Effect of 20-OH ecdysone and methyl farnesoate on histomorphology of the Y-organ during late intermoult and postmoult stages in the freshwater crab Travancoriana schirnerae Bott, 1969 (Crustacea: Gecarcinucidae)

Abstract

This study investigated the effects of 20-OH ecdysone (20E) and methyl farnesoate (MF) administration on histomorphology of the Y-organ (YO) during late intermoult (C3 and C4) and early postmoult stages in the edible freshwater crab Travancoriana schirnerae Bott, 1969, widely distributed in the wetlands of Wayanad, Kerala, India. Histomorphological analyses of the 20E and MF administered crabs revealed that both 20E and MF were effective in inducing significant changes in YO during the late intermoult (P < 0.001) and early postmoult (P < 0.05) stages, as evidenced from a significant rise in the YO index (YO index = wet weight of YO/body weight of crab × 100), size of the gland and lobules, height of the lobular epithelium, cellular hypertrophy, presence of secretory vesicles and abundance of hemocytes. The results also indicated that the effects were more pronounced during the late intermoult stages than the postmoult stage and in the 20E administered crabs than the MF administered individuals. The percent increments in YO index, length and width of the gland and lobules and thickness of the lobular epithelium in 20E and MF injected crabs during the late intermoult stages were 25.95, 20.04, 27.22, 52.15, 67.24, 114.50 % and 16.19, 9.09, 14.45, 21.87, 23.97, 65.25 %, respectively while those of early postmoult were 13.63, 24.04, 25.73, 23.86, 26.13, 38.01 % and 9.09, 15.27, 19.85, 14.95, 19.65, 26.54 %, respectively. In conclusion, 20E and MF administration provide an excellent option for stimulation of YO, thereby inducing ecdysis and growth during the inactive stages (intermoult and postmoult) of the moult cycle.

Keywords
20-OH ecdysone; histomorphology; intermoult; methyl farnesoate; postmoult

INTRODUCTION

The Y-organs (YOs) are paired glands of ectodermal origin, located in the cephalothorax and are analogous to the prothoracic glands of insects in location, external form and function (Chang et al., 1993; Lachaise et al., 1993; Covi et al., 2012; Techa, 2014). This endocrine gland synthesizes and secretes ecdysteroids, which play a crucial role in moulting and growth of crustaceans (Huberman, 2000; Mykles, 2011; Jayasankar et al., 2020). Y-organ removal studies in isopods, amphipods and penaeids have confirmed the moult regulating function of YO (Maissiat, 1970; Burghause, 1975; Bourguet et al., 1977). Generally, in crustaceans, the circulating levels of ecdysteroids remained low during intermoult, reached its peak during early premoult and dropped down during late premoult, prior to ecdysis (Chang and Bruce, 1980; Synder and Chang, 1991; Hopkins, 2009; Shyamal et al., 2014; Mykles and Chang, 2020). The YOs are negatively controlled by an inhibitory neuropeptide, the moult inhibiting hormone (MIH), synthesized and secreted by the X organ-sinus gland (XO-SG) complex of the eyestalk (Nakatsuji et al., 2009; Hopkins, 2012; Techa, 2014; Webster, 2015; Mykles and Chang, 2020). The sensitivity of the YO to MIH varies during different stages of the moult cycle, i.e., highly sensitive during intermoult and postmoult and least sensitive during premoult (Covi et al., 2012).

The structure and activity of the YO have been extensively studied in marine brachyuran and natantian decapods. In Penaeus indicus H. Milne Edwards, 1837, the size and tinctorial affinity of the YO cells varied significantly during the moult cycle (Vijayan et al., 1993). The YO cells of premoult Palaemon paucidens de Haan, 1844 and Astacus astacus (Linnaeus, 1758) showed an increase in the amount of cytoplasm, size of the mitochondria and number of cristae and vesicular smooth endoplasmic reticulum (SER) (Aoto et al., 1974; Birkenbeil and Gersch, 1979). Ultrastructural studies of Bressac (1973) evidenced moult related cyclic changes in the YO of Pachygrapsus marmoratus (Fabricius, 1787). The number of mitochondria, vesicles and electron-dense particles were found increased in the premoult YO of Cancer antennarius Stimpson, 1856 (Hinsch et al., 1980). The intermoult YOs of Portunus sanguinolentus (Herbst, 1783) showed profusely branched lobules with pronounced inter-lobular spaces, indistinct blood sinuses and capillaries while the postmoult YO cells exhibited a degeneration and decrease in the cytoplasmic volume (Babu et al., 1989). Fine structural observations of Carcinus maenas (Linnaeus, 1758) and Hemigrapsus nudus (Dana, 1851) YOs during the moult cycle revealed that the number of SER varied in accordance with the secretion of steroids (Buchholz and Adelung, 1980). Shyamal et al. (2014) reported that the secretory activity of the YO peaked during premoult in Metopograpsus messor (Forskål, 1775). Though many studies detailed the moult and reproductive cycle related changes in histology and/or ultrastructural profile of YO in marine and freshwater decapods, so far, no attempts were made to study the effects of the steroid hormone 20-OH ecdysone (20E) and the sesquiterpenoid methyl farnesoate (MF) on histology/structure of the YO in freshwater crabs.

Exogenous administration of ecdysteroids stimulated the YO in a number of decapods (Rao et al., 1972; Stevenson and Tschantz, 1973; Gilgan and Farquarson, 1977; Jegla and Costlow, 1978; Rao, 1978; Skinner 1985; Songsangjinda and Sumeth, 1988; Sripirom et al., 1991). Gunamalai et al. (2004) noticed a significant reduction in the moult cycle duration of the mole crab Emerita asiatica (H. Milne Edwards, 1837) injected with 20E. The YO of the portunid crab Portunus trituberculatus (Miers, 1876) administered with ecdysterone during intermoult displayed copious free ribosomes, well developed SER, Golgi complexes and electron dense granules (Taketomi and Hyodo, 1986). Repeated injections of 20E led to a significant rise in ecdysteroids and successful induction of ecdysis in Cherax quadricarinatus (Von Martens, 1868) (Shechter et al., 2007). On the other hand, Dell et al. (1999) reported significant reduction in ecdysteroid synthesis following 20E as well as RH-5849 (a non-steroidal ecdysteroid agonist) injection into Orconectes limosus (Rafinesque, 1817) during intermoult. Though numerous studies investigated the moult related histology and/or ultrastructural profile of YO in marine crabs, very few studies detailed the effects of 20E administration on YO histology and/or moulting in freshwater crabs.

Methyl farnesoate, structurally similar to the insect juvenile hormone III (JH III) and secreted by the decapod mandibular organ (MO) (Laufer et al., 1987; Tobe et al., 1989; Qu et al., 2018), has been known to play important roles in regulating moult and reproduction (Borst et al., 1987; Reddy et al., 2004; Nagaraju et al., 2006; Xie et al., 2015; Sudha Devi and Aswani, 2018; Qu et al., 2018). Abundant literature regarding the stimulatory role of MF on moulting and ecdysteroidogenesis (Tamone and Chang, 1993; Wang et al., 2009; Covi et al., 2012; Jayasankar et al., 2020) is available. The moult cycle duration was significantly shortened in the shrimp Penaeus setiferus (Linnaeus, 1767) by implantation of MO from Callinectes sapidus (Rathbun, 1896) (Yudin et al., 1980). Injection of Procambarus clarkii (Girard, 1852) MO extract induced ecdysis in the shrimp Caridina denticulata (De Haan, 1844) (Taketomi et al., 1989). In vitro incubation of the YO with MF resulted in the secretion of significantly high amounts of ecdysteroids in Cancer magister Dana, 1852 (Tamone and Chang, 1993). Ecdysis was accelerated in crayfishes: C. quadricarinatus (see Abdu et al., 2001) and P. clarkii (see Laufer et al., 2005) and the crab Oziothelphusa senex senex (Fabricius, 1798) (Reddy et al., 2004) following MF administration. Introduction of MF stimulated moulting, growth and ovarian maturation in the shrimp Litopenaeus vannamei (Boone, 1931) (Alnawafleh et al., 2014). Tahya et al. (2016) observed increased moulting percentages, moulting simultaneity, growth acceleration and adaptability following injection of MO extract into Scylla olivacea (Herbst, 1796) during intermoult. Dietary supplementation of MF induced moulting in female O. senex senex (see Reddy, 2019). Though voluminous literature is available on the role of MF on crustacean moulting/ecdysteroidogenesis, very few described the effect of MF administration on YO histology/ultrastructure in freshwater crabs.

Travancoriana schirnerae Bott, 1969 is an edible freshwater crab commonly distributed in the wetlands of Wayanad, Kerala, India. Previous studies on ultrastructure of the premoult YO of this species have shown numerous polymorphic mitochondria with tubular cristae, highly anastomosed tubules and vesicles of SER and abundant free ribosomes (Sudha Devi et al., 2015). Further studies on moult cycle-related and destalked crab YO histology in the same species revealed cellular hypertrophy, cytoplasmic granulations and secretory vesicles (Smija and Sudha Devi, 2016). Recently, in the same species, Sudha Devi and Aswani (2019) observed significantly shortened intermoult and premoult intervals and total moult cycle duration and increased incidence of ecdysis following 20E and MF administration during intermoult and early premoult. The current investigation on the effect of administration of 20E and MF on histomorphology of the YO during the late intermoult and early postmoult stages of the moult cycle in T. schirnerae may help to understand the role of YO in the regulation of moulting and growth in freshwater brachyurans.

MATERIAL AND METHODS

Collection and maintenance of animals

Every month, adult male and female crabs in various moult stages (carapace width 4.5-5.0 cm and body weight 35-40 g) were collected over a period of one year (January -December 2019), from the areca plantations of Ondayangady, Wayanad (11°49’N 76°01’E). The collected specimens were transported live to the laboratory and kept in well-aerated circular plastic basins (diameter: 46 cm, depth: 20 cm) (5 individuals per basin) containing freshwater collected from their natural habitat. The pH, turbidity, total dissolved solids and total hardness of water were 7.2, 2.7 NTU, 15.2 mg/L, 14.5 mg/L as CaCO₃ equivalents, respectively. Cooked beef liver, sprouted green gram, boiled egg and decaying aquatic vegetation were given once a day as feed during the period of acclimatization (3-4 days) and experiment. The water was renewed regularly in the morning; the unconsumed food and faeces were removed 2 hrs after feeding to avoid contamination. The temperature (25.0 ± 2.0°C) and photoperiod conditions (12L:12D) were maintained during the course of the experiment.

Preparation of 20-OH ecdysone and trans, trans methyl farnesoate

The stock solutions were prepared by dissolving 1 mg 20E (Sigma Chemicals, USA) and 1 mL trans, trans MF (Echelon Biosciences, Salt Lake City, UT, USA) separately in 1 mL each of absolute alcohol and stored at -20 ^oC. 0.05 µL (50 ng) each of the stock solutions were dissolved in 9.95 µL each of crustacean physiological saline (6.5 g NaCl, 0.42 g KCl, 0.25 g CaCl₂ and 0.2 g NaHCO₃ dissolved in 100 mL distilled water) to get the required concentrations for injection. The test solutions required for the injections were prepared just before administration.

Determination of stages of moult cycle

The transparent edges of the third maxilliped epipodite of male and pleopod endopodite of female were removed and mounted on clean glass slides with a drop of saline and observed under a compound microscope (Olympus, Medical SM 100). The degree of cuticle development, epidermal retraction and setal formation were noted. Morphological characters such as color, texture and rigidity of the exoskeleton were also recorded to determine the moult cycle stages (Nagaraju et al., 2004; Hosamani et al., 2016; Sudha Devi and Aswani, 2019).

Experimental Design

The individuals in C3, C4 and early postmoult stages were divided into 3 groups of 10 each. Group I, which received 10 μL of crustacean physiological saline formed the controls. Group II and III received 50 ng each of 20E and MF in a total volume of 10 μL/injection, respectively on days 1, 7, 14, and 21, through the arthrodial membrane of the coxa of the 3^rd walking leg, and formed the experimental groups. The crabs were identified with waterproof markings on their carapace. The control and experimental animals were sacrificed on 30^th day of the experiment. The carapace width, body weights and moult stages of these animals were recorded at the beginning and end of the experiment, just before dissection.

Histology

The YOs were dissected out from control and experimental crabs under a trinocular stereo zoom dissection microscope (Radical, RSM-9). The dissected tissues were immediately placed in saline, blotted with paper towels after removing from saline and weighed wet on a Shimadzu electronic top loading weighing balance (0.001 g) to calculate the YO index. The size (length and width) of the YO was recorded by placing a measuring scale under the dissection microscope. The YO index was calculated by the equation:

$$\mathrm{Y}\mathrm{O}\mathrm{}\mathrm{i}\mathrm{n}\mathrm{d}\mathrm{e}\mathrm{x}=(\mathrm{w}\mathrm{e}\mathrm{t}\mathrm{}\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{}\mathrm{o}\mathrm{f}\mathrm{}\mathrm{Y}\mathrm{O}/\mathrm{b}\mathrm{o}\mathrm{d}\mathrm{y}\mathrm{}\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{}\mathrm{o}\mathrm{f}\mathrm{}\mathrm{c}\mathrm{r}\mathrm{a}\mathrm{b})\times 100$$

The tissues were fixed in Bouin’s fixative; after 24 hrs of fixation, the tissues were washed, dehydrated in a graded series of ethanol (30 %, 50 %, 70 %, 90 % and absolute), cleared in xylene and embedded in molten paraffin wax (54-56 °C). Sections, 5 µ thickness, were cut (MicroTec CUT4050 rotary microtome, Germany), stained with haematoxylin-eosin and observed under a Nikon ECLIPSE Ni-U Research Microscope. Photomicrographs of the stained sections and measurements of the lobules, lobular epithelium, cells, nuclei and nucleoli were made with a Nikon Y-TV55 camera and Nikon NIS Elements Imaging Software attached to the Nikon ECLIPSE Ni-U Research Microscope.

Statistical analysis

To evaluate the effect of 20E and MF on YO index, size (length and width) of the gland and lobules and thickness of the lobular epithelium, the mean values were compared through one-way analysis of variance (ANOVA) followed by a Tukey post-hoc multiple comparison test using IBM SPSS statistics version 24.0. A probability value of < 0.05 was considered significant statistically. Before applying the ANOVA test, the data were subjected to Kolmogorov-Smirnov and Levene test to check the normality and the homogeneity of variances.

RESULTS

The present study assessed the impact of 20E and MF administration on morphology and histology of the YO during late intermoult (C3 and C4) and early postmoult stages of the moult cycle in the freshwater crab T. schirnerae. The effects of administration were evaluated by comparing the YO index, size of the gland and lobules and thickness of the lobular epithelium of experimental individuals with those of the control individuals.

Effect of 20E and MF administration during C3 stage

The control crabs (100 %) remained in the same moult stage till the end of the experiment. The pale yellow, YO gland measured 3.04±0.30 mm in length and 2.32 ± 0.34 mm in width with a YO index of 0.023 ± 0.006. The YO gland consisted of branched lobules (length 127.14 ± 28.72 µm; width 49.07 ± 5.68 µm) surrounded by a thin monolayered lobular epithelium (9.93 ± 2.25 µm) with prominent inter-lobular spaces. The cytoplasm of the lobular epithelial cells was mildly basophilic with round or oval basophilic nuclei. The homogenous nucleoplasm was encircled by a smooth and distinct nuclear membrane and contained peripherally arranged granular chromatin and 2-5 nucleoli. The lobules contained both smaller (10-15) and larger (23-30) cells. The smaller cells (8.75 ± 0.68 µm), arranged in small patches, have large, oval multi-nucleolated nuclei (7.38 ± 0.60 µm) with granular chromatin and mildly basophilic cytoplasm. The bulk of the lobules were occupied by polygonal larger cells (14.13 ± 2.31 µm) with transparent and homogenous cytoplasm. Their centrally or eccentrically positioned oval to round nuclei (5.83 ± 1.38 µm) possessed peripherally condensed chromatin. A few larger cells (5-10 cells) presented a vacuolated appearance with broken limiting membranes. Blood sinuses and capillaries were not distinct. Very few hemocytes were detected in the inter-lobular hemal spaces (Tab. 1; Figs. 1A, B; 2A-C).

Table 1.
Effect of 20E and MF administration on Y-organ (YO) index, gland and lobule size and height of lobular epithelium during late intermoult and early postmoult stages in Travancoriana schirnerae.

Moult stage	Group	YO index	Gland size		Lobule size		Height of lobular epithelium (µm)
			Length (mm)	Width (mm)	Length (µm)	Width (µm)
Late intermoult (C3 stage)	Control	0.023±0.006	3.04±0.30	2.32±0.34	127.14±28.72	49.07±5.68	9.93±2.25
	20E treated	0.029±0.004*** df = 17 F = 15.877	3.58±0.34*** df = 35 F = 24.667	3.04±0.48*** df = 35 F = 27.846	196.25±16.78*** df = 35 F =72.540	86.25±20.71 *** df = 35 F = 59.258	16.93±2.07*** df = 35 F = 92.389
	MF treated	0.026±0.003* df = 18 F = 4.504	3.24±0.29* df = 37 F = 4.240	2.68 ±0.36** df = 37 F = 10.149	155.33±22.86** df = 37 F = 11.035	59.33±10.60*** df = 37 F = 14.221	12.78±2.25*** df = 37 F = 13.982
Late intermoult (C4 stage)	Control	0.031±0.007	3.36±0.29	2.99±0.37	169.81±27.13	69.87±19.28	40.01±22.27
	20E treated	0.039±0.005*** df = 17 F = 15.922	4.11±0.33*** df = 35 F = 52.818	3.69±0.64*** df = 35 F = 16.946	254.62±21.61*** df = 35 F = 103.562	110.89±11.09*** df = 35 F = 57.067	103.43±29.88*** df = 35 F = 53.256
	MF treated	0.037±0.002*** df = 19 F = 15.703	3.75±0.37*** df = 39 F = 13.691	3.39±0.32*** df = 39 F = 13.381	206.44±30.82*** df = 39 F = 15.913	88.76±6.87*** df = 39 F = 17.025	80.74±14.93*** df = 39 F = 50.789
Early postmoult	Control	0.022±0.004	2.62±0.46	1.36±0.27	111.07±24.26	46.91±15.11	7.76±2.85
	20E treated	0.025±0.004* df = 16 F = 6.180	3.25±0.24*** df = 33 F = 21.424	1.71±0.47* df = 33 F = 7.299	137.57±18.79** df = 33 F = 11.729	59.17±14.30* df = 33 F = 5.658	10.71±3.96* df = 33 F = 6.377
	MF treated	0.024±0.002* df = 17 F = 4.710	3.02±0.32** df = 35 F = 8.621	1.63±0.46* df = 35 F = 4.652	127.68±10.95* df = 35 F = 6.419	56.13±10.31* df = 35 F = 4.329	9.82±3.02* df = 35 F = 4.391

• Data presented as Mean±SD. Asterisks (*) indicate statistical significance for each 20E and MF treated groups related to its control by One-way ANOVA. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001

Figure 1.
Graph showing the effect of 20E and MF administration on YO index, gland and lobule size and height of the lobular epithelium during late intermoult and early postmoult stages in Travancoriana schirnerae. (A) Effect of 20E and MF administration on YO index and gland size. (B) Effect of 20E and MF administration on lobule size and height of lobular epithelium. GL: Gland length; GW: Gland width; LL: Lobule length; LW: Lobule width; LE: Thickness of the lobular epithelium.

Figure 2.
Light micrograph of control Y-organ during C3 stage in Travancoriana schirnerae. (A, B) Small loosely arranged lobules with prominent intra- and inter-lobular spaces. (C) Enlarged view of lobules showing monolayered lobular epithelium, gland cells and vacuoles. ILS: Inter-lobular space; INS: Intra-lobular space; L: Lobule; LC: Larger cell; LE: Lobular epithelium; N: Nucleus; SC: Smaller cell; V: Vacuole.

Eight out of the ten (80 %) crabs administered with 20E reached the next moult stage (C4 stage) at the end of the experiment, with notable changes in morphology and histology of the YO, while 20 % of the individuals remained in the same moult stage without obvious changes in morphology or histology. Histological assessment of the YO of 20E injected crabs revealed significantly increased YO index (0.029 ± 0.004), gland size (length 3.5 8± 0.34 mm; width 3.04 ± 0.48 mm) and the presence of closely arranged branched lobules with a noticeable increase in size (length 196.25 ± 16.78 µm; width 86.25 ± 20.71 µm) compared to the controls (Tab. 1). The multilayered epithelium displayed a considerable increase in thickness (16.93 ± 2.07 µm) than the controls. The percent increments in YO index, length and width of the gland and lobules and thickness of the lobular epithelium in 20E injected crabs were 26.09, 17.76, 31.03, 54.36, 75.77, and 70.49 %, respectively. Small irregular shaped cells without nuclei were seen compactly packed in the inter-lobular spaces (Fig. 3B). 20-OH ecdysone administration did not cause a considerable increase in size of the lobular cells, but effected a considerable increase in their number. The lobules were made up of both smaller (70-80 cells) and larger (40-50 cells) cells. A most important feature of 20E injected crabs of this stage was the proliferation of smaller cells from the lobular epithelium, seen as small patches of closely arranged cells within the lobules (Fig. 3C). The larger gland cells were compactly arranged with clear cellular demarcations. Some larger cells were perceived with centrally or peripherally placed single, large, highly basophilic, round to oval secretory vesicles (Fig. 3C). The cell boundaries of a few cells containing these secretory vesicles were found broken, indicating the release of their contents. The occurrence of fine capillaries and the abundance of hemocytes in the hemal sinuses were characteristic of 20E injected crabs of this stage. Hemocytes were also evident in the spaces between the lobular epithelium and the basal lamina (Tab. 1; Figs. 1A, B; 3A-D).

Figure 3.
Light micrograph of histology of Y-organ of crabs treated with 20E during C3 stage in Travancoriana schirnerae. (A) Closely packed branched lobules with reduced intra- and inter-lobular spaces. (B) Compactly packed, small irregular shaped cells without nuclei in the inter-lobular spaces. (C) Y-organ demonstrating patches of proliferated smaller cells, larger cells carrying secretory vesicles and fine capillaries. (D) Lobules surrounded by multilayered epithelium and inter-lobular hemal spaces with hemocytes. BC: Blood capillary; H: Hemocyte; ILS: Inter-lobular space; INS: Intra-lobular space; L: Lobule; LC: Larger cell; LE: Lobular epithelium; N: Nucleus; SC: Smaller cell; SV: Secretory vesicle, Arrow indicates small irregular shaped cells without nuclei.

Though the MF treated individuals (90 %) remained in the same moult stage till the end of the experiment, they exhibited some marked changes in histomorphology of the YO. Compared to the controls, a significant increase was noticed in the YO index (0.026 ± 0.003), gland size (length 3.24 ± 0.29 mm; width 2.68 ± 0.36 mm) and thickness of the lobular epithelium (12.78 ± 2.25 µm). The YO demonstrated large, branched lobules (length 155.33 ± 22.86 µm; width 59.33 ± 10.60 µm) with reduced inter-lobular spaces. The percent increments in YO index, length and width of the gland and lobules and thickness of the lobular epithelium in MF injected crabs were 13.04, 6.57, 15.52, 22.17, 20.91, and 28.70 %, respectively. Both smaller (40-45 cells) and larger (30-35 cells) cell types were evident in the lobules, arranged closely without inter-cellular spaces. The larger cells exhibited clear cell boundaries and cytoplasmic granularity with a few carrying highly basophilic, round to oval secretory vesicles. Vacuoles were rarely observed. Blood capillaries and sinuses were apparent in the inter-lobular spaces with a few hemocytes in the hemal sinuses (Tab. 1; Figs. 1A, B; 4A-C).

Figure 4.
Light micrograph of histology of Y-organ of crabs treated with MF during C3 stage in Travancoriana schirnerae. (A, B) Large, loosely arranged, branched lobules with reduced intra- and inter-lobular spaces at low and high magnification. (C) Enlarged view of lobules with compactly packed gland cells, secretory vesicles, vacuoles and hemocytes. H: Hemocyte; ILS: Inter-lobular space; INS: Intra-lobular space; L: Lobule; LC: Larger cell; LE: Lobular epithelium; N: Nucleus; SC: Smaller cell; SV: Secretory vesicle, V: Vacuole.

Effect of 20E and MF administration during C4 stage

The control group remained in the same moult stage (C4 stage) till the end of the experiment. Their YO had a pale brown appearance with an average length and width of 3.36 ± 0.29 and 2.99 ± 0.37 mm, respectively, and a YO index of 0.031 ± 0.007. The lobules (length 169.81 ± 27.13 µm; width 69.87 ± 19.28 µm) were closely arranged with reduced intra and inter-lobular spaces (Fig. 5B) and were surrounded by a monolayered epithelium (thickness 40.01 ± 22.27 µm). Their component cells enclosed intensely basophilic, oval to round nuclei with peripherally arranged condensed chromatin and mildly basophilic cytoplasm. The lobules enclosed both cell types. The smaller cells (10-20 cells) (7.95 ± 0.59 μm), arranged in patches, exhibited large, oval to spherical basophilic nuclei (5.13 ± 0.61 μm diameter) and narrow cytoplasm. The larger cells (30-40 cells; 13.08 ± 2.36 µm) with oval to round or spherical, highly basophilic, multi-nucleolated (2-5) nuclei (5.00 ± 1.24 μm diameter), dense peripheral chromatin and mildly basophilic cytoplasm, formed the dominant cell type of this stage. These cells, with their sharp boundaries, were closely packed without inter-cellular spaces. Interconnecting blood capillaries and sinuses with sparsely distributed hemocytes were evident in the YO of this stage (Tab. 1; Figs. 1A, B; 5A-C).

Figure 5.
Light micrograph of Y-organ of control crabs during C4 stage in Travancoriana schirnerae. (A) Closely packed lobules with reduced inter-lobular spaces. (B) Large, branched lobules with closely arranged gland cells. (C) Enlarged view of lobules with compactly packed larger cells. H: Hemocyte; ILS: Inter-lobular space; L: Lobule; LC: Larger cell; LE: Lobular epithelium; N: Nucleus.

By the end of the experimental period, eight out of the ten (80 %) 20E administered individuals attained the D2 stage of early premoult with substantial changes in YO morphology and histology, while one (10 %) seemed to continue in the same moult stage and one (10 %) died during the experimental period. Compared to the controls, a significant rise was seen in the YO index (0.039 ± 0.005) and size of the gland (length 4.11 ± 0.33 mm; width 3.69 ± 0.64 mm) in 20E injected crabs. The organ was composed of closely packed lobules with a noticeable increase in size (length 254.62 ± 21.61 µm; width 110.89 ± 11.09 µm), lacking inter-lobular spaces. The lobular epithelium was multilayered, attained a maximum height of 103.43 ± 29.88 µm, reducing the intra-lobular space (Fig. 6B). The percent increments in YO index, length and width of the gland and lobules and thickness of the lobular epithelium were 25.81, 22.32, 23.41, 49.94, 58.71, and 158.51 %, respectively. The number of smaller (100-200 cells) and larger (40-50 cells) cells increased further. Proliferation of smaller cells, appearing as large patches, was also evident in the lobules (Fig. 7C). The cytoplasm of larger cells appeared granular and mildly basophilic. Some larger cells enclosed large, deeply basophilic, oval to round secretory vesicles, characteristic of the YO of 20E injected crabs of this stage. A small number of these vesicles were found attached to the cell membrane, releasing their contents into the inter-lobular hemal sinus (Figs. 6C, 7B). The presence of fine capillaries and abundance of hemocytes in the inter-lobular hemal sinuses was another characteristic feature of 20E injected crabs of this stage (Tab. 1; Figs. 1A, B; 6A-C, 7A-C).

Figure 6.
Light micrograph illustrating Y-organ of crabs treated with 20E during C4 stage in Travancoriana schirnerae. (A) Compactly packed lobules without inter-lobular spaces. (B) Lobules surrounded by multilayered epithelium. (C) Presence of large number of secretory vesicles in the lobules. BS: Blood sinus; L: Lobule; LC: Larger cell; LE: Lobular epithelium; N: Nucleus; SV: Secretory vesicle.

Figure 7.
Light micrograph of Y-organ of crabs treated with 20E during C4 stage in Travancoriana schirnerae. (A) Abundance of hemocytes in the inter-lobular hemal spaces. (B) Secretory vesicles seen attached to the cell membrane showing the holocrine mode of release of secretion. (C) Proliferated smaller cells seen in large patches. BC: Blood capillary; H: Hemocyte; L: Lobule; LC: Larger cell; LE: Lobular epithelium; N: Nucleus; SC: Smaller cell; SV: Secretory vesicle; Arrow indicates the secretory vesicle attached to the lobular epithelium.

Though MF injection during C4 had no effect on the moult stage, it significantly increased the YO index (0.037 ± 0.002), size of the gland (length 3.75 ± 0.37; width 3.39 ± 0.32) and lobules (length 206.44 ± 30.82; width 88.76 ± 6.87). Histological observations of the YO demonstrated compactly packed large lobules, absence of intra and inter-lobular spaces and thick multilayered lobular epithelium (80.74 ± 14.93) (Figs. 8A, B). The percent increments in the YO index, length and width of the gland and lobules and thickness of the lobular epithelium in MF injected crabs were 19.35, 11.61, 13.38, 21.57, 27.03, and 101.80 %, respectively. The lobules were consistently packed with both smaller (60-100 cells) and larger (40-45 cells) cells. The larger cells enclosed secretory vesicles, which formed the most prominent feature in MF injected crabs of this stage (Fig. 8C). Cell proliferation was another remarkable feature of YO of this stage (Fig. 9B). Hemocytes were frequently encountered in the hemal spaces (Tab. 1; Figs. 1A, B; 8A-C, 9A, B).

Figure 8.
Light micrograph of histological sections of Y-organ of crabs treated with MF during C4 stage in Travancoriana schirnerae. (A) Compactly packed lobules without inter-lobular spaces. (B) Lobules with multilayered epithelium. (C) Larger cells enclosing secretory vesicles. L: Lobule; LC: Larger cell; LE: Lobular epithelium; SV: Secretory vesicle.

Figure 9.
Light micrograph of Y-organ of crabs treated with MF during C4 stage in Travancoriana schirnerae. (A) Y-organ demonstrating abundance of hemocytes and secretory vesicles. (B) Proliferated smaller cells seen in small patches. H: Hemocyte; L: Lobule; LC: Larger cell; LE: Lobular epithelium; N: Nucleus; SC: Smaller cell; SV: Secretory vesicle.

Effect of 20E and MF administration during early postmoult

All the control crabs reached the C1 stage of early intermoult by the end of the experiment. Their YO appeared small (length 2.62 ± 0.46 mm; width 1.36 ± 0.27 mm), pale yellow with a mean YO index of 0.022 ± 0.004. The lobules were small (length 111.07 ± 24.26 μm; width 46.91 ± 15.11 μm), few in number, and loosely arranged with prominent intra and inter-lobular spaces. The lobular epithelium appeared thin (7.76 ± 2.85 μm) and the epithelial cells possessed pycnotic nuclei and indistinct limiting membranes, showing signs of inactivity. The lobules comprised a lesser number of intact cells (8-10 larger cells; width 9.70 ± 1.17 μm), arranged loosely with large intra-lobular spaces. Most of the lobules had a degenerate appearance, characterized by the presence of inactive cells with incomplete limiting membranes, transparent or vacuolated cytoplasm and small deeply stained nuclei (3.81 ± 0.51 μm diameter). Hemal sinuses and capillaries were rarely detected in the inter-lobular spaces (Tab. 1; Figs. 1A, B; 10A, B).

Figure 10.
Light micrograph of histology of control Y-organ during early postmoult stage in Travancoriana schirnerae. (A) Y-organ displaying small, loosely arranged lobules and conspicuous intra- and inter-lobular spaces showing signs of inactivity. (B) Thin, discontinuous lobular epithelium with pycnotic nuclei. ILS: Inter-lobular space; INS: Intra-lobular space; L: Lobule; LE: Lobular epithelium; N: Pycnotic nuclei; V: Vacuoles.

Of the 10 experimental crabs injected with 20E during this stage, 7 (70 %) were able to reach the C2 stage, 2 (20 %) remained unchanged as that of the controls, while one (10 %) died during the experimental period. The YO of 20E treated crabs showed some signs of stimulation, compared to the controls. Both the YO index (0.025 ± 0.004) and gland size (length 3.25 ± 0.24 mm; width 1.71 ± 0.47 mm) increased significantly from those of the controls. Though the lobules were loosely arranged with prominent inter-lobular spaces, statistically significant differences were noted in their size (length 137.57 ± 18.79 μm; width 59.17 ± 14.30 μm) and thickness of the lobular epithelium (10.71 ± 3.96 µm), in comparison to the control crabs. The percent increments in YO index, length and width of the gland and lobules and thickness of the lobular epithelium in 20E injected crabs were 13.64, 24.04, 25.73, 23.86, 26.13, and 38.01 %, respectively. Despite the vacuolated appearance, the lobules accommodated 20-25 larger cells (width 9.70 ± 0.61 µm) with small, strongly basophilic nuclei (width 4.15 ± 0.61 µm), condensed chromatin and transparent cytoplasm. Hemocytes were occasionally spotted in the hemal spaces (Tab. 1; Figs. 1A, B; 11A-C).

Figure 11.
Light micrograph of Y-organ of crabs treated with 20E during early postmoult in Travancoriana schirnerae. (A) Medium sized branched lobules with a continuous lobular epithelium and reduced intra-lobular spaces. Note the prominent inter-lobular spaces. (B) Lobules compactly packed with cells. (C) Enlarged view of lobules with intact lobular epithelium and a few larger cells. ILS: Inter-lobular space; INS: Intra-lobular space; L: Lobule; LC: Larger cell; LE: Lobular epithelium; V: Vacuole; Arrow indicates cell degeneration.

Eight out of the ten (80 %) crabs administered with MF during postmoult attained the C2 stage of early intermoult with noticeable changes in histomorphology of the YO, which were less pronounced compared to those of the late intermoult stages. The remaining 20 % (2) animals appeared as that of the controls without any changes in morphology or histology. Methyl farnesoate administration caused a significant increase in gland size (length 3.02 ± 0.32 mm; width 1.63 ± 0.46 mm) and YO index (0.024 ± 0.002) value compared to the controls. The YO of MF injected crabs displayed loosely arranged, small sized lobules (length 127.68 ± 10.95 μm; width 56.13 ± 10.31 μm) with prominent intra- and inter-lobular spaces. Their lobular epithelium was distinct, composed of a single layer of highly basophilic cells (thickness 9.82 ± 3.02 μm) with pycnotic nuclei. The percent increments in YO index, length and width of the gland and lobules and thickness of the lobular epithelium in MF injected crabs were 9.09, 15.27, 19.85, 14.95, 19.65, and 26.55 %, respectively. The lobules contained very few intact larger cells (10-15 cells). Cell debris and vacuolations were often seen in the lobules. Interconnecting hemal sinuses and capillaries were absent in the YO of MF treated crabs (Tab. 1; Figs. 1A, B; 12A, B).

Figure 12.
Light micrograph of Y-organ of crabs treated with MF during early postmoult in Travancoriana schirnerae. (A) Medium sized, loosely arranged, branched lobules with intra and inter-lobular spaces and lobular epithelial cells with pycnotic nuclei. (B) Thin lobular epithelium and a few intact cells in the lobules. ILS: Inter-lobular space; INS: Intra-lobular space; L: Lobule; LC: Larger cell; LE: Lobular epithelium; N: Pycnotic nuclei; V: Vacuole.

DISCUSSION

The present investigation provided evidence for the stimulatory effect of 20E and MF on histomorphology of the YO during the late intermoult and postmoult stages in the freshwater crab T. schirnerae. The light microscopic observations of the YO of control crabs showed normal structure while 20E and MF administration during the late intermoult and postmoult stages demonstrated significant changes in morphology and histology of the gland, indicated by a significant increase in YO index, size of the gland and lobules and height of the lobular epithelium.

In this study, the crabs that received 20E during C3 and C4 stages of late intermoult reached the C4 and D2 stage of early premoult, respectively, by the end of the experimental period. Further histomorphological analyses of their YO revealed a significant increase in the YO index, size of the gland and lobules, thickness of the lobular epithelium, cellular hypertrophy, presence of secretory vesicles and abundance of hemocytes, which is a positive indication on the excitatory effect of 20E on YO. Earlier research in our laboratory proved that 20E administration during various stages of the intermoult and early premoult caused a significant reduction in the intermoult and premoult intervals and total moult cycle duration and increased incidence of ecdysis (Sudha Devi and Aswani, 2019). Copious free ribosomes, well developed SER, Golgi complexes and electron dense granules were discerned in YO cells of P. trituberculatus, following ecdysterone injection during intermoult (Taketomi and Hyodo, 1986). Similar results were obtained in prawns: Palaemon elegans Rathke, 1837 (Rao et al., 1972; Webster, 1983), Penaeus merguiensis de Man, 1888 (Songsangjinda and Sumeth, 1988), and Penaeus monodon (Fabricius, 1798) (Sripirom et al., 1991), crayfishes: Procambarus simulans (Faxon, 1884) (Lowe et al., 1968), Orconectes obscurus (Hagen, 1870) (Warner and Stevenson, 1972), Orconectes sanborni Faxon, 1884 (Stevenson and Tschantz, 1973) and C. quadricarinatus (see Shechter et al., 2007), lobsters: Homarus americanus H. Milne Edwards, 1837 (Gilgan and Farquarson, 1977) and Panulirus longipes (A. Milne Edwards, 1868) (Dall and Barclay, 1977), the horseshoe crab Limulus polyphemus (Linnaeus, 1758) (Jegla and Costlow, 1978) and the fiddler crab Leptuca pugilator (Bosc, 1802) (Rao, 1978), where 20E/ecdysone administration during intermoult stages significantly reduced the moult cycle duration. In adult male Palaemonetes kadiakensis (Rathbun, 1902), ecdysis was successfully induced by ecdysterone administration during intermoult (Hubschman and Armstrong, 1972). In E. asiatica, individuals that received 20E at C3 stage of intermoult hastened the premoult activities, thereby inducing precocious ecdysis (Gunamalai et al., 2004). On the other hand, in O. limosus, 20E as well as RH-5849 (a non-steroidal ecdysteroid agonist) injection during intermoult stage significantly reduced ecdysteroidogenesis by the YO (Dell et al., 1999). The histomorphological changes in the YO of crabs treated with 20E during the late intermoult stages in the current investigation possibly indicates the synthesis and release of ecdysteroids for the stimulation of moult. It is likely that the high circulating ecdysteroid titer plus the exogenously administered 20E might have superseded the inhibitory effects of the generally low MIH titer observed during C4/early premoult, which in turn stimulated the YO. In support of this, Styrishave et al. (2008) reported high circulating levels of ecdysteroids in C. maenas and Nakatsuji and Sonobe (2004) reported low MIH titers in P. clarkii during C4/early premoult. Another possible explanation is that the YO becomes least sensitive to the inhibitory effects of MIH during C4/early premoult, which is in agreement with the findings of Nakatsuji and Sonobe (2004) in P. clarkii, where the YO showed only 5 % receptiveness to MIH during early premoult. Chung and Webster (2003) observed that the inhibitory effect of MIH decreased by 25 % during early premoult in C. maenas.

Though the exogenously administered 20E during postmoult induced early intermoult (C2) with significant changes in morphology and histology of the YO, evidenced from the significantly increased YO index, size of the gland and lobules and thickness of the lobular epithelium, these changes were less pronounced (P < 0.05) when compared to the late intermoult stages (P < 0.001). Similar observations were made in P. elegans, where administration of higher doses of ecdysterone during postmoult reduced the moult cycle duration (Webster, 1983). The decrease in receptivity of the YO to exogenous 20E during postmoult, compared to the late intermoult stages in the present investigation suggests the significantly high MIH titer that surpasses the effects of the significantly low 20E titer (endogenous plus exogenous) during postmoult, which negatively affects the YO. In support of this, high MIH titers were found in the hemolymph of postmoult C. sapidus (see Lee et al., 1998; Techa, 2014) and in the sinus gland of late postmoult/early intermoult C. maenas (see Chung and Webster, 2003). In L. pugilator, the levels of circulating ecdysteroids remained low during postmoult and at the beginning of C4 and began to increase at the end of C4 (Hopkins, 1983; 1986). The low hemolymph ecdysteroid titer during postmoult was associated with the high MIH mRNA levels indicating the transcriptional regulation of MIH in repressing ecdysteroidogenesis by YO (Chen et al., 2007; Mykles, 2011; Covi et al., 2012; Mykles and Chang, 2020).

As seen from results of the present investigation, there was a significant increase in the YO index, size of the gland and lobules, height of the lobular epithelium, number of gland cells and the presence of secretory vesicles and hemocytes in crabs injected with MF during the late intermoult stages (C3 and C4), indicating the stimulatory effect MF on YO. A number of studies verified the excitatory effects of MF and farnesoic acid (FA) on stimulation of YO (Borst et al., 1987; Laufer et al., 1987; Diwan, 2005). Previous research in our laboratory on injection of multiple doses of MF into intermoult and early premoult crabs significantly shortened the intermoult and premoult intervals and the total moult cycle duration and increased the incidence of ecdysis (Sudha Devi and Aswani, 2019). Implantation of C. sapidus MO shortened the moult cycle duration in the shrimp P. setiferus (see Yudin et al., 1980). Co-incubation of C. magister YO with MO or MF resulted in the secretion of a considerable amount of ecdysteroids into the medium by the YO (Tamone and Chang, 1993). Additionally, ecdysis was induced following MF administration in C. quadricarinatus females (Abdu et al., 2001). In the freshwater crab O. senex senex, a large number of individuals reached the premoult stage on administration of MF during intermoult (Reddy et al., 2004). Injection of MO extract induced moulting in several decapod crustaceans including Charybdis lucifera (Fabricius, 1798) (Allayie et al., 2010) and P. clarkii (see Laufer et al., 2005). Increased moulting percentages, moulting simultaneity, growth acceleration and adaptability were observed by MF administration in intermoult S. olivacea (see Tahya et al., 2016). In O. senex senex, females administered with MF supplemented diet during C4 stage exhibited enhanced growth via moult induction (Reddy, 2019). The possible reason for the stimulatory effects on YO of crabs treated with MF during the late intermoult stages in the current investigation is that the relatively high MF titer (endogenous plus exogenous) overrides the inhibitory effects of MIH. In support of this, high hemolymph MF titers were shown in Macrobrachium rosenbergii(de Man, 1879) (Wilder et al., 1995), P. clarkii (see Laufer et al., 2005) and O. senex senex (see Nagaraju et al., 2006) during intermoult than the postmoult. The presence of secretory vesicles in the YO of crabs injected with MF in the late intermoult stages of the current investigation denoted the production of ecdysteroids. Evidence for the stimulatory effect of MF on YO and ecdysteroidogenesis was shown in C. magister, where in vitro incubation of YO with MO produced significantly higher levels of ecdysteroids into the medium (Tamone and Chang, 1993).

Though MF administration during postmoult demonstrated changes in histomorphology of the YO in terms of YO index, size of the gland and lobules and thickness of the lobular epithelium, these changes were less pronounced than those of the late intermoult stages. In support of this research, in O. senex senex, Nagaraju et al. (2004) observed small sized MOs during postmoult. In M. rosenbergii (see Wilder et al., 1995), P. clarkii (see Laufer et al., 2005) and O. senex senex (see Nagaraju et al., 2006), hemolymph MF concentrations were found to be high during premoult and low during postmoult stage, demonstrating the role of MF in moult regulation. As seen from the results of the present study, MF administration was less effective in stimulating the YO during postmoult when compared to the late intermoult stages, attributed to the high circulating level of MIH that supersedes the effects of the endogenous and exogenous MF titers during postmoult.

Moulting and moult cycle in crustaceans are strictly regulated by the levels of the moulting hormone, ecdysone and the moult inhibiting hormone, MIH. The low hemolymph titer of ecdysteroids and high MIH titer during early intermoult suppresses the activity of the YO (Mattson and Spaziani, 1986; Webster and Keller, 1986). In the present study, the stimulatory effect of 20E on YO during the late intermoult/early premoult (D2) stages could have been due to the quick surge in the circulating levels of 20E contributed by the endogenous and exogenous ecdysteroids and/or due to inhibition of synthesis and release of MIH by the XO-SG complex of the eyestalk, and/or stimulation of synthesis and release of a separate moulting hormone (MH) by the YO. Another possible explanation is that the exogenously administered 20E may override the inhibitory effects of MIH on YO (Passano, 1953; Rao, 1965; Soyez and Kleinholz, 1977). Observations of Chang and Bruce (1980) demonstrated that MIH is not the only regulator of YO ecdysteroidogenesis, and the hemolymph ecdysteroid levels in eyestalk ablated crabs (lacking endogenous MIH) exhibited a pattern similar to that of the intact controls. The above observation specifies a more complex regulation of ecdysteroidogenesis by YO than the simple inhibition by MIH, which needs further investigation. From the present study, it is also clear that MF administration stimulated YO causing ecdysteroidogenesis in YOs, evidenced from the presence of secretory vesicles during the late intermoult stages. In support of this, Wilder et al. (1995), Laufer et al. (2005) and Nagaraju et al. (2006) observed that the MF levels in the hemolymph rise and fall during the premoult and postmoult stages, respectively, in co-ordination with the ecdysteroid level. The mode of action of MF is either direct, through MF receptors on the YO or indirect by inhibiting the synthesis and release of MIH by the eyestalk and/or stimulating the synthesis and release of ecdysteroids by the YO.

To conclude, this study revealed the stimulatory effect of 20E and MF administration on histomorphology of the YO during late intermoult and early postmoult stages in the freshwater crab T. schirnerae. The results also indicated that the effects of 20E and MF administrations were more pronounced in the late intermoult stages than the postmoult stage and in 20E injected crabs than the MF injected individuals. Further gas chromatography-mass spectrometry (GC-MS) studies are required to know the circulating levels of ecdysteroids/MF in 20E and MF administered crabs.

ACKNOWLEDGEMENTS

The financial support provided by the Kerala State Council for Science Technology and Environment (Order No. P 115/2016/KSCSTE, dated 03-05-2016) in carrying out this research is gratefully acknowledged. The authors acknowledge with thanks the anonymous reviewers for the valuable comments/suggestions received in improving the manuscript.

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• Zoobank:

  http://zoobank.org/urn:lsid:zoobank.org:pub:D91A0735-75E4-47B9-BC58-C4048A073E62

Publication Dates

• Publication in this collection
  02 Aug 2021
• Date of issue
  2021

History

• Received
  06 Nov 2020
• Accepted
  22 Apr 2021