General and Comparative Endocrinology 196 (2014) 81–90

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General and Comparative Endocrinology

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The Y-organ secretory activity fluctuates in relation to seasons of moltand reproduction in the brachyuran crab, Metopograpsus messor(Grapsidae): Ultrastructural and immunohistochemical study

0016-6480/$ - see front matter � 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.ygcen.2013.11.016

⇑ Corresponding author. Fax: +91 2243092.E-mail addresses: gopianilkumar@gmail.com, ganilkumar@vit.ac.in

(G. Anilkumar).

Sharmishtha Shyamal a, K. Sudha b, N. Gayathri c, G. Anilkumar a,⇑a School of Biosciences & Technology, VIT University, Vellore 632014, Tamil Nadu, Indiab PG Department of Zoology, Sree Narayana College, Kannur 670007, Kerala, Indiac Department of Neuropathology, National Institute of Mental Health and Neurosciences, Bangalore 560027, India

a r t i c l e i n f o

Article history:Received 2 January 2013Revised 13 November 2013Accepted 15 November 2013Available online 27 November 2013

Keywords:Y-organEcdysteroidImmunohistochemistryBrachyuraUltrastructureMetopograpsus messor

a b s t r a c t

This paper presents a first-time report on the localization, structure and seasonal secretory activity of theY-organ of a grapsid brachyuran crab (Metopograpsus messor). Having exhibited discrete seasonality withreference to the programming of molt and reproduction, this brachyuran crab has offered us an excellentmodel to obtain a clear picture of the fluctuating secretory nature of the Yorgan, all the way through thereproductive (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 peakin premolt crabs during molt–reproduction season (January–May). Interestingly, the Y-organs of theintermolt females that engaged in breeding activity showed higher levels of secretion than those ofthe molt–reproduction inactive season (June–July), implicating the gland’s involvement in reproduction.Immunohistochemical studies using the antiserum raised against 2-succinyl conjugate of ecdysone havedemonstrated the ecdysteroid nature of the secretion from the Y-organ, and results of the quantitativeassay of ecdysteroids (through radioimmunoassay) revealed that the hormone titer fluctuates in conso-nance with the Y-organ’s secretory activity during seasons of molt and reproduction. Pertinently, not onlythat the paper gives us a comprehensive understanding on the secretory activity of the Y-organ in aseason-dependent fashion, it also allows us to have a better insight into the gland’s function related tomolting and reproduction (for the first time) in a grapsid brachyuran crab.

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1. Introduction

The Y-organ (ecdysial gland) of crustaceans has been widely ac-cepted as the seat of growth stimulation, ever since Gabe (1953a,b,1956, 1971) identified a pair of ductless glands in malacostracans,comparable to the one in insects, and Echalier (1954, 1955, 1959)elucidated the role of these glands in molting of the crab Carcinusmaenas. Subsequent investigations involving organ cultureexperiments have identified the crustacean molting hormone asecdysteroids, belonging to polyhydroxylated C27 steroids, and20-hydroxyecdysone as crustecdysone (beta ecdysone), the majormolt-stimulatory principle (Bollenbacher, 1974; Chang, 1993;Chang and Mykles, 2011). Further, it became evident that the Y-or-gan secretes ecdysone (synthesized from cholesterol), which inturn is converted into 20-hydroxyecdysone (Chang and O’Connor,1977; Lachaise, 1990). Later research has unraveled the mecha-

nism of steroid uptake through energy-requiring process involvingNa/K-ATPase (Spindler et al., 1991). Investigations conducted atmolecular levels during the 1990’s and the 2000’s have thrownlight on the receptor-mediated mechanism of (molt) hormone ac-tion (Durica and Hopkins, 1996; Chung et al., 1998a,b; Durica et al.,2002; Chang and Mykles, 2011; Hopkins, 2011; Das and Durica,2013).

Molting in crustaceans is seasonal. The aforementioned series ofexperiments (along with several other works; Chang and Mykles,2011; Nagaraju, 2011) have obviously helped us understand therole of the Y-organ and its secretion in successful accomplishmentof molting in crustaceans. Nevertheless, very little information isavailable on the Y-organ’s involvement in seasonal programmingof molting. Evidently, a clear understanding of the mechanismsinvolved in the seasonal occurrence of a metabolic event wouldrequire a season-dependent study on the endocrine center inquestion and its activity. Being central to the regulation of growth,the Y-organ and its secretory activity need to be assessed through-out the entire annual cycle, in order to obtain a clear view onits involvement (if any) in the seasonal programming of molt.

82 S. Shyamal et al. / General and Comparative Endocrinology 196 (2014) 81–90

Surveying through the literature, we realize that a seasonal assess-ment of the activity of the Y-organ is lacking in crustaceans in gen-eral, and brachyuran crabs in particular.

Significantly, breeding is also another seasonal event that isseen interspersed with molting in its seasonal programming, sug-gestive of physiological interaction between these two high energydemanding metabolic processes (Adiyodi and Adiyodi, 1970; VanHerp and Soyez, 1997; Tsukimura, 2001; Nagaraju et al., 2011;Sudha et al., 2012). That the Y-organ (and its secretion) would haveany influence on crustacean reproduction, is a question that is stillbeing debated (Subramoniam, 2000, 2011; Subramoniam andKirubagaran, 2011; Sudha et al., 2012), although there are sporadicreferences of correlation between ecdysteroid titer and reproduc-tive functions in some crustaceans (Hansen et al., 2008; Nagaraju,2011). On the other hand, there are clear demonstrations on therole of ecdysteroids in successful accomplishment of reproductionin insects, a phylogenic ally of crustaceans (Horn et al., 1966;Chang, 1993; Truman and Riddiford, 1999, 2002; Carney and Bend-er, 2000; Riddiford et al., 2001; Zitnan et al., 2007; Mykles et al.,2010). The amazingly high levels of diversity being exhibited bythe crustaceans (even among close phylogenic groups, some timesin the same species, but occupying different geographical realms)in seasonal programming of molt and reproduction, seems to bea major constraint that does not allow us to draw any generaliza-tions in this regard. Obviously, this is also an area where more re-search is needed.

Keeping these in view, we present the results of our ultrastruc-tural and immunohistochemical studies on the Y-organ of thegrapsid brachyuran crab, Metopograpsus messor, carried outthroughout the annual cycle that involves seasons devoted for ac-tive molting and/or reproduction, and the molt–reproductive inac-tive season. The results are further collated with the season-dependent changes in the ecdysteroid levels.

Inhabiting the intertidal zone of the estuaries of Muzhupilangadin Kannur District of Kerala (India), this grapsid crab (M. messor)exhibits definite patterns of programming of molting andreproduction during various seasons of the year. Accordingly,August–December season is devoted exclusively for breeding.January–May season is for both molting and breeding; while asubset of the population during the season would be involved inpremolt–postmolt changes, the other subset would be involvedin breeding. June–July season is characterized by molt–reproduc-tive repose when the population engages neither in molt norbreeding (Sudha and Anilkumar, 1996, 2007). For the presentstudy, we have considered all these three seasons with a view toassess the Y-organ secretory activity and the fluctuating ecdyster-oid levels in relation to molt and/or reproduction.

2. Materials and methods

2.1. Collection and maintenance of animals

All the females of M. messor (within the size class of 1.8–2.4 cmcarapace width), used for the present study were collected fromthe intertidal zone of the Muzhupilangad estuary (N. Kerala, India)by hand-picking and/or using bait. Soon after collection, the crabswere brought to the laboratory and were either sacrificed immedi-ately, or if required, maintained in cement cisterns for the purposeof further observations.

2.2. Stages of vitellogenesis and molting

The stages of vitellogenesis were characterized by consideringthe color of the ovary, the oocyte diameter (measured using ocularmicrometer to the nearest 1 lm) and the ovarian protein levels as

the criteria (after Sudha and Anilkumar, 1996, 2007). In the presentpaper, however, we have also adopted the terms such as ‘early’,‘mid’ and ‘late’ stages of ovarian growth; the Stage 1 (11–90 lmoocyte diameter) of vitellogenesis (after Sudha and Anilkumar,1996) has been referred to as ‘early’ ovarian growth (in the presentpaper), Stage 2 (91–155 lm oocyte diameter) as the ‘mid’ ovariangrowth, while Stages 3 and 4 (156–330 lm oocyte diameter) to-gether are considered as ‘late’ ovarian growth. Observations onthe texture of the carapace and the microscopic examination ofthe setogenic events of the abdominal pleopods and the maxillipedepipodites were used for characterizing the molt stages (afterSudha and Anilkumar, 1996, 2007; Suganthi and Anilkumar, 1999).

2.3. Dissection and fixation of the Y-organ

Till date, no report was available on the Y-organ of M. messor.Hence, we relied up on previous reviews (Waterman, 1960; Aotoet al., 1974; Lachaise et al., 1993) on the Y-organs of decapod crus-taceans (including the brachyurans), for a reasonable prediction onthe localization of the tissue. After having dissected out (by cuttingopen the carapace), the tissue was immediately fixed in 10% neu-tral buffered formalin (PBS, formaline pH 7.2) for histological andimmunohistochemical studies, and in 3% glutaraldehyde (in phos-phate buffer, pH 7.2) for electron microscopy.

2.4. Histology and immunohistochemistry

10% neutral buffered formalin-fixed tissue was used for histo-logical studies. Paraffin sections 5–7 lm thick, stained in hamat-oxylin-eosin, and were used for light microscopic observations(Pearse, 1968; Humason, 1974; Avwioro, 2011).

Procedure for immunohistochemistry has been adopted fromBirkenbeil (1983), and Seifert and Bidmon (1988). 5 lm thick par-affin sections, mounted on silane-coated slides, were used forimmunohistochemistry. The sections were deparaffinized in xy-lene, rehydrated in descending grades of ethanol and washed inMillipore water. The antigen retrieval steps were carried out inTris–EDTA (pH 8.0) buffer. The buffer was preheated to boilingtemperature for 5 min, then heating with the slides embedded init (at boiling temperature) for 5 min, and subsequently by bringingdown the temperature to 80 and 60 �C for 10 and 5 min respec-tively. The slides were cooled to room temperature and washedin TBS buffer (pH 7.4). Endogenous blocking (5% hydrogen peroxidein methanol) was done for 30 min in the dark and was againwashed in TBST (3% milk in TBS) for 5 min to block the non-specificbinding sites. The sections were then incubated with primary anti-body (2-succinyl conjugate of ecdysone, a generous gift from Pro-fessor Ernest S. Chang, University of California, USA) at 1:500dilutions for 30 min and washed in PBST for 5 min [prior to treat-ment with the antigen, the antiserum was subjected to titer assayto ensure optimal binding sensitivity; 1:500 was found to be themost appropriate dilution for the purpose]. Supersensitive polyHRP (Promega) was used as secondary antibody, in which theslides were again incubated for 30 min. Finally, it was washed inTBST and developed in DAB (Chromogen) and counterstained withhaematoxylin. The sections were dehydrated in ascending alcoholgrades, cleared in xylene and mounted with DPX.

The paraffin sections of the Y-organ, that did not receive theantibody treatment, were used as the controls, for the purpose ofcomparison.

2.5. Transmission electron microscopy

The tissue, after being fixed in 3% glutaraldehyde for 24 h, waswashed thoroughly with 0.1 M phosphate buffer, and post-fixed inosmium 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 (70–90%). Following dehydration in 90% ethanol, the sample was incu-bated in (freshly prepared) 2% ethanolic uranyl acetate (enblocstaining), and was subsequently dehydrated with 100% ethanol,followed by treatment in acetone for complete dehydration ofthe tissue. Propylene oxide was used as the clearing agent. Further,the tissue was left for infiltration for a minimum of 6 h in 1:1 mix-ture of propylene oxide and araldite followed by pure araldite for afurther period of 8 h. The tissue was then embedded in araldite andleft in an oven maintained at 60� undisturbed for 2 days for poly-merization (Williams and Carter, 2009). Semi-thin sections(1 lm), stained in 1% toluidine blue, were used to ensure the exactpositioning of the tissue, prior to ultra-thin (600 Å) sectioning to beused for electron microscopy.

2.6. Ecdysteroid assay

The ecdysteroid titer in the Y-organ and the hemolymph wasestimated through radioimmunoassay (RIA) as per the protocol

Fig. 1. (A) Photomicrograph showing the location and morphology of the Y-organ oHaematoxylin–eosin, depicting the characteristic, closely packed cell arrangements.

Fig. 2. (A) Light photomicrograph of the Y-organ of M. messor during August–December‘‘less dense’’ (vacuolated) zone, (i, ii) inset of the two zones (toluidine blue). (For interprweb version of this article.)

adopted from Chang and O’Connor (1979), Suganthi and Anilkumar(1999) and Sudha and Anilkumar (2007). Samples of the Y-organ(9–10 nos), weighing approximately 0.7–0.8 mg were used for eachassay. The dissected out tissue was quickly transferred to 80%ethanol placed in eppendorf vial maintained in ice. The tissue sam-ple was then homogenized and the homogenate was vacuum-evaporated, and processed for radioimmunoassy. Each of theeppendorf vials (with vacuum evaporated samples) received100 ll borate buffer (pH 8.4) and 100 ll of the radio ligand 3H-ecdysone (�12,000 dpm, Perkin-Elmer). Different aliquots of thestandards, comprising the radio-ligand (3H-edysone), thephosphate buffer and known quantities of the standard ecdysone(Sigma, USA) were also prepared and run along with the unknownsamples, for plotting the standard graph. All the vials (theunknown samples and the standards) were vortexed to allow ade-quate mixing. To each vial, 100 ll antiserum (2-succinyl conjugateof ecdysone) was added [the dilution of the antiserum (1:500) wasdetermined by titer assay as mentioned before]. The mixture wasthen incubated for 12 h at 4 �C, after which 200 ll saturated

f M. messor; (B) photomicrograph of paraffin section of the Y-organ stained in

season, showing clear differentiation between the outer dense zone and the inneretation of the references to colour in this figure legend, the reader is referred to the

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 for20 min to allow complete precipitation. The pellets, resultant ofthe antigen–antibody binding, were separated out by centrifuga-tion at 4800g for 20 min, and washed with 400 ll of 50% ammo-

Fig. 3. Electron micrograph showing the cellular organization of the Y-organ of M. messnucleolus (Nu), pyknotic nucleus (PN), secretory vesicles (SV) and micromitochondmacromitochondria (M).

Fig. 4. Cellular organization of the Y-organ of premolt crabs during January–May; (A) lighseason; (B, C) electron micrograph depicting the intense activity of the gland; note theribosomes (R); (D) Electron micrograph showing basal lamina (BL), intact nuclei (N) and

nium sulfate in borate buffer and centrifuged. The pellets werethen dissolved in 25 ll distilled water and mixed with 500 ll ofscintillation cocktail (0.8% PPO and 0.02% POPOP in a toluene:triton[2:1] mixture) and read using an LKB Liquid Scintillation Counter. A

or during August–December season; (A) overview of the gland with nuclei (N) andria (m); (B) electron micrograph shows golgi (G), micromitochondria (m) and

t micrograph showing the dense cytoplasm, characteristic of the Y-organ during thepresence of golgi (G), micromitochondria (m), macromitochondria (M) and the free

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 and4000 pg ecdysone on a semi-log paper, was used for accurate esti-mation of the ecdysteroid titer in the sample in question.

2.7. Statistical analysis

For assessing the statistical significance (if any) in the fluctua-tion of ecdysteroid levels, the data were subjected to F-test (ANO-VA) and the Students’s t-test using InStat 3 (GraphPad Version 2.0)software. In all the instances, null hypothesis was rejected at 95%confidence interval (p < 0.05).

3. Results

3.1. Localization of the Y-organ, and its morphology

The Y-organ in M. messor was located as minute, oval-shapedtissue (500–850 lm diameter; �X ± SD), between the mandibularexternal adductor muscle at the anterolateral edge of the branchialchamber. Morphologically, the Y-organ of M. messor appears as abi-convex mass of tissue with a characteristic white hue (Fig. 1A).

Fig. 5. Cellular organization of the Y-organ from intermolt females of January–May. (A) Lzone and the less dense central zone; (B) electron micrograph of the peripheral zone of

Fig. 6. (A) Light photomicrograph of the Y-organ of M. messor during June-July (molt–repr(B) electron micrograph showing a pyknotic nucleus.

3.2. Season-dependent fluctuations in gross morphology and structureof the Y-organ

In its gross morphology and size, the Y-organ of M. messorexhibited season-dependent changes. While the population wasnot engaging either in molting or in reproduction (June–July), theY-organ appeared as a minute white mass (540 ± 35.68 lm diame-ter; �X ± SD). The gland attained the maximum size (814 ± 66.49 lmdiameter; �X ± SD) during January–May (molt–reproduction sea-son), and acquired a creamy-yellow colour in premolt crabs. Thegland during the breeding season (August–December) was med-ium-sized (609 ± 45.36 lm in diameter; �X ± SD), but appeared lar-ger than that of the molt–reproductive inactive season. The sizedifferences of the Y-organ existing among these three seasons werefound to be statistically significant (F = 118.09; p < 0.0001).

In paraffin sections, the Y-organ encompassed closely packedcells, with basophilic nuclei (Fig. 1. B), which in some instances dis-played lobulated appearance. Ultrastructurally, the cells were seenarranged in irregular contours with clear cellular demarcations andbasal lamina (BL) (Figs. 3 and 4). The abundance of organelles suchas mitochondria, Golgi, and the secretory vesicles in electronmicrographs was dependent on the season in question at which

ight photomicrograph of the sections of the Y-organ depicting the dense peripheralthe Y-organ showing secretory vesicles (SV).

oduction inactive) season, showing the less dense cells that prevail the entire tissue;

86 S. Shyamal et al. / General and Comparative Endocrinology 196 (2014) 81–90

the tissue was harvested. The season-dependent variations instructure displayed by the Y-organ of M. messor could be describedas follows:

3.2.1. Reproductive (breeding) season (August–December)Almost the entire population is involved in breeding activity

during the season; all the females were seen to possess growingovaries (Stage 1–4, as described in ‘‘Materials and methods’’). His-tologically, during the August–December season, the Y-organ of M.messor was characterised by the occurrence of two distinct zones:(1) an outer zone wherein the cells appeared active, judged by theoccurrence of cytoplasmic basophilia (Fig. 2A, i), and (2) an inner‘‘less dense’’ zone, wherein the cells were with little or no activity,borne out of the less intense basophilia (Fig. 2A, ii). Under electron

Fig. 7. Immunohistochemical staining to demonstrate ecdysteroid synthesis in the Y-orgintense brown coloration; (B) the Y-organ of an intermolt crab of January–May season sAugust–December (breeding) season with medium levels of brown coloration; (D) the Y-othe tissue was not treated with primary antibody.

microscope, the cells of the outer zone appeared active, evinced bythe occurrence of golgi (G), secretory vesicles (SV) and mitochon-dria (M) (Fig. 3); the inner zone, however, displayed the presenceof highly vacuolated area (Fig. 2A, ii). In some instances though,the sections displayed the presence of pyknotic nuclei, a sign ofvery low or no synthetic activity. Significantly, the mitochondriawere of two size-classes; the smaller ones of 0.5–0.8 lm in diam-eter, the micromitochondria, and the larger ones of 1.2–1.8 lm indiameter, referred to as the macromitochondria (Fig. 3A) [namesadopted from Aoto et al., 1974; Lachaise et al., 1993]. The macro-mitochondria displayed the presence of tubular cristae, apparentlyrelated to intense activity. The Y-organ during the season has dis-played the presence of several micromitochondria; macromito-chondria, were also visible (Fig. 3B), although only in smaller

an: (A) the Y-organ of premolt crab during January–May season showing relativelyhowing medium levels of brown pigmentation of the cytoplasm; (C) the Y-organ ofrgan of June–July season with only sparse intensity in staining; (E) controls wherein

S. Shyamal et al. / General and Comparative Endocrinology 196 (2014) 81–90 87

numbers, when compared to the situation during the molt–repro-duction season (January–May).

3.2.2. Molt–reproduction season (January–May)During the season, the female population was seen to be in-

volved in either molt or reproduction. We have considered twomajor cohorts in the population: the one which has already en-tered premolt growth, but with inactive ovaries (with oocyte diam-eter < 11 lm), and the other, at intermolt stage, but havinggrowing ovaries (11–330 lm in diameter). Histologically, the Y-or-gan in general, displayed signs of remarkable activity during theseason. The inner ‘‘less dense’’ zone (as seen during the August–December, Fig. 2), was hardly visible in premolt individuals(Figs. 4A); the intermolt cohort, however, exhibited the presenceof the ‘‘less dense’’ zone (Fig. 5A). Ultrastructurally, the Y-organexhibited signs of activity during the season; in premolt crabs,the cells displayed very profuse presence of free ribosomes (R), gol-gi (G) and macromitochondria (M) (Fig. 4B, C). Among the two co-horts of the mitochondria, macromitochondria were moreprevalent during this season than it were during the August–December (Fig. 4B). In intermolt females, however, the golgi andthe secretory vesicles were less abundant, when compared withthose of the premolt ones (Fig. 5B).

3.2.3. Molt–reproduction inactive season (June–July)The Y-organ displayed the least levels of activity during June–

July season, when the population engaged neither in breedingnor molting [The entire population remained in intermolt, withtheir ovaries appearing as white bands (oocyter diameter < 11 lm),with no signs of yolk deposition during the season]. The cells of theY-organ during June–July season were inactive, judged by theoccurrence of highly vacuolated appearance in the cytoplasm(Fig. 6). The organelles such as free ribosomes, mitochondria andthe golgi, characteristic of the cells involved in synthetic activity,were hardly detectable in the Y-organ during the season.

3.3. Immunohistochemical observations

Immunostaining of the Y-organ to ecdysone revealed weakreaction (Fig. 7D) during June–July (when the population does

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

Hemolymph ecdysteroid levels(pg/ll)

Premolt Intermolt

D2/D3 D4 Ovarian g

January–May 94.75 ± 7.04 (20) 170.83 ± 12.5* (12) 10.35 ± 2August–Decembera – – 17.583 ± 6June–Julyb – – –

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

Y Organ ecdysteroid levels(pg/mg tissue)

Premolt Intermolt

D2/D3 D4 Ovarian grow

January–May 562.899 ± 2.72 (6) 1017.35 ± 5.79 (6) 234.903 ± 10.August–Decembera – – 240.902 ± 6.7June–Julyb – – –

Sample size is in parenthesis.Notes on a and b as in Table 1.

not engage in molt or reproduction), moderate reactivity(Fig. 7E) during August–December (intermolt crabs engaged inreproduction) and intense reaction (Fig. 7C) in premolt crabs(January–May), indicating a spurt of activity of the Y-organ duringthe season. The Y-organ of the intermolt crabs of January–Mayseason (involved in breeding activity), displayed positivity to theperoxidase reaction in fair levels, but less than the levels of premoltcrabs (Fig. 7B).

3.4. Fluctuations in hemolymph ecdysteroid levels

Hemolymph ecdysteroid levels of the female individuals fluctu-ated significantly with respect to various seasons and in responseto physiological parameters. The levels were significantly high dur-ing January–May (molt–reproduction) season, where a sizable pro-portion of the individuals were engaged in molting, attaining thepeak in those crabs at premolt (D2/D3 and D4) stages. The femalecrabs that were engaging in ovarian growth, however, showed asignificantly lower profile of ecdysteroids (p < 0.001), when com-pared with the premolt crabs (Table 1); the levels, however, didnot noticeably fluctuate during the reproductive stages (F = 1.01,p = 0.3044). Significantly, the hemolymph ecdysteroid titers ofthe reproducing females of August–December (breeding season)and those of January–May (molt–reproduction) seasons wereremarkably higher (p < 0.0001) than those during the June–July(molt–reproduction repose) season.

3.5. Fluctuations in ecdysteroid levels of the Y-organ

The fluctuations in ecdysteroid levels of the Y-organ of M. mes-sor with respect to seasons of molt and reproduction were verymuch in tune with those of the hemolymph. During the molt–reproduction season (January–May), the levels of ecdysteroids inthe Y-organ registered significant increase in the premolt crabs(early to late stages); the Y-organ of the late premolt (D4) crabshad the maximum ecdysteroid levels, but it declined thereafter,following exuviation (Table 2). Further, as in the case of hemo-lymph, the ecdysteroid titer of the Y-organ did not show remark-able fluctuation in response to stages of ovarian growth (Table 2)during August–December (breeding season, F = 0.765, p = 0.462)

oduction.

rowth (early) Ovarian growth (mid) Ovarian growth (late) Inactive

.04* (10) 27.12 ± 4.56* (7) 33.88 ± 6.89* (8) –

.09 (6) 27.36 ± 1.02 (6) 32.916 ± 2.18 (6) –– – 7.25 ± 2.91 (6)

tion.

th (early) Ovarian growth (mid) Ovarian growth (late) Inactive

93 (6) 283.899 ± 3.16 (6) 302.210 ± 4.38 (6) –1 (6) 291.688 ± 2.93 (6) 308.46 ± 3.92 (6) –

– – 118.218 ± 7.95 (6)

Table 3Consolidated 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 andreproduction.

Secretory activity ofY-organ

Premolt Intermolt

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.

88 S. Shyamal et al. / General and Comparative Endocrinology 196 (2014) 81–90

and January–May (molt–reproduction season, F = 1.014,p = 0.9591). Significantly, the Y-organ ecdysteroid level declinedfurther (p = 0.0025) during June–July, the season for molt–repro-duction repose (Table 2).

4. Discussion

In this first ever report on the localization, morphology andstructure of the Y-organ in the grapsid brachyuran crab (M. mes-sor), we depict the gland’s seasonal changes at histological, ultra-structural and immunohistochemical levels, supported with thedata on ecdysteroid assay. In almost all the previous occasions,the Y-organ was studied in the context of the cascade of events re-lated to molting. The present paper, on the other hand, adds to ourunderstanding on the gland’s function inasmuch as it describes thechanges in secretory activity through the entire annual cycle,involving molting and reproductive seasons.

In its gross morphology (oval in shape) and general structure (inthe nucleo-cytoplasmic ratio), the Y-organ of M. messor appearscomparable with the ‘‘Carcinus’’ type described by Lachaise et al.(1993), likely to be characteristic of brachyuran crabs in general.The Y-organ of M. messor, shows resemblance to Carcinus sp. inits ultrastructure as well (Lachaise et al., 1993), especially in termsof the characteristic presence of densely packed cells, basal laminaand the lobulated nuclei. It is well established that molting(growth) and reproduction in crustaceans are being successfullyprogrammed in the annual cycle by the up- and the down-regula-tion of the stimulatory as well as the inhibitory hormones(Echalier, 1955; Adiyodi, 1988; Hopkins, 1989, 2011; Subramon-iam and Kirubagaran, 2011). The paucity of information on theexact mechanisms involved in programming of these high en-ergy-demanding processes in a season-dependent fashion, hasprompted us to adopt in this paper, the strategy of assessing thesecretory activity of the Y-organ, to be collated with the ecdyster-oid titer (of the Y-organ and the hemolymph) throughout the an-nual cycle.

Our present study establishes the existence of perceptiblechanges in the Y-organ, in terms of its gross morphology (includingthe gland size) and structure in relation to various seasons of theyear (such as January–May, June–July and August–December sea-sons). The gland attains the maximum size during January–May(F = 118.09; p < 0.0001), the only season when active molting oc-curs among the population. An increase in the cytoplasmic volume

during premolt, as reported in Palaemon serratus (Le Roux, 1977)and Astacus astacus, (Birkenbeil and Gersch, 1979), could be themechanism of such size increase in M. messor as well. The Y-organhas the smallest size during June–July, a season for neither moltnor reproduction. This season-dependent fluctuation in the glandsize is concordant with its secretory activity (and the ecdysteroidproduction, evidenced by the immunohistochemical staining).The ‘‘en-bloc’’ vacuolated appearance of the tissue, coupled withthe pyknotic nuclei, dearth of organelles involved in secretoryactivity, and the lack of brownish hue and prevalence of the bluecolor seen in the immunohistochemical staining during June–July(Fig. 7D), is reminiscent of a total tardiness in the Y-organ activity.This vacuolated appearance seems quite comparable with whatwas reported in Carcinus maenas (Noel et al., 1989) during the stageof its inactivity. During January–May, on the other hand (when thepopulation is actively engaged in molt and reproduction), the glandcells are rich in mitochondria, free ribosomes and golgi, and thesecretory vesicles showing high synthetic activity (Fig. 4). Theoccurrence of the small-sized (micro-) and the large-sized(macro-) mitochondria is interesting, and invites an added atten-tion at this juncture. The profuse occurrence of macromitochondriain premolt crabs (January–May season) strongly supports intenseactivity of the Y-organ during the season. A comparable situationwas found to exist in the freshwater prawn, Palaemon paucidensand the brachyuran crab, Carcinus manaeus wherein the macromi-tochondria were considered to be involved in elevated syntheticactivity (Aoto et al., 1974). The results of our immuno-histochem-ical staining (against anti-ecdysone antibody), not onlydemonstrate that the secretory product of the Y-organ is ecdysone(Fig. 7), but it as well reinforce our findings on the season-dependent cyclicity in secretory activity. Superimposing thedata obtained from the present ultrastructural and immunohisto-chemical studies, on the results of ecdysteroid assay, makes itevident that the seasonal fluctuations in ecdysteroid levels are inconsonance with the seasonal secretory activity of the Y-organ(Table 3).

It is worth noticing that the Y-organ of M. messor demon-strates medium levels of activity during August–December, whenthe entire female population is engaged in breeding activity; thislevel of activity is somewhere between those of January–April(the maximum levels) and June–July (the minimum levels)seasons. The existence of a synthetically active outer zone ofcells (Fig. 2i,) evidenced by the relatively intense cytoplasmic

S. Shyamal et al. / General and Comparative Endocrinology 196 (2014) 81–90 89

basophilia (Fig. 2) and the presence of free ribosomes, golgi andthe seceretory vesicles (Figs. 3 and 4), support such a suggestion.The results of the ecdysteroid assay (Tables 1–3) are also in tunewith this finding. Under immunohistochemical staining, thepresence in medium levels of the characteristic brown color(due to peroxidase reaction) exhibited by the sections of theY-organ harvested from the reproductive females reiterates thiscontention. This finding raises an interesting question on thepossible role of the Y-organ (and its secretion) in crustaceanreproduction, a subject of scarce information. Arguably, theambiguity on ecdysteroids’ role in crustacean reproduction isbeing chiefly driven by the rich diversity existing among evenclosely allied phylogenic groups with respect to the program-ming of molt and reproduction. In brachyuran (true) crabs, forinstance, premolt growth and yolk deposition are mutuallyexclusive events (Bouchon and Souty-Grosset, 1992; Sudha andAnilkumar, 1996, 2007; Sudha et al., 2012); vitellogenesis is re-stricted in general to intermolt crabs only, which could evenimplicate that the premolt titre of ecdysteroids could restrainvitellogenesis. On the other hand, synergistic programming ofmolt and breeding activities is found to be characteristic of pal-aemonid shrimps wherein premolt growth is closely entrainedwith yolk deposition (Phlippen and Webster, 2000; Tsukimura,2001), suggestive of a possible stimulatory role for ecdysteroidsin ovarian growth. Our observation of medium levels of(ecdysteroid) secretory activity in the Y-organ of the intermoltcrabs harvested during the reproductive season (August–Decem-ber) as well as the growth-reproductive season (January–April)(also evidenced by the results of ecdysteroid assay, Table 1),seemingly ascertains that the ecdysteroid secretion by the Y-or-gan does not exert an inhibitory influence on reproduction inthis brachyuran decapod (M. messor). Further, the highly vacuo-lated appearance of the Y-organ during the June–July seasonstrongly suggests for a near-total down regulation of the ecdy-steroid secretion during a time when the population completelyrefrains from reproduction; importantly, the ecdysteroid levels ofthe Y-organ and the hemolymph were at its lowest during June–July season. This in turn suggests for a possible involvement ofthe Y-organ secretion (ecdysone) in reproduction of M. messor.Further, the ecdysteroid secretory activity of the Y-organ ofintermolt females engaging in reproduction, as it exceeds thoseof the reproductively inactive females of June–July season, seemsto suggest that the (medium levels of) activity of the Y-organ(and the circulating ecdysteroid levels) could be related to suc-cessful vitellogenesis in M messor.

5. Conclusion

Having exhibited discrete seasonality with respect to theprogramming of molt and reproduction, this brachyuran crab hasoffered us an excellent model to obtain a clear picture on the fluc-tuating secretory nature of the Y-organ and the ecdysteroid levelsall the way through the reproductive (August–December) as wellas the molt–reproduction active (January–May) and inactive (June– July) seasons. Considerable discrepancy exists in the secretoryactivity (judged from the ultrastructural and immunohistochemi-cal studies) of the Y-organ and the circulating ecdysteroid levels(estimated through radioimmunoassay) of the premolt crabs ofJanuary–May season and the intermolt crabs of the same season.This observation could very well manifest the gland’s involvementin molting. Further, the present seasonal study has also permittedus to touch upon its possible involvement in reproduction. The per-ceptible difference in the ecdysteroid levels and secretory activityof the Y-organ, existing between the intermolt crabs of the breed-ing (August–December) season, and those of the molt–reproduc-

tive inactive (June–July) season, deserves attention in thisrespect. As molting activity is practically nonexistent during Au-gust–December, and as the entire population is engaged in breed-ing, the higher secretory activity during August–December seasonencourages us to argue for the Y-organ’s possible involvement inreproduction. Regulation of crustacean vitellogenesis, by itself, isa topic wherein much ambiguity prevails. Methyl farnesoate (MF)is known to perform gonadotropic function in crustaceans, andthe recent molecular studies have demonstrated the existence ofyolk protein gene and its expression in the ovary (Wilder et al.,2002; Subramoniam and Kirubagaran, 2011). However, it is stillunclear about the possible existence of a putative hormone (otherthan MF) that could activate the yolk protein gene in crustaceans.This is very much pertinent in the context that 20 hydroxyecdy-sone is shown to stimulate the vitellogenin gene (PmVg1) expres-sion in Penaeus monodon (Tiu et al., 2006; Nagaraju, 2011) and thatthe steroids from vertebrates are shown to stimulate vitellogenesisin crustaceans (Yano et al., 2000; Lafont et al., 2005; Brown et al.,2009; Coccia et al., 2010; Nagaraju et al., 2011; Subramoniamand Kirubagaran, 2011). The importance of pursuing the role ofecdysteroids in crustacean reproduction in a more systematicway is being increasingly felt (Nagaraju, 2011; Sudha et al.,2012). Future studies involving (steroid) hormone–receptor–HREcascade should help us address this question very effectively. Per-tinently enough, that the studies using Ribonuclease Protection As-say (RPA) have demonstrated the occurrence of mRNA encodingthe ecdysteroid receptor (UpEcR) in the ovary of the fiddler crab,Uca pugilator (Durica et al., 2002; Sudha and Anilkumar, 2007),seem to suggest that ovary is a putative target for ecdysteroids.Further, through a recent, meticulously designed experiment, Dasand Durica, 2013 have successfully demonstrated the use of RNAifor the knockdown of the ecdysteroid receptor gene (EcR) in U. pug-ilator, for disrupting the mRNA levels, leading to growth arrest.Obviously, such experiments could offer us valuable inputs in fu-ture to demonstrate precisely the involvement (if any) of ecdyster-oids in crustacean reproduction.

Acknowledgments

We express our sincere thanks to Professor Ernest S. Chang,University of California, Davis (USA), for the generous gift of theecdysteroid antibody, Dr. Ramadasan Kuttan and Dr. Girija Kuttan,Amala Cancer Research Center (Kerala, India) for their help for theprovision of the LKB liquid scintillation counter. Thanks are alsodue to Mrs. Hemavathy, Mrs. Sunita (Research fellow) NIMHANS,Bangalore (India) for helping with the electron microscopy andimmunohistochemistry, Aneesh, Helna and Arshad the ResearchFellows of Sree Narayana College, Kannur (Kerala, India), for havinghelped us in procurement of live crabs.

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