Ultrastructural observations of the muscle insertion and modified branchiostegite epidermis in the larval brown shrimp, Penaeus aztecus

TISSUE & C[-;LI, 1972 4 (4) 613--628 Pitblished b), Lon,gnlan Grottp Lid. Prilzled in Great Britain

PRUDENCE TALBOT*, WALLIS H. CLARK*, Jr. and A D D I S O N L. LAWRENCE*

U L T R A S T R U C T U R A L O B S E R V A T I O N S OF THE MUSCLE INSERTION AND M O D I F I E D B R A N C H I O S T E G I T E EPIDERMIS IN THE LARVAL B R O W N S H R I M P , PENAEUS A Z T E C U S ABSTRACT. iFine structural observations on muscle insertion in arthropods are extended to the order Decapoda. The three regions of a muscle insertion (cuticle, epidermis and muscle) are discussed for the larval lbrms or the decapod, Pr, naen.v aztec:us.These regions are cm~trasted to muscle insertionsofinsects and arachnids which have been previously reported in the literature. In addition, a modified epidermal cell located in the branchiostegite o1' the larval shrimp is discussed. The epidermal cell ot" the branchiostegite is modified in a manner similar to the epidermis of the nmscle insertion. The two epidermal cell types are contrasted and the significance of" their modifications is discussed~

digitate with modified epidermal cells. T h e :myo-epidermal interface is characterized by Cvvol~oc~tcv,Lt.Y, the muscle insertions (or a j u n c t i o n a l specialization, usually a desmo~ a t t a c h m e n t s ) of a r t h r o p o d s are interesting some, T h e epidermal cell itself contains regions d e m o n s t r a t i n g e l a b o r a t e modifican u m e r o u s microtubules which are generally tions. T h e muscle cells themselves are conoriented perpendicular to the plane of the nected to the exoskeleton or cuticle by way cuticle. Tile epidermis articulates with the of a highly specialized e p i d e r m a l cell, somecuticle by m e a n s of tonofibrillae (Lai-Fook, times referred to as a °tendon cell' (Lai1967) or muscle a t t a c h m e n t fibers (Caveney, Fook, 1967 a n d Smith et al., 1969). Histo1969). These extend f r o m invaginations of logical i n f o r m a t i o n has been available on the epidermal p l a s m a m e m b r a n e , for example muscle a t t a c h m e n t s for some time and has 7hemidesmosomes or conical h e m i d e s m o been reviewed b y R i c h a r d s (1951). T h e somes, into the cuticle. A s more orders of precise relationship between the cuticle, a r t h r o p o d s h a v e been examined, exceptio~s epidermis a n d muscle in the region o f to the preceding a r r a n g e m e n t have been muscle a t t a c h m e n t s has only been resolved described, e.g. the mite muscle a t t a c h m e n t to within the last decade by electron microscope the exoskeleton ( K u o e t al., 1971) a n d the studies (Bouligand, 1962; Auber, 1963; Laivisceral muscle insertion o n venom-secreting F o o k , 1967; Caveney, 1969 a n d Smith glands in a spider (Smith et al., 1969). To e t al., 1969). date, the ultrastructural descriptions of To s u m m a r i z e briefly f r o m the preceding muscle insertions have dealt primarily with authors, muscle cells adjoin and interthe a r a c h n i d s (Smith et al., 1969 a n d K u o et al., 197l) a n d the insects (Auber, 1963; * Department of Biology, University of Houston, L a i - F o o k , 1967; Caveney, 1969 a n d H a g o Houston, Texas 77004, U.S.A. pian, 1970). The initial fine structural r e p o r t Manuscript received 5 May 1972. Revised naannscript received 14 August 1972. o n a r t h r o p o d muscle insertio:n a n d the only 613 Introduction

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report on this region in crustaceans was made on the copepod, Cyclops, by Bouligand in 1962. Epidermal cells which ultrastrueturally resemble the epidermis of the muscle insertion have been reported in other regions of arthropods. Moulins (1968) has observed what he termed a supporting structure in the lining of the hypopharyngeal cavity of some insects; the epidermal cells of this structure are quite similar to the epidermal cells of muscle insertions. We ihave observed a microtubule-containing epidermal cell in the branchiostegite* of the larval brown shrimp which likewise resembles the epidermal cells of most arthropod muscle insertions. This epidermal cell in the brandaiostegite ihas been mentioned previously in histological reports (Vitzou, 1882 and Travis, 1955L Kthnmel el aL (I 970) have partially described its ultrastructure in Orconectes. However, they were concerned primarily with cuticle formation during a molting cycle and thus showed only the apical surface of the branchiostegite epidermis. Thus, this modified epidermal cell has never been completely described on a fine structural level° From the preceding discussion, it is clear that very little is known concerning the fine structure of the crustacean muscle insertion, and virtually nothing is known regarding the microtubule-containing epidermis of the branchiostegite. Therefore, the purposes of this article may be summarized as follows: (1) to extend ultrastructural observations on muscle insertions to the crustacean subclass Malacostraca; (2) to describe for the t:irst time the muscle insertions in a larval crustacean; (3) to describe the fine structure of a modified epidermis in the crustacean branchiostegite which resembles epidermis in muscle insertions. Materials and Methods

Anima/s-i- were fixed for electron microscopy with S-collidine buffered glutaraldehyde as described by Bell el aL (1969) or by immersion in I% phosphate buffered osmium * T h a t portion of the carapace covering the lale~'al body wall. See [rig. 8. + Larwll brown shrimp i~ protozoea and mysis s~ages were obtail~cd from *he N a t i o n a l Marine Fisheries Service, Galveston, Texas, and from D e w Chemical Compat/y, Freeport, Texas.

CLARK

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LAWRENCE

tetroxide for 1-3 hours at 4'~C. Tissue was dehydrated in an acetone series, infiltrated in an acetone : Maraglas mixture and embedded in Maraglas. Thin sections were made using glass knives on a Porter-Blum MT-2 ultramicrotome and stained with urany/ acetate and lead citrate. Grids were examined on an AEI EM6B electron microscope operating at an accelerating voltage of 40 or 60 kV. For light microscopy, thick Epon sections were stained with toluidine blue. Results (l) Muscle insertio~l

The results to be presented are characteristic for both the protozoea and mysis stages. For orientation, an overview of the decapod muscle insertion is presented in the first schematic diagram (Pig. 1). Beginning on the exterior surface of the shrimp, the three layers comprising the muscle attachment are the cuticle or exoskeleton, the epidermis and Ihe muscle. The myo-epidermal junction is characterized by extensive interdigitation between the epidermal and muscle layers (Figs. 2, 3, 5), and the pattern of this interdigitation may be seen in both cross and longitudinal sections in Fig. 2~ An intermediate junction (zonula adhaerens) is present along this Jnterdigitatio~ except for short segments where no junctional specialization occurs (Fig. 3). The intercellular space between apposed plasma membranes of the muscle and epidermal cells varies in width from 260-510 ~. A moderately electron-dense material appears in the intercellular space of the junction and is never bisected by a denser material, as is the case in some insects (LabFook, 1967 and Caveney, I969) and arachnids (Smilh et aL, 1969). The striated muscle in the brown shrimp resembles that described in the copepod (Bonligand, 1962). The myo:~ibrils are separated from each other by rows of sarcoplasmic reticu/um and mitochondria, The sarcoplasmic reticulum approximates the junctional region at the deepest parl of tim invagination formed by the epidermal cell (Fig. 3). The actin filaments of the l-band illsert on the moderately electron-dense cytoplasmic plaque of the intermediate junction on the muscle side of the junction (Fig. 5).

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17ig, I. S c h e m a l i c d i a g r a m o [ ' a b r o w n s h n n l p muscle insertion. T h e musclc a t t a c l > me~:l( is c o m p o s e d of +t h r e e layers, the muscle (mL the epidermis (rne) a n d the cuticle (cL A highly i n l e r d i g J t a t e d i n t e r m e d i a t e , i u n c t i o n (i i) occurs between the e p i d e r m i s a n d the muscle, Actin filaments o f the l - b a n d (i) insert on the muscle side o f th{s j u n c t i o n . Wilhin the e p k l e r m i s , conical h e m i d c s m o s o m e s (ch) are i<'ormed as i n v a g i n a l i o n s o[ +the apical cell m e m b r a n e (cm) a n d intraculicular' fibers (if') pass from the c o n i c a l hemi+ clesmosomes h'do the ctHicle+ M i c r o t u b u l e s (rot) in the e p i d e r m a l a p e x m a y be freeend{ug oc rnay insert on the conica{ h c m i d e s m o s o m e s , A n apica~ c y t o p l a s m i c b a n d (acbL in which no m i c r o t u b u l c s are observed+ is present a n d c o n t a i n s r i b o s o m e s a n d e u d o p l a s m i c reticulum, C e l l u l a r organelles are packed between a r r a y s of" m i c i o t u b u l e s [ntract~licular rods (h-) mgty be prose]it in the cuticle in the region of' a m u s c l e i n s e r t i o n :

The myosin Iilaments of the A-band are never observed making such an insertion. The epidermal cells of muscle insertions are highly modified (compare unmodified epidermis in Fig. 2 with adjacent epideima/ calls). The most prominent feature of: these tens, in addition to the high]y .interdigitated basal surface ah-eady discussed+ is the lmmerous microtubules which extend I"rom the apical to the basal portion of the epidermal cells (Figs+ 1, 3, 4, 5 and 6). The microtubules measure from 2'5 lo 4+() ~ in

length and over 280 X~ in diameter, Other organelles are packed into rows which are oriented parallel to tile microtubules (Fig° 3). These orgarlelles hmlude a b u n d a n t flee ribosomes and a tEw mitochondria, Some rough (RER) and smooth (SER) endoplasmic reticulum is also packed into this area° Golgi bodies and associated vesicles (Fig, 4) are occasionally seen among the other organdies. In the most apical region of the epidermis, there is a narrow zone of" cytoplasm (abo-ut

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Figo 2~ A montage showing muscle insertions in cross (xs) and longitudinal (Is) section. The three layers comprising a muscle attachment, the cuticle (c), modified epidermis (me) and muscle (m), arc evident at this low magnification, Thc extensive [nterdigitation between lhe modified epiderrnis and muscle is visible in both planes of section. Within the cuticle, intracnticular rods (large arrows) and intraeuticular fibers (small arrows) may be compared in cross section. A portion of an unmodified epidermal cell (e) is present also in the montage. Protozoea. :. 7700.

Fig. 3. Longitudinal section through a muscle insertion at high magnification. A highly interdigitated imcrmediate junction appears bctwecn the modifed epidermis (me) and muscle (m). Occasionally, there are short segments along this interdigitation where an intermediate junction is not evident (between triangles). Elements of sarcoplasmic rcticulum (s) adjoin the iutermediate junction at thc deepest points of the interdigitation of the epidermal cell. Within the cuticle, intracuticular rods (large arrows) are present and extend out to the cuticulin layer (cu)~ Conical hemidesmosomes (ch) occur in the apex of" the epidermal cell and arc surrounded on the epidermal side by an electron-dense material, lntracuticular fibers (small arrows) originate within the conical hemidesmosomes and pass out into the cuticle. Numerous microtubules are prcsent witfiin the epidermis_ Smooth and rough endoplasmic reticulum, ribosomes, mitochondria and Golgi bodies are stacked between microtubules. Protozoea. × 28,00,

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0"2/~ deep) in which only SER, RER, and free ribosomes are found. Microtubules do not extend into this apical zone (Fig. 4). Throughout the rest of this article this region in the epidermis will be referred to as the apical cytoplasmic band (ACB). The apical surface of epidermal cells in the region of the muscle insertion is further modified. The apical plasma lnembrane forms invaginations about 0"8~, deep, and these invagiuations are lined on the adeptdermal side by an electron-dense material. Caveney (1969) has referred to similar modifications as conical hemidesmosomes, and his terminology will be used in this article. Conical hemidesmosomes are depicted in Figs. 1, 3, 4 and 6. In cross section (Fig. 6), the base of a conical hemidesmosome measures about 50 mr< Some microtubules appear to terminate on the electron-dense, cytoplasmic portion of the conical hemidesmosome below the level of the ACB. Many microtubules, however, stop abruptly below the ACB and appear to be free-ending in the apical cytoplasm (Figs. 3, 4). While some of these microtubules cmwe slightly in approachirlg the ACB and may insert on conical hemidesmosomes not in the plane of section, many microtubules

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do not curve at all and stop abruptly at the ACB (Fig. 4). Within the conical hemidesmosomes, electron-dense, amorphous fibers originate and pass out into the cuticle. Similar structures in other animals have been called various names in the literature including: axe osmiophile (Bouligand, 1962); tige de substance opaque (Auber, 1963); tonofibrillae (Lai-Fook, 1967; Talbot and Clark, 1972); Stabe (Ktitnme[ el al., 1970); muscle attachment fibers (Caveney, 1969l and .fibre hm'acuticulaire (Moulins, 1968). The terminology of Moulins, which in translatiml is intracuticular fiber, will be used in the remainder of this article. lntracuticular fibers, which are extracellular to the epidermis, are evident in Figs. 4, 6 and 7. They extend :from the fossa of the conical hemidesmosomes into the cuticle. The fibers extend to the cuticulin layer of the cuticle but do not pass through it to the exterior surface of the cuticle (Figs. 3, 7L IntracuticuIar :fibers are not: extensions of the plasma membrane of the conical hemidesmosomes. In cross section (Fig. 6), the region of low electron density separating the intracuticular fiber and the membranes of the hemidesmosomes is evident.

Fig, 4, A portion of" cuticle (c) and tile apex of an epidet'ma] cell from a muscle insertion, Tile conical bemidesmosomes are formed as invaginations of the apical cell membrane and are s u r r o u u d e d oil their epidermal side by an electrou-dense material (large arrow). W i t h i n the conical benaidesrnosomes, inn~acuticular fibers (white arrows) originate and pass into the clUicle. I-;ree-endhlg microtubnles are present bm/eath the apical cytoplasrnic band ( a c b ) A microtubule is observed inserting on a c o n i c ~ hernld e s m o s o m e at the snmll bJack arrow, Smooth endoplasmic reticulum and ribosomes appear h~ t/~e A C B , Other o r g a n d i e s , i*~cluding a Golgi body (gL are visible betwem~ microtubules. Protozoea. :,:70,00(L Fig. 5. I n t e r m e d i a t e junction between a region of muscle and modified epidermis. Actin filaments of the l-band (i) and microtubules (mr) oCIl~c epidermis insert on tl~e intermediate ,junctiom Some myosin filaments of the A-band (a) are present it1 tile nlierograph. Protozoeao 44,500. Fig. 6, Crosg to oblique section through a muscle insertion. A portion of intermediate jUllCtion (ij) is presenL and microtubulcs (mr) in oblique section insert on the junctiotl. Apical/3< conical hemidcsnlosonms wJtll an elcct~on~,deuse plaque on their epidermal surface a p p e a r in cross section, W i t h i n the conical hemidesmoson'ms is an electror~-dense core, the inttacuticular Iiber (small arrows I), which is separated from the membrane of the conical h e m i d e s m o s o m e by a region of low electron density (see arrow 2h lntracuticular fibers also appear embedded hi the cuticle (large arrow), Protozoon, ;: 28,000, Fig. 7, A muscle insertion showing b r a n c h i n g of the intracuticular fibers (small

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© Fig. 8. Schematic diagram showing a cross section through the cephalothorax oF a larval shrimp. The branchiostegite (b) covers the lateral surface of the animal and forms the branchial chamber (bcL The branchiostegite is composed of an inner (ic) and outer (oc) cuticle and an inner (ie) and outer (co) epidermis, The inner epidermis may be: (1) unmodified, (2) modified for osmoregulation, or (3)modified as supporting epidermis. A hemolymph sinus (h) is present between the two epidermal layers. Inner and outer epidermal cells may adjoin each other to form 'supporting epidermis' (se).

Only one intracuticular fiber extends fronl each conical hemidesrnosome. W i t h i n the cuticle, intracuticular fibers are n o t surr o u n d e d by a space of low electron density a n d thus a p p e a r to be e m b e d d e d directly in the cuticle (Figs. 6, 7), These fibers occasionally b r a n c h within the cuticle ('Fig. 7). In addition to the intracuticular fibers, very thick (1500 ~), electron-dense rods are often seen in the cuticle associated with a muscle insertion. W e will refer to these structures as intracuticular rods. i n cross sections t h r o u g h the animal, the intra~ u u v , uJo~t

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the surface of the cuticle (Fig. 3). They extend to the cuticulin layer, b u t do n o t pass beyond it. Basally, the rods a p p r o a c h the i n n e r m o s t portion of the cuticle, b u t they have not been observed in direct contact with the base of the cuticle or with the epidermal cells. In Fig. 2, which shows muscle insertions in cross a n d longitudinal sections, these intracuticular rods are observed in cross section in one portion of the cuticle only,

These cross sectioned intracuticular rods may be c o m p a r e d with the intracuticular fibers in cross section passing t h r o u g h a different portion of the cuticle in the same m o n t a g e . T h e intracuticular fibers a n d rods in cross section clearly differ from each other in both d i a m e t e r a n d electron density. F u r t h e r m o r e , intracuticular rods are present in various regions of the cuticle a n d their d i s t r i b u t i o n appears to be i n d e p e n d e n t of muscle insertions. (2) Modff7ed carapace epidermis 1,, v . . . . . . . . . a n d mysis stages, +a . . . . i,,,1~ thorax is covered by the carapace. T h e lateral folds of tile carapace, which constitute the outer limiting m e m b e r of the b r a n c h i a l c h a m b e r , are referred to as the b r a n c h i o stegites (Fig. 8) or tergal folds by Snodgrass (1965). T h e branchiostegites are c o m p o s e d of an inner a n d o u t e r cuticle which is in turn u n d e r l i n e d by a n inner a n d outer epidermis (Fig. 8). The two e p i d e r m a l layers are separated by a sinus c o n t a i n i n g h e m o l y m p h .

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The following three types of epidermis have been tentatively identified in the branchiostegite of larva/brown shrimp: (1) unmodified epidermis, i.e. ultrastructurally, it does not show any modifications indicating a specific function; (2) epidermis modified for osmoregulation (see Talbot e t at., 1972b for discussion) and (3) rnicrotubule-containing epidermis, which resembles the epidermis of muscle insertions and is considered in the remainder of this article. On the basis of fine structural observations it is presumed that this tissue has a supportive function will be referred to as 'supporting epidermis', Figure 9 is a low magnification electron micrograph of a cross section through the branchiostegite showing two supporting epidermal cells. Supporting epidermal cells are always observed in pairs, i.e. one cell From the inner and o u t e r epidermis span the ihemolymph sinus and adjoin along their basal surfaces by means of a highly interdigitated intermediate junction. The intermediate junction between the inner and outer supporting epidermal cells is more easily resolved in Figs. l0 mid I la, This junction occurs along only a portion of the basal cell surface, not along the entire surface as in th.e muscle insertion. This is best seen in Fig, 9 where the large arrows denote the cell boundaries of the outer and inner epidermis. In the supporting epidermal cells, microtubules are ;packed into one portion of the cytoplasm. These microtubules extend fl'orn cortical hemldesmosomes in the apical cyto~ plasm of Ihe inner and outer supporting epidermis to the moderately dectron-deuse layer of the intermediate junction, where dley terminate (Figs. 10, 11A). Thus microtubules approach and terminate on the intermediate junctioll from both the inner and outer epbdermis° Apically in the epiderrnal layers, the microtubules often approach the cuticles at an angle (Figs. 10, IFB), and occasionally they appear to spiral and twist out of lhe

ANt)

LAWRENCE

plane of section (not shown). A distinct apical cytoplasmic band as noted in muscle insertions is not seen; most tubules appear to converge on conical hemidesmosomes (Fig. I1B) rather than being free-ending in the apical cytoplasm. Some organe/les, in par,ticular :free ribosomes and a few mitochondria, occur between the arrays of microtubules. Lateral to the microtubular cluster, the cytoplasm contains typical organelles as SER, RER, Golgi bodies and n-dtocbm~dria. In both inner and outer supporting epi-dermis, conical hemidesmosomes occur as invaginations of the apical plasma merebran< and intracuticutar fibers extend from the hemidesmosomes up to the cuticulin layer of the cuticles ('Figs. 10, I tI~). Intracuticular rods often occur along the length of the outer cuticle of the branchiostegite (Fig. 10); they are not observed in the inner cuticle of the branchiostegite, The features of support ing epidermis in the brancbiostegite of a larval decapod are sulnmarized in Fig. 12. Discussion Muscle attachments to the exoskeleton in arthropods are generally quite similar with the exception of the order Acarina (Kuo et al.~ 1971). In this article observations on muscle attachments have been extended to the subclass Matacostraca and to a larval form of a crustacean. In this larval decapod, the fine structure of this region appears very similar in overall organization (it differs in detail) Io most other arthropod nmscle insertions which have been described. In addition, supporting epidermal cells in the decapod branchiostegite are presented in detail fur the first time. These cells show a striking similarity to the epidermal cells of muscle insertions, although one lie/eworthy organizational difference exists between the epidermis in tlnese two regions,

Fig, 9. Cros', section through branchMstegite at low magniqcation. Supporti;~g epidermis is shown spanning t h e h e n l o / y m p h sinus ( h ) A cell from both the inner (ie} and outer (eel epidermis adjoin aJong their basal smfaces by an interdigitated inter-mediate junction (belveeen triangles), The cytoplasm o1" both epi(lernlal cells contains mlnlerous microtubuJes (small arrowa), The four large arrows indicate appro×imately the cell boundaries of the imler mid (rater epidermal cells at their basal smtilces. Notice [ha{ the nlicro'Lubules are clu~tered together ill one portion o f l h e cell~ and h~ cytoplasm lateral to the microlubules, other organclles re,g. mitochondria. RER, I]LICleUS) are present. MysJs. .: 7700,

I N S E R T I O N S A N D B R A N C H I O S T L ; G [ ' I E t-, p I D I-:R M 1S In nmscle insertions, the entire epidermal cell is moditied to articulate with the muscle via an intermediate j u n c t i o n and with the cuticle via a system of conical h e m i d e s m o s o m e s and intracuticular fibers; microtubules are present t h r o u g h o u t the cytoplasrn of this epidermal cell. In the s u p p o r t i n g epidermal cells of the branchiostegite, however, only a segment of each basal cell surface is modified as an intermediate junction. F u r t h e r m o r e , microtubules are clustered into one portiol~ of the s u p p o r t i n g epidermal cell, and o t h e r organelles are contained in the cytoplasm lateral to the m i c r o t u b u l a r cluster. Certain details of the fine structural o r g a n i z a t i o n in the muscle insertion and s u p p o r t i n g epidermis are of interest. In the muscle insertion, the apical aspect of the microtubules is unusual. While some microtubules terminate on the conical hemidesmosomes, many a p p e a r to be free-ending (terminate w i t h o u t a p p a r e n t connection or contact with the h e m i d e s m o s o m e ) below the ACB. It is realized that those w h i c h are freeending may curve and terminate on a n e a r b y h e m i d e s m o s o m e out of the plane of section: however, after e x a m i n a t i o n of n u m e r o u s micrographs, it was concluded t h a t these microtubules are free-ending and do not convergc elsewhere on conical h e m i d e s m o seines. F u r t h e r m o r e , it would not be possible to observe an A C B if these microtubules were to proceed apically and curve. In the supporting epidermis of the branchiostegite, microtubules converge apically on conical hemidesmosomes. They do not a p p e a r to be free-ending or to stop b e n e a t h an ACB. The apical m i c r o t u b u l a r insertions observed in the s u p p o r t i n g epidermis are typical of the pattern ordinarily seen in muscle insertions

625

(Bouligand, 1962; Lai-Fook, 1967; Caveney, 1969 and Hagopian, 1970). T h e free-ending microtubules as observed in the s h r i m p muscle insertion have not been reported previously for a r t h r o p o d muscle insertions. T h e intracuticular fibers are similar ultrastructurally to those observed in o t h e r a r t h r o p o d s , but show one significant difference in that they occasionally b r a n c h wilhin the cuticle. T h e b r a n c h i n g occurs in both the procuticle a n d epicuticle a n d p r o b a b l y aids in a n c h o r i n g the cuticle to the epidermis d u r i n g muscular activity. B r a n c h i n g has n o t been observed in the intracuticular fibers of the branchiostegite s u p p o r t i n g epidermis. In larval shrimp, the intracuticular fibers are e m b e d d e d directly in the cuticle in both the muscle insertions a n d s u p p o r t i n g epidermis; they do not pass out t h r o u g h pore canals as in some adult insects (Caveney, 1969). In fact, pore canals are n o t present in s h r i m p larvae. L a i - F o o k (1967) also' h a s reported t h a t intracuticular libers are e m b e d d e d directly in the cuticle of larval insects. T h e direct e m b e d d i n g of these fibers within the cuticle m a y be characteristic of larval an innals a n d superseded at later stages by passage of the fibers t h r o u g h pore canals. It is possible, therefore, t h a t the i n t r a c u t i c u l a r iibers are in some way involved in initiating or influencing pore canal f o r m a t i o n , l n t r a c u t i c u l a r fibers in s h r i m p larvae do n o t extend b e y o n d the cuticulin layer as they do in the crayfish branchiostegite ( K t h n m e l el al., 1970), T h e literature has b e c o m e very congested with terminology to d e n o t e the intracuticular fibers (designated .fibre i n t r a c u t i c u l a i r e by Moulins, 1968). W e prefer the :nomenclature of Moulins because it describes the position a n d structure of these specializations; they

Fig. 10. High magnification of supporting epidermis. Portions of both tile inner (ie) and outer (oe) epidermis and the intermediate junction (ij) occurring between the epidermal cells are shown. Microtubules are abundant in the cytoplasm of both cells. In tile outer epideJ real ceil, conical hemidesmosomes (ch) are visibie, and intracuticuiar fibers (small arrows) pass from these hemidesmosomes into tile cuticle. The cuticle above the outer epidermis is relatively thick and laminar in structure. Portions of intracuticular rods (large arrow) are also visible in tile outer cuticle. Protozoea° :,: 43,000. Fig. 11. High magnifications of supporting epidermis. A. Microtubules fi'om the inner (ie) and outer (oe) epidermis insert on the intermediate junction (large arrows). Protozoea. x 43,200. B. Conical hemidesmosomes (small arrows) are visible in the apical portion of this inner epidermal cell (ie), and intracuticular fibers (large arrow) extend from the hemidesmosomes into the cuticle. The cuticle (c) above the inner epidermis is very thin and iutracuticular rods are not present in this cuticle. Parallel arrays of microtubules (*) approach and terminate on the hemidesmosomes. Protozoea. ': 39,000.

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bc Fig~ I2, Schematic diagram of the supporting epidermis in the branchiostcgite. |ntracuticular rods (Jr) are present in the outer cuticle (oc) of the branchiostegite. Conical hemidesmosomes occur iI~ both the outer (oe) and inner (ie) epidermis, and intracuticular fibers (if) pass from the hemidesmosomes into the outer (oc) and inner (ic) cuticle. Microtubules insert on the conical hcmidesmosomes and the intcrdigitated intermediate .iu~ction (ij) which occurs between the two epidermal cells~ Hemolymph (h) is presem between the epidermal layers except in regions where supporting epidermis occurs. Most cellular organellcs in the supporting epidermis occur in the cytoplasm lateral to the microtubular cluster. The inner cuticle of the branchiostegite is considerably thinner than the outer cuticle. Tire branchial chamber (be) is indicated.

are fibers, a n d they are f o u n d in the cuticle, except for a small segment occurring within the conical h e m i d e s m o s o m e s . T h e t e r m tonofibrilla which was originally used by the light microscopists is now a m b i g u o u s in its meaning. Finally, terms such as muscle a t t a c h m e n t fiber, as used by Caveney (1969), are t o o restrictive; these fibers clearly m a y originate f r o m epidermis n o t associated with muscle~ Also, there is confusion in the literature c o n c e r n i n g intracuticular fibers a n d the structures which we h a v e t e r m e d intracuticular rods. K 0 m m e l et aL (1970) in their

observations o n the crayfish cuticle h a v e referred to these two structures collectively as Stiibe. I n the larval shrimp, these are two distinct structures which differ f r o m each o t h e r in t h e following respects: (1) intracuticular rods (1500 /~) a n d intracuficu/ar fibers (280 ~ ) vary in diameter a n d in electron density; (2) in longitudinal section, intracuticular rods arc never observed p e n e t r a t i n g the apical region of the epidermal cell or passing into h e m i d e s m o s o m e s as intracuticular fibers always do; (3) intracuticular rods m a y a p p e a r in the cuticle w h e n it is n o t associated with muscle insertions or support-

1NSIiRTIONS

ANI) BRANCHIOS']'I!,GITE EPIDERMIS

ing epidermis; (4l intracuticular fibers may be branched, while rods are not. For these reasons, it is necessary to make a distinction between these two structures. The function of the intracuticular 15bets is probably one of attachment, as stated by previous autlnors (Lai-Fook, 1967 and Caveney, 1969), The rods, on the other ha~d, may give support to the cuticle in regions where it is suhject to stress. The rods, for example, are observed in the outer cuticle of the branchiostegite and may strengthen the cuticle of this structure, which in the larval shrirnp is very thin. The cellular modifications (in particular the microtubules) in the epidermis of the muscle insertion and in the supporting epidermis are interesting when the functions of these two epidermal regions are considered. Presumably, the supporting epidermis belps to brace the cuticles of the attenuated branchiostegite. These cuticles, especially the inner cuticle, are very thin and unstructured in the larval stages. From pilot studies done in our laboratory of the University of Houstom there is reason to believe tlhat brown shrimp in protozoea and mysis stages hypo-osmo~ regulate in their naturalhabitat, m~dtheextensire hyper- and bypo-oslnoregulatory capabilities of older stages of penaeid species has been welPdocumented (see Talbot, el a/., 1972a for references), It is probable then that considerable osmotic pressure is exerted on the branchiostegites and this may occur as early in development as protozoa. Depending on the ambient salinity, this could be an external osmotic pressure on the branchiostegite which would tend to cause it to collapse (hypo-osmo-regulation) or an internal osmotic pressure wlnich would cause the

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branchiostegite to dilate (hyper-osmoregula~ tion), in either case, it would seem that the function of the supporting epidermis is to maintain the lbrm of the branchiostegite and resist tlne effects of an internal or external osmotic pressure. In both hypo- and hyperosmoregulation a pm'lHtN force would be exerted on the epidermal cells. Tlne situation in the muscle insertion is quite different. In this case, during muscular contraction, a pH//iag force is exerted on the epidermal cell as the tension developed by the muscle is transferred to the cuticle. In both situations, however, parallel arrays of microtubules seem to be the key organelle in stabilizing the epidermal cell. Lai-Fook (1967) has suggested tlnat in muscle insel> lions, the epidermis may depend on muscular tension (i.e. pulling) to initiate formation of microtubules. To speculate and extend this point further, tension on epidermal cells due to a difl'erence between the osmotic pressure of the tissue fluids and of the external environment may also elicit microtubule formation, and the supporting epidermis of the brancbiostegite could be such a case. Thus, a force exerted on a cell whether it be due to muscle tension or osmotic pressure, may influence formation of cytoskeletal microtubules within that cell, a point which would be interesting to study experimentally.

Acknowledgements Tile authors wish to thank Dr~ Richard Nea/ and Mr. Harry Cook for making available larval brown shrimp and Miss Candi Frances for preparing the schematic diagrams.

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References AUB~R, J. 1963. U/trastructure de la jonction myodpidermique chez les dipthres. J. Microscol?ie, 2, 325-336. BEt.L, A~ L., BMtNES, S. N. and ANDERSON, K. L. 1969. A fixation technique for electron microscopy which provides uniformly good preservation of the tissue in a variety of marine invertebrates. Biol. Bull mar° biol. Lab. Woods Hole, 137~ 393. BOLIIAGAND, Y. 1962. Les ultrastructures du muscle stri6 et de ses attaches au squelette chez [es cyclops (Crustac6s, Cop6podes). J, Micros'copie, 1, 377-394. CAVENEY,S. 1969. Muscle attachment related to cuticle architecture in Apterygota. d. Cell Sci., 4, 541-559, HAt.SOPIAN, IVl. 1970. Intercellular attachments of cockroach nymph epidermal cells. J. Ultrastrttct. Res., 33, 233 244. Kt~MMI¢L, G., CLASSf~N, H. and KELLER, R. 1970. Zur Feinstruktur yon Cuticula und Epidermis beim Flusskrebs Orco,eetes limosus w~ihrend eines HS_utungszyklus. Z. ZellJbrsch. mikroslc. Anal., 109, 517-551. KUO, J. S.~ McCul_LY, M. E. and HAGGis, G, H. 197 l. The line structure of muscle attachments in an acarid mite Calog@phus myeophagtts (Megnin) (Acarina). 7'issue & Cell, 3, 605 613, L~I-FooK, J. 1967, The structure of developing muscle insertions in insects. J. Morpho[., 123, 503 528, MOtjIdNS, M. 1968, [~tude ultrastructurule d'une formation de soutien 6pidermo-conjonctlve inddite chez le--'~s lnsectes. Z, ZellJbrsch, mikrosk. Anat., 91, 112-134. RICI~ARDS, A. G. 195[, The Integument of Arthropods. University of Minnesota Press, Minneapolis, pp. 237 242. SMCt'H, D. S., JXR~ ~ORS, U. and Rt/SS~