A medium for the maintenance of Chironomus tentans salivary glands in vitro

A MEDIUM FOR THE MAINTENANCE OF CHIRONOMC’S TENTANS SALIVARY GLANDS IN VITRO CONRAD E. FIRLING and BRIAN K. KOBILKA

Abstract-A

synthetic medium based upon the chemical composition of fourth instar C‘h/ronr~/nt~~ hacmolymph was formulated for the br vitro culture of Chironomus tenfut~s salivary glands. Saltvary glands maintained in the medium for up to 4 days appeared morphologrcally normal. Secrettonfree glands. obtained from pilocarpine-treated larvae. accumulated proteinaceous material in the gland lumen and exhibited a 46”,, increase in total gland protein after 14 hr in the medium. Cycloheximide almost totally Inhibited the accumulation of secretion material and the increase in total gland protein by cultured glands. Glands cultured for up to 4 days continued to incorporate ‘JC-leucine into acid-insoluble total protein and “H-uridine into total RNA. but at reduced levels. The mcorporation of both isotopes was almost completely inhibited by cycloheximide. Autoradiographic squash preparations ofglands pulse-labelled with “H-thymidine after 3 days in culture revealed a normal pattern ofasynchronouschromosomal DNA replication. Glandscultured for up to4day\ exhibited “H-uridine incorporation into nucleoli and into distinct chromosomal regrons which corresponded with sites of cytochemically demonstrable acidic protein. The chromosomes of cultured glands appeared morphologically and cytochemically normal. cxccpi for home regression of the Balbiani rings. Addition of ecdysterone to media containing glands preriously cultured for 3 days resulted in puff induction at the IV-Z-B chromosomal locus.

INTRODUCTION THE GIANT polytene chromosomes present in the salivary gland cells of larval dipteran insects represent a unique experimental system for the study of eukaryotic genomic activity, its control, and its interrelations with cellular function and development. A number of investigators have studied these processes in viva or under the controlled conditions of in vitro organ culture. Dipteran salivary glands have been maintained in vitro for up to 24 hr (CANNON, 1964; TULCHIN et al., 1967; ASHBURNER, 1972); however, with the exception of several recent reports (POELS, 1972; FIRLINGand KOBILKA, 1976; SCHIN and MOORE, 1977) their prolonged culture has not been achieved. The principal reasons for the lack of success in this area probably relate to the technical difficulties involved in preventing cellular wounding during gland explanation (KRUEGER. 1966a) and to the lack of specific media designed for the cultivation of salivary glands of particular dipteran species. Most insect culture media have been formulated on the basis of the chemical and physical composition of haemolymph, the only extracellular body fluid present in insects (VAUGHN, 1971). Haemolymph is highly variable even among closely related species and during different developmental stages of the same species (FLORKIN and JEUNIAUX. 1974). Its composition physiological, and biochemical, the reflects morphogenetic events which occur during insect development. Thus, it is usually necessary to design specific media for the culture of the tissues or organs of a specific developmental stage of a particular insect species. This

paper

reports

on

the

formulation

and

evaluation of a synthetic medium designed for the maintenance of Chironomus tentans salivary glands in vitro. The composition of the medium is based upon biochemical analyses of fourth instar Chironomus haemolymph, literature information, and empirical studies. The suitablity of the resulting medium was assessed by examining the capacity of salivary glands in prolonged culture to (1) maintain normal chromosome and gland morphology. (2) replicate chromosomal DNA, (3) synthesize RNA and secretory protein. and (4) respond to stimulation by the insect moulting hormone ecdysterone at the chromosomal level. MATERIALS Experimental

AND METHODS

animuls

tentans were raised in polypropylene tanks containing absorbent cotton and continually aerated 0.069~ NaCl. Animals were fed autoclaved Acer saccharinum leaves and a mixture of nettle powder, yeastolate, and milk powder. Animals were maintained under a 12 hr light, 12 hr dark photoperiod at 19 + 2’C. Mid-to late fourth instar larvae, staged on the basis of chronological age and morphological were used in all appearance (FIRLING, 1977). experiments. Chironomus

Culture

medium

medium of the synthetic The composition formulated in this study is presented in Table I. The medium was prepared from seven separate solutions: (1) r_-aspartic acid. L-cystine, L-tyrosine and Lhomoarginine; (2) CaCI,. and all remaining amino

CONRAD E. FIRLING AND BRIAN K. KOBILKA

94 Table

I, Synthetic

medium

for the maintenance

of Chironon~ustentans salivary

glands

MgilOO ml

Mg/lOO ml Amino acids r_-a-Alanine t_+Alanine L-Arginine.HCI L-Aspardgine.HLO t_-Aspartic acid t_-Citrulline t_-Cysteine L-Cystine L-Glutamic acid t_-Glutamine Glycine L-Histidine.HCI.HZO t_-HomoarginineHCI t_-Hydroxyproline L-Isoleucine t_-Leucine L-Lysine.HCI r-Methionine L-Ornithine,HCl r-Phenylalanine L-Proline L-Serine Taurine L-Threonine L-Tryptophan t.-Tyrosine I.-Valine Carbohydrates Glucose Sucrose Fructose z-Trehalose Maltose Five parts of TC-199 medium medium. The resulting solution

50 I I4 15 8 I 2

I II 15 8 26 2 I 7 8 22 I2

I 8 14 25 2 9 2 I2 13

40 2 2 40 2

Inorganic salts CaCI, MgS0;7HZ0 NaCl NaH,PO;H20 KCI MgCI;6HZ0 Organic acids Malic acid r-Ketoglutaric Succinic acid Fumaric acid Citric acid

33 40 552 36 37 50

4 3 2 2 2

acid

Vitamins ( x IO-“) Biotin Folic acid CholineCI Nicotinamide o-Ca pantothenate Pyridoxal.HCl Thiamine,HCI Riboflavin i-Inositol

2 2 2 2 2 2 2 2 4

Miscellaneous Phenol red Yeastolate Sodium penicillin G Streptomycin sulfate 0-Phosphoethanolamine pH = 7.20-7.25 (0.8 ml I N NaOH)

2 10 3 5 8

(IX) containing Earl’s modified salts and 2 parts FBS are added to 93 parts of the synthetic is sterilized by membrane filtration and stored at 4 C.

except L-glutamic acid and L-glutamine; (3) KCl, yeastolate and sugars; (4) phenol red, organic acids and all remaining inorganic salts except NaCl and NaH2PO;H,0; (5) sodium penicillin G, streptomycin sulphate, L-glutamic acid, t-glutamine and O-phosphoethanolamine; (6) vitamins; (7) NaH,PO;H,O. All solutions were made with double glass distilled deionized water. The solutions were combined in the above order with continuous mixing, the pH was adjusted to 7.20 with a measured volume of 1 N NaOH, NaCl (552 mg, assuming 0.8 ml 1 N NaOH required for pH adjustment) was added to bring the total Na+ content to 105 mM. and the resulting solution was brought up to 1 litre. The medium must be formulated as described in order to prevent the precipitation of calcium and magnesium by phosphate. Ninety-three parts of the above were supplemented with 2 parts of foetal bovine serum (FBS) and 5 parts TC-199 medium containing Earl’s modified salts (Grand Island Biological Co., Grand Island, N.Y.) both of which had been decarbonated by aspirator rotor-evaporation at 0°C for 10 min. The resulting medium was readjusted to pH 7.20-7.25, sterilized with a 0.20 p membrane filter unit (Falcon Plastics, Oxnard, CA) and stored in tightly capped culture bottles at 4°C. acids

in ritro

Organ culture Larvae were kept for 12 hr in sterile 0.06% NaCl containing nettle powder, penicillin (30 pg/ml) and streptomycin (100 pg/ml), rinsed in fresh 0.06% NaCl and placed in culture medium. Animals were decapitated and the delicate ligaments holding the salivary glands within the body cavity were cut with microdissecting scissors to prevent cellular wounding during gland removal. In all experiments, the two sister salivary glands of each larva were removed and rinsed in fresh culture medium prior to their separation and placement in either experimental or control cultures. Cultures consisted of 3-5 glands placed in 0.5 ml of medium contained within the inner well of a 60 x 15 mm organ culture dish (Falcon Plastics, Oxnard, CA). The absorbent ring in the outer well of the dish was wetted with 0.75% NaCl. Cultures were maintained at 19 + 2°C. Pilocarpine-treated

larvae

Larvae were placed for 18-24 hr in 0.06% NaCl containing nettle powder and 0.1 mg/ml pilocarpineHCl. The two glands obtained from individual pilocarpine-treated larvae were separated and placed into experimental and control cultures. Experimental

---

-.--

_-?Jz

k

i

Fig. I. The progressive accumulation of secretion material within the lumen of a salivary gland obtained from a pilocarpine-treated larvae after (A) 15 min. (B) 3 hr. (C) 6 hr. and (D) 24 hr in the culture medium

96

#

*

Fig. 2. Autoradiograph showing the pattern of 3Hkdine (10 &i/ml, 8.0 Ci/mM) incorporation chromosomes and nucleoli of salivary glands pulse-labelled for 30 min after 4 days in organ culture. nucleolus. Scale line represents IO pm.

by N,

97

Fig. 3. Examples of asynchronous DNA synthesis. Autoradiographs of salivary gland chromosomes from glands pulse-labelled (30 min) with H3-methyl thymidine (10 pCi/ml, 2.0 Ci/mM) after 3 days in culture, A. continuous pattern. B, discontinuous pattern. Scale line represents 10 frm.

98

Fig. 4. Example of puff induction at chromosomal locus W-2-B. Gland placed for 1 hr in fresh mediuti supplemented with 10 pg/ml ecdysterone. line represents IO pm.

was cultured for 3 days and then Puff is indicated by arrow. Scale

glands were maintained in culture for 24 hr while control glands were examined immediately. In a second series of experiments, sister glands were separated and placed for 24 hr in normal medium (control cultures) or in medium supplemented with 5 cycloheximide (experimental cultures). Lcg,ml Following culture, the glands were rinsed (2 x) in 0.05 M phosphate buffer (pH 7.2) containing 4.59; sucroseandthendissolvedin 1 NNaOH(30min,50 C). The total protein content of the glands was determined by a modified Lowry procedure (LOWRYef al., 195 1).

Cytochemistr)

Salivary glands were fixed in a 3:l mixture of ethanol and acetic acid (10 min), stained with 2”” orcein in 5O”b acetic acid (40 min), transferred to 5096 acetic acid, and squashed under a siliconized cover slip. The slides were placed on dry ice and the cover slips removed. Slides were subsequently counterstained for 2 days in a mixture of light green FS and orange G at pH 5.0 (CLEVER, 1961). After dehydration in isopropyl alcohol and clearing in xylene, the slides were mounted with a cover slip. Chromosomul

Immediately after dissection, control salivary glands were double-pulse-labelled for 2 hr in medium containing 5 /iCi/ml 3H-uridine (sp. act. 8 Ci/mM) and 5 pCi,ml ‘JC-L-leucine (sp. act. 6 Ci/mM, Schwarz;Mann. Orangeburg. NY). Experimental cultures of sister glands were maintained for l-4 days and then pulsed in the same manner. In a second series of experiments, both experimental and control glands were maintained in culture for l-3 days. Following culture. experimental glands were placed for 6 hr in medium containing 10 pg/ml cycloheximide followed by a two hour pulse in medium containing IjC-L-leucine, 3H-Uridine and cycloheximide. Control glands were treated in the same manner except for exposure to cycloheximide. All pulses were stopped by the addition of 0.5 ml 207” trichloroacetic acid (TCA). Glands were rinsed with 7”,, TCA and then solubilized in 1 N NaOH (1 hr, 40 C). The TCA-insoluble protein and RNA were reprecipitated with 30”,, TCA and the precipitate collected on 0.3 p cellulose filters (Millipore Corp., Bedford. MA) Filters were rinsed with 7% TCA, dehydrated with 95”,, ethanol, dried with chloroform, and then placed in liquid scintillation vials containing PCS Scintillation Fluid (Amersham/Searle. Arlington Heights. IL). Samples were counted in a liquid scmtillation counter using separate scalers to record .3H and ‘-‘C emissions.

Glands were maintained in culture up to 3 days. placed for 2 hr in fresh medium containing 10 pg/ml ecdysterone (Sigma Chemical Co., St. Louis, MO) and then prepared for cytochemical analysis.

RNA und DNA synthesis

Glands were cultured for up to 4 days, pulsed for 30 min in medium containing 10 PCijml 3H,Uridine (sp. act. 8 Ci,‘mM) or 10 /tCi/ml 5 methyl-3H.thymidine (sp. act. 6 Ci/mM), rinsed in cold medium and then processed for autoradiography.

Glands were fixed, stained with acetic orcein, and squashed on slides previously coated with gelatinchrome alum. After cover slip removal and hydration, the slides were covered with Kodak AR-10 autoradiographic stripping film. dried in an air stream and exposed at 4 C in light-tight boxes containing desiccant. After a 5 or 7 day exposure period, the autoradiographs were developed with Kodak D-19 (5 min), rinsed, fixed with Kodak fixer (8 min), dehydrated to absolute ethanol, cleared in xylene, and mounted with a cover slip.

RESULTS Morphological

upprarurzcr of culturrd glands

Salivary glands maintained in the culture medium for up to 4 days appeared morphologically normal when compared with glands freshly removed from larvae. Microscopic observations showed little cytoplasmic blebbing or granulation as a result of prolonged culture. The cytoplasm remained clear and the cells were capable of excluding a 0.19,, solution 01 Trypan blue. Injured cells did not exclude Trypan blue. After 24-72 hr in the medium the gland lumen became totally filled with clear salivary product.

Table 2. In vitro synthesis of total glandular protein by salivary glands obtalned from pilocarpine-treated Chironow~uslarvae*

Exp.

Culture conditions

No. of glands

Mean pg protein per gland 3 S.E.

No culture

25

X.6 i 0.3

24 hr

25

12.7 * 0.3

24 hr + c’heximidel

45

8.6 + 0.4

24 hr

45

13.2 f 0.4

I

P wluet

Increase in protein I”,,)

< 0.01

45.5

< 0.01

41.4

culture

2

*Larvae were kept for 18-24 hr in 0.1 mg/ml pilocarpine before protein determination or culture of glands. +Pair-difference comparison. P-value from Student’s t distribution table. $ Glands were cultured in medium supplemented wth 5 icg’ml cycloheximidc.

AND

CONRAD E. FIRLING

100

BRIAN K. K~RILKA

Table 3. Capacity of Chironomussalivary glands to incorporate ‘VZ-leucine into acidinsoluble total gland protein and 3H-uridine into acid-insoluble total RNA after varying times in the culture medium Days in culture

r4C-leucine incorp. (mean “b of control)

No. of gland pairs

1 2 3 4

21 21 I8 II

ZH-Uridine incorp. (mean “(, of control) 86 42 52 46

88 45 50 35

Salivary glands were double pulse-labelled for 1 hr in media containlng 3H. uridine and 5 &i/ml “C.L-leucine. dis/min CULTURE Iln. dts/mm CONTROL Mean “,, of control = 100 x exp ~~~ N i

Protein synthesis by cultured glands

5 itCi/ml

synthesize protein but at levels equivalent to about 43% of sister unincubated control glands. The addition of 10 pg/ml cycloheximide to media containing glands previously cultured for l-4 days caused an almost total inhibition of “YY.leucine incorporation into total glandular protein (Table 4). These results suggest that the increase in the total protein content of cultured glands is due, at least in part, to de nova protein synthesis. The data do not exclude the possibility that a portion of the total protein content of these glands was protein sequestered from the foetal bovine serum component of the medium. Several investigations have demonstrated the selective uptake, transport, and secretion of haemolymph proteins by Chironomus salivary glands (DOYLE and LAUFER, 1969; SCHIN and LAUFER, 1974). Thus, the culture of glands in medium supplemented with cycloheximide could have resulted in an inhibition of the synthesis of enzymatic protein necessary for protein sequestation by the salivary glands, as well as the synthesis of secretory protein.

The treatment of intact larvae with pilocarpine results in the complete release of secretory material from the salivary gland lumen (BEERMANN, 1961; CLEVER, 1969). When secretion-free glands, obtained from pilocarpine-treated larvae, were cultured for 24 hr in the medium they exhibited a pronounced macroscopic accumulation of material within the gland lumen (Fig. 1). To determine if the increase in lumen size and contents were due to de nova protein synthesis, the uptake of protein from the medium, or an osmotic phenomenon, we examined the capacity of cultured glands to synthesize protein. Gland pairs from individual pilocarpine-treated animals were separated into control and experimental cultures. After 24 hr in normal medium the experimental glands contained 46”/0 more total protein when compared with unincubated control glands (Table 2). In a second series of experiments it was found that glands incubated for 24 hr in normal media contained 477; more total protein than sister glands cultured for the same period of time in media containing 5 pg/ml cycloheximide (Table 2). The synthesis of protein by cultured glands was further investigated by examining the capacity of glands to incorporate r4C.leucine into TCA-insoluble total gland protein after varying periods of time in the medium (Table 3). After one day in culture glands exhibited near normal levels of isotope incorporation, while glands cultured for 2, 3 or 4 days continued to

Total RNA synthesis by cultured glands The capacity of cultured glands to synthesize total RNA was studied by incorporation experiments (Table 3). Glands cultured in the medium for 1 day exhibited near normal levels of incorporation of 3H.uridine into total RNA, while glands cultured for 2. 3 or 4 days synthesized RNA at levels equivalent to 47% of paired control glands. The incorporation of the labelled RNA precursor was almost completely

Table 4. Effect of cycloheximide (10 &ml) on the synthesis of acid-msoluble protein and total RNA by Chrionomus salivary glands after varying times in the culture medium Hours in culture 6 24 72

No. of gland pairs

14C-Leucine incorp. (mean ‘” of control)

20 10 10

4.48 0.97 1.23

‘H-Uridine incorp. (mean I’,>of control) 8.98 1.57 1.93

Cycloheximide (10 mg/ml) was present in the medium 6 hr prior to and during a 2 hr double

pulse with 5 pCi/ml

Mean “/Aof control

“‘CJeucine

= 100 x exp

and 5 /,tCi/ml “H.uridine.

Chirorwmus

salivary glands I, virro

inhibited by exposing the glands to IO pg/rnl cycloheximide 6 hr prior to and during the isotope pulse (Table 4). Qualitative autoradiographic studies described below suggest that nucleolar RNA (ribosomal RNA) is the principal species of RNA synthesized by glands incubated in the medium. There is evidence that cycloheximide represses ribosomal RNA production through its inhibition of RNA polymerase I activity (HORGEN and GRIFFIN. 1971). Thus, the lack of 3H.uridine incorporation into total RNA by glands cultured in media containing cycloheximide probably reflects an inhibition of nucleolar RNA production in this system. In contrast to our findings, POELS (1972) has reported that incorporation of 3H.uridine into RNA by Drosophila salivary glands maintained in vitro is similar in media with or without cycloheximide. at least over a 4 hr incubation period. Autoradiographic DN.4 synthesis

studies of chromosomal

RNA

und

To study chromosomal RNA synthesis, glands were maintained in culture for up to 4 days, pulse-labelled in medium containing 3H.uridine, and prepared for autoradiography. The pattern of isotope incorporation by the chromosomes and nucleoli of cultured glands corresponded well with the pattern exhibited by control glands (Fig. 2). There was diffuse labelling of the chromosomes along with distinct regions of intense “H.uridine incorporation which correspond to sites of cytochemically demonstrable acidic protein. The nucleoli, associated with chromosomes II and III, showed the greatest incorporation of isotope. In viva studies by PELLING (1964) also showed that nucleolar RNA synthesis dominates chromosomal RNA synthesis along chromosomes II and III of this species. To study DNA replication, glands were cultured for up to 3 days and then pulse-labelled in medium supplemented with 3H.thymidine. The two distinct incorporation patterns characteristic of asynchronous DNA replication were observed. Isotope incorporation occurred evenly along the entire length of the chromosomes (continuous pattern, Fig. 3A) or it was restricted to discrete chromosomal sites (discontinuous pattern, Fig. 3B). Intermediate patterns of labelling were also observed. It is generally assumed that these patterns of DNA synthesis represent phases in the normal replication of chromosomal DNA. A number of investigators have reported similar patterns of asynchronous DNA synthesis along the polytene chromosomes of dipteran salivary glands (HOWARD and PLAUT, 1968; RUDKIN. 1972) but never after prolonged organ culture.

Microscopic examination of squash preparations of salivary glands maintained in the medium for up to 4 days showed that the chromosomes and nucleoli were normal in gross morphology and cytochemical appearance. Although the influence of prolonged culture upon chromosomal puff patterns was not specifically examined no gross abnormal puffing was detected. There was, however, some regression in the size of the tissue specific giant puffs or Balbiani rings present on the short fourth chromosome. Figure 4 illustrates the results of an experiment in

IOI

which salivary glands previously cultured f’or 3 days were placed for I hr in medium containing 10 keg/ml ecdysterone. The response was puff induction at chromosomal locus IV-2-B in over 70”,, of the glands examined. Puff formation was not observed at the I-l 8-C chromosomal locus. DISCUSSION Most insect culture media are formulated on the basis of the physicochemical composition of the internal body fluid or haemolymph of the insect whose tissues or organs are to be cultured. The great ecological and physiological diversity among insects makes it difficult to develop synthetic media adapted to the particular requirements of a specific species. The composition of haemolymph varies greatly between species as well as during the development of an individual species. However, a high concentration of free amino acids. which may account for up to 40”,, ot the total osmotic pressure, appears to be a common feature of most haemolymph examined (FLoRlilh and JEUNIAUX, 1974). The amino acid content of the medium formulated in this study is based upon quantitative analyses of the free amino acid pool ot fourth instar C. tentuns haemolymph (FIRLING. 1977). The medium contains 27 amino acids resulting in a total free amino acid concentration of 290 mg/ 100 ml. In many insect culture media, glucose is the onlh carbohydrate energy source. The medium employed in this study contains three carbohydrates qualitatively in C‘. taltoll.\ detected (FIRLING, unpublished) haemolymph: z-trehalose, glucose. and maltose. plus fructose and sucrose. Trehalose. generally present in high concentration in insect haemolymph, is known to be utilized by insect cells in culture (C‘LI.M~NIS and GRACE. 1967). In addition. LAUDERand NAKASL.(IY65) have reported that C. rhummi salivary glands contain high trehalase activity. The amount of glucose was increased over the low levels of this sugar found in quantitative studies of fourth instar (‘. tcrlltrn.s haemolymph (3 mg/lOO ml; FIRL.ING, unpubhshed) Fructose and sucrose are included as additional respiratory substrates since insect cells have been shown to utilize these sugars iu vitro (GHA(.I and BR~OSTOWSKI. 1966). Successful insect culture media generally retlect the concentration or relative proportion of inorganic ions present in haemolymph. The ionic content of this medium is based upon the levels of sodium. potassium. calcium, and magnesium, present in fourth instar Chironomzts haemolymph ( FIRLING. 1970: KROEc;t:R1’1 ul.. 1973; WRIGHT, 1975: SCHIN and MooRI!. 1977). The medium is characterized by a relatively high Na’/K + mole ratio of 21 and a relatively low MgZ+/CaZ.+ mole ratio of 1.4. The medium’s organic. acids, except for citrate. correspond qualitatively. but not quantitatively to those present in the medium of WYATT (1956:1. The high levels of organic acids present in the haemolymph of most insects, the occurrence of tricarboxylic acid cycle metabolism in insect larval tissues (GILMOUR, 1961). and the relatively high malic acid dehydrogenase activity in C. thummi salivary glands (LAUF~R and NAKASE. 1965) suggested that organic acids be included in the medium.

102

CONRAV E.

FIRLINC;AND BRIANK. K~BILKA

The vitamin requirements of insect cells are poorly known. Most insect culture media contain the major vitamins of the B group: biotin, choline, folic acid, nicotinamide, pathothenic acid, pyridoxine. riboflavin, thiamine and inositol. This medium contains the above water soluble vitamins, corresponding to those present in the basal medium (BME) of EAGLE (1955), plus cyanocobalamin (B,Z) and those fat soluble vitamins included as a result of supplementing the basic medium with TC-199 medium (5”)“) and FBS (27,). The addition of TC-199 and FBS also provides glutathione, ATP, purine and pyrimidine bases, lipids. serum protein plus trace quantities of cholesterol. Finally, TC-Yeastolate is included because of its trace metals, peptides, vitamins and general growth promoting properties upon insect cells and tissues in culture (JONES and CUNNINGHAM. 1961). The buffering capacity of the medium depends upon the relatively high concentration of phosphate, amino acids, organic acids and FBS protein. The low titre of bicarbonate in the medium precludes the necessity for use of a controlled gas atmosphere. The osmotic pressure of the medium, determined by freezing point depression. is 260 mOs, a value similar to the mean osmotic pressure of 223 mOs reported for fourth instar C. thunzmi haemolymph (KR~~EGERet ul.. 1973). * Primarily because of the presence of the giant polytene chromosomes in the ceils of dipteran salivary glands, a number of workers have attempted the in vitro culture of these glands. Most studies have been short-term experiments (usually less than several hours) in which the influence of different environmental conditions or chemical agents upon chromosomal puffing has been examined. However. several investigators have reported the culture of salivary glands for periods up to 24 hr or longer. CANNON (1964) developed a chemically-defined medium which supported normal puff formation and DNA synthesis along the polytene chromosomes of Sciara salivary glands after 24 hr of incubation. TULCHIN et al. (1967) observed that Drosopkilu salivary glands cultured for 24 hr in the media of CANNON (1964), GRACE (1962), or JONES and CUNNINGHAM (1961) exhibited normal chromosome morphology and the capacity for DNA synthesis. Recently, SCHIN and MOORE (1977) reported the in vitro maintenance of C. tlzunzn~i salivary glands for 48-72 hr in a basic salt medium supplemented with the growth promoting factors present in the media of Cannon, Grace, or Firling. Gland viability in the three media was measured by gland morphology and the size of the Balbiani rings and nucleoli. An important criterion for the suitability of a medium for the culture of salivary glands is the in vitro demonstration of a physiological response to an insect hormone or pharmacologic agent. CLEVERet ul. (1973) using freshly dissected C. tentans salivary glands, incubated in a modified Cannon’s medium ( RINGBORG et a/., 1970) or an early version ofthis medium, showed that two puffs associated with the moult process are induced by different ecdysone analogs, i.e. puff I-l 8-C is induced by ecdysone while puff IV-2-B is induced by ecdysterone. The induction of specific chromosomal puffs by ecdysteroids has also been demonstrated in short term in vitro experiments in the salivary glands of C. thrtmmi (KR~~EGER,1966b). Sciara (GOODMANet al..

1976) and Drosophila (BERENDES, 1967; ASHBURN~R. 1972; POELS, 1972) but never after prolonged culture. In this study, we have demonstrated that C. tmrum salivary glands maintained in organ culture for up to 3 days have the capacity to respond to ecdysterone with the formation of a puff at the IV-2-B chromosomal locus (Fig. 4). In addition, glands incubated in the medium retain normal chromosomal. cellular. and glandular morphology. and, incorporate radioactive precursors into DNA. RNA, and total gland protein. This medium should be suitable for the investigation of such processes as: DNA replication. RNA synthesis and metabolism. polytene chromosome structure and its relationship with salivary gland function and differentiation, and the influence of hormones and other physiological agents upon the above phenomena in the controlled conditions of in vitro organ culture. Currently, the medium is being utilized in a study of the intluence of ecdysterone upon the ultrastructural morphology of C. tentans salivary glands maintained in vitro.

REFERENCES ASHBURNER M. (1972)

Patterns of puffing activity m the salivary gland chromosomes of DrusophikVI. Induction by ecdysone in salivary glands of D. melunogastrr cultured 38, 255-28 I. Ein Balbiani-Ring als Locus einer Speicheldriisenmutation. Chromowmu 12. l-25.

in vitro. Chromosoma BEERMANN W. (1961)

BERENDESH. D. (I 967) The hormone specific changes in the pattern Drosophila

hydei.

Chromosoma

ecdysone as effector of of gene activities of

22, 274-293.

CANNON G. B. (1964) Culture of insect salivary glands in a chemically defined medium. Scienw, Wash 146, 1063. CLEMENTSA. N. and GRACE T. D. C. (1967) The utilization of sugars by insect cells in culture. .I htvect Ph!~vio/. 13, 1327-1332. CLEVER U. (1961) GenaktivitPten in den Riesenchromosomen von Chironomus tentans and ihre Beziehungen zur Entwicklung--I. Genaktivierungen durch Ecdysonc. Chromosomu

12, 607-675.

CLEVER U. (1969) Chromosome activity and cell function in polytenic cellsPIt. The formation of secretion in the salivary glands of Chironomus. E.upl. Cell Res. 55.3 17-322. CLEVER U., CLEVER I.. STORBECK I. and YOUNC; N. L. (1973) The apparent requirement of two hormones. z-and /jecdysone. for molting induction in insects. Ded. Bid. 31, 47-60. DOYLE D. and LAUFER H. (1969) Sources of larval salivary gland secretion in the dipterdn Chirononlus ren/an.r. J. Cc,// Biol. 40. 61-78. EAOLE H. (1955) Nutrition needs of mammalian cells in tihsuc culture. S&rtcr. Wd. 122. 501-504. FIRLINC; C. E. (1970) The sodium, potassium and calcium concentration of 4th instar Ckironottnc.v IMIWI.\ haemolymph. Am Zoo/. 10, 313. FIRLING C. E. (1977) Amino acid and protein changes in the haemolymph of developing fourth instar Chironomuv tentans.. J.-Insect Phvsioi. 23r 17-22. FIRLING C. E. and K&LKA B. K. (1976) Functional activitv of Chironomus salivary glands maintained in organ culture. Am. Zool. 16, 204. FLORKIN M. and JEUNIAUX C. (1974) Hemolymph composition. In T/W Physiology 01 Insec,/a (Ed. bq RO~KSTEINM.) 5, pp. 255-307. Academic Press. New

York. GILMOUR D. (1961) The Biochemistry

Academic Press. New York.

oJ’Insecrs.

pp. 58-85.

Chironomus salivary R. M.. SCHMIDT E. C. and BENJAMIN W. 9. (1976) The effect of ecdysterone on nonhistone proteins in salivary gland chromosomes of Sciara coprophila. Cell D(ff. 5. 233-246. GRACE T. D. C. (1962) Establishment of four strains of cells from insect tissues grown in vifro. Nafure, Land. 195, 788-789. GRACE T. D. C. and BRZOSTOWSKIH. W. (1966) Analysis of the amino acids and sugars in an insect cell culture medium during cell growth. J. Insect Physiol. 12. 625-633. HORC;ENP. A. and GRIFFIN D. H. (1971) Specific inhibitors of the three RNA polymerases from the aquatic fungus Blastoc~ladiellaemersonii. Proc. Nat. Acad. Sri.. U.S.A. 68, 338-341. HOWARD E. F. and PLAUT W. (1968) Chromosomal DNA synthesis in Drosophila melanogaster. J. Cell. Biol. 39, 4 15-429. JONES B. M. and CUNNINGHAM J. (1961) Growth by cell division in insect tissue culture. Expl. Cell Res. 23, 386-401. KR&C;ER H. (1966a) Micrurgy on cells with polytene chromosomes. In Methods in Cell Phvsiology (Ed. by PRESCOTTD. M.)Z, pp. 61-92. Academic Press, New York. KR~~EGER H. (1966b) Potentialdifferenz und Puff-Muster. Elektrophysiologische und cytologische Untersuchungen an den Speicheldrusen von Chironomus thummi. Erpl. Cell Rcs. 41, 64-80. KR~~EC;ERH.. TR&CH W. and MUELLERG. (1973) Changes in nuclear electrolytes of Chironomus thummi salivary gland cells during development. Expl. Cell Res. 80, 32%339. LA~IFER H. and NAKASE Y. (1965) Salivary gland secretion and Its relation to chromosomal puffing in the dipteran ~‘hironomus rhummi. Proc. Nat. Acad. Sri.. U.S.A. 53, 511-516. LOWRY 0.. ROSEROU(;H N., FARR A. and RANDALL R. (1951) GOODMAN

glands

in l~i!ro

103

Protein measurement with the Folin phenol reagent. J hiol. Chem 193, 365-275. PELLING C. (1964) Ribonukleinsaure-Synthese der Riesenchromosomen. Autoradiographische Untersuchungen an Chironomus tentans Chromosoma 15, 71-112. POELS C. L. M. (1972) Mucopolysaccharide secretion from Drosophila salivary gland cells as a consequence of hormone induced gene activity. Cell D$f: I, 63-78. RINGBORG U.. DANEHOLT B.. EDS~R~~MJ. E.. EC;YH~ZIII E. and RYDLANDER L. (1970) Evidence for transport ot preribosomal RNA from nucleolus to the chromosomes in Chironomus tenrans salivary gland cells. J. motet Biol. 51, 679-686. RUDKIN G. T. f 1972) Replication in polytene chromosomes. In Drwlopmental Studies on Giant Chromosome.\ (Ed. by BEERMANNW.) pp. 58-85. Springer. New York. SCHIN K and LAUFER H. (1974) Uptaken of homologous haemolymph protein by salivary glands of C’hrronomus thummi. J. Insect Ph.vsiol. 20, 405-411. SCHIN K. and Moo~t R. D. (1977) Cation concentrations m the haemolymph of the fly Cttironomus thumm~ during development. J. Insecr PtrM~l. 23, 723-729. TULCHIN N.. MATEYKO G. M. and KOPAC M. J. 11967) Drosophila salivary glands m vitro. J Cell Biot. 34, 891-897. VAUGHN J. L. (1971) Cell culture media and methods. In In?er/c+ra,c Tiy.sul~ C‘ul/urc+(Ed. b> V..\C;CI(’ 1 \‘c~l.I. pp. 340. Academic Press. New York. WRIGHT D. A. (1975) Sodium regulation In the larvae of Chironomus doorsalk (Meig.) and (hnlpr~~~~trin,t~onlzr.s tentans (Fabr.): the effect of salt depletion and some observations on temperature changes. J (2x-p Rio/ 62, 121-l 39. WYATT S. S. (1956) Culture in vifro of tissue from the silkworm. Bon~b~,\ mrwi L .I ,gcrl Ph~.ri~~/39. X-II-X52