Insects

Pupae were obtained from a culture maintained at the New York State Agricultural Experiment Station at Geneva, New York. They were sexed and adults that emerged were held separately in 3.8 l jars. Pupae and adults were kept at 25±1°°°°C under a 16:8 (L:D) photoperiod with ca. 70% humidity provided by a wet paper towel hung inside the jars. All insects used in this study were of the bivoltine E strain that produces a sex pheromone blend containing 99% E and 1% Z isomer of 11–14:OAc (Glover et al. 1987).

Tissue preparation

Ovipositors (abdominal segments 8-10) were extruded by applying slight pressure on the abdomens of the moths and were excised using a razor blade. For light and scanning electron microscopy, the entire ovipositor was immersed in fixative. For transmission electron microscopy, the ovipositors were placed in a drop of a modified Weever's insect saline (Carrow et al. 1981) and the intersegmental membrane between abdominal segments 8 and 9/10 was then dissected out under the saline and placed immediately in fixative.

Routine fixation was accomplished by immersing the tissues directly into 4% glutaraldehyde in 0.1M phosphate buffer (pH 7.4) at 4°C for 24 hr. For scanning and transmission electron microscopy, tissues were postfixed in 1% OsO₄ in 0.1M phosphate buffer for 8 hr at room temperature. After fixation, all tissues were rinsed with 0.1 M phosphate buffer and dehydrated by passing through a graded series of ethanol.

Light Microscopy

Dehydrated tissues were passed through a few changes of xylenes and embedded in Paraplast® (56°C m.p. Fisher Scientific). Sections were cut at 6 μm, stained with hematoxylin and eosin and mounted in Permount® (Fisher Scientific).

Scanning electron microscopy

Dehydrated ovipositors were soaked in three changes of pentane and allowed to air dry in a fume hood (G.T.Baker, personal communication). Dried specimens were mounted on aluminum stubs using double sticky tape, sputter coated with gold-palladium and then examined under a Hitachi S-530 scanning electron microscope at 15 kV.

Transmission electron microscopy

After dehydration, tissues were placed in 1:2, 1:1 and 2:1 Spurr:ethanol. They were then infiltrated with five changes of Spurr resin of 6 hr each and finally embedded in the same at 60°C for 24 hr. Thick sections (1/2–1 μm) were stained with toluidine blue. Thin sections (showing silver interference color) were cut using glass knives and collected on Formvar-coated copper slot grids. After staining with uranyl acetate and lead citrate, sections were examined on a Jeol JEM-100SX transmission electron microscope at 100 kV.

Gland extraction and GC analyses

The intersegmental membrane between abdominal segments 8 and 9/10 was carefully dissected from females during the 5th and 6th hr of their third scotophase. Fat bodies and other extraneous reproductive and digestive tissues were removed and the cuticular membrane was carefully divided into a dorsal and a ventral portion by cutting horizontally at the level of the lateral apophyses. The two portions were transferred separately into 50 μl 2:1 chloroform:methanol containing 200 ng tridecanyl acetate (13:OAc) as an internal standard in small test tubes. Extraction was conducted at −10°C for 24 hr. Tissues were then removed and base methanolysis was carried out according to Bjostad et al. (1987) with slight modifications. Briefly, the extract was dried under a slow stream of N₂ and 0.5M methanolic KOH was added. After 1 hr, the solution was made acidic by the addition of an equal volume of 1N HCl and extracted with hexane. Alcohol moieties in the extract were converted back into their corresponding acetates by treatment with acetyl chloride. Sample analysis was performed on a 30 m StabliWax® capillary column (film thickness 0.25 μm and 0.25 mm I.D.) (Restak, Bellefonte, PA) using N₂ as carrier gas (column head pressure 10 psi) in a HP 5840 gas chromatograph equipped with a flame ionization detector. The column temperature was initially set at 80°C for 1 min and then programmed to rise to 180°C at 7.5°C/min for 20 min. The temperature was then increased to 220°C at 5°C/min. Peak identification was based on direct comparison with the retention times of authentic standards.

External morphology

There are 9 distinguishable abdominal segments in the adult female O. nubilalis. Segments 1–7 are covered with scales. Terminal segments 8–10 form the ovipositor that normally remains telescoped within the preceding segments. The 8th abdominal segment is shield-like and heavily sclerotized. The ostium bursae opens just posterior to the 8th sternum (Fig. 1). Segments 9 and 10 are fused to form the sclerotized papillae anales that are characterized by numerous short, spine-like microtrichae and long setae on their lateral and posterior surfaces (Fig. 1). The hindgut and the median oviduct open respectively at the anus and oviporus between the two lobes of the papillae anales. Arising laterally from the papillae anales and fused to the interseg-mental membrane between abdominal segment 8 and 9/10 is a pair of apophyses that projects anteriad into the hemocoel (Fig. 1). At the place where these apophyses fused to the intersegmental membrane between abdominal segments 8 and 9/10, the entire intersegmental membrane is roughly divided into a dorsal and a ventral half. Muscles from the anterior segments attach to the free ends of the apophyses (Fig. 8). These muscles serve to control the movement of the papillae anales during oviposition and calling.

Figs. 1–5

Scanning electron micrographs of the terminal abdominal segments of the female Ostrinia nubilalis. 1). Lateral view of terminal abdominal segments 8–10 showing the intersegmental membrane between abdominal segments 8 and 9/10; note the smooth surface of the heavily sclerotized 8^th abdominal segment and the location of the ostium bursae (arrow) and also the papillae annales with the long setae. The cylindrical intersegmental membrane is roughly divided into dorsal and ventral halves by an apophysis (arrowheads). The everted oviduct (asterisk) is an artifact that was introduced during tissue preparation. Scale bar = 100 μm. 2). Higher magnification of the dorsal region (above the sclerotized rod) of intersegmental membrane between abdominal segments 8 and 9/10; microtrichae are in aggregates of 7–10. Scale bar = 10 μm. 3) Higher magnification of the ventral region (below the apophysis) of the intersegmental membrane between abdominal segments 8 and 9/10; microtrichae are sparingly located; note the area without microtrichae where several pairs of muscles attach at the hemocoel side. Scale bar = 10 μm. 4). Microtrichae on the lateral region of the intersegmental membrane between abdominal segments 8 and 9/10. Scale bar = 5 μm. 5). High magnification of a microtrichae on the dorsal region of the intersegmental membrane between abdominal segments 8 and 9/10. Scale bar = 1 μm.

Figs. 6–8

Light micrographs of terminal abdominal segments 8 and 9/10 of Ostrinia nubilalis. 6). Sagittal section through abdominal segments 8–10 showing the position of the hypertrophied epidermal cells (arrowheads) in the centrodorsal region of the intersegmemtal membrane; note point of attachment of muscles at the ventral side of the intersegmental membrane. Ov, oviduct; PA, papillae anales. Scale bar = 100 μm. 7). Sagittal section through the dorsal region of the intersegmental membrane between abdominal segmental 8 and 9/10 showing the columnar hypertrophied epidermal cells. Scale bar = 50 μm. 8). Transverse section through the dorsal region of the intersegmental membrane showing the hypertrophied cells. Location of the apophysis is indicated by the arrowhead. Scale bar = 100 μm

Adjoining the 8th segment and the papillae anales is the cylindrical intersegmental membrane that is usually held inverted and hidden underneath the 8th abdominal segment. Eversion of the intersegmental membrane between abdominal segments 8 and 9/10 appears to be caused by an increase in hemolymph pressure. Retraction of the interseg-mental membrane probably is brought about by a reduction in hemolymph pressure together with the contraction of muscles attached to the apophyses and the mid-ventral region of the intersegmental membrane.

Scanning electron microscopy revealed that the entire surface of intersegmental membrane between abdominal segments 8 and 9/10 is wrinkled and decorated with numerous microtrichae (Fig. 1). However, cuticular sculpture varies at different regions of the intersegmental membrane. In the centrodorsal region, the cuticular surface is characterized by the presence of short microtrichia (3–4 μm in length), which often form aggregates of 7–10 (Fig. 2). In contrast, cuticular projections on the rest of the interseg-mental membrane are considerably longer (8–20 μm in length) and are distributed evenly on the intersegmental membrane (Figs. 3, 4). Regardles of their location, none of the microtrichia are porous (Fig. 5). The intersegmental membrane anteriad to the ventral tip of the papillae anales is devoid of any cuticular projections (Fig. 3). This demarcates the point of insertion by several pairs of ventral muscles on the hemocoel side of the intersegmental membrane (Fig. 6).

Histology

At the dorsal region, the intersegmental membrane between abdominal segments 8 and 9/10 consists of a single layer of hypertrophied columnar epidermal cells. These cells measure 60–100 μm in height (Fig. 7). The dorsal epidermis occupies an area of about 450–500 μm X 350–400 μm at the centrodorsal part of the intersegmental membrane. It is surrounded anteriorly and posteriorly by smaller squamous and cuboidal cells (10–20 μm in height) and laterally by squamous epidermal cells (Figs. 6, 8).

Cuticle

The cuticle consists of a dense outer epicuticle, about 0.5 μm thick, overlying the untanned and lamellated endocuticle, about 1.5–3 μm in thickness (Figs. 9, 10). Each lamella measures 0.25–0.5 μm in thickness. The number of lamellae in the endocuticle, generally ranging from 3–7, varies considerably in different regions of the intersegmental membrane. In most cases, the lamellated appearance of the endocuticle is more distinct in sagittal sections than in trans-verse sections. In the region where the cuticle is underlined by columnar hypertrophied cells, numerous pore canals transverse the endocuticle in helicoidal fashion to terminate just beneath the dense epicuticle (Fig. 9). These pore canals contain epicuticular filaments measuring 15–20 nm in diameter (Fig. 11). The distribution of pore canals in the ventral cuticle is not as extensive as that in the dorsal cuticle.

Figs. 9–12

Electron micrographs of the intersegmental membrane between abdominal segments 8 and 9/10 of Ostrinia nubilalis. 9). Apical region of the epidermal cells at the dorsal side of the intersegmental membrane. Numerous pore canals transverse the endocuticle (arrows). En, endocuticle; Ep, epicuticle. Scale bar = 1 μm. 10). Scanning electron micrograph of the cuticular covering of dorsal intersegmental membrane between abdominal segments 8 and 9/10. The intersegmental membrane was fixed and dehydrated as described in materials and methods and cryofractured under LN₂. Note that only the cuticular covering of the intersegmental membrane is shown because the epidermis was probably lost during the processing. En, endocuticle; Ep, epicuticle. Scale bar = 1 μm. 11). High magnification of a pore canal showing the epicuticular filaments (arrow). En, endocuticle. Scale bar = 0.5 μm 12). Cuticular covering of the epidermal cells of the intersegmental membrane between abdominal segments 8 and 9/10 showing the accumulation of lipid droplets (LD) 2-3-days after adult emergence. En, endocuticle; Ep, epicuticle. Scale bar = 0.5 μm.

In 2-3-day-old females, cuticular lining over the dorsal columnar hypertrophied cells contains numerous spherical lipid droplets, ranging from 0.2 to 1.5 μm in diameter (Fig. 12). These lipid droplets contain a homogeneous electron-lucid substance and, in some cases, they are bounded by a thin layer of electron-dense material (Fig. 12). Lipid droplets are absent in all the cuticular lining of the intersegmental membrane and the dorsal cuticular lining in younger females (Fig. 16).

Dorsal Epidermis

In 2-3-days-old females, the dorsal epidermis is made up of tall columnar cells (80–100 μm in height and 20–30 μm in width) (Fig. 7). The cells rest on a electron-dense basement membrane with a uniform thickness of about 0.2 μm throughout the entire dorsal epidermal region (Fig. 13). The basement membrane apposes closely to the basal plasma membrane of the cells, and there are tracheae interposed between the epidermal cells and the basement membrane (Fig. 13).

Figs. 13–15

Transmission electron micrographs of epidermal cells at the dorsal intersegmental membrane of abdominal segment 8 and 9/10 of Ostrinia nubilalis. 13) Fully developed adult (2-3-day old) epidermal cells of the dorsal region of the intersegmental membrane between abdominal segments 8 and 9/10. Note numerous lipid droplets present in the cytoplasm. Scale bar = 0.5 μm. 14) Numerous free ribosomes and mitochondria form the matrix of the cytoplasm of the epidermal cells. Some of the mitochondria are closely associated with the lipid droplets (arrow). Scale bar = 0.6 μm. 15) Apical region of hypertrophied epidermal cells showing the tightly packed membrane foldings (arrowheads). Note electron-dense plaques accumulate at the tips of the membrane foldings (open arrows) and numerous pore canals transverse the cuticular covering (white arrow). The space in the cuticle (asterisk) is an artifact. Scale bar = 0.8 μm.

Immediately beneath the cuticular lining, the apical plasma membrane is characterized by the formation of tightly packed membrane foldings, measuring 0.3–0.5 μm in depth and 50 nm in width (Fig. 15). At the tips of the apical folds, electron dense material appears as dark plaques along the cell-endocuticle interface.

At the apical ends of the cells, plasma membrane of adjacent cells extensively interdigitate (Fig. 15). Desmosomes occur occasionally between neighboring cells along the interdigitated cell membranes. More basal along the lateral margins of the cells, intercellular boundaries are smooth and the cell membranes remain parallel towards the bases of the cells (Fig. 16).

Figs. 16–19

Transmission electron micrographs of epidermal cells at the intersegmental membrane between abdominal segments 8 and 9/10. 16) Epidermal cells of the dorsal region of the intersegmental membrane between abdominal segments 8 and 9/10 in young females. The cells are much smaller than the epidermal cells found in older females. Microvilli at the apical membrane are not very well developed. Sandwiched between the apical region of the epidermal cells and the cuticular covering is a granular amorphous matrix (asterisk). En endocuticle; Ep = epicuticle. Scale bar = 3.6 μm. 17) Epidermal cells of the dorsal region of the intersegmental membrane between abdominal segments 8 and 9/10 in young females. The lateral cell membrane is characterized by extensive interdigitations, numerous desmosomes between the cell boundaries (arrowheads). Scale bar = 0.4 μm. 18) Epidermal cells lining the ventral region of the intersegmental membrane. The cells are cuboidal to squamous and the microvilli at the apical cell membrane is not well developed; an amorphous matrix normally present between the cells and the cuticular covering (asterisk). Scale bar = 1 μm. 19) Numerous smooth endoplasmic reticula (arrows) in the form of vesicles usually present in the cytoplasm of epidermal cells at the dorsal region of the intersegmental membrane. M, mitochondria. Scale bar = 0.5 μm.

Abundant free ribosomes form the ground cytoplasmic matrix. The most distinct feature in the cytoplasm the spherical lipid droplets (0.5–1.5 μm in diameter) containing a homogeneous electron-lucid material (Fig. 13). Coalescing of small lipid droplets to form larger ones is seen, especially near the apical parts of active glandular cells. Numerous mitochondria with tightly packed cristae and an electron-dense matrix are scattered throughout the cytoplasm (Fig. 14). They exist in a variety of shapes, ranging from sausage-shaped to long branched forms. Close associations of mitochondria with the lipid droplets occur frequently in the cytoplasm (Fig. 14). The cytoplasm also contains a rich array of microtubules, with diameters of about 30 nm, oriented parallel to the long axis of the cell (Fig. 15). Golgi complexes and rough endoplasmic reticulum are infrequent. Smooth endoplasmic reticulum in the form of vesicles, though not very well developed, is found scattered sparingly in the cytoplasm (Fig. 19). The oval or slightly lobulated nucleus occupies the central portion of the cell.

In pharate and young adult females (within 4 hr of adult eclosion), the dorsal epidermis is strikingly different in morphology to that in older females (Fig. 16). The columnar epidermal cells at this stage are much smaller (35–40 μm in depth and 5–10 μm in width). Sandwiched between the cuticle and the apical plasma membrane is a granular amorphous matrix (Fig. 16). The apical plasma membrane folds are not as elaborated as that seen in 2-3-day-old females (Figs. 15, 16). The lateral plasma membranes are highly convoluted throughout the length of the cell (Fig. 17). Basally, the plasma membrane exhibits extensive invaginations while remaining closely apposed to the basement membrane. The cytoplasmic matrix contains fewer free ribosomes, microtubules and mitochondria. The latter are usually globular or in the form of short rods. Furthermore, lipid droplets, Golgi complexes and smooth and rough endoplasmic reticula are absent. The oval nuclei, however, resemble those in older dorsal epidermal cells (Fig. 16).

Ventral Epidermis

The ventral epidermis is composed of simple cuboidal and squamous cells (Fig. 18). These cells vary considerably in size in different regions of the intersegmental membrane. The cuboidal cells (about 10 μm in height and 8 μm in width) are located anteriad and posteriad to the dorsal epidermis and also along the basal region of the intersegmental membrane (Fig. 6). The squamous cells, on the other hand, generally occur lateral to the dorsal epidermis and they measure 6–8 μm in height and 10–12 μm in width (Fig. 8). These epidermal cells rest on a thin basement membrane, about 50 nm thick. Intercellular boundaries of these epidermal cells, especially those of the squamous epidermis, are highly convoluted. Although apical infoldings are present in these cells, they are loosely formed and poorly organized (Fig. 18). An amorphous matrix similar the one seen in dorsal epidermal cells in younger females often occur between the cells and the cuticular lining (Fig. 18).

Despite the differences in size and shape, both cuboidal and squamous epidermal cells share similar cytological characteristics. The cytoplasm contains a moderate number of mitochondria of various sizes and shapes. Ribosomes, unattached or in small aggregates, are randomly dispersed in the cytoplasm. Short lengths of rough endoplasmic reticulum and vesicular smooth endoplasmic reticulum are sometimes encountered in the cytoplasm and other organized membrane structures are rare. Lipid droplets are lacking in any of the sections examined. Nuclei are round in cuboidal epidermal cells whereas the squamous cells contain slightly lobulated nuclei.

Fatty Acids Analysis

Gas chromatographic analysis of base methanolysed and acetyl chloride-treated chloroform/methanol extracts of the intersegmental membrane reveals the presence of the sex pheromone components, E11-14:OAc and its immediate fatty acyl precursors, methyl (Z)-11- and methyl (E)-11- tetradecenoate (Z11- and E11-14:Me) only in the dorsal inter-segmental membrane (Fig. 20), and no detectable amounts in the ventral intersegmental membrane. Both dorsal and ventral halves of the intersegmental membrane contain large amounts of the common acids, methyl tetradecanoate (14:Me), methyl hexadecanoate (16:Me), methyl (Z)- 9-hexadecenoate (Z9-16:Me), methyl octadecanoate (18:Me), methyl (Z)- 9-octadecenoate (Z9-18:Me), methyl (Z,Z)- 9,12-octadecadienoate (Z9,Z12-18:Me) and methyl (Z,Z,Z)-9,12,15-octadecatrienoate (Z9,Z12,Z15-18:Me). The dorsal intersegmental membrane contains a large amount of methyl (Z)-11-hexadecenoate (Z11-16:Me), which could not be detected in its ventral counterpart.

Fig. 20

Gas chromatogram of base methanolysed and acetyl chloride-treated chloroform/methanol extracts of the dorsal and ventral portions of the intersegmental membrane between abdominal segments 8 and 9/10 of Ostrinia nubilalis. Equal amounts of the final extracts from the dorsal and ventral intersegmental membrane were injected into the gas chromatograph for comparison. Note the absence of E11-14:OAc, Z/E11-14:Me and Z11-16:Me in the ventral portion of the intersegmental membrane. The amount of the internal standard, 13:OAc, shown on both tracings correspond to 200 ng. Refer to text for a full description of the abbreviations.