Cell differentiation during the ontogeny of larval salivary glands of the fly, Telmatoscopus albipunctatus

J. Insect Physiol.,

1977, Vol. 23, pp. 843 to 854. Peryarnorl Press. Printed in Great Britain.

CELL DIFFERENTIATION DURING THE ONTOGENY OF LARVAL SALIVARY GLANDS OF THE FLY, TELMATOSCOPUS ALBIPUNCTATUS L. C. G. SIM~ES,* A. JURAND~, and S. S. SEHGAL$ University of Edinburgh. Institute of Animal Genetics. West Mains Road. Edinburgh (Received

20 October

1976: revised 7 January

EH9 3JN. Scotland

1977)

Abstract--Using the larvae. pharate pupa. and pharate adults of the moth fly. Telrnaroscopus aNpunctutus, histological and ultrastructural features of the salivary glands were investigated. The gland lumen contains 5 milky secretion from the first instar. This secretion continues to occur at all subsequent developmental stages: with the onset of the pharate pupal stage, however. the secretion becomes transparent and rather viscous. Histochemical tests revealed that it is mainly proteinaceous. Glands from the same developmental stage may respond differently to PAS-reaction. Various cell organelles were compared at consecutive stages of larval development and of secretory activity of the salivary glands. In first and second instar larvae autophagic vacuoles are virtually absent in I he salivary gland cells. They were occasionally found in the third instar, when they appear to be engaged in the process of organelle turnover. Histolysis of the larval glands is initiated towards the close of the fourth instar when the number of autophagic vacuoles starts to increase. Simultaneously. the cytoplasm, previously full of ribosomes and endoplasmic reticulum. starts losing these structures. At the beginning of the pharate adult stage, the cytoplasm becomes practically devoid of all structures other than those engaged in autophagy. Polyteny of the chromosomes during ontogeny of the larval salivary glands is also discussed.

1 VTRODUCTION

SALJVARY glands in Diptera develop during the embryonic stage and grow in size without further cell division during larval life. The chromosomes replicate, but the ccmponent threads always remain together. resulting in the formation of polytene chromosomes. In all cases so far studied, the larval salivary glands histolyse at the end of larval life or during early pupal development. BODENSTEIN(1943) has established that certain hormones play an important rble in initiating cell lysis. After this process has set in. subsequent intracellular events lead the salivary gland cells to takl: on the responsibility for further histolytic processes. The fine structure of the salivary glands of various Diptera has been studied by several (JACOB and JURAND, 1963. 1965; BERENDESand DEBRUYN, 1963; PHILLIPS and SWIFT, 1965; BERENDES.1965; MACGREGOR and MACKIE. 1967; KLOETZEL and LAUFER. 1969, 1970; LANE et ul., 1972; JURAND and PAVAN, 1375. and many others). In each investigation special features have been described in different organisms. -___ ___* Permanent address: Departamento de Biologia, Inst. Bioci&n. Universidatle de SBo Paulo, SZo Paula, Brazil. t Send offprint requests to Dr. A. Jurand. University of Edinburgh, Institute of Animal Genetics, West Mains Road, Edinburgh EH9 3JN. Scotland. $ Permanent addrzss: Department of Zoology. University of Delhi. Delhi 7, India. 843

Although the basic trends are similar in most Diptera, certain details vary from species to species. Some authors have attempted to correlate the ultrastructural changes observed during pre-metamorphic stages with the state of the polytene chromosomes and the occurrence of puffs. The present paper reports the results of investigations on ultrastructural changes in the cytoplasm during the larval, pharate pupal, and pharate adult stages of a psychodid fly. Telrnutoscopus ulbipunctatus. The observations are compared with those at the light microscopic level, and include the state of the polytene chromosomes at each stage studied. Further, an account of the fine structure of the larval salivary glands of this species is given for the first time. Also. an attempt is made to describe histochemical changes in the gland cells during larval development.

MATERIALS

AND

METHODS

The materials used in the present investigations were larvae. pharate pupae. and pharate adults which were maintained in this laboratory. They were divided from an initial population of several adult flies originally brought from the University of Sgo Paulo (Brazil) where cultures have been maintained since 1967. A female normally lays about 200 to 300 eggs which hatch after 2 to 3 days. Larval life lasts from 18 to 20 days, followed by pupation. At 22 k 1°C.

L. C. G. SIMBES.A. JURAND,AND S. S. SEHGAL

844

the entire life cycle takes about 25 to 30 days (AMABIS and SIMdES. 1972). After hatching, the larvae were allowed to remain in the same container. to which 1.Oml diluted sugar syrup (1 part of Lyle’s golden syrup + 3 parts water) was added. At the same time, about l.Og of fresh yeast was placed on the paper. Water was sprinkled regularly every two days or whenever necessary. Rations of fresh yeast were given weekly. and the amount of yeast was increased gradually as the larvae grew older. No further sugar syrup was added. In the stock culture. the diet can be supplemented with finely ground dog biscuits, In this investigation larvae and pupae from all stages were used. They will be referred in the text using the terminology as defined by HINTON (1976): Stage in the development

Stage No.

1st and 2nd instar 3rd and early 4th instar Middle and late 4th instar Early pharate pupa Late pupa or early pharate ____

1

adult

2 3 4 5

For cytological examination. the salivary glands were dissected in insect Ringer and immediately transferred to a drop of mixture composed of glacial acetic acid. 85”,> lactic acid, and distilled water (9:5:6). Following the addition of a small drop of lacto-acetic orcein. the material was squashed between the slide and the coverslip after one minute in the solution. For histological examination, the salivary glands were fixed in 2.5”, glutaraldehyde in cacodylate buffer. After processing in the usual manner. sections 8 kern thick were stained with methyl green pyronin. Whole mounts of glands were also made after they had been fixed in glutaraldehyde, rinsed in buffer. and stained. The glands to be studied with other staining methods were fixed in ethanol acetic acid (3:l). They were squashed either in 60”” acetic acid or in the lactic acid mixture mentioned before. The coverslips were removed after freezing in dry ice. The staining methods used were (i) Feulgen. (ii) Periodic acid_Schiff (PAS). (iii) triple stain (HIMES and MORIBER. 1956). (iv) Sudan black B and (v) Nile blue. The last two methods were used after fixation in lo”, formol-calcium. For electron microscopy. the salivary glands were fixed in lp, osmium tetroxide (1 part of insect Ringer and I part of ?“c, OsOj) for 45 min at room temperature. Ultrathin sections were stained with l”, potassium permanganate solution containing 2.5”, uranyl acetate.

RESULTS Ohserrntions

on srcretory

ucticitJ

The salivary glands of Te/fnutoscoptrs nlhip~cncratus are paired organs. In the early larval instars they are V-shaped. From the apex of each gland arises a thin

salivary duct which joins with the duct from the opposite side to form a median common salivary duct. which extends cephalad and opens at the base of the pre-oral cavity. Each salivary gland consists of 25 to 30 polyhedron-shaped cells. These cells represent a specialized type of epithelial gland cell; they are arranged in a single layer. surrounding a central. rather narrow lumen. The cells located at both the extremities are larger than the average cells. and their chromosomes show a higher degree of polyteny. The cells which make up the wall of the ducts are much smaller than those of the gland. The lumina of the ducts are lined with a cuticular intima which is formed into taenidia or spiral thickenings. At the origin of the paired ducts are some small cells arranged in the form of rings which become prominent during later larval development. These cells probably represent the anlage of the imaginal salivary glands. At the junction of the gland with the duct. there is a crescent formed by 5 small cells. the chromosomes of which show a low degree of polyteny. Except in the cells of glands from pharate pupa (stage 4). the methyl green-pyronin preparations revealed nucleolar activity in the salivary gland cells at all stages examined. Four nucleoli which sometimes fuse together, are seen in the polytene chromosomes. Since the polytene chromosomes during the early stages of development are too small for precise cytological investigation. the origin of the nucleoli observed at these stages could not be traced to any specific chromosome. RNA is always present in the cell cytoplasm, which is intensely pyroninpositive. The 5 cells of the crescent at the junction with the duct contain smaller nucleoli. Their cytoplasm is only weakly pyronin-positive (Fig. 1). From the first instar to the early fourth instar (stages 1 and 2). the gland lumen is filled with a milky secretion; externally the glands can be seen distinctly as white streaks along the lateral sides of the trunk. behind the head. In the late fourth instar just before the onset of the pharate pupa stage, (stages 3 and 4). the milky secretion is no longer seen. having been replaced by a transparent viscous liquid. The shape of the glands changes considerably during the last stage of larval development. Besides increasing in size. the earlier narrow middle part of the gland becomes wider. The gland loses its V-shaped form and becomes more or less like an elongate sac. Except for a few scattered PAS-positive granules (Fig. 2) that occur in most of the cells. the salivary gland cells of early larval instars (stages I and 2) are generally PAS-negative. Later on the large cells at the extremities and the cells opposite the crescent become strongly PAS-positive. Regardless of stage. it was observed that. in several instances, groups of glands derived from larvae at the same developmental stage and fixed and stained at the same time and under the same conditions. stained differently. The majority of the glands were PAS-

845

Fig. 1. Salivary gland of the fourth-instar larva. stained irr r~ro with methyl greenpyronin. showing the duct (D). lumen (L) and the large gland cells (G). The lightly stained area (arrow) is the crescent with smaller nuclei. Fig. 2. Salivary gland of the late second stage stained with Azur A and PAS. PAS-positive granules are seen in the cells towards the lower extremity. The middle part, which is PAS-negative, is completely devoid of granules.

846

x47

Fig. 3. Electron micrograph showing the cytoplasm of the salivary gland at stage 1. The microvilli (MV) on I he apical side project into the lumen of the gland which contains secretory granules (SC). Groups of endoplasmic reticulum cisternae profiles form dark patches (arrows) against the lighter background of the cytoplasmic matrix. Fig. 4. Arrows

indicate

septate desmosomes between adjacent salivary gland cells at stage endoplasmic reticulum (ER) and mitochondria (M).

1 containing

Fig. 5. Basal area of a stage-l salivary gland cell. Covered with a thick extra-cellular basement membrane (BM), the cell membrane underneath forms deep in foldings (IF). Mitochondria (M) display no special orientation in the cell; Golgi areas are not very frequent. Fig. 6. Lcaw power electron micrograph of a gland cell of the first-stage into the lumen of the gland. The nucleus is elongate. the chromosomes inner nuclear membrane (NM). The large. electron dense nucleolus Fig. 7. Elztron The cytoplasm

larva. Microvilli (MV) project (CH) tend to adhere to the (NU) is centrally located.

micrograph showing the appearance of lysosomes (LY) in the cytoplasm is completely filled with rough endoplasmic reticulum and irregularly mitochondria (M).

at stage 2. distributed

Fig. 8. At stage

2, apart

Fig.

10. A large

number

Fig.

11. Autophagic

from the rough endoplasr mc reticulum (arrow) and cells contain conspicuous g roups of lysosomes (LY).

Fig. 9. Morphologically of autophagic

vacuoles

altered

mi tochondrion

(M). the

at stage 3. Y 60.000.

vacuoles (A,V) in the cytoplasm (stage 4).

(AV) at stage 4 containing whorls lipid droplets (arrows).

mitochondria

at the pharate

of membranes

and

pupa

electron

stage dense

**a *

.

013 Fig.

I?. The chromosomes

pharate chromosomes,

threads Salivary

chromosomes

salivary

cells

5. Note the together.

pharate

stage.

tend the

1OOpm

I

f

Fig. 14. Electron Figs. 15-16.

micrograph

of pycnotic

polytene

chromosomes

(CH) at stage 5 adhering

to the folded

nuclear membrane (NM). Depletion of the cytoplasmic matrix at the final stages of histolysis. In Fig. 16 autophagic vacuoles show membranous remnants of the lysed organelles.

Fig. 17. Electron

translucent cytoplasm at tht: basal region containing scattered endoplasmic reticulum at the final stage of histolysis.

vesicular

profiles

of

Cell differentiation of salivary glands negative, but some glands had a few cells on the extremities which wet’e definitely PAS-positive. Others. besides having such cells also had a group of cells opposite the duct junction which were PAS-positive. Still another group of glands was found in which the entire gland was evenly but lightly PAS-positive. The significance of thi.; variability is not known. Results obtained using the HIMES and MORIBER (19561 triple-stain technique show the cells at the extremities taking a brownish stain. whereas all other cells are stained deep yellow with naphthol yellow S. This indicates that the secretion is of a proteinaceous nature. The nuclei are stained blue with Azur A. The secretion i? the lumen and the cell cytoplasm also show Sudan Black B and Nile Blue positive staining at all the stages studied.

Stalls I. The first instar larvae have very small salivary glands in which the polytene chromosomes are barely visible. The banding pattern is not well defined in orcein preparations. During the early larval stages, the chromosomes in squash preparations tend to stick to each other and are frequently linked by thread-like chromatin material. This leads to difficulties in preparing good squashes. Later. when the chromosomes are larger. they spread well when squashed. possibly because the adhesion of chromosomes is overcome by the pressure exerted on the coverslip. In larvae sacrificed about 1 hr after hatching, the salivary gland cells seen at the ultrastructural level already contain differentiated cytoplasm. Endoplasmic reticulum cisternae form disconnected clumps so that. at low magnification. they are seen in sections as dark patches on the lighter background of the cytoplasmic matri:c (Fig. 3). The adjacent cell boundaries display shallow interdigitations with septate desmosomes between the cell membranes (Fig. 4). At the apical border. 1he gland cells form numerous microvilli (Fig. 3) which project into the lumen of the gland. The basement membrane (basal lamina) covers the entire gland acd forms an interphase between the haemolymph and the gland cells. Under the basal lamina the basal cell membrane forms deep infoldings (Fig. 5). Lysosomej and autophagic vacuoles are not present. The abundant mitochondria are irregular in shape and do not display any special orientation within the cell (Figs. 3 and 5). Golgi areas are present but are not very frequent. Vacuoles are randomly scattered in the cytoplasm (Fig. 3). The nucleus is either round or slightly elongated. depending on the position of the cell in the gland. The terminal large cells have round r.uclei. whereas those closer to the duct have elongated nuclei. The chromosomes tend to adhere to the nuclear membrane and show no banding pattern. The nucleolus. which is granular, is probably the result of fusion of the products of different nucleolar zones (Fig. 6). Sometimes chromatinlike material can be observed well inside the nucleolar mass.

851

The rest of the nucleus presents no special features except for occasional darker spots which are either associated with the chromosomes or lying close to them. The salivary glands of the second instar although more developed in size. show no special distinctive external characteristics. Even the nature of the secretion found in the lumina is the same. Stnge 2. The polytene chromosomes at this stage increase in size, but the process of polytenization is slow or may even appear to be interrupted. In comparison with other Diptera, polyteny is very low in relation to the time elapsed from hatching (7 to 10 days). That is why chromosomes at this stage are not yet suitable for cytological investigations. The organization of the cytoplasm at this stage shows an increasing degree of complexity. The number of vacuoles increases and the previously patchy cytoplasm. due to the endoplasmic reticulum forming separate clumps, becomes uniformly filled with rough endoplasmic reticulum (Fig. 7). Golgi areas become more extensive. The lumen of the gland and the plasma membrane remain the same as in the previous stage. Conspicuous additions to the cytoplasmic contents are lysosomes (Fig. 8) and autophagic vacuoles. At the beginning of the fourth instar these vacuoles become much more frequent and often contain other cell organelles. Stage 3. In squash preparations the chromosomes at this stage are found to be distinctly banded. with some asynaptic sections and puffs (Fig. 12). However. all the major puffs occur only at the end of the fourth instar or during the pharate pupal stage. As the larva reaches the middle of the fourth instar approaching the pharate pupal stage. the composition of the cytoplasm is radically changed. Although still full of ribosomes and rough endoplasmic reticulum, it contains a large number of mitochondria which are morphologically altered (Fig. 9). At this stage. the autophagic vacuoles increase in number and organelles like mitochondria and electron dense bodies (lipid droplets) can be observed in the vacuoles. Golgi areas are larger and more widespread. Inside the nucleus. the chromosomes appear distinctly as separate structures still adhering to the nuclear membrane. At this stage they are often surrounded by electron dense material. probably RNP granules. Stage 4. At the early pharate pupal stage. the secretion in the gland lumen is no longer milky. but transparent and viscous. The cells are larger in size. and so are the polytene chromosomes, which at this stage undergo their last round of replication. The nucleolus is present during the early part of pharate pupal life, but regresses as the time of larval-pupal ecdysis approaches. In the cytoplasm there are large numbers of autophagic and other vacuoles (Fig. 10). Inside the autophagic vacuoles it is possible to see parts of other cell organelles being digested (Fig. 11). Autophagy. as seen in the electron microscope, sets in at this stage and undoubtedly it marks the beginning of the process of gland histolysis: it can no

x51

L. C. G. SIM~ES,A. JURAND.AND S. S. SEHGAL

longer be regarded merely as a routine organelle turnover as in earlier stages. The mitochondria are in most cases altered in shape. frequently appearing swollen. Sometimes two parts of the same mitochondrion are seen to be linked by narrow bridges as shown in Fig. 8. The endoplasmic reticulum seems to be less organized than before, and the cytoplasm shows large empty areas which probably were formerly occupied by extensive endoplasmic reticulum (Fig. 11). Ribosomes are seen free in the cytoplasm and inside the autophagic vacuoles. Srage 5. After the larval-pupal ecdysis the gland lumen appears more and more empty. Observations on pupal development show that when the last larval cuticle is cast off, a white pupa emerges, which lasts for about five hours. During this period the polytene chromosomes are still unchanged. except for the nucleolus which has already regressed. The next stage is the yellow pupa. when the cuticle develops yellow pigmentation. At the beginning of this stage the polytene chromosomes are still visible but they no longer spread easily in squash preparations and tend to clump together as in the early larval periods. Long chromatin threads can be seen linking the chromosomes (Fig. 13). The individual homologues are more dissociated than those of the late pupal stage (Fig. 13) and are more pycnotic (Fig. 14). The light yellow pupa is followed by a dark yellow pupa. Salivary gland lysis is completed at this time. Later. the pharate adult becomes darker. with black spots appearing on the head and along the body. The spots increase in size and fuse to make the pharate adult completely black before emergence. The whole process of metamorphosis takes about 4 to 5 days at 22 k 1 C. The final lysis of the salivary glands takes place approximately during the first 30 hr of the pupal and pharate adult period. During the pupal stage the salivary glands are apparently devoid of any secretion but the gland cells are still large. Fixation of the glands at the stage of lysis is difficult, because the gland cells break apart as soon as they are touched by the dissecting needle or taken into a pipette. The general organization of the cytoplasm is basically the same as in the late pupal stage, except that the whole process of histolysis is far more advanced towards the final stage of degradation (Figs. 15 to 17). The nuclear membrane was observed to form folds (Fig. 14). and the nuclei. previously round, become rather convoluted. The folding of the nucleus can be traced back to the late fourth instar larva, but it becomes more prominent at this stage. Adjacent cells of salivary glands of Diptera are sometimes disparate in their development. Next to an almost intact cell, a nearly lysed one may be found. DISCUSSION Our investigation of the ultrastructure vary gland cells shows that. structurally.

of the salithese cells

are similar in many respects to actively synthesizing cells of the salivary glands in other Diptera so far studied. They also resemble the cells of mammalian exocrine pancreas (CARO and PALADE. 1964; PALADE. 1975). The results reported here suggest that the fine structural organization of the endoplasmic reticulum, the Golgi complex and associated granules may be related to the secretory activity of the salivary gland. In Trlmntoscopus, the glands seem to show secretory activity at all larval stages, since they are always filled with a milky or transparent secretion. The presence of numerous microvilli on the apical side of the cells contributes to greater secretory potential of the cells in the same way as in the last larval instars of Drosophi/n (BERENDES,1965: LANE ef irl., 1972) and Chironontns (KLOETZEL and LAWER. 1970) when the glands are most active. The secretory activity of the gland cells can be readily observed; when the larva is held firmly with the forceps at the time of disssection. it discharges orally a large amount of milky secretion into the surrounding medium. In such cases. after dissection the glands are usually found to be empty. The polarization of the gland cells is typical for epithelial tissues. The basal cell surface is different morphologically and functionally from the apical surface. The deep infolding plasmalemma seems to be instrumental in the transport of substances from the surrounding haemolymph to the interior of the cells. Such infoldings are commonly seen in epithelia engaged in water transport (PEASE, 1956). Strangely enough, microtubules are not abundant in the gland cells of Telntntoscopus. Their presence in the cytoplasm has been reported to be correlated with the movement of materials (FREED, 1965). Further. in Telt~llltoscopLfs, the mitochondria are generally numerous: they are present as large structures until the late pupal stage when the process of organelle sequestration by autophagic vacuoles starts to bring about cell degeneration. Autophagic vacuoles containing organelles can also be observed at earlier stages. when sequestration can be interpreted as a routine turnover of organelles rather than as cell lysis. The phenomenon of cellular autophagy is an important factor in the cell cycle. It may occur in several types of cells and may have physiogical. developmental. or pathological significance. The origin of the lysosomes is thought to be connected with the Golgi saccules or, in some instances, the endoplasmic reticulum (NOVKOFF and SHIN, 1964). These authors discuss the possibility that autophagic vacuoles may have different origins in different cell types. It is possible that, in salivary gland cells. the lysosomes observed during the early developmental stages differ in origin from those which appear during the later stage. The process of autophagy is an essential step in development, because the salivary glands, along with other organs and tissues, are completely histolysed during metamorphosis, The work of BODENSTEIN(1943)has established that

Cell differentiation of salivary glands some basic factor:1 regulate salivary gland lysis in Drohe was the first to suggest that histolysis is under hormonal control. The basic facts established by Bodenstein can usually be applied to other Diptera and to other organisms which undergo similar processes. The exact mechanism by which cell lysis is controlled is not yet known. By incubating salivary glands of Rhynchosciara in ritru, one of us (L.C.S.) found that the response of these glands to incubation varies. depending on whether the glands are incubated before. during. or after the onset of the histolysis. The glands may survive or degenerate in aitro, depending on the timing of the beginning of incubation. This suggests that the stimuli for the production of lytic enzymes in rioo are released gradually or are part of a chain reaction which takes place at a particular time. If the chain reaction is interrupted by explanting the gland before a certain point, cell lysis in rirro cannot take place. If, however, a certain stage in the chain reaction has been attained before explantation, the gland cells will react and the process will not be interrupted by explantation. In Rh~nchoscinra, the threshold po.nt was found to be about 48 hr before gland lysis in viva, after the development of DNA puffs in the polytene chromosomes of the salivary glands (SIM~)ZSet al., 1970; SIM~ES et a[., 1973). This stage corresponds to the point when the salivary gland cells are full of lysosomes and autophagic vacuoles (JURAND and PAVAN, 1975). It was found that even a slight difference in the developmental stage of the glands at the time of explantation is decisive for the fate of the gland. It seems that, although there is a gradual accumulation of lysosomes in the glands, there is probably produced at one stage of the chain reaction a decisive factor which unleashes the final degradation. As stated before, nearly lysed cells are frequently observed next to virtually intact ones. This phenomenon has been obslzrved by different authors in several Diptera. Recentl;!. JURAND and PAVAN (1975) reported a similar situation in Rhynchosciara. CASCIANELLI and CES’rARI (1975). working with salivary gland cells of Rhywhosciura incubated in &ro. found it to be true at the ultrastructural level. They also found that the number of degenerating cells was approximately the same in glands incubated for a few hours and glands incubated for several days, and that there seems to be a constant pattern in the localization of these cells in the salivary glands. PHILLIPS and SWIFT (1965) believe that autophagic vacuoles are induced by certam s.lmuh and do not pre-exist in the cells. The stimuli could be a consequence of normal development or a r’zsponse of living cells to unfavourable environmental conditions. A comparison of degenerating tissues of different species shows many morphological sim larities between the lysosomes involved, although their origin, as well as the final product of the lysis can be quite different, as pointed out by SCJIIN and CLFVER (1965). The presence of lyso-

sophila;

853

somes during early stages of development need not necessarily be related to cell breakdown. These structures are engaged in intracellular processes such as the routine turnover of cell organelles, and they may also serve to degrade or transform proteins which enter the cell from the surrounding haemolymph. The stainability of the salivary glands with naphthol yellow S at all stages studied suggests that the secretion is proteinaceous in nature. The different patterns of PAS staining found in cells from glands at the same stage which had been fixed and stained at the same time and under the same conditions suggest the existence of a polymorphism in the population. This assumption would be in agreement with previous chromosomes studies (AMABISand SIM~ES, 1972; FERNANDESNACCACHE, 1975) which have shown a high degree of chromosomal polymorphism both in natural and laboratory populations of Teln~c~toscopus alhipunctatus.

AcXnowlengemellts-The authors wish to acknowledge the financial support of the British Council. CNPq (FlNEP)-SIP-04/001. and to express gratitude to the Universities of SBo Paulo and Delhi for the leave of absence granted to two of us (L.C.S. and S.S.S.. respectively). We are indebted to Miss A. P. GRAY for the skilful editorial work and to Miss HELEN TAIT for technical assistance.

REFERENCES AMABIS J. M. and SIM~ES L. C. G. (1972) Chromosome studies in a species of Teln~atoscopus sp. Caryologia 25.

199-210. BERENDES H. D. (1965) Salivary gland function and chro-

mosomal puffing patterns in

Drosophila

hydei.

Chromo-

soma 17, 35-17.

BERENDES H. D. and DEBRUYN W. C. (1963) Submicroscopic structure of Drosophila hydei salivary gland cells. Z. Zellforsch. mikrosk. Anat. 59, 142-152. BODENXEIN D. (1943) Factors influencing growth and metamorphosis of the salivary gland in Drosophila. Bio. Bull..

Woods Hole

84, 13-33.

CASCIANELLIF. R. and CESTARI A. N. (1975) Abstract. Microscopia electronica de glandulas salivares de Rkynchosciaru “in oitro” Cien. e Cult. 27, 281. CARO L. G. and PALADE G. E. (1964) Protein synthesis, storage and discharge in the pancreatic exocrine cell. An autoradiographic survey. J. Cell Biol. 20, 473-495. FERNANDES-NACCACHE N. (1975) Sintese de DNA no desenvolvimento larval de Telamatoscopus albipwwtatus. University of SBo Paulo Master’s Thesis. FREED J. J. (1965) Microtubules and saltatory movements of cytoplasmic elements in cultivated cells. J. Cell Biol. 27, 29A. HIMES M. and MORIBER L. (1956) A triple stain for deoxyribonucleic acid. polysaccharides and proteins. Stain Technol.

31, 67-71.

HINTON E. H. (1976) Notes on neglected phases in metamorphosis. and a reply to J. M. Whitten. Ann. ent. Sot. Am. 69, 560-566.

JACOB J. and JURAND A. (1963) Electron microscope studies on salivary glands of Bradysia mycorwn-111. The struc-

ture of the cytoplasm. J.

Insect

Physiol.

9, 849-857.

854

L. C. G. SIMBES, A. JURAND, AND S. S. SEHGAL

JACOB J. and JURAND A. (1965) Electron microscope studies on salivary gland cells.-V. The cytoplasm of Smittiu parthenayrnrticn (Chironomidaeb. J. Inserr Physiol. II, 1337-1343. JURAND A. and PAVAN C. (1975) Ultrastructural aspects of hystolytic processes in the salivary gland cells during metamorphic stages in Ritwciwsciaru Mlaenderi (Diptera. Sciaridae). Crli D[fifi^rrerzriniion 4, 219-236. KLOETZL J. A. and LAUFER H. (1969) A fine structural analysis of larval salivary gland function in Chironormrs thummi. J. ultrasrruct. Rex. 29, 15-36. KLOETZEL J. A. and LALWR H. (1970) Developmental changes in fine structure associated with secretion in larval salivary glands of Chironorms. Ezp. Cell Rex 60. 327-337. LANE N. J., CARTER Y. R., and ASHBLIRNERM. (1972) Puffs and salivary gland function: The fine structure of the larval and pupal salivary glands of Drosoplliln melnr~uuster. W.ilkeltn Roux Arch. EntwMech. Oru. 169. 21G-238. MACGREGOR H. C. and MACKIF S. B. (1967) Fine structure of the cytoplasm in salivary glands of Sinurlitrrn. J. Cell Sci. 2. 137-144.

NOW~~OFFA. B. and SHIN W. Y. (1964) The endoplasmic reticulum in the Golgi zone and its relation to microbodies. Golgi apparatus and autophagic vacuoles in rat liver cells. J. Microscopic 3, 187-206. PALADE G. F. (1975) Intracellular aspects of the process of protein synthesis. Science. Wnslr. 189, 347-358. PEASE D. C. (1956) Infolded basal plasma membrane found in epithelia and noted for their water transport. J. Biopitn. Bioci1rct1. Cm (Suppi.) 2, 203-208. PHILLIPS D. M. and SWIFT R. (1965) Cytoplasmic fine structure of Sciara salivary glands. J. Cdl Biol. 27, 395m-409. SCHIN K. S. and CLEVER U. (1965) Lysosomal and free acid phosphatase in salivary glands of Chirorlormrs IPFItms. Science. Wash. 150, 1053-1055. SIM~ES L. C. G., AMABIS J. M.. and DEBONI J. A. (1970) Studies on the development of a DNA puff in Rhynchostiara SD. Cien. e Cult. 2213). 176-l 8 1. SIM~ES L: C. G., MARTHO G.’ R.. and AMABIS D. C. (1973) Polytene chromosomes in vitro. Genetics 74 (Suppl. 2). 256.