The sex pheromone gland of the female European corn borer moth, Ostrinia nubilalis was studied using light and electron microscopy. The pheromone gland is formed by hypertrophied epidermal cells at the mid-dorsal region of the intersegmental membrane between abdominal segments 8 and 9/10. Active glandular cells contain extensive apical membrane foldings, a single nucleus, many free ribosomes, numerous mitochondria, microtubules and lipid droplets. Smooth endoplasmic reticulum is scanty. In young moths, the glandular cells are smaller in size, the microvilli at the apical membrane are poorly developed and the cytoplasm contains fewer mitochondria, microtubules, and no lipid droplets. The surrounding unmodified epidermal cells are small cuboidal or squamous cells. These cells have ill-defined apical membrane foldings and do not contain lipid droplets in the cytoplasm and the overlying cuticle. Fatty acids analyses revealed the presence of the sex pheromone components, (E)-11-tetradecenyl acetate, and their immediate precursors, methyl (E)-11- and methyl (Z)-11-tetradecenoate, only in the dorsal portion of the cylindrical intersegmental membrane. Results of the present study show that the sex pheromone gland of O. nubilalis is restricted to the dorsal aspect of the intersegmental membrane between segments 8–9/10 and is not a ring-gland.
Sex pheromone glands of female Lepidoptera are commonly found as modified intersegmental membranes between the 8th and 9th abdominal segments (Götz 1951; Percy-Cunningham and MacDonald 1987). They also occur between abdominal segments 7 and 8 in Sesamia nonagrioides (Noctuidae) (Sreng and Sreng 1988), around the ostium bursae in Plutella xylostella (Plutellidae) (Chow et al. 1976), and at the papillae anales in Heliothis virescens Fabricius (Teal et al. 1983). In general, the modified interseg-mental membrane is lined with a glandular epidermis made up of hypertrophied cells. However, the location of the hypertrophied cells on the modified intersegmental membrane varies considerably among different moth species (Percy-Cunningham and MacDonald 1987).
The European corn borer, Ostrinia nubilalis, is a serious agricultural pest in many parts of the world including the United States. Female O. nubilalis uses a mixture of (Z) -11-and (E) -11-tetradecenyl acetate (Z/E11-14:OAc) as their sex pheromone (Klun 1968; Klun and Junk 1970; Kochansky et al. 1975). Using light microscopy, Hammad (1961) reported that the entire intersegmental membrane between the 8th and 9th abdominal segments in female O. nubilalis is modified to form the ring-like sex pheromone gland, with the ventral cells being much larger than those located dor-sally.
Sex pheromone production in female O. nubilalis is regulated by PBAN (pheromone biosynthesis activating neuropeptide)-like factors (Ma and Roelofs 1995). Information on how the PBAN-like factors might affect the ultrastructure of the sex pheromone gland is necessary for a future understanding of the action of the PBAN-like factors. To date, ultrastructural information on the sex pheromone gland of O.nubilalis is lacking.
In the present study, light and electron microscopical studies are conducted to describe the ultrastructural features of the sex pheromone gland of O. nubilalis. In addition, gas chromatographic analysis is used to localize the distribution of the sex pheromone, E/Z11-14:OAc, and its biosynthetic acid intermediates in the intersegmental membrane between abdominal segments 8 and 9/10 to confirm the exact location of the sex pheromone gland of O. nubilalis.
MATERIALS AND METHODS
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).
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.
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.
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.
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).
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).
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.
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).
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).
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).
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).
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.
Present histological examinations of the terminal abdominal segments of female O. nubilalis indicate that only the centrodorsal part of the intersegmental membrane is lined with hypertrophied cells. Ultrastructural observations showed that these epidermal cells possess the general cytological features commonly found in sex pheromone-producing-gland cells in other lepidopteran insects (Percy-Cunningham and MacDonald 1987). These features include the presence of numerous mitochondria, lipid droplets both in the cell and the overlying cuticle, and also development of apical plasma membrane foldings. Such characteristic features are either absent or not well defined in epidermal cells occurring elsewhere in the intersegmental membrane and in other parts of the terminal abdominal segments. Furthermore, the major sex pheromone components, E11-14:OAc, and its immediate fatty acyl precursors, Z/E11-14:Me, are detected only at the dorsal portion of the intersegmental membrane. The latter observation also suggests the presence of a Δ11-desaturase, which is the key enzyme involved in the biosynthesis of sex pheromones in many lepidopterous insects (Bjostad et al. 1987), only in the dorsal part of the intersegmental membrane. Thus, morphological and biochemical observations in the present study strongly indicate that the sex pheromone gland of female O. nubilalis is not a ring gland as previous shown (Hammad 1961), but rather a dorsal scent fold (Götz 1951).
Ultrastructural studies of sex pheromone glands of various species of female Lepidoptera have established that the presence of smooth endoplasmic reticulum is a common morphological feature of the modified glandular cells (Percy-Cunningham and MacDonald 1987). In O. nubilalis, only a few smooth endoplasmic reticulum occur in the active pheromone gland cell. Similar situation was also observed in the silkworm moth, Bombyx mori L. (Bombycidae) (Waku and Sumimoto 1969). The absence of well-developed smooth endoplasmic reticulum in O. nubilalis pheromone gland cells may be a fixation artifact introduced during tissue preparation. In vertebrates such as rat liver cells, distribution and quantity of vesicular type smooth endoplasmic reticulum have been found to undergo extensive diurnal changes (Chedid and Nair 1972). Recently, Fonagy et al. (2001) conducted a study to correlate the changes in cytoplasmic lipid droplets in the pheromone-producing cells of the silkmoth, B. mori, and they showed a daily fluctuation in size and number of lipid droplets during the photophase. In the present study, tissues were obtained from insects 4, 24, and 48 hr after adult eclosion (all within 2 hr of the end of the scotophase). If there is a diurnal change in the amount of smooth endoplasmic reticulum in the pheromone gland cell, the period when the cell exhibits a full development of smooth endoplasmic reticulum may have been missed in the current study.
Although there is an indication that the accumulation of lipid in the pheromone glands of O. nubilalis and other moths correlates with the amount of extractable pheromone from the gland (Waku and Sumimoto 1969; Feng and Roelofs 1977; Percy 1979; Ma and Roelofs unpublished observations), the role of lipid deposits in the pheromone gland cells and in the overlying cuticle in O. nubilalis and other lepidopterous insects is not fully understood (Bjostad et al. 1987). The assumption that this lipid deposits may act as a form of storage of sex pheromone available for immediate secretion, however is based solely on morphological observations and lacks the support of biochemical evidence. In the tortricid moth Argyrotaenia velutinana and in O. nubilalis, the triacylglycerols constitute the main pool of non-membrane-origin lipid, which represents the contents of these lipid spheres (Bjostad et al. 1987; Ma and Roelofs unpublished observations). A large amount of the immediate acyl precursors involved in sex pheromone biosynthesis occurs as triacylglycerols (Bjostad et al. 1987). However, fatty acids mass-labeled with stable isotopes in synthetic triacylglycerols were not incorporated into the sex pheromone components of A. velutinana (Bjostad and Roelofs 1986). These authors suggested that this lipid class is not a donor of the immediate fatty acyl precursors for pheromone bio-synthesis, but instead acts as a “dumping ground” for fatty acids that are not used in pheromone biosynthesis. Hence, the physiological role of lipid deposits in the sex pheromone gland of Lepidoptera requires further clarification.
The cuticle overlying the dorsal epidermis apparently contains more pore canals than does the cuticular covering of epidermis elsewhere on the intersegmental membrane. These pore canals usually contain epicuticular filaments and the latter have been implicated as structures involved in the translocation of the secreted pheromone through the cuticular lining to the exterior (Percy 1974; 1979). A recent study on the sex pheromone gland of Helicoverpa zea by Raina et al. (2000) using low temperature scanning electron microscope have shown the presence of excreted droplets at the tip of cuticular hair on the pheromone gland. These authors also showed the presence of pore canals in the epicuticle (Raina et al. 2000). In O. nubilalis, pore canals were not seen to penetrate the tanned epicuticle and pores were not observed on the external surface of the cuticle. Perhaps more vigorous tissue preparation procedures are required to reveal these structures in O. nubilalis.
The functional role of numerous microtubules found in O. nubilalis pheromone gland cells also is not clear. Micro-tubules have been reported to be important in providing mechanical support for midge follicles (Tucker and Meats 1976; Went 1978). In the hemipteran bug, Rhodnius prolixus Stal, (Reduviidae), microtubules are an important cytological element that is involved in the change of the follicular epithelium during vitellogenesis (Huebner 1976; Abu-Hakima and Davey 1977). The periodic folding and unfolding of the modified membrane during the female's calling activity may impose an extreme mechanical stress on the hypertrophied pheromone gland cells. The microtubules in this case may act as a cytoskeletal system that provides a direct strengthening role for maintaining the structural integrity of the hypertrophied cells.
In summary, current morphological observations and biochemical studies confirmed that the sex pheromone glandular epidermis in O. nubilalis female is located only in the dorsal part of the intersegmental membrane between abdominal segments 8 and 9/10. The modified epidermal cells undergo drastic morphological changes to mature into active glandular cells. The hypertrophied glandular cells exhibit the cytological characteristics found in other lepidopteran pheromone gland cells. Such morphological features are poorly represented in epidermal cells found elsewhere on the intersegmental membrane.
We thank Mary Lou Hessney for rearing the insects used in this study. This research was supported in part by a National Science Foundation Grant No. DBC-9017793 to W.L.R. This is publication No. J9995 of Mississippi Agricultural and Forestry Experiment Station