an eosinophilic homogenous matrix formed by the coalition of the yolk platelets. These oocytes were surrounded by a thin chorion overlying the oolemma, which indicates the end of vitellogenesis (Fig. 3 F ) (Tab. 1).

Figure 3.
Oocytes in different stages of development in Arcithelphusa cochleariformis. A, Oogonia; B, chromatin nucleolus stage; C, perinucleolar stage; D, early vitellogenic stage; E, middle vitellogenic stage; F, late vitellogenic stage (mature oocyte). C, chorion; CN, chromatin nucleolus stage oocyte; EV, early vitellogenic oocyte; FC, follicle cell; FE, follicular epithelium; LU, lucent vesicle; LV, late vitellogenic stage oocyte, MV, middle vitellogenic stage oocyte; N, nucleus; NU, nucleolus; OO, oogonium; PN, perinucleolar stage oocyte; YB, yolk bodies; YG, yolk granule.

Table 1.
Characteristic features of oocytes in various stages of development in the freshwater crab Arcithelphusa cochleariformis.

Stage of development	Cell diameter (μm)	Nuclear diameter (μm)	Characteristics
Oogonium	9.12±2.30	7.07±1.90	Small oval cells; large, spherical nucleus with granular chromatin; distinct nuclear envelope and a narrow rim of mildly basophilic ooplasm with indistinct oolemma.
Chromatin nucleolus stage	189.57±5.71	27.17±11.56	Oval cells with moderately basophilic cytoplasm; large, centrally situated round nuclei with a network of fibrillar chromatin and peripherally arranged highly basophilic nucleoli (2-6) (3.01 ± 0.01 μm). A few elliptical, basophilic follicle cells (5.87 ± 2.31 μm) seen scattered around the oocyte.
Perinucleolar stage	324.44±24.56	57.54±1.23	Round oocyte with an appearance of small, basophilic yolk granules (5.15 ± 1.12 μm) and lucent vesicles (12.14 ± 1.23 μm) in the peripheral ooplasm; multinucleolated nucleus. Basophilic follicle cells seen arranged loosely around the oocytes.
Early vitellogenic stage	489.76±97.05	67.21±3.45	Spherical cells; large number of dense, highly basophilic mature yolk granules (19.40 ± 1.23 μm) and lucent vesicles (16.22 ± 2.16 μm); nucleus with enlarged nucleoli (2-3) (7.56 ± 1.10 μm). Follicle cells organized to form a complete layer around the oocytes.
Middle vitellogenic stage	840.98±15.50	26.23±3.11	Numerous large lucent vesicles (25.31 ± 3.02 μm) and mature yolk granules (30.08 ± 0.15 μm); shrunken and condensed nuclei; many mature yolk granules integrated into eosinophilic yolk bodies (19.45 ± 5.61 μm) in the peripheral ooplasm. A ring of follicular epithelium produced by the follicle cells formed around the oocytes.
Late vitellogenic stage	1453.12±90.79	Nucleus indistinct	Largest of the oocytes (mature oocyte) characterized by the absence of a visible nucleus and follicular epithelium; ooplasm filled with an eosinophilic homogenous matrix formed by the coalition of yolk platelets. Oocytes surrounded by a thin chorion overlying the oolemma indicating the end of vitellogenesis.

Arcithelphusa cochleariformis breeds once in a year, accommodating a single ovarian cycle. The annual oogenic cycle of A. cochleariformis was classified into three phases: proliferation (PRO), previtellogenic (PRE), and vitellogenic (VIT) (Minagawa et al., 1993; Islam et al., 2010; Smija and Sudha Devi, 2015). The VIT phase was further divided into primary (PV, October-November), secondary (SV, December-January), and tertiary vitellogenic (TV, February) stages. The TV stage was followed by spawning which occurred at the end of March. After spawning, the females underwent a period of rest (spent or oosorption phase) in which the unspawned oocytes undergo degeneration and dissolution. The spent stage is followed by the proliferation phase. In A. cochleariformis, the ovary exhibited a synchronous pattern of development. The monthly percentage of females in each phase of oogenesis is depicted in Fig. 4. The SR and the vagina demonstrated spectacular changes in histology in conjunction with the stages of development of the ovary.

Figure 4.
Percentage of females in proliferation, previtellogenic, and vitellogenic phases in Arcithelphusa cochleariformis by month.

Proliferation phase (March-May) - Out of the 60 crabs studied, 56 (93.33%) were in the PRO phase followed by 4 (6.67%) in the VIT phase. During this phase, the ovaries (length 8-9 mm; width 2-3 mm) appeared small, translucent and white with a generally low mean ovarian index and oocyte diameter values (0.15 ± 0.01 and 75.03 ± 3.19 μm, respectively; P < 0.05). The germinal zone of the ovary was occupied by clusters of oogonia (57%) and CN (18%) oocytes. In addition, large number of shrunken follicles (12%), pycnotic follicle nuclei (7%), and atretic oocytes (6%) were found dispersed in the ovary (Fig. 5 A ).

Previtellogenic phase (June-September) - Out of the 80 specimens assessed, 73 (91.25%) were in the PRE phase and the remaining 7 (8.75%) in the PRO phase. Compared to the previous phase, a significant increase (P < 0.05) was observed in the mean ovarian index (0.24 ± 0.03) and oocyte diameter (312.75 ± 3.19 μm) values. The cream-colored ovaries (length 10-12 mm; width 4-5 mm) were predominantly distributed with PN (65%) followed by CN (14%) oocytes and oogonia (9%). The number of shrunken follicles (6%), pycnotic follicle nuclei (4%), and atretic oocytes (2%) were found diminished than the earlier phase (Fig. 5 B ).

Vitellogenic phase (October-February) - Out of the 100 specimens studied, 90 (90%) were in the VIT phase and the remaining 10 (10%) in the PRO phase. In the PV stage of VIT phase, the ovaries (length 12-14 mm; width 6-8 mm) attained a pale yellowish color with a significantly high (P < 0.01) mean ovarian index and oocyte diameter values (0.42 ± 0.12 and 545.32 ± 29.42 μm, respectively). The EV oocytes accounted for 75% of the measured oocytes while the oogonia, CN stage oocytes along with a few shrunken follicles and atretic oocytes accounted for 10, 9, 4, and 2%, respectively (Fig. 5 C ). During the SV stage, the ovaries (length 15-18 mm; width 9-10 mm) turned into a slight orange color with a two-fold rise (P < 0.01) in the mean ovarian index (0.97 ± 0.23) and oocyte diameter values (879.44 ± 50.19 μm). A major portion of the ovary was occupied by MV oocytes (67%) with minor proportions of EV (23%) and PN (10%) oocytes (Fig. 5 D ). The ovaries (length 19-20 mm; width 11-12 mm) demonstrated a bright orange color through the TV stage with significantly high (P < 0.001) values for mean ovarian index and oocyte diameter, reaching their maximum (2.21 ± 0.19 and 1435.39 ± 75.03 μm, respectively). The ovary at this stage was fully laden with LV (91%) oocytes followed by a few MV (5%), PN (3%), and CN (1%) oocytes (Fig. 5 E ).

Figure 5.
Light micrographs of Arcithelphusa cochleariformis ovary (sagittal section) in proliferation, previtellogenic and vitellogenic phases. A, Proliferation phase ovary occupied by clusters of oogonia and chromatin nucleolus stage oocytes (April); B, previtellogenic phase ovary, predominantly distributed with PN and CN oocytes (July); C, ovary in primary vitellogenic stage of vitellogenic phase with large number of EV stage oocytes, few PN and CN stage oocytes (October); D, ovary in secondary vitellogenic stage of vitellogenic phase packed with a large number of MV oocytes and small number of EV and PN oocytes (December); E, ovary in tertiary stage of vitellogenic phase laden predominantly with LV oocytes (mature oocytes) and small percentages of MV, PN and CN stage oocytes (February). AO, atretic oocyte; CN, chromatin nucleolus stage oocyte; EV, early vitellogenic oocyte; LV, late vitellogenic oocyte; MV, middle vitellogenic oocyte; OO, oogonium; PN, perinucleolar stage oocyte; SF, shrunken follicle.

Histo-anatomy of the genital ducts during various phases of oogenesis

During the proliferation phase, the small (length 9-10 mm; width 3-4 mm), pale yellow, oval to elongate SR had a collapsed appearance with a relatively low SR index (0.19 ± 1.05) (Fig. 2 A ). Histologically, the SR was divided into dorsal and ventral regions, the wall of which was composed of an outer thin loose connective tissue layer (9.90 ± 0.12 μm thick) surrounding an inner layer of epithelium (55.40 ± 2.10 μm thick). The connective tissue layer was composed of several strata of wavy collagen fibers (Fig. 6 A , B). The epithelium of both dorsal and ventral regions consisted of a single layer of tightly arranged, columnar secretory epithelial cells containing homogenous, granular, mildly eosinophilic cytoplasm, and basally located irregular, highly basophilic, lobed nuclei with heterochromatin (5.35 ± 1.80 μm) (Fig. 6 B , C) (Tab. 2). The epithelium of the ventral region of the SR was lined by a thin cuticle apically (not clearly visible in the figure Fig. 6 B , due to fixation error). Infoldings of the connective tissue and columnar epithelium were obvious at some places in the dorsal and ventral regions (Fig. 6 D ). The epithelium was adjacent to the lumen, loaded with type I secretion and a few residual spermatozoa with irregular outlines and indistinct acrosomes. Dissolution and degeneration of residual spermatozoa were also evident in some parts of the lumen (Fig. 6 E ).

Figure 6.
Light micrographs of the SR (sagittal section) during proliferation (PRO) phase in Arcithelphusa cochleariformis (April). A, General view of the dorsal region of the SR; B, C, enlarged view of the dorsal and ventral regions of the SR showing columnar epithelial cells with irregular, lobed nuclei and lumen. Note the epithelium of the ventral region lined by a thin cuticle apically (due to some fixation error, the cuticle lining was not clearly visible in this figure); D, infolding of the connective tissue and epithelium in the dorsal region; E, part of the dorsal region of the SR displaying wall and lumen with degenerating residual spermatozoa. C, cuticle; CE, columnar epithelium; CT, connective tissue layer; DR, dorsal region; I, infolding; L, lumen; N, nucleus; RS, residual sperm mass.

The vagina was of the simple type and appeared circular or oval in cross section. The wall of the vagina was lined by a layer of closely packed columnar epithelium (82.46 ± 2.76 μm in height) covered externally by a connective tissue layer (18.21 ± 0.31μm in height) and internally by a thin cuticle (not clearly visible in the figure Fig. 7A-D, due to fixation error). The connective tissue layer was composed of many parallel collagen fibers (Fig. 7 A -D). The epithelial cells had large, basally located, basophilic elongate nuclei (9.46 ± 3.12 μm) with centrally or peripherally located nucleoli and weakly basophilic, granular cytoplasm (Fig. 7 A -D). The lumen presented spermatozoa and some residual basophilic secretion towards the periphery (Fig. 7 E ) (Tab. 2).

Figure 7.
Light micrographs (sagittal and transverse sections) of Arcithelphusa cochleariformis vagina during proliferation (PRO) phase (April). A, B, Sagittal sections of the vagina depicting connective tissue layer, columnar epithelium, cuticle and lumen; C-E, Transverse sections of the vagina, wall and lumen containing spermatozoa. BS, basophilic secretion; C, cuticle; CE, columnar epithelium; CT, connective tissue layer; L, lumen; N, nucleus; SZ, spermatozoon.

The SR in the previtellogenic phase appeared as a white, transparent, swollen, and kidney-shaped structure with a significant increase (P < 0.001, F = 251.28, df = 59) in size (length 13-15 mm; width 6-7 mm) and index (0.53 ± 0.03) (Fig. 2 B ). The SR of this phase was characterized by a relatively thick columnar epithelium (75.19 ± 1.34 μm) and connective tissue layer (13.12 ± 0.29 μm) (Fig. 8 A -C). The epithelial cells encompassed moderately basophilic, multi-nucleolated (2-5) lobed nuclei (7.98 ± 0.02 μm), scattered chromatin granules and ample granular cytoplasm (Fig. 8 B , C) (Tab. 2). The apical regions of the epithelial cells facing the lumen had small lucent vesicles, signifying a merocrine mode for release of the secretion (Fig. 8 B , C). The lumen was filled with many oval spermatophores embedded in large amounts of the highly basophilic type II secretion, which indicated a recent insemination (Fig. 8 A , D). The spermatophores (34.20 ± 19.48 μm) were packed with large numbers (25-100) of spermatozoa (5.40 ± 0.50 μm) embedded in a granular, weakly eosinophilic secretion. Some of these spermatophores have their walls disrupted, releasing large numbers of free spermatozoa, immersed in an agranular eosinophilic secretion (Fig. 8 D ). Infoldings of the connective tissue and epithelium were not evident in the SR of this phase (Fig. 8 A ).

Figure 8.
Light micrographs of the SR (sagittal section) during previtellogenic (PRE) phase in Arcithelphusa cochleariformis (July). A, General view of the SR portraying large number of spermatophores (red arrowhead) embedded in the basophilic type II secretion (seminal plasma, black arrowhead) indicating recent insemination; B, connective tissue layer, epithelial cells displaying multi-nucleolated, lobed nuclei and lumen presenting a homogenous layer of type I (intrinsic) secretion immediately beneath the epithelium. Note the presence of small lucent vesicles at the apical region of the epithelial cells, signifying a merocrine mode of release of secretion; C, magnified view of the connective tissue layer and epithelium from B; D, part of the lumen depicting spermatophores surrounded by the highly basophilic type II secretion. Note the spermatophores with disrupted walls, releasing large numbers of free spermatozoa immersed in the agranular eosinophilic secretion. CE, columnar epithelium; CT, connective tissue layer; DR, dorsal region; L, lumen; N, nucleus; SPH, spermatophore; SZ, spermatozoon; TI, type I secretion; TII, type II secretion; V, lucent vesicle; VR, ventral region.

The connective tissue layer and the epithelium supporting the wall of the vagina had a thickness of 28.19 ± 1.45 and 96.89 ± 1.64 μm, respectively (Fig. 9 A ). The columnar epithelial cells were found active, evidenced by large, basophilic nuclei (10.46 ± 4.40 μm) with several centrally or peripherally located nucleoli (1-5), a network of chromatin fibrils and weakly basophilic, voluminous granular cytoplasm (Fig. 9 B ). The luminal diameter was extended further (295.90 ± 2.32 μm), packed with free sperm masses embedded in the basophilic secretion (Fig. 9A -C). No intact spermatophores were observed in the lumen (Tab. 2).

Figure 9.
Light micrographs of Arcithelphusa cochleariformis vagina (transverse section) during previtellogenic (PRE) phase (July). A, Cross section of the vagina displaying thick connective tissue, epithelium and lumen packed with free sperm masses and basophilic secretion; B, a portion of the vaginal wall enlarged; C, lumen displaying large number of free sperm masses along with basophilic secretion. BS, basophilic secretion; CE, columnar epithelium; CT, connective tissue layer; L, lumen; N, nucleus; SZ, spermatozoon.

The SR of the vitellogenic phase showed the same morphological features as those of the earlier phase, except a slight reduction in size (length 12-14 mm; width 5-6 mm) and an insignificant decrease (P < 0.001, F = 34.60, df = 59) in the SR index (0.50 ± 0.02). However, considerable changes were noticed in the histo-anatomical features. During the PV and SV stages, a marked increase was noticed in the thickness of the epithelium (80.25 ± 0.89; 82.21 ± 0.41 and 78.49 ± 0.12 μm, respectively) and the connective tissue layer (18.18 ± 0.32 and 19.98 ± 0.22 μm, respectively), whereas a thin connective tissue layer was noticed in the TV stage (11.18 ± 0.32 μm) (Tab. 2) (Figs. 10A-C; 11A, D). Infoldings of the connective tissue and epithelium were observed in both dorsal and ventral regions of the SR (Figs. 10A, 11D). The epithelial cells showed high secretory activity during this phase as evidenced by the presence of abundant, granular cytoplasm and many lucent vesicles and a homogenous layer of type I secretion immediately beneath the apical region in the lumen (Figs. 10D, E, G, H, 11B, E). The epithelial cells exhibited a basophilic basal half and an eosinophilic distal half in the PV stage. During the PV and SV stages, though both type I and II secretions were apparent in the lumen, a decline was noticed in the amount of the type II secretion. No intact spermatophores were observed, but a large number of spermatozoa were seen dispersed as independent entities in the type I secretion (Fig. 10 F , I).

Figure 10.
Light micrographs of the SR (sagittal section) during primary (PV) and secondary (SV) stages of vitellogenic phase in Arcithelphusa cochleariformis. A, General view of the SR during PV stage (October); B, C, dorsal and ventral regions of the SR in PV stage depicting wall and lumen; D, wall of the dorsal region and lumen illustrating lucent vesicles and a homogenous layer of type I secretion beneath the epithelium during PV stage; E, enlarged view of the dorsal connective tissue layer and epithelial cells demonstrating basophilic basal half and eosinophilic distal half during PV stage (October); F, lumen of the dorsal region of the SR showing a decline in the amount of the type II secretion in PV stage; G, dorsal region of the SR presenting the connective tissue layer, columnar epithelium and lumen during SV stage (December). Note the apical region of cells with large lucent vesicles (indicating secretory activity) and type I secretion as a homogenous layer; H, enlarged view of the wall and lumen of the dorsal SR in SV stage; I, lumen of the ventral SR showing a decline in the type II secretion in SV stage. CE, columnar epithelium; CT, connective tissue layer; I, infolding; L, lumen; N, nucleus; SZ, spermatozoon; TI, type I secretion; TII, type II secretion; V, lucent vesicle.

During the TV stage, the amount of type II secretion was found reduced further in the lumen, detected as small to large condensed islands bounded by large amounts of the more transparent type I secretion (Fig. 11 C ). An enormous number of freely dispersed spermatozoa were found concentrated near the luminal border in the more ventral regions (Fig. 11 E , F). The spermatozoa (5.40 ± 0.50 μm) were spherical in shape, characterized by the presence of a deeply basophilic acrosomal vesicle covered by an operculum and with a centrally placed elongated perforatorial chamber (Fig. 11 E ) (Tab. 2).

Figure 11.
Light micrographs of the SR (sagittal section) in tertiary stage (TV) of vitellogenic phase in Arcithelphusa cochleariformis (February). A, Dorsal region of the SR displaying a slim connective tissue layer, columnar epithelium and lumen; B, enlarged view of the epithelial cells of the dorsal region revealing granular cytoplasm, large lucent vesicles at their apical regions and type I secretion in the lumen. Note a decline in type II secretion in the lumen; C, condensed islands of type II secretion bounded by type I secretion in the lumen of the dorsal region; D, part of the ventral region of the SR; E, enlarged view of the wall and lumen displaying enormous number of freely dispersed spermatozoa at the ventral region. Inset: mature spermatozoa; F, bulk of spermatozoa concentrated at the ventral region of the SR, close to the vagina. Acrosomal vesicle (black arrowhead); CE, columnar epithelium; CT, connective tissue layer; DR, dorsal region; I, involution; L, lumen; N, nucleus; Operculum (red arrow); Perforatorial chamber (white arrowhead); SZ, spermatozoon; TI, type I secretion; TII, type II secretion; VG, vagina; VR, ventral region.

Table 2.
Different phases of oogenesis in Arcithelphusa cochleariformis showing the SR index, width of the muscle layer, columnar epithelium and lumen of the SR and vagina.

Phases of oogenesis	Genital duct	SR index	Wall (μm)		Width of lumen (μm)
			Height of muscle/ connective tissue layer	Height of columnar epithelium
Proliferation	SR	0.19±1.05	9.90±0.12	55.40±2.10	-
	Vagina	-	18.21±0.31	82.46±2.76	231.15±10.12
Previtellogenic	SR	0.53±0.03* F = 322.04 df = 34	13.12±0.29* F = 455.06 df = 34	75.19±1.34* F = 339.67 df = 34	-
	Vagina	-	28.19±1.45* F = 539.21 df = 34	96.89±1.64* F = 491.21 df = 34	295.90±2.32* F = 487.55 df = 34
Vitellogenic	SR	0.50±0.02 F = 436.43 df = 34	Primary: 18.18±0.32* F = 630.01 df = 34 Secondary: 19.98±0.22* F = 750.94 df = 34 Tertiary: 11.18±0.32* F = 879.40 df = 34	Primary: 80.25±0.89* F = 512.22 df = 34 Secondary: 82.21±0.41* F = 644.43 df = 34 Tertiary: 78.49±0.12* F = 809.05 df = 34	-
	Vagina	-	29.18±2.88* F = 969.01 df = 34	98.56±1.96* F = 978.02 df = 34	299.11±10.52* F = 642.18 df = 34

• Note: * indicates P < 0.001.

The thickness of the connective tissue (29.18 ± 2.88 μm) and the columnar epithelium (98.56 ± 1.96 μm) of the vagina during VIT phase remained the same as those in the PRE phase (Fig. 12 A ). The columnar epithelial cells possessed basophilic, oval or elongate, granular nuclei (10.49 ± 4.21 μm) with several nucleoli (1-5) and ample granular cytoplasm (Fig. 12 A , B) (Tab. 2). The lumen (299.11 ± 10.52 μm) was observed completely filled with a large number of free spermatozoa embedded in the basophilic secretion (Fig. 12 A , C). These spermatozoa (5.40 ± 0.50 μm) have the same morphology as those found in the SR at this stage (Fig. 12 C ).

Figure 12.
Light micrographs of Arcithelphusa cochleariformis vagina (transverse section) during tertiary stage (TV) of vitellogenic phase (February). A, Cross section of the vagina (February); B, vaginal wall with connective tissue layer and epithelial cells having multi-nucleolated, basally located granular nuclei; C, part of the lumen showing large number of free spermatozoa. CE, columnar epithelium; CT, connective tissue layer; L, lumen; N, nucleus; SZ, spermatozoon.

DISCUSSION

This study evaluated the histo-morphological variations in the ovary and the genital duct during the oogenic cycle of A. cochleariformis. The H-shaped female reproductive system of this species displayed the general eubrachyuran pattern with paired ovaries, SRs, vaginae, and gonopores (Hartnoll, 1968; Johnson, 1980; Diesel, 1989; Sant’Anna et al., 2007; McLay and Lopez-Greco, 2011; de Souza et al., 2011; McLay and Becker, 2015). In contrast, the typical H-shaped pattern is not followed by heterotremes like Maja brachydactyla Balss, 1922 (Rotllant et al., 2007), Leurocyclus tuberculosus (H. Milne Edwards and Lucas, 1842), and Libinia spinosa Guérin, 1832 (Gonzalez-Pisani et al., 2012). Similarly, the female reproductive system is X-shaped in members of the family Pinnotheridae (Thoracotremata) (Becker et al., 2011). In some heterotremes like Cancer magister Dana, 1852 (Jensen et al., 1996), Limnopilos naiyanetri Chuang and Ng, 1991 (Klaus et al., 2014) and the thoracotreme Percnon gibbesi (H. Milne Edwards, 1853) (Kienbaum et al., 2018), an accessory sperm storage structure, the bursa, was observed between the SR and the vagina, whereas members of the family Dorippidae have a cuticular valve in addition to the bursa, between the oviduct and the SR (Vehof et al., 2017).

In A. cochleariformis, the paired gonopores were non-operculate, situated on the sixth thoracic sternite, and conforming to the coxal-male and sternal-female pattern found in heterotremes (Gonzalez-Pisani et al., 2012; Antunes et al., 2016; Farias et al., 2017). On the other hand, in thoracotremes, the gonopores are sternal in position, covered with opercula in both the sexes (Becker et al., 2011; de Souza et al., 2011; 2016; Vehof et al., 2016).

In the current study, the attachment of the oviduct with the SR is ventral, in agreement with the pattern found in most heterotremes such as Inachus ranulate (Fabricius, 1775) (Diesel, 1989), Chionoecetes opilio (Fabricius, 1788) (Beninger et al., 1988; Sainte-Marie and Sainte-Marie, 1998), Cancer gracilis Dana, 1852 (Orensanz et al., 1995), C. magister (see Jensen et al., 1996), Metacarcinus edwardsii (Bell, 1835) (Pardo et al., 2013), Stenorhynchus seticornis (Herbst, 1788) (Antunes et al., 2016), Dorippe sinica Chen, 1980, Dorippe quadridens (Fabricius, 1793) (Vehof et al., 2017), and various thoracotremes (Lopez-Greco et al., 1999; 2009; Sant’Anna et al., 2007; de Souza and Silva, 2009; Becker et al., 2011; McLay and Lopez-Greco, 2011; Kienbaum et al., 2018). Conversely, the connection is dorsal or intermediate in a few heterotremes like Callinectes sapidus Rathbun, 1896 (Johnson, 1980), L. tuberculosus, Li. Spinosa (see Gonzalez-Pisani et al., 2012), Ovalipes trimaculatus (De Haan, 1833) (Vallina et al., 2014), Chaceon chilensis Chirino-Gálvez and Manning, 1989 (Pardo et al., 2017), and Callinectes danae Smith, 1869 (Assugeni et al., 2021). In polyandrous species (multiple-male mating species), the site where the oviduct opens to the receptacle strongly influences the potential for sperm competition. For example, in species with the ventral type connection, the spermatozoa preferentially used in fertilizing oocytes are those from the last copulation, while in the dorsal type SR, sperms from the first copulation were used for fertilizing the oocytes; and, in species with the intermediate type connection, spermatozoa from neither the last nor the first copulation are favored (Diesel, 1991; McLay and Lopez-Greco, 2011). This view was further supported by the absence of cilia and conspicuous muscles lining the SR for mixing spermatozoa in species with a multiple mating strategy (McLay and Becker, 2015). According to Diesel (1991), the ventral-type connection is usually observed in hard-shelled mating species. Even though the oviduct connects the SR ventrally in A. cochleariformis, studies in our laboratory have shown that in A. cochleariformis, mating occurred once annually, between the soft-shelled females and the hard-shelled males (Sudha Devi, unpublished data). Thus, in the present study, the connection of the oviduct with the SR has no role in determining which batch of spermatozoa fertilize the ova as there will only be a single batch of spermatozoa in the SR.

In A. cochleariformis, based on cell and nuclear diameter, appearance of nucleus and degree of yolk deposition, the oocyte stages were classified into six stages (oogonia, chromatin nucleolus stage, perinucleolar, early, middle, and late vitellogenic oocytes). A comparable classification of oocyte stages was done by Leach, 1815, Cancer pagurus Linnaeus, 1758, and Eriocheir sinensis H. Milne Edwards, 1853, but the criteria used for the classification of oocytes was found to be highly variable between brachyuran species. For example, based on changes in the cytoplasmic granules, nucleus to cytoplasm ratio, position of nuclei, and reaction to staining, four oocyte stages were observed in Potamon dehaani (currently Geothelphusa dehaani (White, 1847)) (Otsu, 1963), Goniopsis cruentata (Latreille, 1803) (de Souza and Silva, 2009), Cardisoma guanhumi Latreille in Latreille, Le Peletier, Serille and Guérin, 1828 (Shinozaki-Mendes et al., 2012), Ca. sapidus (see Carvalho-Saucedo et al., 2015) and Sinopotamon henanense (= Sinopotamon honanense, currently Longpotamon honanense (Dai, Song, He, Cao, Xu and Zhong, 1975)) (Sun et al., 2018). Based on the degree of yolk deposition (vitellogenesis), the oocyte developmental stages were divided into three in Uca rapax (Smith, 1870) (Castiglioni et al., 2007). Previous morphological, histological, and ultrastructural analyses documented four oocyte developmental stages in the freshwater crab Sylviocarcinus pictus (H. Milne Edwards, 1853) (Silva et al., 2012), five in Ca. danae, Callinectes ornatus Ordway, 1863 (Keunecke et al., 2009), Armases rubripes (Rathbun, 1897) (Santos et al., 2009), Aratus pisonii (H. Milne Edwards, 1837) (Nicolau et al., 2012), Portunus pelagicus (Linnaeus, 1758) (Ravi et al., 2011), Oziotelphusa senex senex (currently Spiralothelphusa senex (Fabricius, 1798)) (Swetha et al., 2015), and the sesarmid crab Episesarma singaporense (Tweedie, 1936) (Sudtongkong et al., 2021) and six in Portunus trituberculatus (Miers, 1876) (Wu et al., 2007). The oocytes of the freshwater crabs: Potamon koolooense (currently Hymalayapotamon koolooense (Rathbun, 1904)) (Joshi and Khanna, 1982) and Sodhiana iranica Sharifian, Kamrani and Sharifian, 2014 (Sharifian et al., 2015) were categorized into seven stages, based on cell size, chromatin arrangement, and the number of lipid vesicles. Ten different developmental stages were identified in Ranina ranina (Linnaeus, 1758) (Minagawa et al., 1993) and Travancoriana schirnerae Bott, 1967 (Smija and Sudha Devi, 2015), based on cell size, appearance of nucleus, and degree of yolk accumulation. Arcos-Ortega et al. (2019) distinguished 11 oocyte stages in the king crab Lithodes santolla (Molina, 1782), based on differences in cellular characteristics. All of the above findings, including the present results, clearly indicate that there is little consistency in the classification of oocyte stages among brachyurans.

In A. cochleariformis, the follicle cells organized to form a complete layer around the perinucleolar and early vitellogenic oocytes (maturing oocytes), while they were absent around the fully mature late vitellogenic oocytes. In agreement with this finding, many authors reported follicle cells around the immature oocytes in brachyurans (Johnson, 1980; Joshi and Khanna, 1982; Minagawa et al., 1993; Castiglioni et al., 2007; Quintitio et al., 2007; Islam et al., 2010; Silva et al., 2012; Arcos-Ortega et al., 2019). Ravi et al. (2011) observed follicle cells surrounding the previtellogenic and late vitellogenic oocytes in P. pelagicus, whereas Sharifian et al. (2015) noticed follicle cells surrounding the late vitellogenic oocytes in S. iranica. Disintegration of follicle cells encircling the ripe oocytes (late vitellogenic stage III) was reported in O. senex senex (see Swetha et al., 2015). In contrast, follicle cells were not found in any of the oocyte stages in Po. Dehaani (see Ando and Makioka, 1999). Varadarajan and Subramoniam (1980) have shown that the follicle cells around the oocytes facilitated the entry of lipoproteins into the oocytes. Similarly, many authors reported the involvement of crustacean follicle cells in ovarian yolk production (Kodama et al., 2004; Islam et al., 2010; Silva et al., 2012; Sharifian et al., 2015). The presence of follicle cells around the early vitellogenic oocytes of A. cochleariformis also possibly indicates the transport of yolk proteins into the oocytes, and their absence or disintegration in the late vitellogenic (mature) oocytes indicates the completion of yolk deposition.

The presence of a few atretic oocytes along with Immature oocytes In the ovarian stroma during the proliferation phase (after spawning) in A. cochleariformis was supported by the findings of Minagawa et al. (1993), Keunecke et al. (2009), Silva et al. (2012), Swetha et al. (2015), Smija and Sudha Devi (2015), Sun et al. (2018) and Arcos-Ortega et al. (2019) in R. ranina, C. danae, C. ornatus, S. pictus, O. senex senex, T. schirnerae, S. honanense, and L. santolla, respectively. In contrast, atretic oocytes were not found in the spent stage of ovarian development in P. koolooense (see Joshi and Khanna, 1982) and U. rapax (see Castiglioni et al., 2007). Atretic oocytes are the leftover senescent oocytes observed in the ovary after spawning, which gradually degenerate in the succeeding phase. Keunecke et al. (2009) and Sun et al. (2018) reported that after spawning, empty spaces were observed in the ovarian stroma, along with a considerable amount of atretic oocytes. All these observations indicate that the presence of a few atretic oocytes in the ovary after spawning is common among brachyurans.

As reported for heterotremes such as Ca. sapidus (see Johnson, 1980), Ch. Opilio (see Beninger et al., 1988), I. ranulate (see Diesel, 1989), M. edwardsii (see Pardo et al., 2013) and Medorippe lanata (Linnaeus, 1767) (Vehof et al., 2017), and thoracotremes such as N. ranulate (see Lopez-Greco et al., 1999), Ucides cordatus (Linnaeus, 1763) (Sant’Anna et al., 2007) and C. guanhumi (see de Souza et al., 2011), the SR in A. cochleariformis was observed as a single chamber with dorsal and ventral regions. On the contrary, the existence of a velum or cuticular fold separating the SR into two morphological and functional units (dorsal sperm storage chamber and ventral insemination chamber) has been recorded in many heterotremes (Beninger et al., 1988; Diesel, 1989; Sainte-Marie and Sainte-Marie, 1998; Gonzalez-Pisani et al., 2012; Antunes et al., 2016; Pardo et al., 2017) and a few thoracotremes (de Souza et al., 2016; Vehof et al., 2017). All the above indicate that the histoarchitecture of the SR is not constant in the majority of the heterotremes and the thoracotremes studied so far.

Unlike the situation found in the majority of heterotremes (Sainte-Marie and Sainte-Marie, 1998; Sal Moyano et al., 2010; Zara et al., 2014; Antunes et al., 2016; de Souza et al., 2016; Assugeni et al., 2021) and some thoracotremes (Castilho-Westphal et al., 2013; de Souza et al., 2016; Vehof et al., 2016), where stratified epithelium lines the dorsal SR, the dorsal and ventral regions of the SR in A. cochleariformis are lined with a mono-layered columnar epithelium showing secretory activity. In support of this, many authors reported the presence of a mono-layered columnar epithelium in the SR of thoracotremes such as U. cordatus (see Sant’Anna et al., 2007), C. guanhumi (see de Souza et al., 2011) and Pe. Gibbesi (see Kienbaum et al., 2018). According to McLay and Becker (2015), the type of epithelium lining the SR was found to be varied from squamous, cylindrical, cuboidal to elongated in both heterotremes and thoracotremes. They also suggested that the stratified epithelium was the first lining of the SR, originating from the oviduct and the ovary, while the mono-layered epithelium is an apomorphy in thoracotremes that has replaced the stratified glandular epithelium (McLay and Becker, 2015). It appears to be the same case in A. cochleariformis, where the stratified epithelium (found in the majority of heterotremes) is replaced by the mono-layered columnar epithelium.

Our study revealed a merocrine mode for the release of secretion by the columnar epithelium in the dorsal and ventral regions of the SR. A similar feature was noted in the SR dorsal epithelium of Ch. Opilio (Sainte-Marie and Sainte-Marie, 1998). Alternatively, several authors documented the holocrine nature of the SR dorsal epithelium in heterotremes belonging to the families: Portunidae, Carcinidae, Cancridae (Spalding, 1942; Zara et al., 2014; Assugeni et al., 2021), Majidae (Hartnoll, 1968; Beninger et al., 1988; Sal Moyano et al., 2010; Gonzalez-Pisani et al., 2012) and Leucosiidae (Hayer et al., 2015), and in thoracotremes belonging to the family Cryptochiridae (Vehof et al., 2016). An apocrine mode for the release of secretion was reported in some thoracotremes (Pinnotheridae) (Becker et al., 2011) and heterotremes (Dorippidae) (Hayer et al., 2016). It is therefore understood that the mode of release of secretion of the SR epithelium is highly varied among heterotremes and thoracotremes.

The SR In A. cochleariformis was enveloped by a loose connective tissue layer formed of collagen fibers, similar to that described for C. guanhumi by de Souza et al. (2011). Collagen fibers were also noted in the SR of Ca. sapidus (see Johnson, 1980), U. cordatus (see Sant’Anna et al., 2007), Ce. Chilensis (see Pardo et al., 2017), and Ar. Cribrarius (see Zara et al., 2014). Beninger et al. (1988) also observed elastic fibers in the SR of Ch. Opilio. The SR was additionally lined externally by considerable amounts of muscle fibers in L. tuberculosus, Li. Spinosa (see Sal Moyano et al., 2010; Gonzalez-Pisani et al., 2012), Ocypode quadrata (Fabricius, 1787) (Lopez-Greco et al., 2009), Danielethus crenulatus (A. Milne-Edwards, 1879) (Farias et al., 2017), Ce. Chilensis (see Pardo et al., 2017), and Pe. Gibbesi (see Kienbaum et al., 2018). It is possible that the presence of a connective tissue layer in the SR of A. cochleariformis gives more flexibility to the SR and helps to maintain the shape.

Our study confirmed the presence of two types of secretions in the SR lumen: eosinophilic secretion produced by the columnar epithelial cells of the SR (intrinsic secretion) and the basophilic (extrinsic) secretion (produced by the male-the seminal plasma) in which the spermatophores were seen embedded in and transferred along with to the female SR during copulation. The presence of two types of secretions is recorded in the SR of almost all the heterotremes investigated so far: St. seticornis (see Antunes et al., 2016), L. tuberculosus, Li. Spinosa (see Gonzalez-Pisani et al., 2012), Ar. Cribrarius (see Zara et al., 2014), and the thoracotreme Pe. Gibbesi (see Kienbaum et al., 2018). In contrast, the SR of thoracotremes like C. guanhumi (see de Souza et al., 2011) and U. cordatus (see Sant’Anna et al., 2007) contained only a single type of secretion, i.e., the eosinophilic secretion without seminal plasma. Three types of secretions (acidophilic, weakly, and strongly basophilic) were identified in the SR lumen of the heterotreme Ca. danae (see Assugeni et al., 2021). Previous studies have pointed out that the secretions in the SR were composed of proteins and lipids (Anilkumar and Adiyodi, 1977; Jeyalectumie and Subramoniam, 1987). In C. guanhumi, de Souza et al. (2016) detected polysaccharides and proteins as the main components of the SR secretions. In Ar. Cribrarius (see Zara et al., 2014) and Ca. danae (see Assugeni et al., 2021), the eosinophilic secretion contained protein and neutral polysaccharides, while the basophilic secretion contained glycoprotein and acid polysaccharides. Seminal receptacle secretions plays important multiple roles in spermatophore dehiscence and spermatozoa maintenance (Diesel, 1989; Sal Moyano et al., 2010), killing microbes (Jensen et al., 1996; Zara et al., 2014; Antunes et al., 2016) and elimination of dead spermatozoa from the SR lumen (Sal Moyano et al., 2010). It is assumed that the eosinophilic and basophilic secretions in the SR of A. cochleariformis may have similar composition and functions as mentioned above.

As reported in Ch. Opilio (see Beninger et al., 1988), N. ranulate (see Lopez-Greco et al., 1999) and L. spinosa (see Sal Moyano et al., 2010; Gonzalez-Pisani et al., 2012), this study noticed partial dehiscence of spermatophores in the SR during the PRE phase. These results are in contradiction with the observations in some majoids (Sainte-Marie and Sainte-Marie, 1998), where intact spermatophores were stored for more than a year in the SR. No spermatophores were found in the SR of Macrophthalmus hirtipes (currently Hemiplax hirtipes (Hombron and Jacquinot, 1846)) (Jennings et al., 2000) and C. guanhumi (see de Souza et al., 2011). The presence of spermatophores with intact walls during the PRE phase of the present investigation indicates recent insemination and the presence of spermatophores with partially dehisced walls indicates the occurrence of earlier insemination.

The current study observed histo-morphological changes in the wall and luminal contents of the SR in accord with the annual mating event and ovarian cycle. Before mating (in the PRO phase), the SR appeared small and collapsed, enclosing small amounts of secretions, while it appeared swollen containing large amounts of spermatophores, free spermatozoa, and type II secretion (seminal fluid) after mating (in the PRE phase). The SR had a withered appearance in the PV, SV, and TV stages, and with decreased amounts of seminal plasma and few freely dispersed spermatozoa concentrated in the ventral region during the TV stage. Several authors reported changes in the SR appearance correlated with the stage of development of the ovary and sperm load (Sainte-Marie and Sainte-Marie, 1998; Pardo et al., 2013; Zara et al., 2014). Parallel observations were made in heterotremes like Ar. Cribrarius (see Zara et al., 2014) and Ca. danae (see Assugeni et al., 2021). In these works, the SR had a shrunken appearance, filled only with luminal secretion during the juvenile stage (pre-copulatory period); became enlarged and packed with a sperm plug containing large amounts of seminal fluid and spermatophores in the rudimentary stage (post-copulatory period), and finally flaccid with a decrease in the amount of seminal fluid with the advancement of ovarian maturation. The SR in recently mated M. edwardsii contained large amounts of seminal fluid and spermatozoa packed within spermatophores, while the SR of mature ovaries showed a reduction in the seminal contents (Pardo et al., 2013). Further, in Paratelphusa hydrodromous (currently Spiralothelphusa hydrodromous (Herbst, 1749)) (Anilkumar and Adiyodi, 1977), high and low secretory activity of the SR epithelium was recorded during early and late stages of vitellogenesis, respectively. In contrast, in U. cordatus (see Sant’Anna et al., 2007), the columnar epithelium of the SR continued to produce secretion independent of the stage of development of the ovary. From the above observations, it is concluded that the annual mating and ovarian development related histo-morphological changes in the SR of A. cochleariformis agree with the general pattern found in heterotremes.

In A. cochleariformis, large masses of spermatozoa were found displaced towards the ventral region of the SR in the TV (fully mature) stage, in agreement with the heterotreme families, Portunidae and Carcinidae: Carcinus maenas (Linnaeus, 1758) (Spalding, 1942), Ar. Cribrarius (see Zara et al., 2014) and Ca. danae (see Assugeni et al., 2021). Sant’Anna et al. (2007) suggested that the secretions produced by the dorsal epithelium of the SR in U. cordatus contributed to the movement of the spermatozoa to the fertilization spot. The large amounts of eosinophilic secretion (intrinsic secretion) produced during the VIT phase (PV, SV and TV stages) in the current study seemed to be responsible for the displacement of sperm masses towards the ventral region of the SR in the TV stage in order to fertilize the oocytes entering the ventral fertilization zone.

Hartnoll (1968) classified the eubrachyuran vaginae into simple (round in transverse section) and concave types (concave in transverse section), based on the appearance of the lumen. The vaginae of A. cochleariformis present as simple, following the pattern reported for the majority of the heterotremes (Johnson, 1980; Jensen et al., 1996; Sainte-Marie and Sainte-Marie, 1998; Pardo et al., 2013; 2017). In contrast, the vaginae are of the concave pattern in some heterotreme families: Majoidae (Hartnoll, 1968; Diesel, 1989), Leucosiidae (Hayer et al., 2015), Pilumnidae (Hartnoll, 1968), Gecarcinidae (de Souza et al., 2011), Hymenosomatidae (Klaus et al., 2014) and Dorippidae (Vehof et al., 2017), and all thoracotremes studied (Becker et al., 2011; de Souza et al., 2016; Vehof et al., 2016; Kienbaum et al., 2018). Ewers-Saucedo et al. (2016) reported an intermediate type vagina in calappid heterotremes. According to Hartnoll (1968), the simple type of vagina was the more primitive form and the concave one evolved from the simple type. Hartnoll reported that the simple type vagina is found in heterotremes with soft-shelled mating females, where the newly calcified cuticle closes the vulva and therefore mating is only possible shortly after moulting (when the females are in a soft shelled condition), and is in agreement with the findings in the present species.

In conclusion, the morphology of the female reproductive system of A. cochleariformis followed the general pattern observed for Eubrachyura. The SR of A. cochleariformis exhibits a novel combination of both heterotreme and thoracotreme patterns. Since this crab currently belongs to the Heterotremata, the majority of the histo-morphological features of the female reproductive system followed the common heterotreme pattern-such as the coxal male and sternal female position of the gonopore, the ventral connection of the oviduct with the SR and the simple type vagina. The mono-layered columnar secretory epithelium lining both the dorsal and ventral regions of SR in this species (in contradiction with the dorsal stratified and the ventral columnar epithelium normally found in heterotremes) follows the thoracotreme pattern. From observations of the female reproductive system of eubrachyurans, including the present study, it is understood that among eubrachyurans, heterotremes display varying patterns, while it is rather more uniform in thoracotremes. The discrepancies found in the SR and the female reproductive system among heterotremes, including the current species, may reflect their paraphyletic or polyphyletic origin, in contrast to the monophyletic origin of thoracotremes. Observations in the present study also reveal histo-morphological changes in the SR and vagina associated with the annual mating event and ovarian growth. Further fine structural studies are essential to know more about the secretory activity of the SR epithelium and vagina in relation to ovarian growth.

ACKNOWLEDGEMENT

We wish to thank Prof F.J. Zara (Departamento de Biologia Aplicada, Faculdade de Ciências Agrárias e Veterinárias, Jaboticabal, Brazil) for helpful conversations on seminal receptacles and for interpretation of figures.

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• Consent for publication
  The authors declare that they have reviewed the content of the manuscript and gave their consent to submit the document.
• Data availability
  All study data are included in the article.
• Funding and grant disclosures
  The financial support provided by the University Grants Commission in the form of Junior Research Fellowship for the first author (order No. 20/12/2015(ii) EU-V dated 25 October 2016) is thankfully acknowledged.
• Study association
  Not applicable.
• Study permits
  Not applicable.
• ZOOBANK:
  http://zoobank.org/urn:lsid:zoobank.org:pub:8A96E773-DD7B-4CF4-9006-FF5806B9EE9C

Edited by

• Associate Editor:
  Christopher Tudge

• Editor-in-chief:
  Christopher Tudge

Data availability

All study data are included in the article.

Publication Dates

• Publication in this collection
  07 Apr 2025
• Date of issue
  2025

History

• Received
  17 Mar 2024
• Accepted
  13 June 2024