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Histochemistry of the cuticle of the crab Menippe rumphii (Fabricius) (Crustacea: Brachyura) in relation to moulting
J. Exp. Mar. Biol. Eeoi., 1985, Vol. 88,
pp. 129-144
129
Elsevier
JEM470 HISTOCHEMISTRY
OF THE CUTICLE OF THE CRAB MENZPPE
RUMPHZZ (Fabricius) (CRUSTACEA : BRACHYURA)
IN RELATION
TO
MOULTING
D. ERRI BAN, K. HANUMA~HA
RAO, K. SHYAMASUNDARI
and D.V. UMA
DEVI debarment
of Zoology, Andhru Un~~ersj~, ~a~air-~3~ 003, India
(Received 6 August 1984; revision received 6 February 1985; accepted 8 February 1985)
Abstract: Histological and histochemical methods have been employed to study the formation and growth of the exoskeleton in relation to the moulting cycle of the crab Menippe rumphii (Fabricius). In the premoult condition the epidermal cells secrete a two-layered cuticle. Later these layers are widened by the secretions coming from the reserve cells, tegumental glands, and the Leydig cells. The fully formed cuticle of the intermoult crab is divisible into four layers, epicuticle, exocuticle, mesocuticle, and endocuticle. Histochemical observations on different cells have revealed that the tegumental glands secrete both neutral and acid mucopolysaccharides. The reserve cells are positive to PAS, BPB, Sudan Black B and Alizarin Red S techniques indicating the presence of carbohydrates, proteins, lipids, and mineral calcium. The Leydig cells are loaded with enzymes, including alkaline phosphatase, acid phosphatase, lipase, and phenoloxidase. Other histochemical tests have been employed to investigate the formation of different layers of the cuticle. Key words: histochemistry; cuticle; moulting; crab; Menippe rump&i
INTRODUCTION
In crabs and other crustaceans it is the outer exoskeleton which gives shape to the animal. This exoskeleton is frequently shed and reformed during the moulting cycle, which enables the animal to grow in size. Moulting is a physiological phenomenon during which period many changes take place both in the integument as well as in the internal organs. The moulting cycle is divided into live principal stages, Stage A, B, C, D, and E (Drach, 1939); the first four stages are again subdivided. With regard to the structure and formation of the exoskeleton, there is scanty literature on the brachyuran crustaceans. Travis (1955, 1957) has given some detailed information on the formation of the integumentary tissue in Pam&us. The role played by the hepatopancreas during this period has also been discussed in these papers. Dennell (1960) has reviewed the integument and exoskeleton of different crustaceans. The details of the structure and metabolism of the integumentary tissue in Gecarcinus lateralis has been given by Skinner (1962). Hackman (197 1) has illustrated the types of cuticles in different arthropods and their chemical nature. Aspects on the tanning process during moulting in different crustaceans have been studied in detail by both 0022”0981/85/$03.30 0 1985Elsevier Science Publishers B.V. (Biomedical Division)
130
D.ERRIBABU~~~~.
histochemical and biochemical methods (Stevenson & Adomako, 1967; Summers, 1967; Vacca& Fingerman, 1975; Barnes & Blackstock, 1976; Nellaiappan et al., 1982). The histolo~c~ details of the cuticle in the crab ~alline~t~s ~ffpi~~~have been studied by Johnson (1980). Recently Doughtie & Rao (1982) while studying the rosette glands in the gills of the shrimp Puluemonetes pugio, have illustrated the secretory activity of the glands in relation to moulting and also their probable role in the secretion of phenoloxidase. There is scanty info~ation on the histochemical details of the integumentary tissue, its formation and the composition of the tegumental glands. Although structurally these tegumental glands look like those of the salivary and intestinal glands (Erri Babu et al., 1979) they differ considerably in their composition as well as activity. The present paper deals with the details of the formation of the exoskeleton at different stages of moulting, the histoche~s~ of the different layers of the integument, and the role played by different gland cells in secretory activity as well as in the tanning of the newly forming cuticle.
MATERIAL AND
METHODS
Menippe rumphii, (Fabricius) males measuring 35 to 45 mm in carapace width were collected during the moulting season, April to July, and maintained in the laboratory in individual plastic troughs at a temperature of 22-24 “C. They were fed fish muscle and pieces of liver. The moulting stages of the crabs were assessed according to Drach (1939). The integumentary tissues were sampled in the premoult (Stages, Dl, D2, D3, and D4), postmoult (Stages Al, A2, Bl and B2) and intermoult periods (Stages Cl, C2, C3, and C4). Tissues were preserved in various fixatives such as Susa, Bouin’s, neutral buffered formalin, formol calcium, and processed (Humason, 1965; Culling, 1974). The exoskeleton of the intermoult crab was decalcified in Jenkin’s decalcifying fluid. Sections 8 to 10 pm thick were cut for all histological and h~stochemic~ preparations. For histological observations the sections were stained with Mallory’s tripple, Heidenhain’s Azan, and Delafield’s Haematoxylin/Eosin (Humason, 1965). A series of tests was made to study the histochemistry of cuticle formation and growth, including the role played by the tegumental glands and other cells associated with the cuticular epithelium. For histochemical studies (see Table I) on the enzymes, sections were cut at - 20 “C in a Cryocut, after fixing in chilled acetone and chilled formalin.
RESULTS
The intermoult exoskeleton is divisible chiefly into four layers: an outer epicuticle (0.006 mm thick), followed by exocuticle (0.069 mm thick), mesocuticle (0.003 mm thick) and endocuticle (0.432 mm thick) (Fig. 1). Underneath the exoskeleton is the
THE CUTICLE OF MEh’IPPE
RUMPHII
131
epidermis consisting of a single layer of cells and below this there is a thin layer of basement membr~e, which separates the epidermis from the body cavity. In M. mmphii the epicuticle is divisible into two layers. The outermost layer is very thin and is faintly stained with Azocarmine and Acid Fuchsin. This layer also shows positive reaction to SchitI’s reagent after periodic acid oxidation. The exocuticle is also divisible into two layers on the basis of staining qualities. The outer layer of exocuticle is faintly stained with Aniline Blue, whereas the inner layer is somewhat more darkly stained. The latter layer is intensely stained with Luxol Fast Blue in the copper phth~ocy~in reaction for phospholipids. These are represented as lon~tudinal rods. In contrast to this the upper layer lacked phospholipids and did not show any rod-like features. This quantity of phospholipid is not represented anywhere in the exoskeleton except for a trace in the endocuticle. The exocuticle and the endocuticle are separated by a very thin layer of mesocuticle which is highly positive to Azocarmine stain. The endocuticle was stained with Aniline Blue in both Mallory’s tripple and Heidenhain’s Azan techniques. Moreover this layer is again divisible into two layers on the mode of ~~gement of the protein filaments. The upper wider zone is represented by lamellae which are spaciously arranged and the inner layer is represented by lamellae which are more or less compact. On the other hand, the filaments of the wider zone are longer when compared with the filaments of the inner zone. The lamellae take a regular pattern in their arrangement. These lamellae show positive reaction towards proteins. Beneath the endocuticle there is a single layer of epidermis. It is represented by long columnar type of cells with a central nucleus. This layer is separated from the connective tissue of the interment by a thin basement membrane. Within the connective tissue the most conspicuous parts observed are the tegumental glands and the muscles. The tegumental glands are mostly localized in particular regions, although they may occur sparsely in other areas. Regardless of their location these glands are either rounded or oval. They typically contain six cells, although in some cases eight cells are present. All the cells are arranged in a rosette form. A duct leads from the centre of the gland to the epicuticle and opens there. The muscular arrangement in the connective tissue is also quite interesting. Some muscles are long and go through the basement membr~e and epidermal cells and are directly attached to the endocuticle. The other muscle tibres are small and are attached directly to the basement membrane. In addition to these structures there are setae which are regularly represented on the epicuticle. The setae are present on specific grooves, are spiny in nature, and are sometimes branched. These setae and the branching nature of them is particularly conspicuous during the premoult condition. The grooves are filled with secretions. There is a duct leading from the epidermal cell and it opens at the base of the seta. In addition to this there are other pore canals which traverse through the exoskeleton and open onto the epicuticle.
132
D. ERR1 BABU ETAL.
FORMATION OF THE INTEGUMENT
DURING THE PREMOULT PERIOD
The new cuticle begins to grow in the premoult period. At fast a thin layer of tissue is secreted just above the epidermis. This layer is positive to Schifl’s reagent, Azocarmine and Acid Fuchsin. Later this layer becomes enlarged and a two-layered structure is noticed. The widening process of the cuticle is taken up by a special type of cells of the connective tissue called the reserve cells. Some PAS positive material from these cells is seen to migrate through the epidermis as small granules. Later these granules are laid down on the newly forming epicuticle. These granules appear as a brush border on the epicuticle (Figs. 2,3). They are PAS positive and are digested by the salivary amylase in the early stages, suggesting the inclusion of glycogen; at a later stage these granules are resistant to saliva digestion. In subsequent stages tanning of these layers is seen. This is facilitated by another type of cells, called Leydig cells, which are also present in the connective tissue. These cells are mostly rounded in shape, but are smaller in size than the reserve cells. These cells contain two layers and a central lumen (Fig. 4). The lumen contains a homogeneous substance, which is faintly positive to PAS, ferric fe~cyanide reaction for S-H groups, Millon’s reaction for tyrosine, Permanganate~Alcian Blue reaction for S-S groups and copper phthalocyanin reaction for phospholipids. But in this reaction the walls are more positive than the lumen. The amino-acid tyrosine is important for quinone tanning of the cuticle. Leydig cells which are first present in the connective tissue and are loaded with their secretions, slowly migrate to the epidermal region, then they pass through the epidermis and are seen to liberate their contents by apical breakage (Fig. 4). At this stage the granular nature of the newly forming cuticle becomes softened and homogeneous, after the addition of the required tanning agents. Another important feature of the premoult cuticle is the formation of the spines or setae. The spines seem to form from the two-layered wall of the Leydig cells, after the release of their contents. So the base of the spines appear cup-shaped initially and to be two-layered. A connecting link appears between the setae and the epidermal cells as a canal, through which the epidermal secretions pass and are deposited at the base of the setae, which is sticky. It may be presumed that the tegumental glands also release some of their secretory material into the grooves of the spines, because these secretions give positive reactions to PAS and Alcian Blue at 2.5 pH, which are thought to be neutral mucopolysaccharides. The reserve cells gave positive reactions to proteins, lipids, carbohydrates, and mineral calcium. In the premoult period fust glycogen granules are deposited from the reserve cells over the newly forming cuticle. Later this glycogen is transformed into chitin, as glycogen is the precursor for chitin synthesis (Hackman, 1971). Above this chitin layer, proteins are deposited. Some of the glycogen may be combined with protein to form glycoproteins, as evidenced by a positive reaction towards Congo Red. In addition to this, some neutral mucopolysaccharides are also observed in the premoult cuticle, mostly in the upper layer. This neutral mucopolysaccharide is derived from the tegumental glands.
a e
#
I
Fig. 1. Transverse section of intermoult exoskeleton (Azan): ENC. endocuticle; EPC, epicuticie; EXC, exocuticle; MEC, mesocuticle. Fig. 2. Transverse section ofpremoult integument(PAS): ENC,endocuticle;EPC, epicuticle;GG, glycogen granules; PC, pore canal. Fig, 3. Transverse section of premoult integument (Azau): RC, reserve cell. Fig.4. Transverse section of earlypostmoult integument(Mallory):LC, Leydigcell; see the apicalbreakage of the Leydigcell (arrow) at the region of the brush border,
TABLE I Histochemical
tests applied to the integument: + + + , intensely positive; moderately positive; 5, traces; - , negative.
Histochemical test applied
Reacting substance
Red
Heidenhain’s Azan
AB pH 2S/PAS AB/Safranin Aldehyde Fuchsin Aldehyde Fuchsin/AB
ABjactive methyl&m/ saponification T&dine Blue
Congo Red Bromophenol Blue Millon’s reaction p-DMAB-nitrite KMnO,/AB Ferric ferricyanide
Glycoproteins Basic proteins Tyrosine Tryptophan s-s groups S-H groups
Copper phthalocyanin Sudan Black B Alizarin Red ‘S Von Kossa AB with 0.06 M MgCl,
Phospholipids Lipids Calcium Calcium Carboxyl and sulphated mucosubstances Weakly and strongly mucous substances Strongly sulphated mucous substances Highly sulphated connective tissue Keratin sulphate mucous Alkaline phosphatases
AB with 0.3 M M&l, AB with 0.5 M MgCl, AB with 0.5 M MgCl, AB with 0.9 M MgCl, Gomori’s method for alkaline phosphatase Gomori’s method for acid phosphatase Gomori’s method for lipase Smyth’s method for phenoloxidase
Blue
Red
Blue
Red
f + + * Purple
+ + + + Purple
Purple
Purple
_
+
Carboxylated mucins Sulphated and carboxylated mucous substance Sulphated, carboxylate or acid mucosubstance Metachromasia
AB/mild methyl&ion AB/active methylation
Orange.
? _
+
Glycogdn
Acid mucopolysaccharides Acid mucopolysaccharides Acid and neutral mucopolysaccharides Acid and neutral mucopolysaccharides Strongly acid mucopolysaccharides Sialomucins Sulphated and carboxylated mucous substances
Endocuticle
_ _ Blue
Carbohydrates
+ Purple Purple
Violet (wet) blueviolet (dry)
_ f _
+ Blue
_ _
_
* _
_
_
_
Violet
Blue
++ ++
+ _ _
Redviolet (wet) violet (dry) + _ * _ _ _ +++ +++ +++ +++
_ * +
_ _
+ +
+ +
+++ +++
_ _
+ f
Acid phosphatase
_
Lipase Phenoloxidase
+
+,
EXOCUtitle
blue Orange. blue + + + + Purple/ blue Purple/ blue _ _ Blue
Red
Mallory’s triple Periodic AcidiSchiff (PAS) PAS/saliva Al&n Blue (AB) pH 1.0 AB pH 2.5 AB pH l.O/PAS
Epicutitle
+ + , strongly positive;
_ _
_
*
_
_
_ _
_ ++
_ _
++
_
++ *
+ _
_ _
THE CUTICLE
Premoult Eprcutitle
Endocuticle
Red Red
Leydig cells
Blue
Red
Red
Blue
Red
Red
+++ ++
t + Blue
i+ + + Purple
Blue
Purple
++ +
Blue
Blue
_ _
Light violet (wet) blue (dry) + + _ _ _ _
+ + _
Deep purple Deep purple _
Light purple Light purple _
+ Purple
+ Purple
_ _
_ _
_
_
++ ++ _ _ ++
_ _
++ ++ ++ ++
_ +
+
+
_
+ + + + +
+
+ + _ _ _
*
_
_
f
_
_
_ +
++
+
++
+ +
+ +
_
_
Epicutitle
Exocutitle
Endocuticle
Reserve cells
Red
Red
Orange
Blue
Red
Humason
(1965)
Red
Red
Light blue + + + _
Light blue t
Red
Humason
(1965)
Purple
Light purple Light purple _
Fegumental glands
+ + +++ ++ Blue/ purple Blue/ purple Blue ++ Purple and blue + +
++ ++ _ * Blue Purple _ _
Purple/ blue _ _
Blue
t t _
Blue
_
+ +
_
Reference
Purple
Peruse Pearse Peruse Pearse Mowry
Purple
Mowry & Winkler (1956)
_
(1968) (1968) (1968) (1968) & Winkler (1956)
_ _
Spicer ef al. (1967) Spicer & Meyer (1960) Spicer & Meyer (1960)
_
Spicer (1960) Spicer er al. (1967)
_ _
_
Spicer & Lillie (1959) Violet
Blue
Red and violet
(wet) light violet (dry) + +
135
_ Blue
Blue
Red
_ _
RUMPHII
Postmoult
-
Reserve cells
-
OF MENIPPE
Blue
Pearse (1968)
Blue
(wet) light violet + _ _ _
++
(dry) i _
t tt
+
+
t
t t
t
?r + ++ + t
_
+ _
+t
_
tt +t t+ +t
++
* -I +
++
+
_
_
*
*
_
+ +
*
+ +
*
_
* + t ++ +t _
Pearse (1968) Mazia et al. (1953) Baker (1956) Adams (1957) Arvy & Gabe (1962) Chevremont & Frederic (1943) Kliiver & Barrera (1953) Chiflle & Putt (195 I) Bancroft (1975) Bancroft (1975) Bancroft (1975) Bancroft
(1975)
_
BancroR (1975)
_
Bancroft
_
(1975)
++
?
&
tt
_
Bancroft (1975) Culling (1974)
++
*
*
tt
_
Culling (1974)
_ _
Culling (1974) Smyth (1954)
+ ++
++
t t
t t
D. ERR1 BABU ETAL.
136 TEGUMENTAL
GLANDS
Striking features of the premoult integumentary tissue are the tegumental glands and their staining qualities. There are both large and small tegumental glands. Generally the larger glands are positive to PAS and smaller glands to Alcian Blue at 1.0 and 2.5 pH. Out of the six cells in each gland three cells secrete neutral mucopolysaccharides, and the remaining three secrete acidic mucous substances. In a majority of the glands the acid mucopolysaccharide secreting cells alternate with the neutral mucopolysaccharide secreting cells. Furthermore, some of the glands are completely secreting acid mucopolysaccharides, whereas some are secreting neutral mucopolysaccharides. These secretions are conveyed by fine ducts to the epicuticular region. The gland cells have vacuolar nature in their cytoplasm. The secretions appear as granules in the cells. So vacuoles and granules are simultaneously observed in the cells. FORMATION
OF THE EXOSKELETON
DURING
THE POSTMOULT
PERIOD
Immediately after ecdysis (Stages A and B) the thickening of the cuticle is noticed by further addition of chitin and protein. Even during the postmoult period (Stage Al) glycogen granules are seen initially but not later. Protein depositions are greater in the succeeding stages. As a result the size of the reserve cells decreases. In addition, calcification of the integument appears following ecdysis; calcium is deposited over the epicuticle, exocuticle, and the forming endocuticular layers of the postmoult exoskeleton. In Stage Cl a thin layer of chitin and protein are formed above the epidermis. This layer is not calcified and represents the membranous layer. The widening process of the membranous layer takes place in further stages, C2, C3. During Stage C2 another thin layer is formed over the exocuticle. This layer is negative to protein tests. It stains deep red in both Heidenhain’s Azan and Mallory’s tripple stains, indicating its nature to be of chitin. This layer is formed in a similar way as that of the premoult epicuticle, i.e., deposition of glycogen granules initially from the reserve cells and conversion of these into chitin. The exocuticle of the postecdysial integument is formed during the late part of Stage Cl and early part of C2. This layer is negative to protein tests, as is the epicuticle, but it gives a strong reaction to phospholipids. This phospholipid comes from the reserve cells of the connective tissue. The deposition of phospholipid is not uniform and as a result an uneven layer of phospholipid is formed over the exocuticle. Heavy calcification is also noticed in this layer. In the endocuticular region of the postmoult exoskeleton, the homogeneous nature slowly vanishes after the deposition of protein as lamellae. HISTOCHEMISTRY
The various histochemical results are presented in Table I. In the Periodic Acid Schiff (PAS) reaction for carbohydrates, unsaturated fatty acids, and phospholipids the premoult epicuticle and endocuticle, the epicuticle, exocuticle, and the endocuticle of the
THE CUTICLE OF MENIPPE
RUMPHII
137
postmoult and intermoult interments respond positively but with different intensities. The reserve cells of the connective tissue in the premoult condition stain intensely {see Fig. 3), whereas the same cells in the postmoult condition respond only moderately. A moderate positive reaction was also noticed in the tegumental glands. The PAS positivity in all the regions is resistant to saliva digestion, except for a change in intensity of staining in the reserve cells of the premoult condition, suggesting the presence of some glycogen in these cells. In addition, some glycogen granules are also observed over the newly forming epicuticle of the premoult animal. A strong positivity towards Alcian Blue at both 1.0 and 2.5 pH was noticed in the tegumental glands, suggesting the presence of rich qu~tities of mucopolysacch~ides. In the PAS/Alcian Blue reaction, (Fig. 5), for studying acid and neutral mucopolysaccharides, some of the glands were completely stained with Alcian Blue, but the majority showed mixed staining, indicating the presence of both neutral and acid mucopolysaccharides. With the Aldehyde Fuchsin/Alci~ Blue reaction for sulphated and carboxylated mucous substances, the glands stained purple or blue indicating both carboxylated and sulphated mucous substances. In the Safranin/Alcian Blue reaction the glands were alcianophilic, indicating the absence of strongly acid mucopolysaccharides. These various tests for mucopo@saccharides have been confirmed by employing Alcian Blue after mild methylation, active methylation followed by saponilication, and Alcian Blue with graded increase in magnesium chloride concentration in sodium acetate buffer at 5.8 pH. With the Toluidine Blue reaction for metachromasia all three types of metachromasia have been noticed. The metachromatic nature was more conspicuous in wet conditions rather than after dehydration. The tegumental glands stained red in both wet and dry sections, suggesting the presence of gamma metachromasia (Fig. 6). The epicuticle of the premoult condition was, however, light violet in wet conditions, but after dehydration turned blue (orthochromasia). The epicuticle of the postmoult and intermoult crabs was violet in wet conditions (beta metachromasia~ and blue in dry conditions. The exocuticle, however, showed beta metachromasia in both wet and dry conditions. Other regions showed orthochromasia. The pore canals stained blue but within the pore canals red and violet stained granules were present which were being conveyed to the epicuticle. These granules were seen at the base of the setae and in the grooves in which the setae were present. In the Congo Red reaction for amyloids, the epicuticle, endocuticle, and the reserve cells responded positively, but the epicuticle of the intermoult and postmoult crabs did not. In the Bromophenol Blue reaction for basic proteins the endocuticle of the intermoult and postmoult periods responded strongly together with the reserve cells. The epi- and endocuticle of the intermoult and postmoult periods responded strongly together with reserve cells. The epi- and endocuticle of the newly forming exoskeleton responded moderately to this reaction. With the ferric ferrocyanide reaction for S-H groups the endocuticle in all stages of moulting, the Leydig cells of the premoult integument, and the reserve cells gave a positive reaction. In the permanganate/Alcian Blue reaction for S-S groups the Leydig cells, the tegumental glands, the epi- and endocuticle of the
138
D. ERR1 BABU ETAL.
Fig. 5. Transverse se&on of integument &owing tegumental glands (PAS/Al&n Blue). Fig. 6. Transverse section of the integument showing gamma metachromasia (Tnluidine Blue). Fig. 7. Exocutick showing phospholipids (copper phthalocymin). Fig. S. Transverse section uf the postnauft integument showing alkaline phosphatase activity, Fig. 9. Transverse section of the postmault integument: LC, Leydig cells with lipase.
THE CUTICLEOF ~E~IPPE
RU~PHII
139
postmoult integument responded positively, whereas the epi-, exo- and endocuticle of the intermoult crabs did not respond to this reaction. With Millon’s reaction for tyrosine, a positive reaction was noticed in the Leydig cells and the exo- and endocuticle of the postmoult integument. Traces of tyrosine were also observed in the epi- and exocuticle of the intermoult crabs. Tryptophan was totally absent in all regions as indicated by a negative p-dimethylamino-benzaldehyde nitrite reaction. An intense reaction was noticed with copper phthalocyanin in the exocuticle of the intermoult crabs (Fig. 7) while the epicuticle is completely lacking any phospholipid. Less intense reaction was noticed in the postmoult exocuticle. An intense reaction was also noticed in the reserve cells of the premoult integument. No positive reaction was noticed for calcium in either the epi- or endocuticle of the premoult condition, but rich quantities were noticed in the reserve cells. The endocuticle and the exocuticle of the intermoult crab showed greater amounts of calcium deposits. Alkaline and acid phosphatase activity was found to be more pronounced in the premoult condition (Fig. 8). Lipase activity was also observed in the epi- and endocuticle layers of the premoult integument and in the exo- and endocuticular layers of the postmoult and intermoult crabs. The enzyme, phenoloxidase, was also noticed in the early postmoult period ofthe moult cycle. All the enzymes were found in the Leydig cells only (Fig. 9) and they were later conveyed to the epicuticular region for their activity. The movement of the Leydig cells and their breakage was also observed during the postmoult period. DWXSSION
In the exoskeleton ofthe late intermoult condition i.e. Stages C3-C4 of Drach (1939) there are four principal layers, a thin epicuticle, the exocuticle, thin mesocuticle, and a wider endocuticle. Beneath these layers, the basement membrane, epidermal layer and the connective tissue are found. Within the connective tissue there are large reserve cells, small Leydig cells and the flower-like tegumental glands. The epicuticle contains the spines or setae which are connected by pore canals from the epidermal cells. In addition to these pore canals there are other pore canals coming from the tegumental glands. These are very import~t for conveying the secretory material from the te~ment~ glands as well as from the epidermal cells. The epicuticle is stained red both with Heidenhain’s Azan and Mallory’s tripple staining procedures indicating its nature to be of chitin (Humason, 1965). On the other hand, this layer does not show any positive reaction to either proteins or lipids, but a moderate positivity is noticed with the PAS and Alcian Blue reaction for neutral mucopolysaccharides. The chitin nature of epicuticle has demonstrated earlier by Krishnan et al. (1955) and Krishnan (1956) in the arthropods Palamnaeus and Scolopendra. Hackman (1971) is of the view, however, that the epicuticle does not contain chitin. The presence of spines or setae in the epicuticle has been reported by several authors in different crustacean species (Travis, 1955; 1957; Dennell, 1960; Skinner, 1962; Hackman, 1971). The characteristic feature
140
D. ERR1 BABU ET&.
observed in Menippe ~~~~ii is that the spines are branched at the base (Fig. lo), although some are unbranched. This branching of the spines has not been reported earlier. The second layer is the exocuticle which is stained orange in Azan and Mallory’s triple techniques. This layer contains rich quantities of phospholipids which are distributed vertically. The layer does not contain carbohydrates or proteins except a moderate quantity of glycoproteins, as evidenced by a positive reaction to Congo Red. The presence of exocuticle in crustaceans has been indicated by Travis (1955), Dennell (1960), Skinner (1962), and Hackman (1971). Hackman (1971) reported protein and chitin in the exocuticle, however this layer was also calcified. In M~nip~ ~~~~~i the exocuticle is also calcified but no protein is observed in this layer. Instead there is a heavy concentration of phospholipid which has not been found in other crustacean species. The exocuticle is followed by a very thin layer of mesocuticle. This separates the exo- and endocuticles. The mesocuticle is stained deep red with Azocarmine and Acid Fuchsin. The presence of mesocuticle in crustaceans and its positivity towards Acid Fuchsin stain was described by Hackman (1971). The fourth layer is the endocuticle; this contains protein tibrils arranged in a laminated fashion. Three-quarters of this layer in the upper region are calcitied whereas the lower one-fourth region is not. As already indicated this non-calcified part represents the membranous layer. In Menr&e ~~~~ii this membranous layer starts its appearence as a thin membrane in Stage Cl and continues in further stages (Stages C2 and C3). According to Travis (1955,1957) the membr~ous layer or non-calcified layer does not apparently begin to be deposited before the third week following moult (Stage C3 of Drach) and by Stage C4 the membranous layer is fully formed. Yamaoka & Scheer (1971) have indicated that during Stage C3 the completion of the exoskeleton takes place and the membranous layer is deposited, this period lasting for 2-5 wk. A similar phenomenon was also reported in Astucus (Welinder, 1975). Observations on Menippe mnphii have shown that the origin and completion of the membranous layer varies significantly depending upon the age of the animal. The arrangement of protein fib& in these two zones is also not similar. The laminae of the endocuticle have been worked out earlier in ~o~ff~~ g~~~u~s (Drach, 1953). Dennell (1960) has given the structure of the laminae in Astucus. In M~nippe ~~p~ii glycoproteins are prominent in the endocuticle. The glycoprotein nature of the endocuticle in crustaceans has also been noted by Hackman (1971). The histochemical account of the formation of the different layers of the integument indicates that from Stage D2 onwards there is the formation of a two-layered cuticle initially secreted by the enlarged epidermal cells. Furthermore, these layers are strengthened by the addition of secretions from the reserve cells, Leydig cells, and the tegumental glands. Earlier studies on different crustaceans have already indicated the secretion of the epicuticle in the premoult condition and the formation of the epi-, exo-, meso-, and endocuticles during further stages of moulting, but the histophysiolo~c~ studies on the formation of these layers is very meagre. Initi~ly, the epi- and endocuticle
THE CUTICLE
OF MENZPPE
RUMPHZZ
141
of the premoult crabs are PAS-positive. The outer layer is more positive than the inner layer. Glycogen granules are seen on the epicuticle, which appears as a brush border; this glycogen is the precursor for chitin synthesis. The streaming of glycogen particles onto the epicuticle may be a fuation artifact, as reported by Johnson (1980). On the other hand, some of the glycogen may be combined with protein to form glycoprotein which also exists in these two layers, The reserve cells of the premoult integument are very big and are arranged nearer to the epidermis. These cells contain carbohydrates, proteins, lipids, and calcium (see Table I). Initially, carbohydrates are deposited on the epicuticle, then proteins are deposited and, in the early postmoult period, phospholipids are also deposited on the exocuticle. As a result the size of the reserve cells greatly decreases. In further stages calcium is also deposited (Figs. 11 and 12). Calcium granules are seen to migrate from the reserve cells through the epidermal cells and finally
Fig. 10. Transverse
section ofthe premoult
integument showing branching nature ofthe spines (Azan): ST’, spine. Fig. 11. Transverse section of the late premoult integument showing the migration of calcium granules through the epidermal cells (Von Kossa): CA, calcium. Fig. 12. Deposition of calcium in the exocuticle during the postmoult period (Von Kossa).
142
D. ERR1 BABU ETAL.
to the cuticular layers; that is why calcium granules are seen in the epidermal cells of the early postmoult crabs. After the deposition of glycogen and protein, the Leydig cells of the connective tissue pass to the exterior and deposit their contents over them. Leydig cells give positive reactions to tyrosine, cystine, and phenoloxidase which are involved in tanning of the newly forming exoskeleton. The addition of the Leydig cell secretions are mostly found during the postmoult period. As indicated earlier the spines are branched even in the premoult condition. The difference between the premoult and intermoult condition is that during the premoult condition the spines are present in detinite grooves or depressions formed in the epicuticle, but in the intermo~t condition the epicuticle is fully stretched and as a result the spines are not seen in grooves. Each spine is rather hollow, the outer wall shows orthochromasia, but the lumen and the branches show beta metachromasia. The grooves of the premoult epicuticle show some granular depositions which are stained violet with Toluidine Blue. These secretions perhaps come from the mucous glands present in the connective tissue. The spines present on the epicuticle may serve as mechanoreceptors in perceiving the movements of water, wave direction etc., which are of immense importance to the crab during the postmoult period. Nerve junctions were also observed between the epithelial cell and the nerve cell in the region of the spine. During moulting the crab does not come out of the protection of rocks and so avoids predators. That is why the spines are active in the early postmoult period; the branching of the spines and the granular secretions are also observed in excess during the early postmoult period. The grarmlar secretions at the base of the spines are not so conspicuous in the intermoult condition as the carapace is very hard and thick. The tegumental or mucous glands of the connective tissue are other prominent structures in the premoult and early postmoult crabs. These glands secrete acid mucopolysaccharides, which are both sulphated and carboxylated in nature. In addition, some of the gland cells also secrete neutral mucopolysacch~des. These secretions are conveyed through the pore canals, which are different from the pore canals of the spines. The secretions from the tegumental glands are observed only on the epi- and exocuticles. The acid mucous substances provide the siippery nature of the outer integument. The tegumental glands display gamma and beta metachromasia. The epi-, exo- and, endocuticle of the intermoult cuticle show beta metachromasia, whereas the endocuticular layer of the premoult cuticle shows gamma metachromasia. In the early postmoult condition only the exocuticle is betachromatic, whereas the outer layers are not metachromatic. Such tegumental giands have also been described by Yonge (1932), Travis (1955, 1957), Skinner (1962), and Dennell (1960) in the integument of arthropods, but the histochemical nature of these glands has not been studied. The presence of gamma metachromasia in the membranous and principal layers of PunuZirusargus during the premoult condition was, however, observed by Travis (1955). The structure of the tegumental gtands in the connective tissue of the integument is similar to that in the oesophageal and intestinal glands that are present in the connective tissue of the oesophagus and hindgut (Erri Babu et al., 1982). Erri Babu et al. (1979)
THE CUTICLE OF MENIPPE RUMPHII
143
observed the presence of acid and neutral mucopolysaccharides in the oesophageal glands of ~e~~~~e ~~p~jj. Although there is some similarity in structure, the number of cells in the oesophageal glands vary from 4 to 10, whereas the number of cells is restricted to a maximum of 8 in the tegumental glands. Regarding calcium deposition, in the premoult condition there is no calcium deposition of the two-layered integument, but the reserve cells are loaded with calcium deposits. In the postmoult condition calcium deposits could be seen first at the base of the epidermal cells, later these migrate to the apex and then move to the various layers for calcification (see Fig. 11). Only the exo- and endocuticles are highly calcified. The inner layer of the endocuticle is not calcitied. These observations are in agreement with those on Punulirus argus (Travis, 1955, 1957), and various other crustacean species (Dennell, 1960; Hackman, 1971). The presence of alkaline and acid phosphatase was observed during the tanning of the cuticular layers as well as during calcium deposition. ACKNOWLEDGEMENT
The first author is grateful to the Council of Scientific and Industrial Research for the financial assistance during the tenure of this work. REFERENCES ADAMS,C. W.M., 1957. A pdimethylamino benzaldehyde nitrite method for the histochemical demonstration of tryptophan and other related compounds. J. Clin. Pathol., Vol. 10, pp. 56-62. ARVY, L. & M. GABE, 1962, Histochemistry of the neurosecretory products of the pars intercerebralis of pterygote insects. In, Neurosecr~~on, edited by H. Heller & R.B. Clark, Academic Press, New York, pp. 33 l-344. BAKER,J. R., 1956. Histochemical recognition of phenols, especially tyrosine. Q. J. Microsc. Sci., Vol. 97, pp. 161-164. BANCROFT,J.D., 1975. Histochemical techniques. Butterworths, London, 348 pp. BARNES,H. & J. BLACKSTOCK,1976. Further observations on the biochemical composition of the cement of Lepasfascicula~s Ellis & Solander; electrophoretic examination of the protein moieties under various conditions. J. Exp. Mar. Biol. Ecot., Vol. 25, pp. 263-271. CHEVREMONT,M. & J. FREDERIC, 1943. Une nouvelle methode histochemique de mise en evidence des substances a fonction sulthydrille. Application a l’epiderme en poil et a la lavure. Arch. Biol., Vol. 54, pp. 589-605. CHIFFLLE,T. L. & F. A. PUTT, 1951. Propylene and ethylene glycol as solvents for Sudan IV and Sudan Black B. Stain Technol., Vol. 26, pp. 51-56. CULLING,C. F. A., 1974. Handbook of ~~topathoia~~a~and histochemicai techniques. Butterworths, London, 712 pp. DENNELL,R., 1960. Integument and exoskeleton. In, The physjolo~ of Crustacea, Vol. 1, edited by T. H. Waterman, Academic Press, New York, pp. 449-472. DOUGHTIE,D. G. & K. R. RAO, 1982. Rosette glands in the gills of the grass shrimp Palaemonetes pugio. 1. Comparative morphology, cyclical activity and innervation. J. Morphol., Vol. 171, pp. 41-67. DRACH,P., 1939. Mue et cycle d’intermue chez les Crustaces dtcapodes. Ann. Inst. Oceanogr. (Paris), N.S., Vol. 19, pp. 103-391. DRACH, P., 1953. Structure des lamelles cuticulaires chez les Crustacts. C. R. Acad. Sci., Vol. 237, pp. 1172-1774. ERRI BABU, D., K. SHYAMASUNDARI & K. HANUMANTHARAO, 1979. The structure and histochemistry of the oesophageal glands in the crab Menippe rumphii (Fabricius) (Crustacea : Brachyura). Proc. Indian Acad. Sci., Vol. 88B, pp. 217-285.
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D. ERRI BABU ETAL.
ERRI BABU, D., K. SHYAMASUNDARI & K. HANUMA~THARAO, 1982. Studies on the digestive system of the crab Men~pe rumphii (Fab~cius) (Crustacea: Brach~ra). J. Exp. Mar. Biot. Ecot., Vol. 58, pp. 175-191. HACKMAN,R. H., 1971. The integument of Arthropoda. In, Chemical zootoglr, Vol. 6. Arthropoda, part B, edited by M. Florkin & B.T. Scheer, Academic Press, New York, pp. l-62. HUMASON,G.L., 1965. Animal tissue techniques. W.H. Freeman & Co., London, 569 pp. JOHNSON, P.T., 1980. Histology of the blue crab, Callinectes sapidus: a model for the Decapoda. Praegar Publishers, New York, 456 pp. KLCIVER,H. & B. BARRERA,1953. A method for the combined staining of cells and fibres in the nervous system. .I. Neuropathot. Exp. Neurot., Vol. 12, pp. 400-402. KRISHNAN,G., 1956. The nature and composition of the epicuticle of some arthropods. Physiot. Zool., Vol. 29, pp. 324-337. KRISHNAN,G., G.N. RAMACHANDRAN & M. S. SANTANAM,1955. Occurrence of chitin in the epicuticle of an arachnid Patamneus swammerdami. Nature (London), Vol. 176, pp. 557-558. MAZIA, D., P. A. BREWER& M. ALFERT,1953. The cytochemical staining and measurement ofprotein with mercuric bromophenol blue. Biol. BUN. (Woods Hole, Mass.), Vol. 104, pp. 57-67. MOWRY,R. W. & C.H. WINKLER, 1956. The coloration of acidic carbohydrates of bacteria and fungi in tissue sections with special reference to capsules of C~~tococcus neoformans, Pneumococcus and Staphytococcus. Am. J. Puthot. Vol. 32, pp. 628-629. NELLAIAPPAN,K., T. THANGARAJ& K. RAMALINGAM,1982. Phenoloxidase activity and its rBle in cuticular sclerotization in a mole crab Emerita asiatica Milne Edwards. J. E-q. Mar. Biot. Ecot., Vol. 6 1, pp. 75-83. PEARSE,A. G. E., 1968. Hisfochemistry, theoretical and applied, Yal. 1. Churchill Livingstone, London, 3rd edition, 998 pp. SKINNER,D. M., 1962. The structure and metabolism of a crustacean integumentary tissue during a molt cycle. Biot. Bull. (Woods Hole, Mass.), Vol. 123, pp. 635-647. SMYTH, J. D., 1954. A technique for the histochemical demonstration of phenoloxidase and application to egg shell formation in helminth and bysus formation in Mytitus. Q. J. Mtcrosc. Ski., Vol. 95, pp. 139-152. SPICER,S. S., 1960. A correlative study ofthe histochemical properties of rodent acid mucopolysaccharides. 1. H~~ochem. Cyrochem., Vol. 8, pp. 18-35. SPICER, S. S., R. G. HORN, & T. J. LEPPI, 1967. H~~ochern~t~: of connective tissue mucopolysaccharides, symposium on connective &sue research methods, Vot. I?. The William & Wilkin Co. Baltimore, pp. 251-302. SPICER, S. S. & R. D. LILLIE, 1959. Saponification as a means of selectively reversing the methylation blockage of tissue basophilia. J. Histochem. Cytochem., Vol. 7, pp. 123-12.5. SPICER, S. S. & D. B. MEYER, 1960. Histochemical differentiation of acid mucopolysaccharides by means of combined Aldehyde Fuchsin/Alcian Blue staining. Am. J. Clin. Pathot., Vol. 33, pp. 453-460. STEVENSON,J. R. & ADOMAKO, 1967. Diphenoloxidase in the crayfish cuticle. Localisation and changes in activity during the moulting cycle. J. Insect Physiol., Vol. 13, pp. 1803-1811. SUMMERS,N.M., 1967. Cuticle sclerotisation and blood phenoloxidase in the fiddler crab, Uca pugnax. Camp. B&hem. Physiol., Vol. 23, pp. 129-138. TRAVIS, D. F., 1955. The mouhing cycle of the spiny lobster Panutirus argus Latrelle. II. Preecdysial histological and histochemical changes in the hepatopancreas and integumental tissues. Biot. Bull. (Woods Hole, Muss.), Vol. 108, pp. 88-112. TRAVIS, D.F., 1957. The moulting cycle of the spiny lobster Panutirus argus Latrelle. IV. Postecdysial histological and histochemical changes in the hepatopancreas and integumental tissues. Biot. Bull. (Woods Hole, Muss.), Vol. 113, pp. 451-479. VACCA,L. L. & M. FINGERMAN,1975. The mechanism of tanning in the fiddler crab, Uca pugilator. II. The cyclic appearance of tanning agents and attached carrier proteins in the blood during the moulting cycle. Comp. ~joehem. Physioi., Vol. 5 1B, pp. 483-487. WELINDER,B.S., 1975. The crustacean cuticle-II. Deposition of organic and inorganic material in the cuticle of Astacusfluviatfifs in the period after moulting. Comp. Biochem. Physiot., Vol. 51B, pp. 409-416. YAMAOKA,L. H. & B. T. SCHEER,1971. Chemistry of growth and development in crustaceans. In, Chemical zoology, Vol. 5, edited by M. Florkin & B.T. Scheer, Academic Press, New York, pp. 321-341. YONGE, C. M. 1932. On the nature and permeability of chitin. 1. The chitin lining the foregut of decapod Crustacea and the function of the tegumental glands. Proc. R. Sot. London, Vol. 3, pp. 298-329.
pp. 129-144
129
Elsevier
JEM470 HISTOCHEMISTRY
OF THE CUTICLE OF THE CRAB MENZPPE
RUMPHZZ (Fabricius) (CRUSTACEA : BRACHYURA)
IN RELATION
TO
MOULTING
D. ERRI BAN, K. HANUMA~HA
RAO, K. SHYAMASUNDARI
and D.V. UMA
DEVI debarment
of Zoology, Andhru Un~~ersj~, ~a~air-~3~ 003, India
(Received 6 August 1984; revision received 6 February 1985; accepted 8 February 1985)
Abstract: Histological and histochemical methods have been employed to study the formation and growth of the exoskeleton in relation to the moulting cycle of the crab Menippe rumphii (Fabricius). In the premoult condition the epidermal cells secrete a two-layered cuticle. Later these layers are widened by the secretions coming from the reserve cells, tegumental glands, and the Leydig cells. The fully formed cuticle of the intermoult crab is divisible into four layers, epicuticle, exocuticle, mesocuticle, and endocuticle. Histochemical observations on different cells have revealed that the tegumental glands secrete both neutral and acid mucopolysaccharides. The reserve cells are positive to PAS, BPB, Sudan Black B and Alizarin Red S techniques indicating the presence of carbohydrates, proteins, lipids, and mineral calcium. The Leydig cells are loaded with enzymes, including alkaline phosphatase, acid phosphatase, lipase, and phenoloxidase. Other histochemical tests have been employed to investigate the formation of different layers of the cuticle. Key words: histochemistry; cuticle; moulting; crab; Menippe rump&i
INTRODUCTION
In crabs and other crustaceans it is the outer exoskeleton which gives shape to the animal. This exoskeleton is frequently shed and reformed during the moulting cycle, which enables the animal to grow in size. Moulting is a physiological phenomenon during which period many changes take place both in the integument as well as in the internal organs. The moulting cycle is divided into live principal stages, Stage A, B, C, D, and E (Drach, 1939); the first four stages are again subdivided. With regard to the structure and formation of the exoskeleton, there is scanty literature on the brachyuran crustaceans. Travis (1955, 1957) has given some detailed information on the formation of the integumentary tissue in Pam&us. The role played by the hepatopancreas during this period has also been discussed in these papers. Dennell (1960) has reviewed the integument and exoskeleton of different crustaceans. The details of the structure and metabolism of the integumentary tissue in Gecarcinus lateralis has been given by Skinner (1962). Hackman (197 1) has illustrated the types of cuticles in different arthropods and their chemical nature. Aspects on the tanning process during moulting in different crustaceans have been studied in detail by both 0022”0981/85/$03.30 0 1985Elsevier Science Publishers B.V. (Biomedical Division)
130
D.ERRIBABU~~~~.
histochemical and biochemical methods (Stevenson & Adomako, 1967; Summers, 1967; Vacca& Fingerman, 1975; Barnes & Blackstock, 1976; Nellaiappan et al., 1982). The histolo~c~ details of the cuticle in the crab ~alline~t~s ~ffpi~~~have been studied by Johnson (1980). Recently Doughtie & Rao (1982) while studying the rosette glands in the gills of the shrimp Puluemonetes pugio, have illustrated the secretory activity of the glands in relation to moulting and also their probable role in the secretion of phenoloxidase. There is scanty info~ation on the histochemical details of the integumentary tissue, its formation and the composition of the tegumental glands. Although structurally these tegumental glands look like those of the salivary and intestinal glands (Erri Babu et al., 1979) they differ considerably in their composition as well as activity. The present paper deals with the details of the formation of the exoskeleton at different stages of moulting, the histoche~s~ of the different layers of the integument, and the role played by different gland cells in secretory activity as well as in the tanning of the newly forming cuticle.
MATERIAL AND
METHODS
Menippe rumphii, (Fabricius) males measuring 35 to 45 mm in carapace width were collected during the moulting season, April to July, and maintained in the laboratory in individual plastic troughs at a temperature of 22-24 “C. They were fed fish muscle and pieces of liver. The moulting stages of the crabs were assessed according to Drach (1939). The integumentary tissues were sampled in the premoult (Stages, Dl, D2, D3, and D4), postmoult (Stages Al, A2, Bl and B2) and intermoult periods (Stages Cl, C2, C3, and C4). Tissues were preserved in various fixatives such as Susa, Bouin’s, neutral buffered formalin, formol calcium, and processed (Humason, 1965; Culling, 1974). The exoskeleton of the intermoult crab was decalcified in Jenkin’s decalcifying fluid. Sections 8 to 10 pm thick were cut for all histological and h~stochemic~ preparations. For histological observations the sections were stained with Mallory’s tripple, Heidenhain’s Azan, and Delafield’s Haematoxylin/Eosin (Humason, 1965). A series of tests was made to study the histochemistry of cuticle formation and growth, including the role played by the tegumental glands and other cells associated with the cuticular epithelium. For histochemical studies (see Table I) on the enzymes, sections were cut at - 20 “C in a Cryocut, after fixing in chilled acetone and chilled formalin.
RESULTS
The intermoult exoskeleton is divisible chiefly into four layers: an outer epicuticle (0.006 mm thick), followed by exocuticle (0.069 mm thick), mesocuticle (0.003 mm thick) and endocuticle (0.432 mm thick) (Fig. 1). Underneath the exoskeleton is the
THE CUTICLE OF MEh’IPPE
RUMPHII
131
epidermis consisting of a single layer of cells and below this there is a thin layer of basement membr~e, which separates the epidermis from the body cavity. In M. mmphii the epicuticle is divisible into two layers. The outermost layer is very thin and is faintly stained with Azocarmine and Acid Fuchsin. This layer also shows positive reaction to SchitI’s reagent after periodic acid oxidation. The exocuticle is also divisible into two layers on the basis of staining qualities. The outer layer of exocuticle is faintly stained with Aniline Blue, whereas the inner layer is somewhat more darkly stained. The latter layer is intensely stained with Luxol Fast Blue in the copper phth~ocy~in reaction for phospholipids. These are represented as lon~tudinal rods. In contrast to this the upper layer lacked phospholipids and did not show any rod-like features. This quantity of phospholipid is not represented anywhere in the exoskeleton except for a trace in the endocuticle. The exocuticle and the endocuticle are separated by a very thin layer of mesocuticle which is highly positive to Azocarmine stain. The endocuticle was stained with Aniline Blue in both Mallory’s tripple and Heidenhain’s Azan techniques. Moreover this layer is again divisible into two layers on the mode of ~~gement of the protein filaments. The upper wider zone is represented by lamellae which are spaciously arranged and the inner layer is represented by lamellae which are more or less compact. On the other hand, the filaments of the wider zone are longer when compared with the filaments of the inner zone. The lamellae take a regular pattern in their arrangement. These lamellae show positive reaction towards proteins. Beneath the endocuticle there is a single layer of epidermis. It is represented by long columnar type of cells with a central nucleus. This layer is separated from the connective tissue of the interment by a thin basement membrane. Within the connective tissue the most conspicuous parts observed are the tegumental glands and the muscles. The tegumental glands are mostly localized in particular regions, although they may occur sparsely in other areas. Regardless of their location these glands are either rounded or oval. They typically contain six cells, although in some cases eight cells are present. All the cells are arranged in a rosette form. A duct leads from the centre of the gland to the epicuticle and opens there. The muscular arrangement in the connective tissue is also quite interesting. Some muscles are long and go through the basement membr~e and epidermal cells and are directly attached to the endocuticle. The other muscle tibres are small and are attached directly to the basement membrane. In addition to these structures there are setae which are regularly represented on the epicuticle. The setae are present on specific grooves, are spiny in nature, and are sometimes branched. These setae and the branching nature of them is particularly conspicuous during the premoult condition. The grooves are filled with secretions. There is a duct leading from the epidermal cell and it opens at the base of the seta. In addition to this there are other pore canals which traverse through the exoskeleton and open onto the epicuticle.
132
D. ERR1 BABU ETAL.
FORMATION OF THE INTEGUMENT
DURING THE PREMOULT PERIOD
The new cuticle begins to grow in the premoult period. At fast a thin layer of tissue is secreted just above the epidermis. This layer is positive to Schifl’s reagent, Azocarmine and Acid Fuchsin. Later this layer becomes enlarged and a two-layered structure is noticed. The widening process of the cuticle is taken up by a special type of cells of the connective tissue called the reserve cells. Some PAS positive material from these cells is seen to migrate through the epidermis as small granules. Later these granules are laid down on the newly forming epicuticle. These granules appear as a brush border on the epicuticle (Figs. 2,3). They are PAS positive and are digested by the salivary amylase in the early stages, suggesting the inclusion of glycogen; at a later stage these granules are resistant to saliva digestion. In subsequent stages tanning of these layers is seen. This is facilitated by another type of cells, called Leydig cells, which are also present in the connective tissue. These cells are mostly rounded in shape, but are smaller in size than the reserve cells. These cells contain two layers and a central lumen (Fig. 4). The lumen contains a homogeneous substance, which is faintly positive to PAS, ferric fe~cyanide reaction for S-H groups, Millon’s reaction for tyrosine, Permanganate~Alcian Blue reaction for S-S groups and copper phthalocyanin reaction for phospholipids. But in this reaction the walls are more positive than the lumen. The amino-acid tyrosine is important for quinone tanning of the cuticle. Leydig cells which are first present in the connective tissue and are loaded with their secretions, slowly migrate to the epidermal region, then they pass through the epidermis and are seen to liberate their contents by apical breakage (Fig. 4). At this stage the granular nature of the newly forming cuticle becomes softened and homogeneous, after the addition of the required tanning agents. Another important feature of the premoult cuticle is the formation of the spines or setae. The spines seem to form from the two-layered wall of the Leydig cells, after the release of their contents. So the base of the spines appear cup-shaped initially and to be two-layered. A connecting link appears between the setae and the epidermal cells as a canal, through which the epidermal secretions pass and are deposited at the base of the setae, which is sticky. It may be presumed that the tegumental glands also release some of their secretory material into the grooves of the spines, because these secretions give positive reactions to PAS and Alcian Blue at 2.5 pH, which are thought to be neutral mucopolysaccharides. The reserve cells gave positive reactions to proteins, lipids, carbohydrates, and mineral calcium. In the premoult period fust glycogen granules are deposited from the reserve cells over the newly forming cuticle. Later this glycogen is transformed into chitin, as glycogen is the precursor for chitin synthesis (Hackman, 1971). Above this chitin layer, proteins are deposited. Some of the glycogen may be combined with protein to form glycoproteins, as evidenced by a positive reaction towards Congo Red. In addition to this, some neutral mucopolysaccharides are also observed in the premoult cuticle, mostly in the upper layer. This neutral mucopolysaccharide is derived from the tegumental glands.
a e
#
I
Fig. 1. Transverse section of intermoult exoskeleton (Azan): ENC. endocuticle; EPC, epicuticie; EXC, exocuticle; MEC, mesocuticle. Fig. 2. Transverse section ofpremoult integument(PAS): ENC,endocuticle;EPC, epicuticle;GG, glycogen granules; PC, pore canal. Fig, 3. Transverse section of premoult integument (Azau): RC, reserve cell. Fig.4. Transverse section of earlypostmoult integument(Mallory):LC, Leydigcell; see the apicalbreakage of the Leydigcell (arrow) at the region of the brush border,
TABLE I Histochemical
tests applied to the integument: + + + , intensely positive; moderately positive; 5, traces; - , negative.
Histochemical test applied
Reacting substance
Red
Heidenhain’s Azan
AB pH 2S/PAS AB/Safranin Aldehyde Fuchsin Aldehyde Fuchsin/AB
ABjactive methyl&m/ saponification T&dine Blue
Congo Red Bromophenol Blue Millon’s reaction p-DMAB-nitrite KMnO,/AB Ferric ferricyanide
Glycoproteins Basic proteins Tyrosine Tryptophan s-s groups S-H groups
Copper phthalocyanin Sudan Black B Alizarin Red ‘S Von Kossa AB with 0.06 M MgCl,
Phospholipids Lipids Calcium Calcium Carboxyl and sulphated mucosubstances Weakly and strongly mucous substances Strongly sulphated mucous substances Highly sulphated connective tissue Keratin sulphate mucous Alkaline phosphatases
AB with 0.3 M M&l, AB with 0.5 M MgCl, AB with 0.5 M MgCl, AB with 0.9 M MgCl, Gomori’s method for alkaline phosphatase Gomori’s method for acid phosphatase Gomori’s method for lipase Smyth’s method for phenoloxidase
Blue
Red
Blue
Red
f + + * Purple
+ + + + Purple
Purple
Purple
_
+
Carboxylated mucins Sulphated and carboxylated mucous substance Sulphated, carboxylate or acid mucosubstance Metachromasia
AB/mild methyl&ion AB/active methylation
Orange.
? _
+
Glycogdn
Acid mucopolysaccharides Acid mucopolysaccharides Acid and neutral mucopolysaccharides Acid and neutral mucopolysaccharides Strongly acid mucopolysaccharides Sialomucins Sulphated and carboxylated mucous substances
Endocuticle
_ _ Blue
Carbohydrates
+ Purple Purple
Violet (wet) blueviolet (dry)
_ f _
+ Blue
_ _
_
* _
_
_
_
Violet
Blue
++ ++
+ _ _
Redviolet (wet) violet (dry) + _ * _ _ _ +++ +++ +++ +++
_ * +
_ _
+ +
+ +
+++ +++
_ _
+ f
Acid phosphatase
_
Lipase Phenoloxidase
+
+,
EXOCUtitle
blue Orange. blue + + + + Purple/ blue Purple/ blue _ _ Blue
Red
Mallory’s triple Periodic AcidiSchiff (PAS) PAS/saliva Al&n Blue (AB) pH 1.0 AB pH 2.5 AB pH l.O/PAS
Epicutitle
+ + , strongly positive;
_ _
_
*
_
_
_ _
_ ++
_ _
++
_
++ *
+ _
_ _
THE CUTICLE
Premoult Eprcutitle
Endocuticle
Red Red
Leydig cells
Blue
Red
Red
Blue
Red
Red
+++ ++
t + Blue
i+ + + Purple
Blue
Purple
++ +
Blue
Blue
_ _
Light violet (wet) blue (dry) + + _ _ _ _
+ + _
Deep purple Deep purple _
Light purple Light purple _
+ Purple
+ Purple
_ _
_ _
_
_
++ ++ _ _ ++
_ _
++ ++ ++ ++
_ +
+
+
_
+ + + + +
+
+ + _ _ _
*
_
_
f
_
_
_ +
++
+
++
+ +
+ +
_
_
Epicutitle
Exocutitle
Endocuticle
Reserve cells
Red
Red
Orange
Blue
Red
Humason
(1965)
Red
Red
Light blue + + + _
Light blue t
Red
Humason
(1965)
Purple
Light purple Light purple _
Fegumental glands
+ + +++ ++ Blue/ purple Blue/ purple Blue ++ Purple and blue + +
++ ++ _ * Blue Purple _ _
Purple/ blue _ _
Blue
t t _
Blue
_
+ +
_
Reference
Purple
Peruse Pearse Peruse Pearse Mowry
Purple
Mowry & Winkler (1956)
_
(1968) (1968) (1968) (1968) & Winkler (1956)
_ _
Spicer ef al. (1967) Spicer & Meyer (1960) Spicer & Meyer (1960)
_
Spicer (1960) Spicer er al. (1967)
_ _
_
Spicer & Lillie (1959) Violet
Blue
Red and violet
(wet) light violet (dry) + +
135
_ Blue
Blue
Red
_ _
RUMPHII
Postmoult
-
Reserve cells
-
OF MENIPPE
Blue
Pearse (1968)
Blue
(wet) light violet + _ _ _
++
(dry) i _
t tt
+
+
t
t t
t
?r + ++ + t
_
+ _
+t
_
tt +t t+ +t
++
* -I +
++
+
_
_
*
*
_
+ +
*
+ +
*
_
* + t ++ +t _
Pearse (1968) Mazia et al. (1953) Baker (1956) Adams (1957) Arvy & Gabe (1962) Chevremont & Frederic (1943) Kliiver & Barrera (1953) Chiflle & Putt (195 I) Bancroft (1975) Bancroft (1975) Bancroft (1975) Bancroft
(1975)
_
BancroR (1975)
_
Bancroft
_
(1975)
++
?
&
tt
_
Bancroft (1975) Culling (1974)
++
*
*
tt
_
Culling (1974)
_ _
Culling (1974) Smyth (1954)
+ ++
++
t t
t t
D. ERR1 BABU ETAL.
136 TEGUMENTAL
GLANDS
Striking features of the premoult integumentary tissue are the tegumental glands and their staining qualities. There are both large and small tegumental glands. Generally the larger glands are positive to PAS and smaller glands to Alcian Blue at 1.0 and 2.5 pH. Out of the six cells in each gland three cells secrete neutral mucopolysaccharides, and the remaining three secrete acidic mucous substances. In a majority of the glands the acid mucopolysaccharide secreting cells alternate with the neutral mucopolysaccharide secreting cells. Furthermore, some of the glands are completely secreting acid mucopolysaccharides, whereas some are secreting neutral mucopolysaccharides. These secretions are conveyed by fine ducts to the epicuticular region. The gland cells have vacuolar nature in their cytoplasm. The secretions appear as granules in the cells. So vacuoles and granules are simultaneously observed in the cells. FORMATION
OF THE EXOSKELETON
DURING
THE POSTMOULT
PERIOD
Immediately after ecdysis (Stages A and B) the thickening of the cuticle is noticed by further addition of chitin and protein. Even during the postmoult period (Stage Al) glycogen granules are seen initially but not later. Protein depositions are greater in the succeeding stages. As a result the size of the reserve cells decreases. In addition, calcification of the integument appears following ecdysis; calcium is deposited over the epicuticle, exocuticle, and the forming endocuticular layers of the postmoult exoskeleton. In Stage Cl a thin layer of chitin and protein are formed above the epidermis. This layer is not calcified and represents the membranous layer. The widening process of the membranous layer takes place in further stages, C2, C3. During Stage C2 another thin layer is formed over the exocuticle. This layer is negative to protein tests. It stains deep red in both Heidenhain’s Azan and Mallory’s tripple stains, indicating its nature to be of chitin. This layer is formed in a similar way as that of the premoult epicuticle, i.e., deposition of glycogen granules initially from the reserve cells and conversion of these into chitin. The exocuticle of the postecdysial integument is formed during the late part of Stage Cl and early part of C2. This layer is negative to protein tests, as is the epicuticle, but it gives a strong reaction to phospholipids. This phospholipid comes from the reserve cells of the connective tissue. The deposition of phospholipid is not uniform and as a result an uneven layer of phospholipid is formed over the exocuticle. Heavy calcification is also noticed in this layer. In the endocuticular region of the postmoult exoskeleton, the homogeneous nature slowly vanishes after the deposition of protein as lamellae. HISTOCHEMISTRY
The various histochemical results are presented in Table I. In the Periodic Acid Schiff (PAS) reaction for carbohydrates, unsaturated fatty acids, and phospholipids the premoult epicuticle and endocuticle, the epicuticle, exocuticle, and the endocuticle of the
THE CUTICLE OF MENIPPE
RUMPHII
137
postmoult and intermoult interments respond positively but with different intensities. The reserve cells of the connective tissue in the premoult condition stain intensely {see Fig. 3), whereas the same cells in the postmoult condition respond only moderately. A moderate positive reaction was also noticed in the tegumental glands. The PAS positivity in all the regions is resistant to saliva digestion, except for a change in intensity of staining in the reserve cells of the premoult condition, suggesting the presence of some glycogen in these cells. In addition, some glycogen granules are also observed over the newly forming epicuticle of the premoult animal. A strong positivity towards Alcian Blue at both 1.0 and 2.5 pH was noticed in the tegumental glands, suggesting the presence of rich qu~tities of mucopolysacch~ides. In the PAS/Alcian Blue reaction, (Fig. 5), for studying acid and neutral mucopolysaccharides, some of the glands were completely stained with Alcian Blue, but the majority showed mixed staining, indicating the presence of both neutral and acid mucopolysaccharides. With the Aldehyde Fuchsin/Alci~ Blue reaction for sulphated and carboxylated mucous substances, the glands stained purple or blue indicating both carboxylated and sulphated mucous substances. In the Safranin/Alcian Blue reaction the glands were alcianophilic, indicating the absence of strongly acid mucopolysaccharides. These various tests for mucopo@saccharides have been confirmed by employing Alcian Blue after mild methylation, active methylation followed by saponilication, and Alcian Blue with graded increase in magnesium chloride concentration in sodium acetate buffer at 5.8 pH. With the Toluidine Blue reaction for metachromasia all three types of metachromasia have been noticed. The metachromatic nature was more conspicuous in wet conditions rather than after dehydration. The tegumental glands stained red in both wet and dry sections, suggesting the presence of gamma metachromasia (Fig. 6). The epicuticle of the premoult condition was, however, light violet in wet conditions, but after dehydration turned blue (orthochromasia). The epicuticle of the postmoult and intermoult crabs was violet in wet conditions (beta metachromasia~ and blue in dry conditions. The exocuticle, however, showed beta metachromasia in both wet and dry conditions. Other regions showed orthochromasia. The pore canals stained blue but within the pore canals red and violet stained granules were present which were being conveyed to the epicuticle. These granules were seen at the base of the setae and in the grooves in which the setae were present. In the Congo Red reaction for amyloids, the epicuticle, endocuticle, and the reserve cells responded positively, but the epicuticle of the intermoult and postmoult crabs did not. In the Bromophenol Blue reaction for basic proteins the endocuticle of the intermoult and postmoult periods responded strongly together with the reserve cells. The epi- and endocuticle of the intermoult and postmoult periods responded strongly together with reserve cells. The epi- and endocuticle of the newly forming exoskeleton responded moderately to this reaction. With the ferric ferrocyanide reaction for S-H groups the endocuticle in all stages of moulting, the Leydig cells of the premoult integument, and the reserve cells gave a positive reaction. In the permanganate/Alcian Blue reaction for S-S groups the Leydig cells, the tegumental glands, the epi- and endocuticle of the
138
D. ERR1 BABU ETAL.
Fig. 5. Transverse se&on of integument &owing tegumental glands (PAS/Al&n Blue). Fig. 6. Transverse section of the integument showing gamma metachromasia (Tnluidine Blue). Fig. 7. Exocutick showing phospholipids (copper phthalocymin). Fig. S. Transverse section uf the postnauft integument showing alkaline phosphatase activity, Fig. 9. Transverse section of the postmault integument: LC, Leydig cells with lipase.
THE CUTICLEOF ~E~IPPE
RU~PHII
139
postmoult integument responded positively, whereas the epi-, exo- and endocuticle of the intermoult crabs did not respond to this reaction. With Millon’s reaction for tyrosine, a positive reaction was noticed in the Leydig cells and the exo- and endocuticle of the postmoult integument. Traces of tyrosine were also observed in the epi- and exocuticle of the intermoult crabs. Tryptophan was totally absent in all regions as indicated by a negative p-dimethylamino-benzaldehyde nitrite reaction. An intense reaction was noticed with copper phthalocyanin in the exocuticle of the intermoult crabs (Fig. 7) while the epicuticle is completely lacking any phospholipid. Less intense reaction was noticed in the postmoult exocuticle. An intense reaction was also noticed in the reserve cells of the premoult integument. No positive reaction was noticed for calcium in either the epi- or endocuticle of the premoult condition, but rich quantities were noticed in the reserve cells. The endocuticle and the exocuticle of the intermoult crab showed greater amounts of calcium deposits. Alkaline and acid phosphatase activity was found to be more pronounced in the premoult condition (Fig. 8). Lipase activity was also observed in the epi- and endocuticle layers of the premoult integument and in the exo- and endocuticular layers of the postmoult and intermoult crabs. The enzyme, phenoloxidase, was also noticed in the early postmoult period ofthe moult cycle. All the enzymes were found in the Leydig cells only (Fig. 9) and they were later conveyed to the epicuticular region for their activity. The movement of the Leydig cells and their breakage was also observed during the postmoult period. DWXSSION
In the exoskeleton ofthe late intermoult condition i.e. Stages C3-C4 of Drach (1939) there are four principal layers, a thin epicuticle, the exocuticle, thin mesocuticle, and a wider endocuticle. Beneath these layers, the basement membrane, epidermal layer and the connective tissue are found. Within the connective tissue there are large reserve cells, small Leydig cells and the flower-like tegumental glands. The epicuticle contains the spines or setae which are connected by pore canals from the epidermal cells. In addition to these pore canals there are other pore canals coming from the tegumental glands. These are very import~t for conveying the secretory material from the te~ment~ glands as well as from the epidermal cells. The epicuticle is stained red both with Heidenhain’s Azan and Mallory’s tripple staining procedures indicating its nature to be of chitin (Humason, 1965). On the other hand, this layer does not show any positive reaction to either proteins or lipids, but a moderate positivity is noticed with the PAS and Alcian Blue reaction for neutral mucopolysaccharides. The chitin nature of epicuticle has demonstrated earlier by Krishnan et al. (1955) and Krishnan (1956) in the arthropods Palamnaeus and Scolopendra. Hackman (1971) is of the view, however, that the epicuticle does not contain chitin. The presence of spines or setae in the epicuticle has been reported by several authors in different crustacean species (Travis, 1955; 1957; Dennell, 1960; Skinner, 1962; Hackman, 1971). The characteristic feature
140
D. ERR1 BABU ET&.
observed in Menippe ~~~~ii is that the spines are branched at the base (Fig. lo), although some are unbranched. This branching of the spines has not been reported earlier. The second layer is the exocuticle which is stained orange in Azan and Mallory’s triple techniques. This layer contains rich quantities of phospholipids which are distributed vertically. The layer does not contain carbohydrates or proteins except a moderate quantity of glycoproteins, as evidenced by a positive reaction to Congo Red. The presence of exocuticle in crustaceans has been indicated by Travis (1955), Dennell (1960), Skinner (1962), and Hackman (1971). Hackman (1971) reported protein and chitin in the exocuticle, however this layer was also calcified. In M~nip~ ~~~~~i the exocuticle is also calcified but no protein is observed in this layer. Instead there is a heavy concentration of phospholipid which has not been found in other crustacean species. The exocuticle is followed by a very thin layer of mesocuticle. This separates the exo- and endocuticles. The mesocuticle is stained deep red with Azocarmine and Acid Fuchsin. The presence of mesocuticle in crustaceans and its positivity towards Acid Fuchsin stain was described by Hackman (1971). The fourth layer is the endocuticle; this contains protein tibrils arranged in a laminated fashion. Three-quarters of this layer in the upper region are calcitied whereas the lower one-fourth region is not. As already indicated this non-calcified part represents the membranous layer. In Menr&e ~~~~ii this membranous layer starts its appearence as a thin membrane in Stage Cl and continues in further stages (Stages C2 and C3). According to Travis (1955,1957) the membr~ous layer or non-calcified layer does not apparently begin to be deposited before the third week following moult (Stage C3 of Drach) and by Stage C4 the membranous layer is fully formed. Yamaoka & Scheer (1971) have indicated that during Stage C3 the completion of the exoskeleton takes place and the membranous layer is deposited, this period lasting for 2-5 wk. A similar phenomenon was also reported in Astucus (Welinder, 1975). Observations on Menippe mnphii have shown that the origin and completion of the membranous layer varies significantly depending upon the age of the animal. The arrangement of protein fib& in these two zones is also not similar. The laminae of the endocuticle have been worked out earlier in ~o~ff~~ g~~~u~s (Drach, 1953). Dennell (1960) has given the structure of the laminae in Astucus. In M~nippe ~~p~ii glycoproteins are prominent in the endocuticle. The glycoprotein nature of the endocuticle in crustaceans has also been noted by Hackman (1971). The histochemical account of the formation of the different layers of the integument indicates that from Stage D2 onwards there is the formation of a two-layered cuticle initially secreted by the enlarged epidermal cells. Furthermore, these layers are strengthened by the addition of secretions from the reserve cells, Leydig cells, and the tegumental glands. Earlier studies on different crustaceans have already indicated the secretion of the epicuticle in the premoult condition and the formation of the epi-, exo-, meso-, and endocuticles during further stages of moulting, but the histophysiolo~c~ studies on the formation of these layers is very meagre. Initi~ly, the epi- and endocuticle
THE CUTICLE
OF MENZPPE
RUMPHZZ
141
of the premoult crabs are PAS-positive. The outer layer is more positive than the inner layer. Glycogen granules are seen on the epicuticle, which appears as a brush border; this glycogen is the precursor for chitin synthesis. The streaming of glycogen particles onto the epicuticle may be a fuation artifact, as reported by Johnson (1980). On the other hand, some of the glycogen may be combined with protein to form glycoprotein which also exists in these two layers, The reserve cells of the premoult integument are very big and are arranged nearer to the epidermis. These cells contain carbohydrates, proteins, lipids, and calcium (see Table I). Initially, carbohydrates are deposited on the epicuticle, then proteins are deposited and, in the early postmoult period, phospholipids are also deposited on the exocuticle. As a result the size of the reserve cells greatly decreases. In further stages calcium is also deposited (Figs. 11 and 12). Calcium granules are seen to migrate from the reserve cells through the epidermal cells and finally
Fig. 10. Transverse
section ofthe premoult
integument showing branching nature ofthe spines (Azan): ST’, spine. Fig. 11. Transverse section of the late premoult integument showing the migration of calcium granules through the epidermal cells (Von Kossa): CA, calcium. Fig. 12. Deposition of calcium in the exocuticle during the postmoult period (Von Kossa).
142
D. ERR1 BABU ETAL.
to the cuticular layers; that is why calcium granules are seen in the epidermal cells of the early postmoult crabs. After the deposition of glycogen and protein, the Leydig cells of the connective tissue pass to the exterior and deposit their contents over them. Leydig cells give positive reactions to tyrosine, cystine, and phenoloxidase which are involved in tanning of the newly forming exoskeleton. The addition of the Leydig cell secretions are mostly found during the postmoult period. As indicated earlier the spines are branched even in the premoult condition. The difference between the premoult and intermoult condition is that during the premoult condition the spines are present in detinite grooves or depressions formed in the epicuticle, but in the intermo~t condition the epicuticle is fully stretched and as a result the spines are not seen in grooves. Each spine is rather hollow, the outer wall shows orthochromasia, but the lumen and the branches show beta metachromasia. The grooves of the premoult epicuticle show some granular depositions which are stained violet with Toluidine Blue. These secretions perhaps come from the mucous glands present in the connective tissue. The spines present on the epicuticle may serve as mechanoreceptors in perceiving the movements of water, wave direction etc., which are of immense importance to the crab during the postmoult period. Nerve junctions were also observed between the epithelial cell and the nerve cell in the region of the spine. During moulting the crab does not come out of the protection of rocks and so avoids predators. That is why the spines are active in the early postmoult period; the branching of the spines and the granular secretions are also observed in excess during the early postmoult period. The grarmlar secretions at the base of the spines are not so conspicuous in the intermoult condition as the carapace is very hard and thick. The tegumental or mucous glands of the connective tissue are other prominent structures in the premoult and early postmoult crabs. These glands secrete acid mucopolysaccharides, which are both sulphated and carboxylated in nature. In addition, some of the gland cells also secrete neutral mucopolysacch~des. These secretions are conveyed through the pore canals, which are different from the pore canals of the spines. The secretions from the tegumental glands are observed only on the epi- and exocuticles. The acid mucous substances provide the siippery nature of the outer integument. The tegumental glands display gamma and beta metachromasia. The epi-, exo- and, endocuticle of the intermoult cuticle show beta metachromasia, whereas the endocuticular layer of the premoult cuticle shows gamma metachromasia. In the early postmoult condition only the exocuticle is betachromatic, whereas the outer layers are not metachromatic. Such tegumental giands have also been described by Yonge (1932), Travis (1955, 1957), Skinner (1962), and Dennell (1960) in the integument of arthropods, but the histochemical nature of these glands has not been studied. The presence of gamma metachromasia in the membranous and principal layers of PunuZirusargus during the premoult condition was, however, observed by Travis (1955). The structure of the tegumental gtands in the connective tissue of the integument is similar to that in the oesophageal and intestinal glands that are present in the connective tissue of the oesophagus and hindgut (Erri Babu et al., 1982). Erri Babu et al. (1979)
THE CUTICLE OF MENIPPE RUMPHII
143
observed the presence of acid and neutral mucopolysaccharides in the oesophageal glands of ~e~~~~e ~~p~jj. Although there is some similarity in structure, the number of cells in the oesophageal glands vary from 4 to 10, whereas the number of cells is restricted to a maximum of 8 in the tegumental glands. Regarding calcium deposition, in the premoult condition there is no calcium deposition of the two-layered integument, but the reserve cells are loaded with calcium deposits. In the postmoult condition calcium deposits could be seen first at the base of the epidermal cells, later these migrate to the apex and then move to the various layers for calcification (see Fig. 11). Only the exo- and endocuticles are highly calcified. The inner layer of the endocuticle is not calcitied. These observations are in agreement with those on Punulirus argus (Travis, 1955, 1957), and various other crustacean species (Dennell, 1960; Hackman, 1971). The presence of alkaline and acid phosphatase was observed during the tanning of the cuticular layers as well as during calcium deposition. ACKNOWLEDGEMENT
The first author is grateful to the Council of Scientific and Industrial Research for the financial assistance during the tenure of this work. REFERENCES ADAMS,C. W.M., 1957. A pdimethylamino benzaldehyde nitrite method for the histochemical demonstration of tryptophan and other related compounds. J. Clin. Pathol., Vol. 10, pp. 56-62. ARVY, L. & M. GABE, 1962, Histochemistry of the neurosecretory products of the pars intercerebralis of pterygote insects. In, Neurosecr~~on, edited by H. Heller & R.B. Clark, Academic Press, New York, pp. 33 l-344. BAKER,J. R., 1956. Histochemical recognition of phenols, especially tyrosine. Q. J. Microsc. Sci., Vol. 97, pp. 161-164. BANCROFT,J.D., 1975. Histochemical techniques. Butterworths, London, 348 pp. BARNES,H. & J. BLACKSTOCK,1976. Further observations on the biochemical composition of the cement of Lepasfascicula~s Ellis & Solander; electrophoretic examination of the protein moieties under various conditions. J. Exp. Mar. Biol. Ecot., Vol. 25, pp. 263-271. CHEVREMONT,M. & J. FREDERIC, 1943. Une nouvelle methode histochemique de mise en evidence des substances a fonction sulthydrille. Application a l’epiderme en poil et a la lavure. Arch. Biol., Vol. 54, pp. 589-605. CHIFFLLE,T. L. & F. A. PUTT, 1951. Propylene and ethylene glycol as solvents for Sudan IV and Sudan Black B. Stain Technol., Vol. 26, pp. 51-56. CULLING,C. F. A., 1974. Handbook of ~~topathoia~~a~and histochemicai techniques. Butterworths, London, 712 pp. DENNELL,R., 1960. Integument and exoskeleton. In, The physjolo~ of Crustacea, Vol. 1, edited by T. H. Waterman, Academic Press, New York, pp. 449-472. DOUGHTIE,D. G. & K. R. RAO, 1982. Rosette glands in the gills of the grass shrimp Palaemonetes pugio. 1. Comparative morphology, cyclical activity and innervation. J. Morphol., Vol. 171, pp. 41-67. DRACH,P., 1939. Mue et cycle d’intermue chez les Crustaces dtcapodes. Ann. Inst. Oceanogr. (Paris), N.S., Vol. 19, pp. 103-391. DRACH, P., 1953. Structure des lamelles cuticulaires chez les Crustacts. C. R. Acad. Sci., Vol. 237, pp. 1172-1774. ERRI BABU, D., K. SHYAMASUNDARI & K. HANUMANTHARAO, 1979. The structure and histochemistry of the oesophageal glands in the crab Menippe rumphii (Fabricius) (Crustacea : Brachyura). Proc. Indian Acad. Sci., Vol. 88B, pp. 217-285.
144
D. ERRI BABU ETAL.
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