FINE STRUCTURE AND MINERALIZATION OF THE GASTRIC MILL … st… · glutaraldehyde with 0.1 M cacodylate buffer (pH 7.4) at 48C for 2 h, and postﬁxed with 1% osmium tetroxide in

FINE STRUCTURE AND MINERALIZATION OF THE GASTRIC MILL IN THE

CRAYFISH PROCAMBARUS CLARKII DURING INTERMOLT STAGE

Hideki Chisaka and Yukishige Kozawa

Department of Anatomy, Nihon University Graduate School of Dentistry at Matsudo,

2-870-1, Sakae-cho Nishi, Matsudo, Chiba, 271-8587 Japan

(HC, correspondence: [email protected]); (YK: [email protected])

A B S T R A C T

This paper aims to elucidate the structure and characteristic mineralization of the gastric mill cuticle in the

crayfish Procambarus clarkii (Girard) using light and transmission electron microscopy. Structure and

mineralization of the lateral teeth of the gastric mill in the intermolt stage are documented. The lateral tooth is

composed of two types: incisor-like teeth and molar-like teeth. The tooth structure is divided into three

regions: ‘‘large-cusp region’’ is the protuberance region in the incisor-like teeth, ‘‘small-cusp region’’ is the

protuberance region in the molar-like teeth, and ‘‘marginal region’’ is the surrounding region. The cuticle is

divided into epicuticle, exocuticle, endocuticle, and membranous layer. The cuticle of the large-cusp region

lacks epicuticle and endocuticle, while the cuticle of the small-cusp region lacks the epicuticle. The cuticle of

the marginal region is composed of epicuticle, exocuticle, endocuticle, and membranous layer.

Ultrastructurally, the exocuticle of the mill is composed of an electron-lucent matrix, pore canals, and

electron-densematrix surrounding the pore canals. The exocuticle in both cusp regions differs from that in the

marginal region by the presence of a lamellar structure in the marginal region, which is composed of packed

lamellae. During mineralization, needle-shaped crystals are deposited in the endocuticle. These crystals are

found to consist of hydroxyapatite by electron diffraction andEDX. In conclusion, the structure of the gastric

mill differs from that of the integumental cuticle, and the differences occur mainly in the exocuticle.

The gut of the crayfish Procambarus clarkii(Girard, 1852) is composed of a foregut (anesophagus, anterior chamber and posteriorchamber), midgut, and hindgut. The gastric mill,composed of a median tooth and two lateralteeth, is observed in the setal screen of theanterior chamber of most crayfishes (Icely andNott, 1992). McLaughlin (1983) has reportedthat the foregut and hindgut of crustaceans arederived from embryonic ectoderm, and thelumen is covered with a cuticle resembling theintegument. The integumental cuticle is dividedinto four layers: epicuticle, exocuticle, endocu-ticle, and membranous layer (Richards, 1951,quoted inHegdahl et al., 1977c). These structureshave been discussed by many scientists (Travis,1955; Locke, 1961; Travis and Friberg, 1963;Green and Neff, 1972; Hegdahl et al., 1977a–c;Roer and Dillaman, 1984; Felgenhauer, 1992).However, the histological features of the gastricmill have been rarely documented (Shmitz andScherrey, 1983). The integumental cuticle ofmost crustaceans possesses various crystals; forexample, calcite crystals of CaCO3 are found inCancer pagurus (see Hegdahl et al., 1977c),Carcinus maenas (see Roer andDillaman, 1984),and Lirceus brachyurus (see Hawkes and

Schrare, 1973). The other minerals reportedinclude magnesium carbonate (Chave, 1954)and amorphous calcium phosphate (Lowenstamand Weiner, 1989). Hegdahl et al. (1977a–c)have reported that mineralization occurs in porecanals of the epicuticle, in the interprismatic areaof the exocuticle, and around the chitin-proteinfibers of the endocuticle in Cancer pagurus.These studies also investigated the integumentalcuticle of the dorsal carapace.Calcified tissues are found in the stomach,

including frameworks of calcified ossicles in thecuticle (Icely and Nott, 1992). Simkiss andWilbur (1989) and Lowenstam and Weiner(1989) reviewed mineralization in the carapacesof many species. However, there are few reportsconcerning mineralization of the stomach.Chisaka et al. (2000) reported that the mineralin the gastric mill is hydroxyapatite, whichdiffers from the integumental cuticle. Theresearch reported in this paper aims to elucidatethe detailed structure and characteristic miner-alization form of the gastric mill.

MATERIALS AND METHODS

Adult crayfish Procambarus clarkii were obtained froma commercial source (Kapuasu Ltd., Matsudo, Japan). They

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JOURNAL OF CRUSTACEAN BIOLOGY, 23(2): 371–379, 2003

were kept in shallow aquaria at room temperature. Bothmales and females at intermolt stage, and with carapacesmeasuring approximately 20–30 mm in length, wereinvestigated. The gastric mills were dissected from crayfishthat were anaesthetized by ice.

Light Microscopy (LM)

The dissected gastric mills were fixed with 2.5%glutaraldehyde with 0.1 M cacodylate buffer (pH 7.4) at48C for 2 h, and postfixed with 1% osmium tetroxide in thesame buffer (2 h at 48C). Specimens were dehydrated in anethanol series and then exchanged with propylene oxide.They were embedded in Quetol 812 resin. Semi-thinsections were stained by 0.1% toluidine blue as describedby Chisaka et al. (1999) and observed under a lightmicroscope. Calcified region was stained with von Kossatechnique as described by Hawkes and Schrare (1973).

Transmission Electron Microscopy (TEM)

Specimens were prepared in the same way as for LM.Ultra-thin sections were stained with uranyl acetate and leadcitrate and observed using a JEM1010.

Electron Diffraction Analysis and X-ray Analysis (EDX)

The dissected gastric mills were rinsed in distilled water,dried, and embedded in Quetol 812. Ultra-thin sections wereused for these analyses. The EDX was obtained using QX-2000 (Link Analytical, Ltd.) and a JOEL 1200EX. Theelectron diffraction pattern was taken and analyzed in thesame area of the section. The diffraction pattern wascompared with the pattern of a fluorapatite standard. Theinterplanar distances were calculated, and the d-spacing wascompared with that of hydroxyapatite given in the JCPDFindex.

RESULTS

The gastric mill found in the anterior chamberof most crayfish is comprised of a median toothand two lateral teeth (Fig. 1a). The lateral teethof the gastric mill were classified into anincisor-like tooth and molar-like tooth (Fig. 1b).

Based on the structure of the cuticle, the teethwere divided into three regions: ‘‘large-cuspregion’’, protuberance region in the incisor-liketooth; ‘‘small-cusp region’’, protuberance re-gion in the molar-like tooth, and ‘‘marginalregion’’, the surrounding region.

Light Microscopic Structure

The structure of the large-cusp region of thegastric mill at the intermolt stage is schemati-cally presented in Fig. 2. The gastric millcuticular structure is composed of an epicuticle,two kinds of exocuticle, an endocuticle, anda membranous layer (Fig. 2). The characteristicof the mill is found mainly in the exocuticlebecause the cusp of tooth is formed by theexocuticle. The exocuticular matrix of the cuspregion is extended in the direction of the base ofthe cuticle. The three-dimensional form of theexocuticular matrix is cone-shaped. As a result,this exocuticle occupies a wide area in the cuspregion (Fig. 2).

Incisor-like Tooth.—The incisor-like tooth had alarge-cusp region and amarginal region (Fig. 3a).The cuticle of the large-cusp region is composedof an outer exocuticle and an inner membranouslayer. The cuticle of the marginal region iscomposed of an epicuticle, exocuticle, endocu-ticle, and a membranous layer (Fig 3a). Theexocuticle was scarcely stained with toluidineblue in the large-cusp region but was darklystained in the marginal region. In the junctionalarea between the large-cusp region and marginalregion, the cuticle on the epithelium wasobserved to have a five-layer structure as follows:

Fig. 1. The gastric mill and lateral teeth in Procambarus clarkii. (a) The gastric mill consists of a median tooth (m) and twolateral teeth (l). (b) Scanning electron micrograph of the lateral teeth, dorsal view. The lateral teeth are classified into incisor-like teeth (it) and molar-like teeth (mt).

372 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 23, NO. 2, 2003

an epicuticle, two kinds of exocuticle, anendocuticle, and a membranous layer (Fig. 3a).

Molar-like Tooth.—The molar-like tooth iscomposed of a small-cusp region and a marginalregion (Fig. 3b). The cuticle of the small-cuspregion is composed of an outermost exocuticle,an endocuticle, and a membranous layer. Thecuticle of the marginal region had the samestructure as that described for the incisor-liketooth (Fig. 3b). In the junctional area betweensmall-cusp region and marginal region, thecuticle of the molar-like tooth had the samestructure as that in the incisor-like tooth (Fig. 3b).

Ultrastructure

A thin outer membrane and a thicker inner onewere observed in the epicuticle, which resem-bled the integumental epicuticle. A few porecanals leaned toward the lumen of the anteriorchamber in the inner membrane (Fig. 4).Two different kinds of exocuticle were

observed in the cusp and marginal regions (Figs.5, 6). Figure 5 shows the exocuticle in the twocusp regions, and Fig. 6 shows the exocuticle inthe marginal region. In the exocuticle of the cuspregions, an electron-dense matrix was observedalong the pore canals, which leaned toward thelumen of the anterior chamber in the electron-lucent matrix (Fig. 5). The exocuticle in themarginal region was composed of stackedelectron-dense matrix in the electron-lucentmatrix like an integumental exocuticle showing

a lamellar structure, and the pore canals alsoleaned toward the lumen. The thickness of theexocuticular lamellae is about 400 nm (Fig. 6).The endocuticle is composed of stacked

lamellae resembling the integumental endocu-ticle, and the thickness of the endocuticularlamellae was about 4.5 lm (Fig. 7). Amembranous layer was observed between theendocuticle and epithelium and was composedof electron-dense matrix (Fig. 8). The lamellarthickness in the membranous layer was about350 nm (Fig. 8).

Calcification

Figures 9a, b show toluidine blue and vonKossa staining, respectively, of the gastric mill.Mineralization occurs mainly in the endocuticle(Fig. 9b). The crystals are needle-shaped and areseen by TEM to be deposited in the endocu-ticular matrix (Fig. 10).The electron diffraction pattern of the needle-

shaped crystals showed one type of hydroxy-apatite, according to the JCPDF index (Fig. 11,Table 1). The EDX revealed a high calcium andphosphorus content (Fig. 12). The mean for theCa/P molar ratio is about 1.4.

DISCUSSION

Structure

Shmitz and Scherrey (1983) reported that theforegut (esophagus, anterior chamber, andposterior chamber) and hindgut in crustaceans

Fig. 2. Schematic drawing of the incisor-like tooth at intermolt stage in Procambarus clarkii. (a) Vertical section shows thefive kinds of cuticles: epicuticle (p); exocuticle (x) of the cusp region; exocuticle (e) of the marginal region; endocuticle (n);and membranous layer (m). The cuticle of the large-cusp region is composed of epicuticle (p), exocuticle (x), andmembranous layer. (b) Horizontal section along the solid line of Fig. 2a shows the exocuticle (x) of the cusp regionsurrounded by the exocuticle (e) of the marginal region and endocuticle (n) and the outermost epicuticle layer (p).

373CHISAKA AND KOZAWA: STRUCTURE, MINERALIZATION OF GASTRIC MILL

all have the same origin as the integument; theforegut cuticle molts at ecdysis along with theexoskeleton. As quoted in Hegdahl et al.(1977c), Richards (1951) reported that theintegumental cuticle is divided into four layers:epicuticle, exocuticle, endocuticle, and mem-branous layer. This study used their division ofthe cuticle. Babu et al. (1989) studied thestructure of the foregut cuticle histochemically,suggesting that the cuticle was divided into twolayers: a keratin layer and collagen layer. Thestructural studies of the cuticle in foregut havenot been reported after this study. Icely and Nott(1992) mentioned the outer structures of thegastric mill in crustaceans. However, thecomponents of the cuticle have not beenmentioned. This research may become the basalstudy to elucidate a diversity of cuticle and thereason that the gastric mill has the complex andcharacteristic outer structure in crustaceans. Thecrayfish Procambarus clarkii was used for

a model of Astacidae in crustaceans. In theview of the structure of the cuticle, we concludethat the cuticle has five features as follows:

(1) Exocuticle in Both Cusp Regions.—Theexocuticle of the cusp region was identified inthis study by LM and TEM observations. Thespecific characteristics of the exocuticle in thecusp region show the exocuticle deeply relates tothe function of the gastric mill. The gastric millcuticle becomes thick by the distribution andform of the exocuticle in the cusp region, and theexocuticle may make the outer structure morecomplex. In the structure, for instance, theexocuticle of integument in Cancer pagurus(see Hegdahl et al., 1977b) and marginal regionof the cusp region inProcambarus clarkii (Fig. 6)is composed of stacked lamellae. This exocuticleof integument and marginal region may includea hard region and soft region, as the remarkabledifference of the electron density was observed in

Fig. 3. Light microscopic observation of a vertical section of the incisor-like tooth (a) and the molar-like tooth (b) inProcambarus clarkii. The cuticle is composed of an epicuticle (p), two kinds of exocuticle (e and x), an endocuticle (n), anda membranous layer (m) lined with epithelium (i). (a) The cuticle of the large-cusp region is composed of an exocuticle (x)and a membranous layer (m). The cuticle of the marginal region is composed of an epicuticle (p), an exocuticle (e), anendocuticle (n), and a membranous layer (m). (b) The cuticle of the small-cusp region is composed of an exocuticle (x), anendocuticle (n), and a membranous layer (m). The cuticle of marginal region is composed of an epicuticle (p), an exocuticle(e), an endocuticle (n), and a membranous layer (m).

Figs. 4–8. Transmission electron micrographs, Procambarus clarkii. Fig. 4. The epicuticle, lined with exocuticle (e), iscomposed of a thin electron-dense outer layer (arrows) and an electron-lucent inner layer (p). A pore canal (arrowhead) isobserved in the electron-lucent inner layer. Fig. 5. The exocuticle of both cusp regions. Arrows show the pore canals,surrounded by electron-dense matrix. The pore canals lean toward the lumen of anterior chamber. Fig. 6. The exocuticle inthe marginal region of the mill. Packed lamellae are observed in this region. The width of each lamella is about 400 nm(arrowheads). Arrows show the pore canals, surrounded by the electron-dense matrix. The pore canals lean toward the lumenof anterior chamber. Fig. 7. The endocuticle in the small-cusp and marginal regions of the mill. Packed lamellae are observedin this region. The width of each lamella is about 4.5 lm. Pore canals (arrows) are observed in the lamellae. Fig. 8. Themembranous layer in the mill. The membranous layer is lined with epithelium (i). Packed lamellae (arrowheads) are observedin this region. The pore canals (arrows) lean toward the lumen of anterior chamber.

!



the exocuticle. However, the exocuticular struc-ture of the cusp region is not a lamellar structure,suggesting that the hardness of the exocuticularmatrix might comparatively be uniform andstable in comparing the matrix of the exocuticleof the integument andmarginal region.With sucha reason, the exocuticular structure of the cuspregion might prevent the matrix from being lostrapidly by the function that the gastric mill chewsand masticates the food.

(2) Exocuticle in Marginal Region.—Roer andDillaman (1984) reported a lamellar thicknessof approximately 2.2 lm in the integumentalexocuticle of Carcinus maenas. In the speciesinvestigated in the present study, the thickness

of the exocuticular lamellae is approximatelyone-sixth that of the integumental exocuticularlamellae. The exocuticle of the marginal regionmay support the exocuticle of the cusp regionwith stacks of thin lamellae, and support thefunction of the cusp regions of the teeth inmasticating food.

(3) Diversity of the Cuticle.—Although thegastric mill cuticle has the same origin as theintegumental cuticle, its structure differs. Re-garding the diversification of the cuticle,Andrews and Dillman (1993) reported the lossof endocuticle in the gill for the decapodProcambarus clarkii, and Hegdahl et al.(1977a) reported a thicker epicuticle in the

Figs. 9–11. The gastric mill of Procambarus clarkii. Fig. 9. Light microscopic observation of transverse sections of a molar-like tooth of the mill. The cuticle layer is composed of epicuticle (p), two kinds of exocuticle (e and x), endocuticle (n), andmembranous layer (m), lined with epithelium (i). (a) Stained by toluidine blue. (b) Von Kossa staining shows mineralizationin endocuticle. Fig. 10. Transmission electron micrograph of needle-shaped crystals in the endocuticle of the mill. Needle-shaped crystals (arrows) are electron-dense. Fig. 11. Electron diffraction pattern of needle-like crystals in endocuticle (leftside) of the mill and fluorapatite standard sample (right side).


spine of the carapace for the decapod Cancerpagurus. Andrews and Dillman (1993) sug-gested that the diversification might depend onthe cuticular function. In the present study, thisdiversification occurs in the exocuticle, suggest-ing that the cuticle of the cusp region mightdiffer from that of the marginal region in thespeed of the cuticular development or in thelength of the periods of each stage of the moltcycle. In other word, the former shows that ifthe cuticle in the cusp and marginal regionsbegin to be formed at the same time, the speedof the cuticular development in the cusp regionmay become faster than that in the marginalregion because the cuticle in the cusp region isthicker than that in the marginal region. And thelatter shows that the cuticle in the cusp regionmay begin to be formed earlier than that inmarginal region do because the cuticle of thecusp region is thicker than that of marginalregion. Authors think that the latter may bemore suitable. Because Andrews and Dillman(1993) reported that in the length of the periodsin each stage of molt cycle, the cuticle of gilldiffers from that of the integument.

(4) Loss of the Epicuticle in Both Cusp Regionsat Intermolt Stage.—The vertical section of thegastric mill shows a loss of the epicuticle inboth cusp regions during the intermolt stage(Fig. 3a, 3b). Because the epicuticle is located atthe occlusal surface, this loss may result frommastication. As a result of the loss of epicuticle,the exocuticle is exposed in the lumen.

(5) Loss of Endocuticle in Large Cusp Region,and the Junctional Regions Between CuticularLayers.—The endocuticle is usually observed inthe layer between the exocuticle and membra-nous layer at intermolt stage; however, theendocuticle in this large cusp region was notobserved in the area between the exocuticle andmembranous layer, which shows that after thefirst exocuticular matrix of the large-cusp regionbegins to be deposited, the matrix may continueto form until the matrix of the membranous layerbegins to be deposited. In other words, the exo-cuticular matrix in the large-cusp region maybe formed while the endocuticular matrix ofthe marginal and small-cusp regions is formed.The reasons we used the term ‘‘exocuticle’’ in thelarge- and small-cusp regions are that the exo-cuticle has been observed between the epi-cuticle and endocuticle in the small cusp regionand include the pore canals surrounded by the

electron-dense matrix. The junctional regionsbetween the cuticular layers were stained bytoluidine blue. These regions may possess thematrix with a different organization from that ofthe other cuticular regions, because its functionis to connect them. The other conclusion is asfollows.

The Relationship Between Electron-dense Ma-trix and Chitin-protein Fibers.—The exocuticleand endocuticle in integumental cuticle arecomposed of the stacked lamellae by chitin-protein fibers (Hegdahl et al., 1977b, c; Roerand Dillaman, 1984). In this report, the term‘‘chitin-protein fiber’’ was not used, as thechitin-protein fiber has not been defined clearlyin crustaceans. However, if the chitin-proteinfiber is defined, the electron-dense matrixobserved in the exocuticle and endocuticle ofthe gastric mill may be composed of the chitin-protein fiber, because the electron-dense matrixhave been sometimes observed as the fiber-likestructure.

Why Was the Endocuticle of the Cusp RegionNot Stained by Toluidine Blue?.—The endocu-ticle showed the complex structure of the

Table 1. D-space of hydroxyapatite (ASTM9-432) and ofelectron diffraction pattern of needle-shaped crystals shownin Fig. 11.

Hydroxyapatite (ASTM 9-432) Needle-shaped crystal

hkl d(A) I d(A)

002 3.44 40 3.41102 3.17 11210 3.08 17211 2.81 100 2.82112 2.78 60 2.77300 2.72 60 ND202 2.63 25 ND301 2.53 5212 2.30 7310 2.26 20 ND221 2.23 1311 2.15 9302 2.13 3113 2.07 7400 2.04 1203 2.00 5 1.99222 1.94 30 1.94312 1.89 15320 1.87 5213 1.84 40 1.82321 1.81 20 ND410 1.78 11402, 303 1.75 15004, 411 1.72 20 1.70

ND: not detected.


stacked lamellae by the von Kossa staining andTEM observation, which was also observed bythe metachromatic staining of toluidine blue(not shown). However, the metachromaticstaining was lost soon after the staining.

Calcification

Many reports suggest that the mineral in theintegumental cuticle of crustaceans is calciumcarbonate, with calcite as the crystalline form(Travis, 1963; Hawkes and Schrare, 1973;Yano, 1975; Hegdahl et al., 1977c; Roer andDillaman, 1984; Lowenstam and Weiner, 1989;Simkiss and Wilbur, 1989). The results of thisstudy showed that the needle-shaped crystal ishydroxyapatite in the endocuticle of the gastricmill of Procambarus clarkii. However, thisstudy could not show an X-ray diffractionpattern and the structure of the needle-shapedcrystal because the crystal is very small and thewidth of crystals is very narrow. With sucha result, we propose that the crystal is in facthydroxyapatite. If this crystal were hydroxyap-atite, this would lead to the assumption thatdeposition of hydroxyapatite is related to thefunction of the mill, which is mastication, thusdiffering from that of the integument. In otherwords, the hydroxyapatite, which is a hardercrystal than the calcite in the integumentalcuticle, is deposited in the endocuticle of themill, which may show that the gastric mill can

always keep withstanding the pressure ofmasticating food. Regarding the reason thatthe exocuticle of the mill is not calcified, ifa calcium salt is deposited in the exocuticle, thecalcium salt may be dissolved by the gastricjuice. Therefore, to protect the calcified areafrom the gastric juice, the calcified endocuticleof the gastric mill may be covered with the thickand uncalcified exocuticle.

ACKNOWLEDGEMENTS

The authors thank associate professor Toshiro Sakae andDr. Hiroyuki Mishima for helpful suggestions and usefulcomments on electron diffraction. This study was supportedby a Grant from the Ministry of Education, Culture, Sports,Science and Technology to promote 2001-MultidisciplinaryResearch Projects (in 2001–2005).

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RECEIVED: 8 January 2002.ACCEPTED: 12 August 2002.


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FINE STRUCTURE AND MINERALIZATION OF THE GASTRIC MILL … st… · glutaraldehyde with 0.1 M cacodylate buffer (pH 7.4) at 48C for 2 h, and postﬁxed with 1% osmium tetroxide in