A comparison of cuticle deposition during the pre- and posteclosion stages of the adult weevil, Anthonomus grandis Boheman (Coleoptera : Curculionidae)

Int. J. InsectMorphol. & Embryol., Vol.21, No. 1. pp. 37-62, 1992

0020-7322/92 $5.00+ .00 Pergamon Pressplc

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A COMPARISON OF CUTICLE DEPOSITION DURING THE PRE- A N D POSTECLOSION STAGES OF THE A D U L T WEEVIL, A N T H O N O M U S GRANDIS BOHEMAN (COLEOPTERA • CURCULIONIDAE)* ROGER A . L E O P O L D , t SAMUEL M . N E W M A N t a n d GARY HELGESON~ § ~U.S. Department of Agriculture, Agricultural Research Service, Biosciences Research Laboratory and ~Department of Mathematical Sciences, North Dakota State University, Fargo, ND 58105, U.S.A. (Accepted 11 October 1991)

A b s t r a c t - - A n ultrastructural analysis of cuticle deposition before and after adult eclosion of the cotton boll weevil, Anthonomus grandis Boheman (Coleoptera : Curculionidae), is made to monitor the intra- and extracellular events that accompany the shift in cuticle architecture. A typical 5-layered epicuticle and a multi-lamellate procuticle are deposited during the pharate adult stage. Secretion of the epicuticle and the procuticle begins about 2 and 4 days after pupation, respectively. Following eclosion, a lattice-like endocuticle is secreted in the form of layers of parallel rod- or beam-shaped macrofibers. Deposition of endocuticle over the first week after emergence, is at a rate of 3 or 4 layers per day. The imaginal endocuticle accounts for the major portion of the cuticle mass as there is about a 14-fold increase in sclerite thickness and an overall 4-fold increase in non KOHextractable exoskeletal mass during the first week after emergence. Extensive cytoskeletal and surface remodeling plus a change in secretory product packaging occurs at the apical region of the epidermal cells upon shifting to deposition of the endocuticle. Intralayer orientation of the macrofibers is under cellular control and is accomplished by the formation of templates consisting of membrane placque-bearing, canal-like depressions on the apical surface extending across cell borders. Comparisons of cuticle sections to simulated plots drawn via computer graphics, show that each successive layer of macrofibers is rotated with respect to the overlying layer by an angle of about 72 °. Except for vertical columns of cuticular fibers that support pore canals, microfibril orientation within the procuticle/exocuticle generally follows the Bouligand model for a typical lamellate arthropod cuticle. Direct cellular control over the interlaminar orientation of the microfibrils forming the procuticle could not be discerned in this study.

Index descriptors (in addition to those in title): Insecta, development, integument, endocuticle, fiber orientation, TEM, SEM, computer modeling.

* Mention of a trademark or a proprietary product does not constitute endorsement, a guarantee or warranty of the product by the U.S. Department of Agriculture, and does not imply its approval to the exclusion of other products that may also be suitable. § Current address: MTS Systems Corp., Eden Prairie, MN 55343, U.S.A. Abbreviations: A Z = assembly zone; BM = basement membrane; D = duct; D C V = dark core vesicle; D E = dense epicuticle; En = endocuticle; Ep = epicuticle; Ex = exocuticle; GI = glycogen; G o = Golgi body; H = hemocoele; L = lysosome; M = matrix; MF = macrofiber; MVB = multivesiculate body; MW = membrane whorl; N = nucleus; P = procuticle; PC = pore canal; SD = septate desmosome; Se = seta; SV = secretory vesicle; T Z = transition zone; V = vesicle; Z A = zonula adherens. 37

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INTRODUCTION THE ARTHROPODintegument is a multi-layered structure, consisting of an epithelium and noncellular fibrous cuticular components. Fiber orientation within the lamellae forming the noncellular components of the cuticle has been the subject of study for decades. Interpretations differ on how the lamellae are constructed and to what extent the underlying epidermal cells are involved in the patterning of the cuticular fibers. Drach (1953) and Locke (1960), in ultrastructural studies of crustacean and insect cuticle, originally concluded that the fiber orientation exists usually as microfibrils (mrs) arranged in parabolic arcs that extend from layer to layer. Subsequently, Bouligand (1965) interpreted this arcuate appearance to be an artifact caused by oblique sectioning, and proposed that the lamellae were constructed of sheets of mrs mostly running horizontal to the outer surface of the integument that rotated in a helicoidal manner. However, the generally accepted Bouligand model does not fit laminar cuticles displaying vertical mrs or those with discrete separated lamellae, such as those found in various crustaceans and insects (Dennell, 1973; Hepburn and Ball, 1973; Mutvei, 1974; Dalingwater, 1975). Weis-Fogh (1970) devised a model whereby the horizontal sheets of mrs could gradually rotate vertically to accommodate cuticles with vertical components, but it does not allow for cuticles with separate lamellae. Further clouding the issue on the construction of the cuticular lamellae, are the arthropods that have cuticles which show a marked departure from the typical arcuate appearance of fiber orientation. Insects, such as those producing daily growth layers of cuticle, may alternate one or more helicoidal lamellae with a layer of unidirectional, parallel-running mrs (Neville and Luke, 1969). Other arthropods display areas of the endocuticle constructed of layers of crossed macrofibers (MFs) or "Balken", such as have been described for diplopods, tarantulas, and some beetles and crustaceans (Richards, 1951). While Biedermann (1903) correctly assumed the MFs to be constructed of smaller fibers, a German biologist (Meyer, 1842) was the first to describe microscopically visible fibrous aggregates in the cuticle of a rhinoceros beetle, and compared them to beams or to the wooden rafters that support a roof. Hence, the German word, "Balken", has been used in the early literature to refer to similar configurations found in arthropod cuticle. Other early workers observed that the axes of the MFs (or "Balken") appeared to cross those of adjacent layers at regular angles of 45 °, 60° or 90° in the cuticles of a variety of arthropods (Haekel, 1864; Biedermann, 1903; Schulze, 1913; Kuhnelt, 1928; Frey-Wyssling, 1938). Similar to this arrangement, is the "plywood" endocuticle of certain hemipteran and coleopteran insects, which has been characterized by Zelazny and Neville (1972a) as layers of parallel mrs crossing adjacent layers in a pseudoorthogonal manner. The mechanisms controlling orientation of the mrs that form the various architectural patterns observed in arthropod cuticles have yet to be resolved, but several theories suggesting cellular involvement (Locke, 1967; Neville and Luke, 1969), self-assembly systems (Weis-Fogh, 1970; Neville, 1975) and post-secretory mechanical stresses (Richards, 1951) have been advanced. Further, the temporal changes occurring within and on the surface of the epidermal cells secreting the helicoidal lamellar cuticle have been extensively documented by Locke and his coworkers (see review of Locke (1985)), but it is not clear whether the same sequence of events occurs in cells secreting cuticle in the form of macrofibers. During the pharate adult stage, just before eclosion, the epidermal cells underlying the

A Comparison of Cuticle Deposition of the Adult Weevil

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sclerites of the cotton boll weevil, Anthonomus grandis, cease formation of the helicoidal type of lamellar cuticle in preparation to switching to the formation of an endocuticle, constructed of layers of the large rod- or bar-shaped MFs (Leopold et al., 1985). The epidermis of this weevil secretes this type of cuticle after eclosion, and in some areas of the exoskeleton, the thickness may increase up to 20-fold during the first 2 weeks of adult life. This report represents a detailed study of cuticle deposition occurring in the integument during the pupal and adult stages of the boll weevil. In it, we document the architectural design of the adult cuticle and compare the pre- and posteclosion epidermal cell activity during differentiation of the two cuticle patterns.

MATERIALS

AND

METHODS

Insects The weevils used in this study were of the ebony strain (Bartlett, 1967) and were obtained from a colony maintained at this laboratory. The environmental conditions for rearing consisted of a fluctuating temperature regime which cycled from a low of 22°C during lights off to a high of 32°C which was held for 7 hr during the middle of a 16 hr photophase. Relative humidity was held at 55 + 5%. The posteclosion adults were maintained at a temperature of 29 + 2°C, 60 + 5% RH and a 16 hr photophase. Additional information on rearing and the diets used can be obtained from Earle et al. (1970) and North et al. (1981). Microscopy Portions of the integument were dissected from the center of the second ventral abdominal sclerite of adult weevils at various times pre- and posteclosion. The tissue samples were immediately placed into 3% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2) either overnight or for up to 2 days following dissection. Tissues were postfixed in 1% OsO4 (1 hr, 4°C) in the same buffer and dehydrated in a graded series of ethanol washes. Samples to be examined with transmission electron microscopy (TEM) were placed in 3 changes of propylene oxide, propylene oxide : Spurr's resin (1 : 1) overnight, and then embedded in Spurt T M low viscosity resin (Spurr, 1969). The resin was cured for 8 hr at 70°C before sectioning. Thin sections were stained with saturated aqueous uranyl acetate for 45 min and then in lead citrate for 5 min (Venable and Coggeshall, 1965). TEM was performed on a Phillips 300T M electron microscope operated at 60 kV. Sections of about 1 Ixm were also cut and used for light microscopical examination and stained with an aqueous solution of 1% azure II B, 1% methylene blue and 1% sodium borate. The angle of rotation of each layer of MFs was estimated by tracing the images projected through a camera lucida attached to a Zeiss T M compound research microscope. Alternate samples, to be examined with scanning electron microscopy (SEM), were processed similar to those for TEM through ethanol dehydration following which, they were critical-point dried with liquid COz in an Autosamdri 810T M (Tousimis Corp., Rockville, Maryland). The dried tissues were then coated with a 25-30 nm layer of gold/palladium using a Hummer V TM (Anatech Ltd, Alexandria, Virginia) and viewed with an Amray AMR 1000T M scanning electron microscope operated at 20 kV. Some samples of weevil integument were deproteinated in hot alkali (10% KOH for 20 min at 100°C) and then processed for SEM as described above. In most cases, observations were made on 10-20 tissue samples for TEM studies and 8-10 samples for SEM. Computer simulation A program was devised for the graphic simulation of the adult boll weevil endocuticle, requiring an IBM XT/AT TM or compatible computer equipped with a color graphics adapter and accompanying monitor. The plots generated by this program represent various cross-sectional views of the layered endocuticular MFs crossing each other at predetermined fixed angles. The program was conceived and implemented as a simple 3-dimensional graphics manipulation tool, and was created by using the 3-dimensional transformation techniques as outlined by Newman and Sproull (1979). It contains only specific information on MF orientation within the endocuticle of the boll weevil. The primary implementation language is "C" with some limited use of 8088/8086 assembler for the low level screen I/O routines. The program is compiled and linked with the DeSmet C T M compiler, assembler and linker. Copies of this program and/or source code can be obtained by sending a 51/4 or 31/2 in. diskette to one of the authors.

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4EPICUTICLEI. ~PROCUTICLE~ [ 4ENDOCUTICLE~.

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FI6.1. Diagram illustrating chronology of events as related to cuticle deposition during the pupal and early adult stages of the cotton boll weevil.

RESULTS

Preeclosion cuticle deposition The time course of the cellular events that occur during formation of the adult boll weevil cuticle, is depicted in Fig. 1. The pupal stage, under the rearing conditions used in this study, is approximately 6 days long, as compared with about 4 days when a constant temperature of 28°C is used. Apolysis of the pupal cuticle begins during the white-eyed stage, about 18-24 hr after the larval-pupal transformation, and extends into the beginning of the yellow-eyed stage. Secretion of the outer layer of the epicuticle, the first indication of the onset of adult cuticle deposition, occurs during the yellow-eyed stage (c. 40-48 hr after larval-pupal transformation). Deposition of the procuticle (putative exocuticle following eclosion) begins about 48 hr before eclosion and ceases prior to emergence. The condition of the epidermal cells during secretion of the epicuticle is essentially similar to what has been described for other insects. Figure 2 shows a photomicrograph of a typical cell after apolysis at the beginning of adult epicuticle deposition. The elongate cells contain rounded nuclei which are located in the basal region. There are numerous filamentous mitochondria primarily located near the apical surface of the cells. Interspersed among the mitochondria are small deposits of glycogen, numerous ribosomes, and small coated vesicles and larger vesicles containing a flocculentappearing material. Occasionally, Golgi complexes are found in the apical region of the cells, but more often they are located closer to the basally positioned nucleus. Flattened cisternae of rough endoplasmic reticulum (RER) are abundant and are mostly located perinuclear as are large concentrations of glycogen that usually surround membranelimited vacuoles. The lateral plasma membranes of adjacent cells are joined at the apical surface by belt desmosomes (zonula adherens) and below that by septate desmosomes. Very few gap junctions are present. The lateral surfaces of these cells are usually seen in close apposition down to their basal surfaces, but do display some intercellular spaces. The typical, continuous basal lamina is not always present at this stage, especially in

A Comparison of Cuticle Deposition of the Adult Weevil

FIGS 2-5. Cellular configurations that occur during secretion of the epicuticle. Figure 2 shows a

portion of an epithelial cell from a weevil in yellow-eyed pupal stage after apolysis. The elongate cell bears slender apical microviUi (dark arrow), has basal concentrations of glycogen and perinuclear flattened RER cisternae (white arrow) plus clusters of mitochondria (asterisk) and Golgi bodies in the apical region. Figure 3 is an enlarged view of apical microvilli secreting the beginnings of an outer epicuticle (arrow). Figure 4 shows layers comprising epicuticle at the beginning of the dark-eyed pupal stage. Upper arrow denotes superficial layer, next 3 arrows mark trilaminate outer epicuticle, and black on white bars show extent of the dense epicuticle. Figure 5 is a lower magnification view of an area similar to that of Fig. 4, showing low club-shaped villi (arrows) and cellular junctions.

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areas where fat body cells and oenocytes are closely apposed. The epidermal cells appeared to be separated from these cells by only their plasma membranes at this stage of cuticle formation. The epicuticle is the first layer of the new cuticle that is deposited and, depending upon the stage and/or species involved, it consists of 2-5 layers (Hadley, 1986). During the initial stage of the formation of the epicuticle in the weevil, the apical surfaces of the cells bear clumps of elongated, very slender microvilli (Fig. 3). The epicuticular layer first appears as dark patches of material associated with one or more of the microvilli. This layer is very electron-dense and probably corresponds to the beginnings of the "cuticulin" layer (Locke, 1966) or the alternative "outer epicuticle", as denoted by WeisFogh (1970). Subsequently, the microvilli shorten and become more numerous as this 3-layered structure is deposited. Two dense laminae are separated by a lighter area, which altogether are about 20 nm thick (Fig. 4). The process of depositing this trilaminate structure in this insect, is apparently very rapid. We were unable to obtain examples that were devoid of portions of the inner epicuticle--even when the latter portion of the yellow-eyed and early black-eyed stages were sampled at 4-5 hr intervals. The inner epicuticle (dense layer of Locke (1961)) is a homogeneous, granularappearing layer and it varies in thickness from 0.1 to 2 Ixm over the sclerites. During formation of the inner epicuticle, the microvilli at the apical plasma membrane are short and in the shape of club-shaped mounds. The membrane placques are not visible (Fig. 5). Also at this time, a thin layer (10 nm) appearing as an accumulation of fine fibers, is deposited on the surface of the outer epicuticular layer (Fig. 4). This layer is partially obscured by aggregations of coarse fibrous material located in the space between the ecdysial membrane and the surface of the new cuticle. Further, this layer disappears after eclosion of the adult. The origin of this "superficial layer" (Filshie, 1970; Zacharuk, 1972) is obscure but it possibly derives from the epidermal cells, by diffusing through the outer epicuticle through minute pores (Locke, 1966). When newly formed, the innermost layer of the epicuticle seems to merge with the first lameUa of the procuticle with no real evidence of change in electron density, and except for the appearance of the fibers it is difficult to identify where the procuticle begins. Where pore canals are present, this zone is more evident because the canals appear to terminate where the inner epicuticular layer merges with the first lamella of the procuticle (Fig. 6). However, after emergence, the sclerotization and pigmentation of the more superficial layers of the exocuticle cause a definite change in the appearance of the relative electron density of these layers. At this latter stage, the inner epicuticular layer has lower electron density than either the outer epicuticle or the exocuticle (Fig. 7). Also, the trilaminate appearance of the outer epicuticle is not evident in the postemergent insects. The outer epicuticle now appears as a single 20 nm thick layer. Deposition of a typical lamellate procuticle begins during the black-eyed stage about 2 days prior to eclosion and continues until emergence. During this time, the apical surface of the cells is configured into numerous, often closely apposed folds or ridges (Figs 8, 9). The tops of these folds bear membrane placques which provide fibrous constituents of the cuticular layers, while the valleys between the folds show evidence of transport of material from associated vesicles. The interface between the cell surface and the new cuticle is the presumptive site of the assembly zone (Delbecque et al., 1978) where the precursors of the cuticle are assembled or undergo self-assembly into the lamellae. The

A Comparison of Cuticle Deposition of the Adult Weevil

FIGs 6-9. Views of epicuticle/procuticle and procuticle/cellular interfaces. Figure 6 shows an

epicuticle/procuticle interface of a preecdysial pharate adult. Arrow denotes trilaminate outer epicuticle. Also shown is electron-opaque, dense epicuticle and a pore canal terminating at dense epicuticle. Figure 7 is similar to Fig. 6, except that it is from a postecdysial adult. Trilaminate structure is no longer visible and dense epicuticle is less electron-opaque. Figure 8 shows a procuticle/cellular interface of a preecdysial adult. In assembly zone, loosely arranged laminae are visible which arise from membrane placques (arrow) on folds at apical cell surface. Figure 9 shows an oblique section of the procuticle/cellular interface. Asterisks mark assembly zone next to more dense procuticle, and arrows mark tops of folds where laminae are assembled.

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cell-cuticle interface represents a span of about 0.5-0.6 ~m and contains masses of fibrous material from which the organized lamellae above it are formed. This material is less dense than the lamellae and frequently has the form of loosely assembled laminae. The membrane placques appear to serve as assembly initiation sites for these laminae because continuous fibrous connections between placques are often observed (Figs 8, 12). Between 18 and 24 lamellae are constructed before the procuticle deposition ceases. We found that the number of lamellae deposited varies with location within the sclerite and also between weevils. Often those areas within the sclerite which require structural modification, such as an accommodation for a duct, sensory hair or muscular attachments, show a departure from the number of lamellae existing in areas without such modifications (Fig. 10). Further, there is a gradual increase in the thickness of the lamellae as procuticle deposition proceeds to completion (Fig. 22). The lamellae immediately beneath the epicuticle are about 0.1 ~m thick as opposed to 0.16 IJ,m for those overlying the endocuticle. The thickness of the ventral abdominal sclerites at emergence ranges from 1.7 to 2.6 Ixm. The elongated shape of the epithelial cells during deposition of the epicuticle is retained during the initial stages of procuticle formation. Then the cells become progressively shorter as deposition of procuticle approaches completion. The massive stores of glycogen within the cells become reduced in size during procuticle deposition, and are generally located at the apical region in the form of small aggregates (Fig. 10). The perinuclear Golgi bodies, with most vesicular cisternae, remain as numerous as they were during deposition of the epicuticle, then begin to diminish in number about midway into deposition of the procuticle. Vesicles of medium electron density and ranging from 40 to 60 nm in diameter are associated with the Golgi bodies and are also dispersed throughout the apical cytoplasm. Smaller coated vesicles are also present in the apical cytoplasm, but not in the abundance seen during epicuticle formation. Homogeneous large vesicles (1.0-2.0 ~m) ranging from medium to high electron density are present in small numbers and are mostly located in the basal area of the cells. Vesicles of similar size, but of extremely different content, are also in the apical cytoplasm. These are the multivesicular bodies as described by Locke (1966). They contain varying amounts of small empty vesicles and a mixture of granular and fibrous materials (Fig. 11). A few cells, during epicuticle formation and noticeably more towards the end of the formation of the procuticle, show considerable departure in cytoplasmic organization from that just described. The most noticeable difference is in the rough endoplasmic reticulum. It is extensively vesiculate in both the apical and basal regions of the cells. These cells also have fewer ribosomes, and the nuclei have less densely staining nucleoplasm. Membrane whorls are often seen, while the other organelles, vesicles, and inclusions seem to be of normal abundance and condition. Exocytosis apparently occurs unabated in these cells because no differences are evident in cuticular assembly or structure at the cell-cuticle interface when they are compared to the adjacent more typical cells (Figs 12, 13). The numerous pore canals that penetrate the procuticle are formed around unipolar extensions of the cells. These canals run perpendicular to the lamellae, and appear as vertical columns with fibrous cores surrounding small groups of densely staining filaments (Fig. 14). No obvious coiling of the pore canals is seen, unlike those of the

A Comparison of Cuticle Deposition of the Adult Weevil

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1.0pro FIGS 10 and 11. View of integument of pharate adults at dark wing stage. Figure 10 shows a portion of procuticle containing a large duct of a gland which disrupts arrangement of lamellae (asterisk). Arrows denote array of different sized secretory vesicles. Figure 11 shows the array of organelles present during formation of procuticle. Asterisk marks folding of apical surface of cell, and arrow a coated vesicle,

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FIGS 12 and 13. Apical views of cells having vesiculate ER during early and late stages of procuticle deposition. Figure 12 depicts the early stage showing membrane placques (arrow) connected by laminar fibrils, a multivesiculate body, and atypical vesicular RER (asterisks). Figure 13 depicts the late stage, also showing vesicular RER (asterisks) plus membrane-bound secretory vesicles, microtubules (arrows), and membrane whorls.

A Comparison of Cuticle Deposition of the Adult Weevil

FIG. 14. Latter stage of procuticle deposition showing 2 pore canals being formed by unipolar cellular extensions (dark arrows) and containing pore canal filaments (white arrow). FIG. 15. Switch-over from exocuticle to endocuticle formation with an intervening transition zone. White arrows mark limits of a 'canal' on apical surface of a cell where an endocuticular macrofiber is formed. Dark arrow marks a dark core vesicle. FI6. 16. An example of increased cell membrane interdigitation occurring in teneral adult. Arrows denote septate desmosomes and a unidirectional grouping of microtubules surround asterisk. FI6.17. Golgi body and dark core vesicles (arrows).

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larval cuticle (micrograph not shown). Several hours preceding emergence, the epithelial cells of the pharate adult begin changing from depositing a typical lamellate procuticle to that of the imaginal endocuticle. At the cuticle--cell interface, the folds in the plasma membrane become taller and the laminar arrangement of the material secreted into the assembly zone is replaced by a fibrous cuticle without any apparent laminar ordering. This area is a transition zone (Fig. 15) of varying thickness with an oblique fiber orientation, which marks the site of changeover from the helicoidal to the crossed macrofiber type of cuticle deposition.

Posteclosion endocuticle deposition Following emergence, the epithelial cells of the teneral adult retain the low cuboidal shape with rounded or slightly flattened nuclei. Basal intercellular spaces appear narrower than in previous stages and may extend up to two-thirds of the way toward the apical surface. The arrangement and type of cellular junctions remain essentially the same as in previous stages, although they appear to increase in complexity as the plasma membranes are often observed to become increasingly convoluted and interdigitated (Fig. 16). As in the beginning of the procuticle deposition, the cells exhibit the typical appearance of cells maintaining a high rate of synthesis and export. However, in addition to the moderate changes in overall cell configuration, there are also some differences in cell content. The RER is noticeably more evenly distributed throughout the cells and the mitochondria are exceedingly more filamentous than in previous stages of cuticle deposition. Although considerably more numerous in the apical region, more mitochondria are also seen in the basal region of the cells than previously. The apparent primary secretory product is a Golgi-derived, dark core vesicle (Fig. 17), which has a diameter of about 0.15 Ixm. These dark core vesicles move from the perinuclear Golgi to the apical surface. The mechanism by which the contents of the dark core vesicles cross the apical plasma membrane and are incorporated into the structure of the cuticle was not evident in our preparations. Also in the apical region, but less common, are larger vesicles with a diameter of about 0.5 Ixm and containing a sparse amount of flocculent material. Least common are vesicles with diameters up to 0.7 txm and containing a moderately electron-dense material. They are primarily located basally and around the nuclei. Glycogen stores are as evident in the basal area as before, but rarely are they seen in the area between the nucleus and the apical surface (Fig. 18). The epithelial cells of the teneral adult also undergo marked surface alterations. The apical surfaces of the cells become patterned into clusters of villi arranged into parallel rows of canal-like depressions, which are continuous across cellular boundaries (Figs 15, 19, 20). The tips of the clustered villi with their membrane placques are directed toward the interior of each canal. Each canal serves as the template for a MF as the assembly of unidirectional mrs is initiated here. The orientation of the canals and ultimately the first layer of MFs, is parallel to the longitudinal axis of the insect. Grouped along the canals are numerous dark core vesicles. Numerous microtubules are oriented both parallel and perpendicular to the canals (Fig. 19). This type of ordering of the microtubules differs from that seen earlier (Fig. 8). Before fabrication of the first layer of MFs is complete, a second begins to form in a

A Comparison of Cuticle Deposition of the Adult Weevil

FIG. 18. Integument of a one day postemergent adult with all 3 layers of cuticle present. White arrow marks epicuticle. Endocuticle displays 4 layers of macrofibers at various levels of assembly. Large dark arrows mark filamentous mitochondria. Small dark arrows indicate secretory vesicles. Basal area exhibits abundant amounts of glycogen surrounding vacuoles.

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FIGS 19 and 20. Enface and cross sections of macrofibers during deposition. Figure 19 shows the two layers visible at apical cell surface. One layer is seen to span border (dark arrows) of cells. Dark core vesicles and microtubules (open arrows) accumulate along membrane adjacent to macrofibers next to membrane placques (white arrows). Asterisk marks a branch in macrofiber. Figure 20 depicts 4 layers of macrofibers (1-4) with associated cellular extensions (dark arrows). Membrane placques line a canal-like depression in apical surface (white arrows). Layers 3 and 4 are comparable to the 2 layers of macrofibers in Fig. 19.

A Comparison of Cuticle Deposition of the Adult Weevil

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parallel plane but at an altered angle to the one above it, and before either is finished, a third layer is initiated. In this fashion, usually 3 and sometimes 4 layers are observed to be in various stages of deposition at one time (Figs 18, 20). The accretative process by which the MFs are constructed, apparently requires that the cell membrane placques be in close proximity to the site of accretion, because elaborate villiform extensions of the cell are developed to accommodate the completion of the upper layers. However, the cell surface canals encasing the components of the most recently initiated layer often bear placques attached to villi that are much reduced in height. Most of these membrane placques are regularly spaced at intervals of about 0.13 p~m and are closely apposed to the surface of each nascent MF (Figs 19, 20). The assembly zone for the MFs bears little resemblance to that of the lamellar cuticle. It undergoes a progressive change in size and content. The transformation of this zone accompanies the increase in size of the MFs. The assembly of the second and subsequent layers of MFs starts as a loose accumulation of mrs having a unidirectional orientation. Each nascent MF usually attaches to the MFs in the above layer at the points where they cross. The core of each MF becomes less electron-dense as the component mrs appear to condense. Thus, the assembly zone often appears as a darkened semi-circle surrounding the lighter, but probably more compact collection of mrs. The darkened area of the assembly zone then becomes progressively smaller as the MF nears completion, and the surfaces of the membrane placques become more closely apposed to these compact bundles of mrs (Figs 18, 19). In addition to the attachment sites where each MF is joined to the adjacent MFs above and below, examination by SEM of KOH-treated endocuticle revealed that numerous fibrous struts arise in a criss cross fashion at these intersections (Fig. 21). These struts are not as clearly evident in the TEM preparations, and in the outermost layers they are obscured by the fibrous matrix filling the area between the MFs. The manner by which these struts are fabricated was not discerned in our preparations. As mentioned above, filling the spaces between the MFs is a matrix having a fibrous appearance, but unlike the MFs, it is extractable with KOH. This presumably proteinaceous material shows no obvious repetitive inter- or intralayer ordering (Fig. 22). Moreover, while the MFs exhibit varying degrees of electron density, depending upon the angle at which the section was cut, the apparent density of the fibrous matrix is consistent regardless of the angle of sectioning. Membrane placques are present at the time of the deposition of matrix between the MFs, but it could not be determined whether they were the source of the matrix fibers or were forming the interconnecting struts. This matrix also supports the numerous pore canals that continue outward and are continuous with those formed in the lamellate portion of the cuticle prior to eclosion (Figs 22, 23). As in the lamellate cuticle, the canals are formed as a collection of thin (20--40 nm) electron-dense filaments, but in the endocuticle they are not associated with a similarly dense fibrous core. Following the pore canals over a distance of more than 3 or 4 layers was not possible in our preparations. Most of the segments of canals that we were able to follow appeared to be relatively straight. This suggests that the interlayer alignment of the MFs is such that most pore canals are able to traverse the endocuticle with only minor deviations. However, near the junction of the MFs with the exocuticle, a few canals appear to follow a zigzag route through the endocuticular layers (Fig. 23). Further, no obvious helical

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FIG. 21. SEM of 3 layers of macrofibers (1-3) which had been subjected to K O H digestion. Arrows denote struts running between macrofibers and asterisk marks bifurcation of a fiber. FIG. 22. Pore canals show columns of vertical microfibers (white asterisks) in exocuticle. Black arrow marks a bifurcation of pore canal filaments in endocuticle surrounded by a matrix, which starts at transition zone. Black asterisks compare widths of lamellae at outer and inner exocuticle. FIG. 23. Black arrows mark serpentine route pore canal filaments follow through macrofiber layers. White asterisk marks cuticutar column and black asterisk a branching pore canal.

A Comparisonof Cuticle Depositionof the Adult Weevil

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coiling of the canals, comparable to that reported by other workers (Richards, 1951; Neville et al., 1969; Neville and Luke, 1969) was seen in the endocuticle of this insect. Branching of the MFs can be seen in both the TEM and SEM preparations (Figs 19, 21). Usually, the branching is in the form of a bifurcation. While MF branching most often occurs in areas where some structural modification of the endocuticle is required (e.g. at the boundaries of the sclerite and around ducts, sensory components and setae), it is also seen in areas where the need for a change in direction is not readily apparent. A difference in tensibility is observed when the layers of the most recently fabricated MFs are compared to those formed earlier. A SEM examination of the adult cuticle of 3 day postemergent weevils, transected with a razor blade, shows that the 3 or 4 innermost layers of the endocuticle are cut cleanly, while the rest of the layers are torn or broken unevenly (Fig. 24). Interestingly, the exocuticle also cuts cleanly with no tearing. Further, a gradual change in the size and cross-sectional shape of the MFs occurs as the new layers form beneath older ones (Fig. 26). Next to the arcuate layers of cuticle, the MFs have a roughly rounded cross-sectional shape with a diameter of about 0.8 p,m, while deeper into the endocuticle they have an ovoid or rectangular shape with dimensions as great as 1.2 I~m × 2.7 p,m. The thickness of the second abdominal ventral sclerite at 7 days postemergence ranges from 33 to 42 ~m, of which the endocuticle makes up about 80%. Thus, the increase in sclerite thickness over the 7 day postemergence period is about 14-fold.

Macrofiber orientation The endocuticle sections cut en face for light microscopy and SEM, suggest that each layer of MFs is oriented at a regularly fixed angle relative to the layer immediately below it, and also, except for the first layer, to the one above it (Fig. 25). The angles formed by the crossing of the MFs were measured with the aid of a camera lucida on light microscopy sections of cuticle cut en face. The mean of the layer-to-layer shift in MF orientation was 71.9 ___ 2.4 ° (25 measurements, 5 insects). Further, many of the TEM micrographs we examined seemed to indicate that there is a repetitive orientation of the MFs, which appears every 6th layer (Fig. 26). While this 72° rotation of the MF orientation (i.e. 360° - 5) in the TEM micrographs was the most prevalent implication, there also appeared to be other repetitive situations that were not indicative of a 72° angle of rotation. We subsequently suspected that the angle at which the cuticle was sectioned could possibly present an appearance of a different pattern. To investigate this possibility, a computer program was designed to plot simulated patterns of boll weevil endocuticle having cross-sectional configurations that could be varied on the x and/or y axes. The plane of the graphics screen represents the angle at which the microtome knife had passed through the cuticle specimen. In this program each layer of parallel MFs is assigned a 72° angle relative to the layer above it. Thus, the program creates a viewing window showing a portion of endocuticle where the intersection points of the layers of MFs define the appearance of each cut end of a MF on the screen. When the 3-dimensional schematic representation of the boll weevil endocuticle that was stored in the program is manipulated by rotation about its own axes, the intersection of the lines defining the cut edges of the MFs with respect to the graphics screen is recomputed and replotted. This causes the image to change and each time the image is rotated about one of its axes, it is as though the microtome knife had taken the

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R . A . LEOPOLD et al.

FIGS 24-26. Comparison of physical/morphological nature of postecdysial adult exo- and endocuticle. Figure 24 is a SEM view of integument of a 3-day postemergent adult sliced with a razor blade. White arrows mark limits of 12 layers of macrofibers, black arrows delineate inner 3 layers that cut without tearing. Figure 25 shows an oblique slice through endocuticle showing relative orientation of 6 layers of macrofibers. Layers 1 and 6 have same orientation. Figure 26 is a T E M view of area similar to Fig. 24. Bars delineate exo- and endocuticle. White asterisks mark layers having same orientation (Nos 1, 6, and 11).

A Comparison of Cuticle Deposition of the Adult Weevil

x=90 y=45

55

° °

B FIGS 27A and B. Computer simulated plots for 2 TEM views of adult integument cut at different angles on y axis.

56

R.A. LEOPOLOet al. 24

K 22 28

7

18-

(4

14O 12

Id

A

Z 5 (4 I~J 4.J Ld X 3 t4 0 X 2~d

4

I

k. 0

8

~

14 OilY5

POSTEHERGENCE

FIG. 28. Comparisonof number of endocuticularlayers vs. exoskeletalweight measured over a period of 14 days:0, layers;O, weight.

corresponding path through the specimen. Comparison of the computer-generated plots with certain TEM micrographs, in which the interlayer fiber orientation appeared to differ from the 72° angle of rotation, confirmed that it did not (cf. Fig. 27A, B). Rate o f cuticle deposition The time course of deposition of the endocuticular layers of the second abdominal sternites of weevils during the 14 day period after adult emergence is depicted in Fig. 28. During the first week, about 3 or 4 layers are deposited per day under a 16 hr photophase and a temperature of 28°C. During the second week postemergence, cuticle deposition proceeds at a much slower rate. Total exoskeletal mass generally mirrors the increase seen in the number of endocuticular layers of abdominal sternites. The mass increases during the first week and then levels off. Additional studies indicated that deposition of the cuticle in this weevil is not under circadian control (data not shown).

DISCUSSION Under the rearing and maintenance conditions used in this study, the time frame required for deposition of a definitive abdominal cuticle for this insect is nearly 2 weeks. While deposition of imaginal cuticle occurs both before and after eclosion, the major thickening of the abdominal sclerites occurs during the first week after ecdysis. During the first 7 days of the posteclosion phase of cuticle synthesis, there is an average 14-fold increase in sclerite thickness, which also translates into nearly a 4-fold increase in the non KOH-extractable exoskeletal mass.

A Comparison of Cuticle Deposition of the Adult Weevil

57

Zelazny and NeviUe (1972a) examined the rate of deposition of the endocuticular layers over the time of linear growth in 5 species of beetles. It ranged from 22 hr/layer in Olethrius insularis to 44 hr/layer in 3 Scarabaeidae species. During the first week following eclosion, the boll weevil deposits an average of 1 layer every 8 hr. Thus, while the total number of pronotal endocuticular layers produced by the adult scarab Orcytes rhinoceros in the study by Zelazny and Neville (1972a) was nearly the same as the boll weevil, the period of linear increase was only about one week for A. grandis as opposed to 40 days for O. rhinoceros. It should be mentioned, however, that the endocuticle of the beetles surveyed in the Zelazny and Neville study is not entirely comparable to that of the boll weevil. All beetles examined by Zelazny and Neville had the plywood-type of endocuticle, unidirectional layers of mfs separated by intervening layers of helicoidally arranged mrs. In the areas we examined, ecdysis not only marks the beginning of a significant increase in the quantity of cuticle to be formed by the boll weevil, but also a switch in structural design. However, the link between adult ecdysis and change in structural design is not firmly established among the beetles. In O. rhinoceros, Zelazny and Neville (1972a) found that in certain anatomical areas up to 3 layers of endocuticle are deposited before adult ecdysis, while in T. molitor adult endocuticle begins in the elytra 1 day after ecdysis. With the rhinoceros beetle, the sites of the preecdysial deposition of endocuticle were related to darkening of pharate adult exocuticle. Determining a possible explanation for diversion of a considerable amount of energy stores by the boll weevil during the first 7-10 days of imaginal life to the construction of a thickened cuticle equipped with criss crossing MFs was beyond the scope of this study. However, it is reasonable to assume that this is a means for the weevil to provide itself with a durable protective exoskeleton, which aids it in surviving the extremes of its environment for periods lasting up to 6 months (Mitchell et al., 1973).

Preeclosion cuticle structure The ultrastructural configuration of the 2 principal components of the cuticle formed prior to eclosion, the epicuticle and procuticle, are basically similar to what has been previously described for a variety of insects (GaUeria and Colpodes--Locke (1961, 1966); Locusta--Rinterknecht and Levi (1966); Nezara--Filshie and Waterhouse (1969); Lucilia--Filshie (1970); Drosophila--MitcheU et al. (1971); Ctenicera, Limonius and Hypolithus--Zacharuk (1972); Tenebrio--Delachambre (1970) and Delbecque et al. (1978)). These studies show evidence of taxonomic, developmental, and individual regional body variation in the number of epicuticular layers among the insects thus far examined. For the sake of clarity and uniformity in describing the boll weevil epicuticle, we have used terminology which is essentially that suggested by Weis-Fogh (1970) and recently redefined by Wigglesworth (1990). Thus, the epicuticle of the abdominal sclerites consists of an extracuticular superficial layer lying on top of the osmiophilic outer epicuticle which lies upon the thickest layer, the inner epicuticle. The fine structure of the boll weevil procuticular lamellae generally has the typical arcuate appearance, which is widely accepted to be indicative of parallel layers of mrs arranged in a helicoidal manner as first proposed by Bouligand (1965). One departure from this basic design for arthropods, is the presence of columns of mfs which traverse the lamellae in a direction perpendicular to surface. These columns provide a system of routes for the pore canal filaments to carry the cellullar materials across the lamellae

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R . A . LEOPOLD et al.

which are subsequently utilized in sclerotization, maintenance, and repair of the outer layers of the cuticle (Locke, 1961). Most often, these filaments follow coiled paths across the lamellae, which apparently rotate in phase with the helicoidal arrangement of the mrs (Neville et al., 1969). However, Delachambre (1971) described cuticular columns of mrs associated with pore canal filaments in the adult exocuticle (preecdysial procuticle) of Tenebrio molitor which appear to be the same or similar to that of the boll weevil. Further, vertically arranged chitin fibers that are continuous between layers of arcuate cuticle have been observed in other insects as well as decapods (cf. review of Hepburn (1985)). The presence of vertical mfs in the midst of an apparent primary helicoidal arrangement, as found in the exocuticle of the boll weevil, introduces some uncertainty as to how the Bouligand model can accommodate this dichotomy of mf orientation. Others have pointed out the inability of this model to account for the double spiral orientation of mrs, such as observed within the lamellae of the corneal cuticle of Boreus californicus (Gordon and Winfree, 1978). Since Hepburn (1985) has thoroughly reviewed the various difficulties associated with the Bouligand model, they will not be restated here.

Posteclosion cuticle structure Prior observations on the structure of the endocuticle of arthropods having layers of parallel MFs crossing adjacent layers at regularly fixed angles, like that of the cotton boll weevil, have primarily been made using the light microscope. The use of the TEM and SEM microscopes and computer modeling in the present study has provided a means to gain a more comprehensive understanding of the deposition of MFs and the integral microfibrillar orientation. The central theme for arthropod cuticle attributed to be regularly rotating layers of mostly parallel chitin fibers, be it mfs within a helicoidal lamellar cuticle or MFs within certain adult endocuticles, is generally affirmed by our studies. However, there are exceptions to this central theme as we and others have observed, and these exceptions relate to both the primary and secondary orientation of the mrs. For example, with regard to primary orientation within the endocuticle of the boll weevil abdominal sclerites, one complete rotation of the parallel MFs is achieved every 5 layers. However, except for the endocuticle of the dorsal pronutum, which appears to be identical to the ventral abdominal sclerites, other areas of the sclerotized exoskeleton were not surveyed. It is possible that some of the sclerites of the boll weevil do not have endocuticle that is constructed of layers of MFs crossing regularly at angles of about 72°. Early observers examining the elytra of certain lucanids and cicindelids, stated that the MFs (Balken) crossed at angles of 60° (Schulze, 1913; Stegemann, 1929), while Biedermann (1903) maintained that the first 2 layers cross at 45 ° and then change to 90° at the same location in Orcytes nasicornis. Dennell (1976), when comparing what was apparently MF orientation in 2 phasmids, found that the MFs of Eurycnema versiruba were orthogonaUy arranged, while those of Extaosoma tiaratum crossed at varying angles, because the MFs were formed into randomly disposed horizontal arcs. Further, Zelazny and Neville (1972b), in studying fiber orientation of the plywood-type endocuticle, found significant locational differences when measuring the angle of rotation between successive layers of the unidirectional "preferred" mrs of O. rhinoceros. The bifurcations of the MFs observed in the boll weevil endocuticle are also

A Comparison of Cuticle Deposition of the Adult Weevil

59

an indication of the small deviations which occur in the unidirectional norm and angle of rotation. When considering the secondary orientation, it is clearly evident that most of the MFs of the endocuticle of this insect are connected, above and below, to the adjacent crossing MFs. These attachments require some shifting in the orientation of the integral mfs from a horizontal to a perpendicular direction to accommodate making the connections at these sites. Further, the presence of the struts which interconnect the adiacent and crossing MFs is irrefutable evidence that mf orientation within boll weevil endocuticle often deviates from the unidirectional orientation within horizontally rotating layers. The susceptibility of the interstitial fibrous matrix to KOH extraction suggests that, unlike the MFs, chitin is not present. The matrix is presumably composed of protein fibers, which support the many pore canal filaments that traverse the interstices formed by the criss crossing MFs. The presence of cuticular columns around pore canal filaments in the exocuticle and not in the endocuticle of this insect is as Delachambre (1971) described for T. molitor. Since this situation is not found in the larval and nymphal insects, Delachambre suggested that the cuticular columns were characteristic of only highly sclerotized cuticles. Preeclosion vs. posteclosion cuticle deposition By following the process of cuticle deposition occurring before and after adult eclosion, we were able to identify several conspicuous cellular and extracellular events, which are presumably responsible for producing qualitative and quantitative differences existing between the 3 main morphologically distinct components (epicuticle, procuticle/ exocuticle and endocuticle). Further, while we found these events often encompassed widespread changes in cell configuration and content, most of the more conspicuous events were found to occur near or at the apical plasma membrane of the cell and at the cuticle-cell interface. Epicuticle formation prior to eclosion is characterized by a 2 step change in microvillar configuration and an abundance of coated and uncoated vesicles which appear to associate with the spaces between the villi. Initially, elongated microvilli secrete the trilaminate membrane portion, while the shorter, club-shaped versions having no or quiescent membrane placques are associated with deposition of the homogeneous inner epicuticle. These events appear identical to what occurs in other Coleoptera (Delachambre, 1971; Zacharuk, 1972) and, in general, for all insects thus far studied (cf. review of Locke (1985)). The origin of the transitory superficial layer was not evident in our preparations, but the timing of its appearance and disappearance suggests that it may be connected to the digestion of the old pupal cuticle. Zacharuk (1972) suggested that it may protect the new layers of the epicuticle from the digestive process. Further, the loss of the preecdysial trilaminate appearance by the increased osmophilia of the outer epicuticle, which we observed immediately after eclosion of the adult boll weevil, was also reported by Zacharuk (1972) in the sclerites of wireworms. The increase in osmophilia may indicate that there is an addition, via the pore canals, of waterproofing materials, such as sclerotin and lipids (Wigglesworth, 1985). From the appearance of the cross and en face sections, we have interpreted the apical surface of the cells secreting the procuticle to be configured into a series of folds rather than into a surface typically covered with microvilli. To our knowledge, this type of

60

R.A. LEOPOLDet al.

surface configuration has not been noted by other investigators, although in some insects the cross-sectional appearance of cell apices at this stage of cuticle deposition resembles that of the boll weevil (cf. Fig. 16--Zacharuk (1972); Plate 5 - - L o c k e (1967)). The secretion of the procuticle during the latter portion of the pharate adult stage apparently demands an increased cellular activity, because we observed an increase in the small Golgi-derived vesicles, an enhancement of the amount of RER, and a subsequent rise in the quantity of the larger lytic-type bodies right before adult ecdysis. The latter organelles appear to be multivesiculate bodies, as described by Locke (1966) for the epithelial cells of Calpodes, and evidently participate in membrane turnover and auto- and heterophagic activity (Locke, 1985). Since the deposition of an adult endocuticle requires a considerable increase in the apical cell surface to accommodate the simultaneous assembly of several layers of MFs, the cells may already be processing membrane components prior to ecdysis to meet the demands that occur immediately after eclosion. Posteclosion cuticle deposition not only demands an increase in apical membrane surface, but also a substantial remodeling of the cytoskeleton which supports the cellular extensions that bear the membrane placques. The ordered arrangement of the microtubules associated with the canal-like surface depressions which form the templates for the MFs is circumstantial evidence for microtubular involvement with MF orientation. Richards (1951) suggested that mechanical stresses acting after secretion were responsible for the orientation of the crossed fiber structure of the endocuticle, while Wigglesworth (1948) observed an apparent relationship between how cells were arranged and fiber direction in adult cuticle of T. molitor. Regardless of whether there is microtubular involvement in the orientation of the MFs, it is evident that the epidermal cells of the boll weevil exert direct control over the direction in which the MFs are aligned by modification of the surface contours. It is unclear whether the cells have direct control over how the mrs will be configured into a series of lameUae consisting of rotated, overlapping planes of fibers. If totally under cellular control, the architecture of the cuticle to be deposited may relate to the placement of the plasma membrane placques. Locke (1967) noted a direct relationship of the axial orientation of the placque-bearing, elongated microvilli with the alignment of mrs during bristle formation in Calpodes. Thus, the orientation of the mrs forming each lamina of helicoidally arranged cuticle may also relate to the positioning of the membrane placques on cuticle-depositing cells. Coordinated rotation of the placques on the apices of the microvilli or in the case of the weevil, folds, could account for the rotation of the overlapping planes of fibers. Alternately, the placques could remain stationary, while the surface contours of the cells located beneath a segment or sclerite rotated in a coordinated fashion. Neville (1967) presented a convincing argument for the existence of extracellular physical factors, such as ionic concentration, pH changes, degree of hydration, and regulation of protein-chitin cross-linking in the control of macromolecular morphogenesis. The semicircular arrangement of the membrane placques directed by the surface contours of the cells forming the MFs of the weevil endocuticle, and the maintenance of cellular extensions over several layers, indicates that the cells take an active role in the initial ordering and final shaping of these structures. If assembly of the MFs was totally under the control of physical factors existing at the time of deposition in the extraceUular assembly zone, then there would be no need for the elaborate structuring of the surface

A Comparison of Cuticle Deposition of the Adult Weevil

61

c o n t o u r s o f t h e cells t h a t s e c r e t e t h e i m a g i n a l e n d o c u t i c l e . W e f o u n d t h e first e n d o c u t i c u l a r l a y e r o f M F s to b e p a r a l l e l to t h e l o n g i t u d i n a l axis o f t h e insect, w h i c h is s i m i l a r to w h a t is o b s e r v e d in O. r h i n o c e r o s ( Z e l a z n y a n d N e v i l l e , 1972b) a n d in T. m o l i t o r ( C a v e n e y , 1973). Z e l a z n y a n d N e v i l l e (1972b) s u g g e s t e d t h a t a cell p o l a r i t y g r a d i e n t m a y d e t e r m i n e t h e d i r e c t i o n o f t h e first l a y e r o f fibers since this o r i e n t a t i o n o f t e n m a t c h e d t h e b i s y m m e t r i c a l p a t t e r n of c u t i c u l a r bristles. C a v e n e y (1973), b y a n a l y z i n g r o t a t e d grafts o f i n t e g u m e n t , also c o n c l u d e d t h a t t h e p o l a r i t y of e n d o c u t i c u l a r u n i d i r e c t i o n a l f i b e r d e p o s i t i o n was u n d e r cell g r a d i e n t c o n t r o l . I n his s t u d y , p r o g r a m m i n g o f t h e e p i t h e l i a l cell p o l a r i t y was f o u n d to c o i n c i d e with t h e o c c u r r e n c e o f cell division in t h e p r e v i o u s s t a g e o f d e v e l o p m e n t . I n t e r e s t i n g l y , t h e angles o f crossing o f M F s , in s u c c e e d i n g l a y e r s r e l a t i v e to t h o s e in t h e first l a y e r , was n o r m a l in t h e r o t a t e d grafts. W h i l e a c e l l u l a r g r a d i e n t c o u l d b e r e s p o n s i b l e for t h e a l i g n m e n t o f t h e first l a y e r o f M F s in t h e b o l l w e e v i l , it is difficult to i m a g i n e g r a d i e n t c o n t r o l o v e r s u c c e e d i n g l a y e r s w h e n s e v e r a l o f t h e s e l a y e r s a r e b e i n g d e p o s i t e d at once. O t h e r levels o f c o n t r o l , such as c o o r d i n a t i o n o f M F o r i e n t a t i o n across cell b o r d e r s , t h e i r d i v e r s i o n a r o u n d i n t e g u m e n t a l s t r u c t u r e s , a n d m a i n t e n a n c e o f t h e fixed angles o f crossing, a r e also still o p e n t o s p e c u l a t i o n . Acknowledgements--We thank Drs Edwin P. Marks, Ridge, Maryland, and John G. Riemann, Fargo, North

Dakota, for their helpful suggestions and comments on earlier versions of this manuscript. Special thanks are due to Ms Jane Knoper, U.S. Bureau of Mines, Albany, Oregon, for providing several of the micrographs gained from the early stages of this study.

REFERENCES BARTLETr,A. C. 1967. Genetic markers in the boll weevil. J. Hered. 58: 159-63. BIEDERMANN,W. 1903. Geformte Sekrete. Z. Allg. Physiol. 2: 395-481. BOULIGAND,Y. 1965. Sur une architecture torsad6e r6pandue dans de nombreuses cuticules d'Arthropodes. C. R. Hebd. S~ances Acad. Sci., Ser. D 261: 3665--68. CAVENEY,S. 1973. Stability of polarity in the epidermis of a beetle Tenebrio molitor L. Dev. Biol. 30: 321-35. DAUNOWArER,J. E. 1975. SEM observations on the cuticles of some decapod crustaceans. Zool. J. Linn. Soc. 56: 327-30. DELACHAMBRE,J. 1970. Etudes sur l'epicuticle des Insectes. I: le d6veloppement de l'6picuticule chez l'adulte de Tenebrio molitor L. Z. Zellforsch. Mikrosk. Anat. 108: 380-96. DELACHAMBRE, J. 1971. La formation des canaux cuticulaires chez l'aduite Tenebrio molitor L. Etude ultrastructurale et remarques histochimiques. Tissue Cell 3: 499-520. DELBECQUE,J.-P., M. HIRN, J. DELACHAMBREand M. DE REC,61. 1978. Cuticular cycle and molting hormone levels during the metamorphosis of Tenebrio molitor (Insecta Coleoptera). Dev. Biol. 64: 11-30. DENNELL,R. 1973. The structure of the cuticle of the shore-crab Carcinus maenas (L.). Zool. J. Linn. Soc. 52: 159-63. DENNELL,R. 1976. The fine structure of the cuticle of some Phasmida, pp. 177-91. In H. R. HEPBURN(ed.) The Insect Integument. Elsevier, Amsterdam, The Netherlands. DRACH,P. 1953. Structure des lamelles cuticulaires chez les Crustaces. C. R. Hebd. S~ances Acad. Sci., Set. D 237- 1772-74. EARLE, N. W., I. PADOVANI,M. J. THOMPSONand W. E. ROBBINS.1970. Inhibition of larval development and egg production in the boll weevil following ingestion of ecdysone analogues. J. Econ. Entomol. 63: 1064-69. FILSHIE, B. K. 1970. The fine structure and deposition of the larval cuticle of the sheep blow fly, Lucilia cuprina. Tissue Cell 2: 479-98. FILSHIE, B. K. and D. F. WATERnOUSE. 1969. The structure and development of the green vegetable bug, Nezara viridula. Tissue Cell 1: 367-85. FREY-WYSSLING, A. 1938. Submikroskopische Morphologie des Protoplasmas und Seiner Derivate. Protoplasma Monographien series, vol. 15. Borntrager, Berlin. GORDON,H. and A. T. WINFREE.1978. A single spiral artifact in arthropod cuticle. Tissue Cell 10- 39-50. HADLEY,N. F. 1986. The arthropod cuticle. Sci. Amer. (1): 104-12.

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