The Y-Organ Secretory Activity Fluctuates in Relation to Seasons Of

General and Comparative Endocrinology 196 (2014) 81–90

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The Y-organ secretory activity ﬂuctuates in relation to seasons of molt and reproduction in the brachyuran crab, Metopograpsus messor (Grapsidae): Ultrastructural and immunohistochemical study ⇑ Sharmishtha Shyamal a, K. Sudha b, N. Gayathri c, G. Anilkumar a, a School of Biosciences & Technology, VIT University, Vellore 632014, Tamil Nadu, India b PG Department of Zoology, Sree Narayana College, Kannur 670007, Kerala, India c Department of Neuropathology, National Institute of Mental Health and Neurosciences, Bangalore 560027, India article info abstract

Article history: This paper presents a first-time report on the localization, structure and seasonal secretory activity of the Received 2 January 2013 Y-organ of a grapsid brachyuran crab (Metopograpsus messor). Having exhibited discrete seasonality with Revised 13 November 2013 reference to the programming of molt and reproduction, this brachyuran crab has offered us an excellent Accepted 15 November 2013 model to obtain a clear picture of the fluctuating secretory nature of the Yorgan, all the way through the Available online 27 November 2013 reproductive (August–December) as well as the molt–reproduction active (January–May) and inactive (June–July) seasons. Ultrastructural studies revealed that the secretion of the Y-organ was at its peak Keywords: in premolt crabs during molt–reproduction season (January–May). Interestingly, the Y-organs of the Y-organ intermolt females that engaged in breeding activity showed higher levels of secretion than those of Ecdysteroid Immunohistochemistry the molt–reproduction inactive season (June–July), implicating the gland’s involvement in reproduction. Brachyura Immunohistochemical studies using the antiserum raised against 2-succinyl conjugate of ecdysone have Ultrastructure demonstrated the ecdysteroid nature of the secretion from the Y-organ, and results of the quantitative Metopograpsus messor assay of ecdysteroids (through radioimmunoassay) revealed that the hormone titer fluctuates in consonance with the Y-organ’s secretory activity during seasons of molt and reproduction. Pertinently, not only that the paper gives us a comprehensive understanding on the secretory activity of the Y-organ in a season-dependent fashion, it also allows us to have a better insight into the gland’s function related to molting and reproduction (for the first time) in a grapsid brachyuran crab. Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction nism of steroid uptake through energy-requiring process involving Na/K-ATPase (Spindler et al., 1991). Investigations conducted at The Y-organ (ecdysial gland) of crustaceans has been widely ac- molecular levels during the 1990’s and the 2000’s have thrown cepted as the seat of growth stimulation, ever since Gabe (1953a,b, light on the receptor-mediated mechanism of (molt) hormone ac- 1956, 1971) identiﬁed a pair of ductless glands in malacostracans, tion (Durica and Hopkins, 1996; Chung et al., 1998a,b; Durica et al., comparable to the one in insects, and Echalier (1954, 1955, 1959) 2002; Chang and Mykles, 2011; Hopkins, 2011; Das and Durica, elucidated the role of these glands in molting of the crab Carcinus 2013). maenas. Subsequent investigations involving organ culture Molting in crustaceans is seasonal. The aforementioned series of experiments have identiﬁed the crustacean molting hormone as experiments (along with several other works; Chang and Mykles, ecdysteroids, belonging to polyhydroxylated C27 steroids, and 2011; Nagaraju, 2011) have obviously helped us understand the 20-hydroxyecdysone as crustecdysone (beta ecdysone), the major role of the Y-organ and its secretion in successful accomplishment molt-stimulatory principle (Bollenbacher, 1974; Chang, 1993; of molting in crustaceans. Nevertheless, very little information is Chang and Mykles, 2011). Further, it became evident that the Y-or- available on the Y-organ’s involvement in seasonal programming gan secretes ecdysone (synthesized from cholesterol), which in of molting. Evidently, a clear understanding of the mechanisms turn is converted into 20-hydroxyecdysone (Chang and O’Connor, involved in the seasonal occurrence of a metabolic event would 1977; Lachaise, 1990). Later research has unraveled the mecha- require a season-dependent study on the endocrine center in question and its activity. Being central to the regulation of growth, the Y-organ and its secretory activity need to be assessed through- ⇑ Corresponding author. Fax: +91 2243092. out the entire annual cycle, in order to obtain a clear view on E-mail addresses: [email protected], [email protected] its involvement (if any) in the seasonal programming of molt. (G. Anilkumar).

Surveying through the literature, we realize that a seasonal assess- the criteria (after Sudha and Anilkumar, 1996, 2007). In the present ment of the activity of the Y-organ is lacking in crustaceans in gen- paper, however, we have also adopted the terms such as ‘early’, eral, and brachyuran crabs in particular. ‘mid’ and ‘late’ stages of ovarian growth; the Stage 1 (11–90 lm Significantly, breeding is also another seasonal event that is oocyte diameter) of vitellogenesis (after Sudha and Anilkumar, seen interspersed with molting in its seasonal programming, sug- 1996) has been referred to as ‘early’ ovarian growth (in the present gestive of physiological interaction between these two high energy paper), Stage 2 (91–155 lm oocyte diameter) as the ‘mid’ ovarian demanding metabolic processes (Adiyodi and Adiyodi, 1970; Van growth, while Stages 3 and 4 (156–330 lm oocyte diameter) to- Herp and Soyez, 1997; Tsukimura, 2001; Nagaraju et al., 2011; gether are considered as ‘late’ ovarian growth. Observations on Sudha et al., 2012). That the Y-organ (and its secretion) would have the texture of the carapace and the microscopic examination of any influence on crustacean reproduction, is a question that is still the setogenic events of the abdominal pleopods and the maxilliped being debated (Subramoniam, 2000, 2011; Subramoniam and epipodites were used for characterizing the molt stages (after Kirubagaran, 2011; Sudha et al., 2012), although there are sporadic Sudha and Anilkumar, 1996, 2007; Suganthi and Anilkumar, 1999). references of correlation between ecdysteroid titer and reproductive functions in some crustaceans (Hansen et al., 2008; Nagaraju, 2.3. Dissection and fixation of the Y-organ 2011). On the other hand, there are clear demonstrations on the role of ecdysteroids in successful accomplishment of reproduction Till date, no report was available on the Y-organ of M. messor. in insects, a phylogenic ally of crustaceans (Horn et al., 1966; Hence, we relied up on previous reviews (Waterman, 1960; Aoto Chang, 1993; Truman and Riddiford, 1999, 2002; Carney and Bend- et al., 1974; Lachaise et al., 1993) on the Y-organs of decapod crus- er, 2000; Riddiford et al., 2001; Zitnan et al., 2007; Mykles et al., taceans (including the brachyurans), for a reasonable prediction on 2010). The amazingly high levels of diversity being exhibited by the localization of the tissue. After having dissected out (by cutting the crustaceans (even among close phylogenic groups, some times open the carapace), the tissue was immediately fixed in 10% neu- in the same species, but occupying different geographical realms) tral buffered formalin (PBS, formaline pH 7.2) for histological and in seasonal programming of molt and reproduction, seems to be immunohistochemical studies, and in 3% glutaraldehyde (in phos- a major constraint that does not allow us to draw any generaliza- phate buffer, pH 7.2) for electron microscopy. tions in this regard. Obviously, this is also an area where more research is needed. 2.4. Histology and immunohistochemistry Keeping these in view, we present the results of our ultrastructural and immunohistochemical studies on the Y-organ of the 10% neutral buffered formalin-fixed tissue was used for histo- grapsid brachyuran crab, Metopograpsus messor, carried out logical studies. Paraffin sections 5–7 lm thick, stained in hamat- throughout the annual cycle that involves seasons devoted for ac- oxylin-eosin, and were used for light microscopic observations tive molting and/or reproduction, and the molt–reproductive inac- (Pearse, 1968; Humason, 1974; Avwioro, 2011). tive season. The results are further collated with the season- Procedure for immunohistochemistry has been adopted from dependent changes in the ecdysteroid levels. Birkenbeil (1983), and Seifert and Bidmon (1988).5lm thick par- Inhabiting the intertidal zone of the estuaries of Muzhupilangad affin sections, mounted on silane-coated slides, were used for in Kannur District of Kerala (India), this grapsid crab (M. messor) immunohistochemistry. The sections were deparaffinized in xy- exhibits definite patterns of programming of molting and lene, rehydrated in descending grades of ethanol and washed in reproduction during various seasons of the year. Accordingly, Millipore water. The antigen retrieval steps were carried out in August–December season is devoted exclusively for breeding. Tris–EDTA (pH 8.0) buffer. The buffer was preheated to boiling January–May season is for both molting and breeding; while a temperature for 5 min, then heating with the slides embedded in subset of the population during the season would be involved in it (at boiling temperature) for 5 min, and subsequently by bringing premolt–postmolt changes, the other subset would be involved down the temperature to 80 and 60 °C for 10 and 5 min respec- in breeding. June–July season is characterized by molt–reproductively. The slides were cooled to room temperature and washed tive repose when the population engages neither in molt nor in TBS buffer (pH 7.4). Endogenous blocking (5% hydrogen peroxide breeding (Sudha and Anilkumar, 1996, 2007). For the present in methanol) was done for 30 min in the dark and was again study, we have considered all these three seasons with a view to washed in TBST (3% milk in TBS) for 5 min to block the non-specific assess the Y-organ secretory activity and the fluctuating ecdyster- binding sites. The sections were then incubated with primary anti- oid levels in relation to molt and/or reproduction. body (2-succinyl conjugate of ecdysone, a generous gift from Pro- fessor Ernest S. Chang, University of California, USA) at 1:500 dilutions for 30 min and washed in PBST for 5 min [prior to treat- 2. Materials and methods ment with the antigen, the antiserum was subjected to titer assay to ensure optimal binding sensitivity; 1:500 was found to be the 2.1. Collection and maintenance of animals most appropriate dilution for the purpose]. Supersensitive poly HRP (Promega) was used as secondary antibody, in which the All the females of M. messor (within the size class of 1.8–2.4 cm slides were again incubated for 30 min. Finally, it was washed in carapace width), used for the present study were collected from TBST and developed in DAB (Chromogen) and counterstained with the intertidal zone of the Muzhupilangad estuary (N. Kerala, India) haematoxylin. The sections were dehydrated in ascending alcohol by hand-picking and/or using bait. Soon after collection, the crabs grades, cleared in xylene and mounted with DPX. were brought to the laboratory and were either sacrificed immedi- The paraffin sections of the Y-organ, that did not receive the ately, or if required, maintained in cement cisterns for the purpose antibody treatment, were used as the controls, for the purpose of of further observations. comparison.

2.2. Stages of vitellogenesis and molting 2.5. Transmission electron microscopy

The stages of vitellogenesis were characterized by considering The tissue, after being fixed in 3% glutaraldehyde for 24 h, was the color of the ovary, the oocyte diameter (measured using ocular washed thoroughly with 0.1 M phosphate buffer, and post-fixed in micrometer to the nearest 1 lm) and the ovarian protein levels as osmium tetroxide for 1–2 h at 4 °C. It was again washed in 0.1 M S. Shyamal et al. / General and Comparative Endocrinology 196 (2014) 81–90 83 phosphate buffer and dehydrated in graded series of ethanol (70– adopted from Chang and O’Connor (1979), Suganthi and Anilkumar 90%). Following dehydration in 90% ethanol, the sample was incu- (1999) and Sudha and Anilkumar (2007). Samples of the Y-organ bated in (freshly prepared) 2% ethanolic uranyl acetate (enbloc (9–10 nos), weighing approximately 0.7–0.8 mg were used for each staining), and was subsequently dehydrated with 100% ethanol, assay. The dissected out tissue was quickly transferred to 80% followed by treatment in acetone for complete dehydration of ethanol placed in eppendorf vial maintained in ice. The tissue sam- the tissue. Propylene oxide was used as the clearing agent. Further, ple was then homogenized and the homogenate was vacuum- the tissue was left for infiltration for a minimum of 6 h in 1:1 mix- evaporated, and processed for radioimmunoassy. Each of the ture of propylene oxide and araldite followed by pure araldite for a eppendorf vials (with vacuum evaporated samples) received further period of 8 h. The tissue was then embedded in araldite and 100 ll borate buffer (pH 8.4) and 100 ll of the radio ligand 3H- left in an oven maintained at 60° undisturbed for 2 days for poly- ecdysone (12,000 dpm, Perkin-Elmer). Different aliquots of the merization (Williams and Carter, 2009). Semi-thin sections standards, comprising the radio-ligand (3H-edysone), the (1 lm), stained in 1% toluidine blue, were used to ensure the exact phosphate buffer and known quantities of the standard ecdysone positioning of the tissue, prior to ultra-thin (600 Å) sectioning to be (Sigma, USA) were also prepared and run along with the unknown used for electron microscopy. samples, for plotting the standard graph. All the vials (the unknown samples and the standards) were vortexed to allow ade- 2.6. Ecdysteroid assay quate mixing. To each vial, 100 ll antiserum (2-succinyl conjugate of ecdysone) was added [the dilution of the antiserum (1:500) was The ecdysteroid titer in the Y-organ and the hemolymph was determined by titer assay as mentioned before]. The mixture was estimated through radioimmunoassay (RIA) as per the protocol then incubated for 12 h at 4 °C, after which 200 ll saturated

Fig. 1. (A) Photomicrograph showing the location and morphology of the Y-organ of M. messor; (B) photomicrograph of parafﬁn section of the Y-organ stained in Haematoxylin–eosin, depicting the characteristic, closely packed cell arrangements.

Fig. 2. (A) Light photomicrograph of the Y-organ of M. messor during August–December season, showing clear differentiation between the outer dense zone and the inner ‘‘less dense’’ (vacuolated) zone, (i, ii) inset of the two zones (toluidine blue). (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) 84 S. Shyamal et al. / General and Comparative Endocrinology 196 (2014) 81–90 ammonium sulfate was added to each vial and incubated at 4 °C for nium sulfate in borate buffer and centrifuged. The pellets were 20 min to allow complete precipitation. The pellets, resultant of then dissolved in 25 ll distilled water and mixed with 500 llof the antigen–antibody binding, were separated out by centrifuga- scintillation cocktail (0.8% PPO and 0.02% POPOP in a toluene:triton tion at 4800g for 20 min, and washed with 400 ll of 50% ammo- [2:1] mixture) and read using an LKB Liquid Scintillation Counter. A

Fig. 3. Electron micrograph showing the cellular organization of the Y-organ of M. messor during August–December season; (A) overview of the gland with nuclei (N) and nucleolus (Nu), pyknotic nucleus (PN), secretory vesicles (SV) and micromitochondria (m); (B) electron micrograph shows golgi (G), micromitochondria (m) and macromitochondria (M).

Fig. 4. Cellular organization of the Y-organ of premolt crabs during January–May; (A) light micrograph showing the dense cytoplasm, characteristic of the Y-organ during the season; (B, C) electron micrograph depicting the intense activity of the gland; note the presence of golgi (G), micromitochondria (m), macromitochondria (M) and the free ribosomes (R); (D) Electron micrograph showing basal lamina (BL), intact nuclei (N) and secretory vesicles (SV). (All other figure legends are as in the previous figures). S. Shyamal et al. / General and Comparative Endocrinology 196 (2014) 81–90 85 standard curve plotted with 25, 50, 125, 250, 500, 1000, 2000 and 3.2. Season-dependent fluctuations in gross morphology and structure 4000 pg ecdysone on a semi-log paper, was used for accurate esti- of the Y-organ mation of the ecdysteroid titer in the sample in question. In its gross morphology and size, the Y-organ of M. messor exhibited season-dependent changes. While the population was 2.7. Statistical analysis not engaging either in molting or in reproduction (June–July), the Y-organ appeared as a minute white mass (540 ± 35.68 lm diame- For assessing the statistical significance (if any) in the fluctua- ter; X ± SD). The gland attained the maximum size (814 ± 66.49 lm tion of ecdysteroid levels, the data were subjected to F-test (ANO- diameter; X ± SD) during January–May (molt–reproduction sea- VA) and the Students’s t-test using InStat 3 (GraphPad Version 2.0) son), and acquired a creamy-yellow colour in premolt crabs. The software. In all the instances, null hypothesis was rejected at 95% gland during the breeding season (August–December) was med- confidence interval (p < 0.05). ium-sized (609 ± 45.36 lm in diameter; X ± SD), but appeared larger than that of the molt–reproductive inactive season. The size 3. Results differences of the Y-organ existing among these three seasons were found to be statistically significant (F = 118.09; p < 0.0001). 3.1. Localization of the Y-organ, and its morphology In paraffin sections, the Y-organ encompassed closely packed cells, with basophilic nuclei (Fig. 1. B), which in some instances dis- The Y-organ in M. messor was located as minute, oval-shaped played lobulated appearance. Ultrastructurally, the cells were seen tissue (500–850 lm diameter; X ± SD), between the mandibular arranged in irregular contours with clear cellular demarcations and external adductor muscle at the anterolateral edge of the branchial basal lamina (BL) (Figs. 3 and 4). The abundance of organelles such chamber. Morphologically, the Y-organ of M. messor appears as a as mitochondria, Golgi, and the secretory vesicles in electron bi-convex mass of tissue with a characteristic white hue (Fig. 1A). micrographs was dependent on the season in question at which

Fig. 5. Cellular organization of the Y-organ from intermolt females of January–May. (A) Light photomicrograph of the sections of the Y-organ depicting the dense peripheral zone and the less dense central zone; (B) electron micrograph of the peripheral zone of the Y-organ showing secretory vesicles (SV).

Fig. 6. (A) Light photomicrograph of the Y-organ of M. messor during June-July (molt–reproduction inactive) season, showing the less dense cells that prevail the entire tissue; (B) electron micrograph showing a pyknotic nucleus. 86 S. Shyamal et al. / General and Comparative Endocrinology 196 (2014) 81–90 the tissue was harvested. The season-dependent variations in microscope, the cells of the outer zone appeared active, evinced by structure displayed by the Y-organ of M. messor could be described the occurrence of golgi (G), secretory vesicles (SV) and mitochon- as follows: dria (M) (Fig. 3); the inner zone, however, displayed the presence of highly vacuolated area (Fig. 2A, ii). In some instances though, 3.2.1. Reproductive (breeding) season (August–December) the sections displayed the presence of pyknotic nuclei, a sign of Almost the entire population is involved in breeding activity very low or no synthetic activity. Signiﬁcantly, the mitochondria during the season; all the females were seen to possess growing were of two size-classes; the smaller ones of 0.5–0.8 lm in diam- ovaries (Stage 1–4, as described in ‘‘Materials and methods’’). His- eter, the micromitochondria, and the larger ones of 1.2–1.8 lmin tologically, during the August–December season, the Y-organ of M. diameter, referred to as the macromitochondria (Fig. 3A) [names messor was characterised by the occurrence of two distinct zones: adopted from Aoto et al., 1974; Lachaise et al., 1993]. The macro- (1) an outer zone wherein the cells appeared active, judged by the mitochondria displayed the presence of tubular cristae, apparently occurrence of cytoplasmic basophilia (Fig. 2A, i), and (2) an inner related to intense activity. The Y-organ during the season has dis- ‘‘less dense’’ zone, wherein the cells were with little or no activity, played the presence of several micromitochondria; macromito- borne out of the less intense basophilia (Fig. 2A, ii). Under electron chondria, were also visible (Fig. 3B), although only in smaller

Fig. 7. Immunohistochemical staining to demonstrate ecdysteroid synthesis in the Y-organ: (A) the Y-organ of premolt crab during January–May season showing relatively intense brown coloration; (B) the Y-organ of an intermolt crab of January–May season showing medium levels of brown pigmentation of the cytoplasm; (C) the Y-organ of August–December (breeding) season with medium levels of brown coloration; (D) the Y-organ of June–July season with only sparse intensity in staining; (E) controls wherein the tissue was not treated with primary antibody. S. Shyamal et al. / General and Comparative Endocrinology 196 (2014) 81–90 87 numbers, when compared to the situation during the molt–repro- not engage in molt or reproduction), moderate reactivity duction season (January–May). (Fig. 7E) during August–December (intermolt crabs engaged in reproduction) and intense reaction (Fig. 7C) in premolt crabs 3.2.2. Molt–reproduction season (January–May) (January–May), indicating a spurt of activity of the Y-organ during During the season, the female population was seen to be in- the season. The Y-organ of the intermolt crabs of January–May volved in either molt or reproduction. We have considered two season (involved in breeding activity), displayed positivity to the major cohorts in the population: the one which has already en- peroxidase reaction in fair levels, but less than the levels of premolt tered premolt growth, but with inactive ovaries (with oocyte diam- crabs (Fig. 7B). eter < 11 lm), and the other, at intermolt stage, but having growing ovaries (11–330 lm in diameter). Histologically, the Y-or- 3.4. Fluctuations in hemolymph ecdysteroid levels gan in general, displayed signs of remarkable activity during the season. The inner ‘‘less dense’’ zone (as seen during the August– Hemolymph ecdysteroid levels of the female individuals fluctu- December, Fig. 2), was hardly visible in premolt individuals ated significantly with respect to various seasons and in response (Figs. 4A); the intermolt cohort, however, exhibited the presence to physiological parameters. The levels were significantly high dur- of the ‘‘less dense’’ zone (Fig. 5A). Ultrastructurally, the Y-organ ing January–May (molt–reproduction) season, where a sizable pro- exhibited signs of activity during the season; in premolt crabs, portion of the individuals were engaged in molting, attaining the the cells displayed very profuse presence of free ribosomes (R), gol- peak in those crabs at premolt (D2/D3 and D4) stages. The female gi (G) and macromitochondria (M) (Fig. 4B, C). Among the two co- crabs that were engaging in ovarian growth, however, showed a horts of the mitochondria, macromitochondria were more significantly lower profile of ecdysteroids (p < 0.001), when com- prevalent during this season than it were during the August– pared with the premolt crabs (Table 1); the levels, however, did December (Fig. 4B). In intermolt females, however, the golgi and not noticeably fluctuate during the reproductive stages (F = 1.01, the secretory vesicles were less abundant, when compared with p = 0.3044). Significantly, the hemolymph ecdysteroid titers of those of the premolt ones (Fig. 5B). the reproducing females of August–December (breeding season) and those of January–May (molt–reproduction) seasons were 3.2.3. Molt–reproduction inactive season (June–July) remarkably higher (p < 0.0001) than those during the June–July The Y-organ displayed the least levels of activity during June– (molt–reproduction repose) season. July season, when the population engaged neither in breeding nor molting [The entire population remained in intermolt, with 3.5. Fluctuations in ecdysteroid levels of the Y-organ their ovaries appearing as white bands (oocyter diameter < 11 lm), with no signs of yolk deposition during the season]. The cells of the The fluctuations in ecdysteroid levels of the Y-organ of M. mes- Y-organ during June–July season were inactive, judged by the sor with respect to seasons of molt and reproduction were very occurrence of highly vacuolated appearance in the cytoplasm much in tune with those of the hemolymph. During the molt– (Fig. 6). The organelles such as free ribosomes, mitochondria and reproduction season (January–May), the levels of ecdysteroids in the golgi, characteristic of the cells involved in synthetic activity, the Y-organ registered significant increase in the premolt crabs were hardly detectable in the Y-organ during the season. (early to late stages); the Y-organ of the late premolt (D4) crabs had the maximum ecdysteroid levels, but it declined thereafter, 3.3. Immunohistochemical observations following exuviation (Table 2). Further, as in the case of hemolymph, the ecdysteroid titer of the Y-organ did not show remark- Immunostaining of the Y-organ to ecdysone revealed weak able fluctuation in response to stages of ovarian growth (Table 2) reaction (Fig. 7D) during June–July (when the population does during August–December (breeding season, F = 0.765, p = 0.462)

Table 1 Ecdysteroid levels in the hemolymph of M. messor in relation to seasons of molt and reproduction.

Hemolymph ecdysteroid levels Premolt Intermolt (pg/ll) D2/D3 D4 Ovarian growth (early) Ovarian growth (mid) Ovarian growth (late) Inactive January–May 94.75 ± 7.04 (20) 170.83 ± 12.5* (12) 10.35 ± 2.04* (10) 27.12 ± 4.56* (7) 33.88 ± 6.89* (8) – August–Decembera – – 17.583 ± 6.09 (6) 27.36 ± 1.02 (6) 32.916 ± 2.18 (6) – June–Julyb – – – – – 7.25 ± 2.91 (6)

Sample size is in parenthesis. a Premolt–postmolt crabs were not seen during August–December. b The crabs were undergoing a period of molt–reproductive repose during June–July. * Adopted from Sudha and Anilkumar, 2007.

Table 2 Ecdysteroid levels in the Y-organ of M. messor in relation to seasons of molt and reproduction.

Y Organ ecdysteroid levels Premolt Intermolt (pg/mg tissue) D2/D3 D4 Ovarian growth (early) Ovarian growth (mid) Ovarian growth (late) Inactive January–May 562.899 ± 2.72 (6) 1017.35 ± 5.79 (6) 234.903 ± 10.93 (6) 283.899 ± 3.16 (6) 302.210 ± 4.38 (6) – August–Decembera – – 240.902 ± 6.71 (6) 291.688 ± 2.93 (6) 308.46 ± 3.92 (6) – June–Julyb – – – – – 118.218 ± 7.95 (6)

Sample size is in parenthesis. Notes on a and b as in Table 1. 88 S. Shyamal et al. / General and Comparative Endocrinology 196 (2014) 81–90

Table 3 Consolidated results of the secretory activity of the Y-organ, and the ecdysteroid levels in the hemolymph and the Y-organ of M. messor in relation to seasons of molt and reproduction.

Secretory activity of Premolt Intermolt Y-organ D2/D3 D4 Ovarian growth (early) Ovarian growth (mid) Ovarian growth (late) Inactive January–May + ++ ± ± ± – August–Decembera –– ±±±– June–Julyb –– – – – – Hemolymph ecdysteroid levels (pg/ll) January–May 94.75 ± 7.04⁄ (20) 170.83 ± 12.52⁄ (12) 10.35 ± 2.04⁄ (10) 27.12 ± 4.56⁄ (7) 33.88 ± 6.89⁄ (8) – August–Decembera –– 17.583 ± 6.09 (6) 27.36 ± 1.02 (6) 32.916 ± 2.18 (6) – June–Julyb –– – – – 7.25 ± 2.91 (6) Y Organ ecdysteroid levels (pg/mg tissue) January–May 562.899 ± 2.72 (6) 1017.35 ± 5.79 (6) 234.903 ± 10.93 (6) 283.899 ± 3.16 (6) 302.210 ± 4.38 (6) – August–Decembera –– 240.90 ± 6.71 (6) 291.688 ± 2.93 (6) 308.46 ± 3.92 (6) – June–Julyb –– – – – 118.218 ± 7.95 (6)

Sample size is in parenthesis. For depicting the Y-organ secretory activity: ++, very high; +, high; ±, medium; –, low. Notes on a, b and ⁄ as in Table 1.

and January–May (molt–reproduction season, F = 1.014, during premolt, as reported in Palaemon serratus (Le Roux, 1977) p = 0.9591). Significantly, the Y-organ ecdysteroid level declined and Astacus astacus,(Birkenbeil and Gersch, 1979), could be the further (p = 0.0025) during June–July, the season for molt–repro- mechanism of such size increase in M. messor as well. The Y-organ duction repose (Table 2). has the smallest size during June–July, a season for neither molt nor reproduction. This season-dependent fluctuation in the gland size is concordant with its secretory activity (and the ecdysteroid 4. Discussion production, evidenced by the immunohistochemical staining). The ‘‘en-bloc’’ vacuolated appearance of the tissue, coupled with In this first ever report on the localization, morphology and the pyknotic nuclei, dearth of organelles involved in secretory structure of the Y-organ in the grapsid brachyuran crab (M. mes- activity, and the lack of brownish hue and prevalence of the blue sor), we depict the gland’s seasonal changes at histological, ultra- color seen in the immunohistochemical staining during June–July structural and immunohistochemical levels, supported with the (Fig. 7D), is reminiscent of a total tardiness in the Y-organ activity. data on ecdysteroid assay. In almost all the previous occasions, This vacuolated appearance seems quite comparable with what the Y-organ was studied in the context of the cascade of events re- was reported in Carcinus maenas (Noel et al., 1989) during the stage lated to molting. The present paper, on the other hand, adds to our of its inactivity. During January–May, on the other hand (when the understanding on the gland’s function inasmuch as it describes the population is actively engaged in molt and reproduction), the gland changes in secretory activity through the entire annual cycle, cells are rich in mitochondria, free ribosomes and golgi, and the involving molting and reproductive seasons. secretory vesicles showing high synthetic activity (Fig. 4). The In its gross morphology (oval in shape) and general structure (in occurrence of the small-sized (micro-) and the large-sized the nucleo-cytoplasmic ratio), the Y-organ of M. messor appears (macro-) mitochondria is interesting, and invites an added atten- comparable with the ‘‘Carcinus’’ type described by Lachaise et al. tion at this juncture. The profuse occurrence of macromitochondria (1993), likely to be characteristic of brachyuran crabs in general. in premolt crabs (January–May season) strongly supports intense The Y-organ of M. messor, shows resemblance to Carcinus sp. in activity of the Y-organ during the season. A comparable situation its ultrastructure as well (Lachaise et al., 1993), especially in terms was found to exist in the freshwater prawn, Palaemon paucidens of the characteristic presence of densely packed cells, basal lamina and the brachyuran crab, Carcinus manaeus wherein the macromi- and the lobulated nuclei. It is well established that molting tochondria were considered to be involved in elevated synthetic (growth) and reproduction in crustaceans are being successfully activity (Aoto et al., 1974). The results of our immuno-histochem- programmed in the annual cycle by the up- and the down-regula- ical staining (against anti-ecdysone antibody), not only tion of the stimulatory as well as the inhibitory hormones demonstrate that the secretory product of the Y-organ is ecdysone (Echalier, 1955; Adiyodi, 1988; Hopkins, 1989, 2011; Subramon- (Fig. 7), but it as well reinforce our findings on the season- iam and Kirubagaran, 2011). The paucity of information on the dependent cyclicity in secretory activity. Superimposing the exact mechanisms involved in programming of these high en- data obtained from the present ultrastructural and immunohisto- ergy-demanding processes in a season-dependent fashion, has chemical studies, on the results of ecdysteroid assay, makes it prompted us to adopt in this paper, the strategy of assessing the evident that the seasonal fluctuations in ecdysteroid levels are in secretory activity of the Y-organ, to be collated with the ecdyster- consonance with the seasonal secretory activity of the Y-organ oid titer (of the Y-organ and the hemolymph) throughout the an- (Table 3). nual cycle. It is worth noticing that the Y-organ of M. messor demon- Our present study establishes the existence of perceptible strates medium levels of activity during August–December, when changes in the Y-organ, in terms of its gross morphology (including the entire female population is engaged in breeding activity; this the gland size) and structure in relation to various seasons of the level of activity is somewhere between those of January–April year (such as January–May, June–July and August–December sea- (the maximum levels) and June–July (the minimum levels) sons). The gland attains the maximum size during January–May seasons. The existence of a synthetically active outer zone of (F = 118.09; p < 0.0001), the only season when active molting oc- cells (Fig. 2i,) evidenced by the relatively intense cytoplasmic curs among the population. An increase in the cytoplasmic volume S. Shyamal et al. / General and Comparative Endocrinology 196 (2014) 81–90 89 basophilia (Fig. 2) and the presence of free ribosomes, golgi and tive inactive (June–July) season, deserves attention in this the seceretory vesicles (Figs. 3 and 4), support such a suggestion. respect. As molting activity is practically nonexistent during Au- The results of the ecdysteroid assay (Tables 1–3) are also in tune gust–December, and as the entire population is engaged in breed- with this finding. Under immunohistochemical staining, the ing, the higher secretory activity during August–December season presence in medium levels of the characteristic brown color encourages us to argue for the Y-organ’s possible involvement in (due to peroxidase reaction) exhibited by the sections of the reproduction. Regulation of crustacean vitellogenesis, by itself, is Y-organ harvested from the reproductive females reiterates this a topic wherein much ambiguity prevails. Methyl farnesoate (MF) contention. This finding raises an interesting question on the is known to perform gonadotropic function in crustaceans, and possible role of the Y-organ (and its secretion) in crustacean the recent molecular studies have demonstrated the existence of reproduction, a subject of scarce information. Arguably, the yolk protein gene and its expression in the ovary (Wilder et al., ambiguity on ecdysteroids’ role in crustacean reproduction is 2002; Subramoniam and Kirubagaran, 2011). However, it is still being chiefly driven by the rich diversity existing among even unclear about the possible existence of a putative hormone (other closely allied phylogenic groups with respect to the program- than MF) that could activate the yolk protein gene in crustaceans. ming of molt and reproduction. In brachyuran (true) crabs, for This is very much pertinent in the context that 20 hydroxyecdy- instance, premolt growth and yolk deposition are mutually sone is shown to stimulate the vitellogenin gene (PmVg1) expres- exclusive events (Bouchon and Souty-Grosset, 1992; Sudha and sion in Penaeus monodon (Tiu et al., 2006; Nagaraju, 2011) and that Anilkumar, 1996, 2007; Sudha et al., 2012); vitellogenesis is re- the steroids from vertebrates are shown to stimulate vitellogenesis stricted in general to intermolt crabs only, which could even in crustaceans (Yano et al., 2000; Lafont et al., 2005; Brown et al., implicate that the premolt titre of ecdysteroids could restrain 2009; Coccia et al., 2010; Nagaraju et al., 2011; Subramoniam vitellogenesis. On the other hand, synergistic programming of and Kirubagaran, 2011). The importance of pursuing the role of molt and breeding activities is found to be characteristic of pal- ecdysteroids in crustacean reproduction in a more systematic aemonid shrimps wherein premolt growth is closely entrained way is being increasingly felt (Nagaraju, 2011; Sudha et al., with yolk deposition (Phlippen and Webster, 2000; Tsukimura, 2012). Future studies involving (steroid) hormone–receptor–HRE 2001), suggestive of a possible stimulatory role for ecdysteroids cascade should help us address this question very effectively. Per- in ovarian growth. Our observation of medium levels of tinently enough, that the studies using Ribonuclease Protection As- (ecdysteroid) secretory activity in the Y-organ of the intermolt say (RPA) have demonstrated the occurrence of mRNA encoding crabs harvested during the reproductive season (August–Decem- the ecdysteroid receptor (UpEcR) in the ovary of the fiddler crab, ber) as well as the growth-reproductive season (January–April) Uca pugilator (Durica et al., 2002; Sudha and Anilkumar, 2007), (also evidenced by the results of ecdysteroid assay, Table 1), seem to suggest that ovary is a putative target for ecdysteroids. seemingly ascertains that the ecdysteroid secretion by the Y-or- Further, through a recent, meticulously designed experiment, Das gan does not exert an inhibitory influence on reproduction in and Durica, 2013 have successfully demonstrated the use of RNAi this brachyuran decapod (M. messor). Further, the highly vacuo- for the knockdown of the ecdysteroid receptor gene (EcR)inU. pug- lated appearance of the Y-organ during the June–July season ilator, for disrupting the mRNA levels, leading to growth arrest. strongly suggests for a near-total down regulation of the ecdy- Obviously, such experiments could offer us valuable inputs in fu- steroid secretion during a time when the population completely ture to demonstrate precisely the involvement (if any) of ecdyster- refrains from reproduction; importantly, the ecdysteroid levels of oids in crustacean reproduction. the Y-organ and the hemolymph were at its lowest during June– July season. This in turn suggests for a possible involvement of Acknowledgments the Y-organ secretion (ecdysone) in reproduction of M. messor. Further, the ecdysteroid secretory activity of the Y-organ of We express our sincere thanks to Professor Ernest S. Chang, intermolt females engaging in reproduction, as it exceeds those University of California, Davis (USA), for the generous gift of the of the reproductively inactive females of June–July season, seems ecdysteroid antibody, Dr. Ramadasan Kuttan and Dr. Girija Kuttan, to suggest that the (medium levels of) activity of the Y-organ Amala Cancer Research Center (Kerala, India) for their help for the (and the circulating ecdysteroid levels) could be related to suc- provision of the LKB liquid scintillation counter. Thanks are also cessful vitellogenesis in M messor. due to Mrs. Hemavathy, Mrs. Sunita (Research fellow) NIMHANS, Bangalore (India) for helping with the electron microscopy and immunohistochemistry, Aneesh, Helna and Arshad the Research 5. Conclusion Fellows of Sree Narayana College, Kannur (Kerala, India), for having helped us in procurement of live crabs. 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