Ultrastructure of sex pheromone gland cells in Lobesia botrana Den & Schiff. (Lepidoptera: Olethreutidae)

B. LALANNE-CASSOU 1.N.R.A.IC.N.R.S. Laboratoire de Recherches sur MPdiareurs Chimique-Domaine de Brouessey-78470 Magny les

Hameaux par St. Rem Les Chevreuses, Paris, France

JEAN PERCY AND J . A. MACDONALD Insect Pathology Research Institute, P.O. Box490, Sault Ste. Marie, Ont., Canada P6A 5M7

Received October 8 , 1976

LALANNE-CASSOU, B., J. PERCY, and J. A. MACDONALD. 1977. Ultrastructure of sex pheromone gland cells in Lobesia botrana Den & Schiff. (Lepidoptera: Olethreutidae). Can. J. Zool. 55: 672-680.

In newly emerged and 48-h-old female adults of Lobesia botrana the sex pheromone gland cells are columnar. Organelles characteristic of lipid-producing cells are very prominent. Smooth tubular endoplasmic reticulum is extensively developed but few lipid droplets are observed. Numerous microbodies are present and are located, along with the Golgi complexes, primarily in the vicinity of the nucleus. The apical plasma membrane is folded. The cuticle overlying the gland cells differs from unmodified cuticle in the number and location of epicuticular filaments. They originate near the bases of the furrows between the apicalfolds.

LALANNE-CASSOU, B., J. PERCY et J. A. MACDONALD. 1977. Ultrastructure of sex pheromone gland cells in Lobesia botrana Den & Schiff. (Lepidoptera: Olethreutidae). Can. J. Zool. 55: 672-680.

Chez les femelles de Lobesia bolrana qui viennent de sortir du cocon et chez les femelles de 48h, les cellules de la glande a pheromone sexuelle sont cylindriques. Les organites caracteristiques des cellules productrices de lipides sont trks apparents. Le eticulum endoplasmique tubulaire lisse est tres developpe. mais on observe peu de gouttelettes de lipides. On voit de nombreux corpuscules situes surtout, 3 I'insrar des apparc~ls de Golgi. dans le voisinage du noyau. La membrane plasrnatique apicale est repliee. La cuticule qui recouvre les cellules glandulaires differe de la cuticule non diffkrenciie par le nombre et la position des filaments Cpicuticulaires; ceux-ci prennent leur oripine pres de la base des sillons. entm les replis apicaux.

[Traduit par le journal]

Introduction This investigation was undertaken to deter-

~ ~ h ~ ~ i ~ ~ ~ ~ l , field, and chemical investigations mine whether there were detectable ultrastruc-

have established the presence of a pheromone in tural modifications in the gland cells that could

female Lobesia botrana. The pheromone has be related to the production of the pheromone.

been isolated and is (E,z)-7,9-dodecadien-1-01 acetate (Buser et al. 1974). Extracts of the aland Materials and Methods

becomex active in beha;ioural bioassays-48 h Lobesia botrana, the grapevine moth, is not a North

before adult emergence and remain so until the American species. A culture was obtained from France and reared in the laboratory on artificial diet (Guennelon

death Of the virgin (Lalanne-Cassouy in et al. 1970). However, very few insects were successfully preparation). reared and only newly emerged and 48-h-old insects were

In nearly all female Lepidoptera so far ex- available. amined the pheromone gland is a modified inter- Killing and initial fixation were accomplished by injec-

tion into the abdomen of cold 5% glutaraldehyde in 0.05 segmental the One which is located M cacodylate buffer pH 7.2, containing 2% sucrose and between the eight and ninth abdominal segments 0.01 M calcium chloride. The pheromone gland was dis- (see Percy and Weatherston (1974) for complete sected out and placed in ice-cold glutaraldehyde to fix list of references). As in other olethreutids overnight. Tissue was postfixed with 1% osmium tetrox-

( ~ ~ ~ l ~ f ~ and F~~~ 1968 ; percy and weatherston ide in 0.05 M cacodylate buffer containing 4% sucrose, stained in block with hot 2% aqueous uranyl acetate,

1971) the gland in L. botrana is a sac dehydrated in an ethanol series, and embedded in Aral- (Lalanne-Cassou, in preparation). dite-(~ocke et a/. 1971). Sections were observed without

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LALANNE-C :ASSOU ET AL. 673

further staining and photographed using a Hitachi HU12A microscope operated at 75 kV.

Results Gland Cells

The gland cells are columnar and about 16 to 25 pm deep. The nucleus, located midcell, is lobed and about 6 to 9 pm in diameter.

In longitudinal section the apical membrane appears to have numerous small disorganized microvilli which extend about 0.5 to 1.5 pm into the endocuticle (Fig. 1). However, circular cross sections, as expected from true microvilli, were never encountered. Therefore, the projections are, in fact, apical folds. Since they are short and disorganized it is unlikely that they extend across the entire apical surface of the cell.

Organelles characteristic of lipid-producing cells are prominent in these cells. There is exten- sive smooth tubular endoplasmic reticulum throughout the cytoplasm (Fig. 1). Its develop- ment greatly exceeds that of rough endoplasmic reticulum. Microbodies, averaging 0.1 pm in diameter, are most numerous in the vicinity of the nucleus (Fig. 2). They are also found in other parts of the cell particularly near the bases of the apical folds (Fig. 1). Microbodies are usually round and often are in continuous chains, indi- cating that they arise as dilations of smooth en- doplasmic reticulum (Fig. 3). Golgi complexes occur near the nucleus in the vicinity of the microbodies (Fig. 2). They have secretory vac- uoles that are similar in size to the nearby micro- bodies but their contents are less dense (Fig. 4).

Few lipid droplets occur in the cytoplasm. When observed they are usually near the base of the cells or occasionally, immediately beneath the apical folds in both newly emerged and 48-h insects. Mitochondria are, at times, intimately associated with these lipid droplets (Fig. 3).

The basal plasma membrane is sometimes in- voluted. A thin basement membrane follows the contours of the plasma membrane (Fig. 2).

Cuticle The cuticle overlying the gland cells is thin

(0.9 to 1.4 pm) and consists of cuticulin (50 A), dense epicuticle (0.5 to 0.9 pm), patches of meso- cuticle, and non-lamellar endocuticle (0.2-0.6 pm) (Fig. 5). The outer waxy layer usually ob- served on insect cuticle is often not present, prob- ably because it is lost during the embedding pro- cedures. Within the endocuticle are gaps between

the microfibrils representing pore canals (Fig. 5, arrows). The pore canals are usually filled with epicuticular filaments which originate at the base of the furrows between the apical folds and ter- minate in clear oval depressions near the outer surface of the epicuticle (Figs. 5,6, and 7). Pores, about 65 A in diameter, are infrequently ob- served in the cuticulin (Fig. 8).

The interpretation of the arrangement of organelles within a gland cell and its relationship with the cuticle is shown in Fig. 8.

Unmodified Cells Unmodified epidermal cells are similar to

those of other insects in that they are flattened, contain moderate amounts of rough endoplasmic reticulum, very little smooth endoplasmic reti- culum, and have small apical folds. The overlying cuticle is about 1.0 to 1.2 pm thick and consists of lamellate endocuticle (0.6 to 1 pm), dense epicuticle (0.3 pm), and cuticulin (Fig. 9).

Discussion The ultrastructure of cells in the pheromone

gland of L. botrana is consistent with the hy- pothesis that they are indeed involved in phero- mone production. Cells from similar glands in other insects also contain abundant smooth en- doplasmic reticulum although the type and the degree of development varies (Steinbrecht 1964; Percy 1974, 1975; Feng and Roelofs, in prepara- tion). In gland cells of Choristoneura furniferana for example, the endoplasmic reticulum is smooth and tubular (Percy 1974) while in cells from Orgyia leucostigrna smooth cisternal endo- plasmic reticulum predominates (Percy 1975). In the former insect the pheromone is a mixture of aldehyde isomers (Weatherston et al. 1971; Sanders and Weatherston 1976), while in the latter it is likely to have a ketonic component (Weatherston, personal communication). The development of smooth endoplasmic reticulum may reflect different methods of biosynthesis of the respective pheromones. Other lipid-secreting cells of insects which contain abundant smooth endoplasmic reticulum include certain cells of sternal glands of various termites (Quennedey 1971, 1972; Stuart and Satir 1968; Noirot and Quennedey 1974), the cells of Gilson's glands in larvae of Phryganea varia (Quennedey 1969), certain cells of the defensive gland of Eleodes longicollis Eisner et al. 1964), and the wax gland of Calpodes ethlius larvae (Locke 1969).

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674 CAN. J . ZOOL. VOL. 55, 1977

F~G. 1. Apical region of adult gland cell. 32 600 x . Apical folds, of; dense epicuticle, [I?; endocuticle, p n ; Golgi compIex, G: mitochondrion, m; microbody, ~ n b ; nuclcus, n; smwth tuhular endoplasmic reticulum, ster. C

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676 CAN. J. ZOOL. VOL. 55, 1977

FIG. 3. Middle to basal region of gland cell showing chains of microbodies and close association of mitochondrion to lipid droplet. 59 000 x . Lipid droplet, L; mitochondrion, m; microbody, mb. Ar- rows indicate chains of microbodies. FIG. 4. Golgi complex located in the vicinity of microbodies. 100 000 x . Golgi complex, G ; mitochondrion, m; microbody, mb; secretory vesicle, so.

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LALANNECASSOU ET AL. 677

FIG. 5. Cuticle overlying gland cells. 65 000 x . Apical fold, af; cuticulin, c ; dense epicuticle, de; epicuticular filaments within apical furrow, ef; mesocuticle, me. FIG. 6. Epicuticular filaments pene- trating dense epicuticle overlying gland. 154 000 x . Cuticulin, c ; dense epicuticle, de; epicuticular filaments, ef; oval depression, od. FIG. 7. Pore in cuticulin overlying gland at termination of epicutic- ular filaments. 154 000 x . Apical fold, of; cuticulin, c ; dense epicuticle, de; epicuticular filaments, ef; pore, indicated by arrow.

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FIG. 8. Interpretation of cuticular structure and the arrangement of organelles within gland cell. Apical folds, af; dense epicuticle, de; Golgi complex, G; lipid droplet, L; mitochondrion, m ; micro- bodies, mb.

mone, pheromone precursors, or enzymes in- volved in the biosynthesis.

Microbodies are a constant feature of lipid- producing cells (Percy (1974) and references therein). In L. botrana their predominant loca- tion is near the nucleus and always in association with Golgi complexes as also occurs in cells of 0. leucostigma (Percy 1975). However, cells of L. botrana, unlike those of 0. leucostigma, also have Golgi-derived vesicles similar in size to the

microbodies but of lesser staining density. In mammalian systems it has been shown that microbodies usually contain at least two en- zymes, catalase (EC 1.1 1.1.6) and a peroxide- producing oxidase (Novikoff and Novikoff 1972 ; Black and Bogart 1973). Possibly they also con- tain products of the Golgi complex necessary for pheromone production.

The structure of the cuticle overlying gland cells of L. botrana certainly differs from that

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LALANNE-CASSOU ET AL. 679

FIG. 9. Epidermal cell from unmodified intersegmental membrane. 18 000 x . Apical projections, ap; basement membrane, bm; cuticulin, c; dense epicuticle, de; epicuticular filaments, ef; endocuticle, en; nucleus, n.

overlying unmodified epidermal cells. In the former the epicuticular filaments have their origin somewhere near the bases of the furrows between the apical folds. A somewhat analogous situation occurs in honeybee wax-secreting cuti- cle where the epicuticular (or wax canal) fila- ments also penetrate the cell, albeit further, while remaining outside the plasma membrane (Locke 1961). On the other hand they originate some- where near the tips of the microvilli in cuticle overlying the gland in C. fumiferana and 0. leu- costigma and also in unmodified cuticle of both these insects, L. botrana, and developing epider- mal cells of Cai'podes ethlius (Locke 1969).

Near the bases of the furrows, but within the cell, microbodies are frequently observed. The contents of the microbodies here may be neces- sary for final synthesis of pheromone near the surface of the cuticle. Outside the cell these contents could be located on, or transported through, the epicuticular filaments (Percy 1974).

Insect pheromone gland cells although exhib- iting considerable variation also exhibit certain fundamental similarities. Hopefully as further studies are carried out on the ultrastructure and cytochemistry of these lipid-secreting cells, a clearer insight will be gained into the relationship of the various organelles to one another and eventually to the direct relationship of the or- ganelles to biosynthesis of pheromones.

Acknowledgments

This study was partly supported by a coopera- tive agreement between Centre Nationale de Recherches SciCntifiques and NRCC under whose auspices funds were provided for educa- tional leave for B. Lalanne-Cassou. This publica- tion represents contribution No. 321 from the Insect Pathology Research Institute. BLACK, V . H . , and B. I. BOGART. 1973. Peroxisomes in the

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