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Uptake of homologous haemolymph protein by salivary glands of Chironomus thummi
r. InsectPhysiol.,1974, Vol. 20, pp. 405 to 411. PergamonPms. Printed in Great Britain
UPTAKE OF HOMOLOGOUS HAEMOLYMPH BY SALIVARY GLANDS OF CHIRONOMUS KISSU
SCHIN
and HANS
PROTEIN THUMMI
LAUFER
Department of Biological Sciences, State University of New York, College of Arts and Science, Plattsburgh, New York 12901; and Biological Sciences Group, University of Connecticut, Storrs, Connecticut 06268, U.S.A. (Received 16 July 1973) Abstract-Radioisotopically labelled homologous protein fractions were injected into the haemolymph of Chtbtontus larvae. Among four different protein fractions, fraction II appears to enter the salivary gland and accumulates in the glandular lumen. Lack of blood pigmentation in the gland and secretion lumen indicates that the haemoglobin is probably not secreted, but a portion of the haemoglobin molecule or a fraction co-precipitating with haemoglobin in fraction II is being transported by the gland. Since only a portion of fraction II is taken up and secreted, the specificity of the process suggests to us a molecular basis for the activity which must ultimately be reflected in the genes of the transporting tissue. INTRODUCTION THE FUNCTION of
the salivary glands of the fly, Chironomus thummi, is the production of secretory proteins that are used as cementing substances to bond particles together to form the larval tube and the pupal cocoon. The evidence indicates that the production of the secretion is under the control of the genome (MECHELKE, 1953; BEERMAN, 1961; GROSSBACH, 1969; DOYLE and LAUFER, 1969a, b). Electrophoretic, enzymatic, and antigenic analysis of secretory products and intracellular proteins and haemolymph showed that certain constituents of the secretion were indistinguishable from some haemolymph proteins (DOYLE and LAUFER,1969a, b; LAUFERand NAKASE, 1965) which suggested that among the functions of the gland cells was the transport of proteins from the haemolymph to the lumen of the gland. In experiments involving injection of the exogenous protein, ferritin, we found that injected protein rapidly entered the lysosomal compartment of glandular cells (SCHIN and CLEVER,1968). Using this procedure we were not able to observe any transport activity of the gland. Both KLOJZTZEL and LAUFER(1970) and DOYLEand LAUFER(1969a, b) used isotopic labels either in conjunction with or without the EM and found evidence in support of transport of blood protein into the gland lumen. In the studies reported here we used fractions of Chironomus blood proteins, labelled with Is11 or lzsI or 3H, to determine whether gland cells are involved in the uptake and transport of specific fractions of homologous proteins. The
406
KISSUSCHINANDHANSLAUFER
experimental conditions used were as close to physiological as possible, in order not to initiate novel processes. The experiments determined whether the transport function was directed toward their own blood components. MATERIALS AND METHODS Animals Chironomus thummi larvae were reared in plastic tanks at 20°C in aerated water on a diet of nettle powder (LAUFERand WILSON, 1970). They were staged according to their external appearance as follows: mid-fourth instar larvae, late fifth instar larvae, early and late pharate pupae (LAUFERand NAKASE,1965).
Procedure for radioiodination of larval blood protein Haemolymph collected from mid- and late fourth instar larvae was centrifuged at 100,000 g. The supernatant was decanted and passed through a S-29 x 42 column of Sephadex G-100 medium (Pharmacia, Uppsala, Sweden) which had been preequilibrated with borate buffer (pH 8). The fractions were collected in 40 to 50 5 ml test-tubes. Two major protein fractions and two less prominent fractions were among those proteins which were individually subjected to the radioiodination. The approximate molecular weight of these protein fractions was determined by the use of Squire’s formula (SQUIRE, 1964), or with the aid of protein markers (Manns Res. Lab., Inc.). The protein samples were designated by the following labels on the basis of molecular weight and the colour of the solutions as follows: I, yellow proteins (mol. wt.: > 67,000); II, haemoglobin containing mixture (mol. wt.: > 25,000- < 45,000); III, unpigmented proteins of moderate size (mol. wt.: <25,000); and IV, unpigmented low molecular size proteins (mol. wt.: ca. 12,400). These proteins were iodinated with carrier-free r311 in the presence of iodine monochloride (HELMKAMPet al., 1960). The amount of i311used was 50 PCi for 0.25 mg of fraction I, 50 $Zi for O-4 mg of fraction II, 100 $Zi for 0.75 mg of fraction III, and 100 &i for 1.0 mg of fraction IV. Radioactivity was measured in an aliquot taken from each preparation and the remainder was passed through Sephadex G-25 to remove free iodine. Protein samples emerging from the gel contained only labelled proteins which could be precipitated with 10% TCA. The protein solutions were rapidly lyophilized and dissolved in 0.154 M NaCl to a desired volume. The radioactivity of each sample was: I, 4.9 x lo4 counts/min per ~1; II, 7.5 x lo4 counts/min per ~1; III, 3.7 x lo4 counts/min per ~1; and IV, 2.7 x lo3 counts/min per ~1. The major protein fraction II, containing haemoglobin, was subjected to acrylamide gel electrophoresis to determine whether or not these proteins were denatured. The haemoglobin which was eluted through Sephadex gel in the presence of borate buffer (pH 8.0) sh owed all major bands which were indistinguishable from those of fresh Chironomus haemoglobin (SCHIN et al., in press, 1973).
UPTAKE OF BLOOD PROTEIN BY INSECT SALIVARY GLANDS
407
Tritiation of larval blood protein 3H-amino acids mixture (1~1) (Schwartz Biochem. Inc., sp. act.: 05 mCi/ml) was injected into early fourth instar larvae. After 18 hr or longer haemolymph was collected from each larva, pooled, and centrifuged at 100,000 g. The supernatant was filtered through a Sephadex gel-25 column in the presence of ammonium bicarbonate buffer (pH 8.2) and then lyophilized. Lyophilized protein, which had a molecular weight greater than 5000 and was freed of amino acids, was resuspended in 0.3 ml of 0.154 M NaCl. Th e activity of the protein fraction after dissolving in the NaCl solution was 6.1 x lo5 counts/min per ml. Injection of labelled homologousprotein 3H-labelled whole blood protein solution (1~1) or 1~1 of 1311-blood protein fraction or 1251-blood protein (for autoradiography) was injected into the haemolymph through the proleg of late fourth instar larvae and of mid- and late pharate pupae of C. thummi. At intervals of 15 min, 30 min, 1 hr, 4 hr, and 18 hr after injection the salivary glands were dissected out and thoroughly rinsed in 0,154 M NaCl solution. They were then prepared for either autoradiography or for scintillation counting. For determination of radioactivity glands were washed with 10% TCA containing an excess of non-radioactive amino acids and measured for the radioactivity using Bray’s solution or Liquiflour in a Packard liquid scintillation counter. For autoradiography, glands were fixed with 1 per cent OsO,-collidine buffer (pH 7.2) for 1 h r and dehydrated in an alcohol series. After embedding in D.E.R. embedding mixture (WINBORN, 1965), 2 pm thick sections covered with NTB-3 (Kodak) were processed for photography after a 1 week exposure. RESULTS
Radioactivity in the salivary glands The loss of radioactivity from the haemolymph after injection of homologous blood protein-aH was characteristically accompanied by an increase in glandular radioactivity. During the first 15 min the haemolymph retained most of the injected radioactivity and few counts/min were recorded from salivary glands (Table 1). Glands dissected out 1 hr after the injection showed increased uptake, and radioactivity in the gland continued to increase during successive periods (4 hr) when the labelling leveled off. Salivary glands after injection of homologous blood protein-1311 1~1 of each of the four different classes of iaiI-blood protein was injected into the old fourth instar larvae, mid- and late pharate pupae. As shown in Table 2 the salivary glands were almost impermeable to proteins I, III, and IV. The small amount of radioactivity detected was not much more than background in many instances (background was about 20-25 counts/min). The length of time following the injection had little or no detectable effect on the uptake of these proteins by the gland. In contrast, glands exposed to iaiI-fraction II displayed significant amounts
KIWJ
408 TABLE
Specimen collected after :
~--RADIOACTIVITY
SCHIN
AND
HANS LAUFER
RECOWRED IN SALIVARY GLANDS AFTER INJECTION
Radioactivity recovered in blood (counts/mm per ml of blood protein)
Radioactivity recovered in salivary glands (counts/min per mg of salivary gland protein)
No. of determinations
Mean
S.E.
No. of determinations
Mean
SE.
9 10 10 10
5289 3268 2680 2670
317.8 170.8 224.1 136.9
10 5 15 9
44 400 692 654
6.7 24.4 41.2 22.3
1.5 min 1 hr 4l-u 18hr
Each larva obtained 1~1 of homologous haemolymph SH-protein (sp. act.: 6.1 x lo6 counts/n-&r per ml). Age of animal : late fourth instar and young pharate pupa. For each determination at least 5 larvae were used. TABLJZ 2-~1o~cmvIT~
(counts/m.in)
lSII-Haemo-
Radioactivity recovered in salivary glands (counts/n& pairs of salivary glands) after :
lymph
protein fractions injected Fraction Fraction Fraction Fraction
I II III IV
No. of 15min determinations Mean SE. 12 10 12 12
39.2 30.0 25.0 19.2
14.3 13.0 10.0 3.3
RECOVERED IN SALIVARY GLANDS AF-IXR INJECTION
1 hr Mean 25.8 165.7 35.0 45.0
S.E. 7.9 14.2 11.5 13.7
4 hr Mean 66.7 415.8 78.7 35.6
S.E. 30.0 67.4 29.7 14.4
per 10
18 hr Mean
270.8
S.E.
19.0
Each larva was injected with 1 ~1 of the following protein fractions : fraction 1: mol. wt. >67,000, sp. act., 4.9 x lo* counts/min per ~1; fraction 2: mol. wt. >25,000-~45,000, sp. act., 7.5 x lo4 counts/mm per ~1; fraction 3: mol. wt. <25,000, sp. act., 3.7 x lo4 counts/mm per ~1; fraction 4: mol. wt. cu. 12,000, sp. act., 2.7 x lOa counts/min per ~1.
of radioactivity (eight times background on the average), though the radioactivity in individual salivary glands varied considerably. The highest radioactivity values were obtained from glands removed at 4 hr after injection of fraction II. Autoradiography of glands dissected out 15 min after the injection of la51labelled fraction II revealed little uptake of protein; however, after 1 hr the number of grains in the cytoplasm had increased. Little radioactivity was noted in the glandular lumen. Glands dissected out 4 hr after injection of fraction II displayed more grains in the lumen of the gland than in their cells. These glands did not show the haemoglobin pigmentation of the fraction from which the radioactivity was derived, despite the accumulation of radioactivity. In contrast to the results obtained with fraction II, none of the glands exposed to radioiodinated protein
UPTAKE OFBLOOD
PROTEINBY
INSECTSALIVARY
409
GLANDS
fractions I, III, or IV showed grains in their cells or glandular lumen. These autoradiographic experiments are consistent with the observations obtained with the scintillation counter, above. The data suggest to us that haemoglobin or a derivative, or another constituent of fraction II, are the only blood constituent which are taken up by the gland specifically and preferentially, and released as part of the secretion into the glandular lumen. Control experiments involving the other fractions or the injection of pepsin-treated fraction IIJslI or free 1311showed that these fragments do not gain entry into the salivary gland cell (Table 3). TABLE%--RADIOACTIVITYRBCOVBREDINSALIVARYGLANDS 4hr AFTERINJECTION
(counts/minperlOpairsofglands),
Radioactivity recovered in salivary glands (counts/mm per 10 pairs of glands 4 hr after injection of: Pepsin-treated fraction IIJ311 (activity: 4.4 x lo4 counts/min per
Free iodinenl (activity: 9.1 x 104/$)
P)
No. of determinations
Mean
S.E.
Mean
10
47.0
15.9
56.0
S.E. 29.9
Each larva was injected either with 1 ~1 of pepsin-treated fraction IIJ311 or free iodine131. Prior to injection, 0.04 ml of 0.154 M NaCl solution containing 0.01 mg of pepsin (Sigma) was added to 0.1 ml (0.5 mg) of fraction II-13r (activity: approximately 6.2 x lo4 counts/mm per ~1). The mixture was then kept at 37°C for 30 min before it was injected into the larva.
Radioactivity in the secretion
Determinations of the amount of radioactivity in the culture medium and in the secretion released from larvae show that both the culture medium and glandular secretion contain radioactivity. In attempts to determine the amount of radioactivity in secretory products, injected larvae were transferred to fresh scintillation vials at intervals of 10 min for the first hour following the injection. Intestinal excretion of injected material no longer appeared after approximately 40 min from the time of injection. At 4 and 8 hr after this action the secretions were collected, thoroughly rinsed in 0.154 M NaCl solution, and measured for radioactivity. The radioactivity in the salivary gland secretions after 8 hr was approximately twice as much as after 4 hr. Despite the low counts/min in the salivary secretion, we found the experiments repeatable and consistent and thus we feel that they are reliable. DISCUSSION It is clear that foreign horse radish peroxidase gland cell (SCHIN 14
and
proteins
such as human
serum
albumin,
ferritin,
and
can enter the salivary gland and gain access to the salivary CLEVER,
1968). Our present data test whether constituents
410
K~ssu SCHIN
AND
HANSLAUFES
of the organism’s own haemolymph proteins enter the salivary gland cell. The small amount of radioactivity detected was not above background for fractions I, III, and IV tested. The data do show that one or more constituents of fraction II which contains the blood pigment, haemoglobin is the major, and probably the only fraction, from which components enter the gland cell and are secreted into the gland lumen. This was demonstrable by two independent assay procedures, autoradiography and scintillation spectrometry. The results were consistent with both procedures. The size-class of constituents of fraction II may play a role in determining which components are transported by the gland. It has been reported that DNase and other proteins found in salivary gland cells and secretions have a striking similarity to those of blood in their immunological properties. This was interpreted as a transport of these proteins from the blood into the glandular lumen (LAUFER,1965 ; LAUFERand NAKASE,1965). Subsequent experiments involving the use of an exogeneous protein, ferritin, showed that this protein entered lysosomes of salivary gland cells (SCHIN and CLEVER,1968). The rapid appearance of exogenous protein within the lysosomes was interpreted to mean that some proteins may be degraded within the lysosomes rather than transported intact into the glandular lumen. Our results showed that despite the occurrence of radioactivity in the cell and secretion products, the majority of the glands investigated did not reveal blood pigmentation in the secretion. In the light of the fact that the salivary gland possesses at least two types of haemoglobin proteases (pH 3.5 and S-S), it is suggested that if the proteins taken up by the glandular cell are haemoglobins then they may be digested before being secreted into the glandular lumen (RODEMSet al., 1969; HENRIKSONand CLEVER,1972). However the alternative, that other proteins of fraction II or part of haemoglobin such as globin without haeme, may be transported to the glandular lumen seems likely in the light of our present results. The salivary gland cells synthesize much of their proteins under the control of their own genome (BEERMANN,1961; DOYLE and LAUFER,1969a, b). The appearance of chromosomal puffing in these cells has been considered to reflect differential synthesis of RNA, possibly m-RNA, which in turn may affect the constituents of glandular proteins. This view has been amplified and expanded in accord with the experimental observations made by LAUFERand NAKA~E(1965) because some of the secretory proteins show immunological properties similar to those of blood proteins, and the suggestion was made that the gland may be functioning sa a transport organ (LAUFER, 1965; LAUFERand NAKASE,1965). Our experiments in which Chironomus’ own blood proteins were used, indicate that the glands are indeed involved in the rather selective uptake of blood proteins, constituents of fraction II, but they do not indicate what proportion of the protein taken up by the cell is transported intact and how much is in part degraded in its passage into the gland lumen. The occurrence of the radioactivity in the gland lumen is most likely to be the results of uptake of constituents of fraction II by the gland, an activity which the cell must regulate ultimately with active genes, and perhaps ‘puff’ loci still to be identified.
UPTAKEOF BLOODPROTEINBY INSECTSALIVARY GLANDS
411
Acknowle&ements-The authors are indebted to Dr. RALPH CLARK at SUNY, Plattsburgh, for his kind criticism and advice in the preparation of the manuscript. Thanks are due also to Dr. T. HOLT for his helpful discussion and laboratory assistance. This work was supported by grants from the NSF, the University of Connecticut Research Foundation, and SUNY Research Foundation. REFERENCES BEERMAN W. (1961) Ein Balbiani-Ring als Locus einer Speicheldruesen-Mutation. Chromosoma 12, l-25. DOYLE D. and LAUFJXRH. (1969a) Sources of larval salivary gland secretion in the dipteran Chironomus tentans. J. Cell Biol. 40, 61-78. DOYLE D. and LAUFER H. (1969b) Requirements of ribonucleic acid synthesis for the formation of salivary gland specific proteins in the larvae Chironomus tentans. Exp. Cell Res. 57, 205-210. GROSSBACH U. (1969) Chromosomen-Aktivitaet und biologische Zelldifferenzierung in den Speicheldruesen von Campto chironomus. Chromosome 28,136-187. HELMKAMPR. W., GOODLAND R. L., BALE W. F., SPAR I. L., and MUTSCHLERL. E. (1960) High specific activity iodination of -globulin with iodine-131 monochloride. Cancer Res. 20,149.5-l 500. HENRIKSONP. A. and CLEVERU. (1972) Protease activity and cell death during metamorphosis in the salivary gland of Chironomus tentans. J. Insect Physiol. 18, 1981-2004. KLOETZELJ. and LAUFERH. (1970) Developmental changes in fme structure associated with secretion in larval salivary gland of Chironomus. Exp. Cell Res. 60,327-337. LAUFERH. (1965) Developmental studies of the dipteran salivary gland-III. Relationships between chromosomal puffing and cellular function during development. In Developmental and Metabolic Control Mechanisms and Neoplasia, 19th Ann. M. D. Anderson Symp. on Fundamental Cancer Res., University of Texas, M. D. Anderson Hospital and Tumor Institute, pp. 237-250. LAUFER H. and NAKASEY. (1965) Salivary gland secretion and its relation to chromosomal puffing in the dipteran, Chironomus thummi. Proc. nut. Acad. Sci. U.S.A. 53, 511-516. LAUFERH. and WILSON M. (1970) Hormonal control of gene activity as revealed by puffing of salivary gland chromosomes in dipteran larvae, in laboratory experiments. In General and Comparative Endocrinology (Ed. by PETER R. E. and GORBMANA.), pp. 185-200. Prentice-Hall, Englewood Clifts, N. J. MECHELKEF. (1953) Reversible Strukturmodificationen der Speicheldruesen-Chromosomen von Acricotopus lucidus. Chromosoma 5, 511-543. RODEMSA. E., HENRIKSONP. A., and CLEVERU. (1969) Proteolytic enzymes in the salivary gland of Chironomus tentans. Experientia 25, 686-687. SCHIN K. S. and CLEVER U. (1968) Ultrastructural and cytochemical studies of salivary gland regression in Chironomus tentans. Z. Zellforsch. micr. Anat. 86, 262-279. SCHIN K., POLUHOWICH J. J., GAMO T., and LAUFERH. (1973) Degradation of haemoglobin in Chironomus during metamorphosis. J. Insect Physiol. In press. SQUIREP. G. (1964) A relationship between the molecular weights of macromolecules and their elution volumes based on a model for Sephadex gel filtration. Archs Biochem. Biophys. 107, 471-478. WINBORN W. (1965) Dow epoxy resin with triallyl cyanurate, and similarly modified Araldite and Maraglas mixtures, as embedding media for electron microscopy. Stain Tech. 40,227-231.
UPTAKE OF HOMOLOGOUS HAEMOLYMPH BY SALIVARY GLANDS OF CHIRONOMUS KISSU
SCHIN
and HANS
PROTEIN THUMMI
LAUFER
Department of Biological Sciences, State University of New York, College of Arts and Science, Plattsburgh, New York 12901; and Biological Sciences Group, University of Connecticut, Storrs, Connecticut 06268, U.S.A. (Received 16 July 1973) Abstract-Radioisotopically labelled homologous protein fractions were injected into the haemolymph of Chtbtontus larvae. Among four different protein fractions, fraction II appears to enter the salivary gland and accumulates in the glandular lumen. Lack of blood pigmentation in the gland and secretion lumen indicates that the haemoglobin is probably not secreted, but a portion of the haemoglobin molecule or a fraction co-precipitating with haemoglobin in fraction II is being transported by the gland. Since only a portion of fraction II is taken up and secreted, the specificity of the process suggests to us a molecular basis for the activity which must ultimately be reflected in the genes of the transporting tissue. INTRODUCTION THE FUNCTION of
the salivary glands of the fly, Chironomus thummi, is the production of secretory proteins that are used as cementing substances to bond particles together to form the larval tube and the pupal cocoon. The evidence indicates that the production of the secretion is under the control of the genome (MECHELKE, 1953; BEERMAN, 1961; GROSSBACH, 1969; DOYLE and LAUFER, 1969a, b). Electrophoretic, enzymatic, and antigenic analysis of secretory products and intracellular proteins and haemolymph showed that certain constituents of the secretion were indistinguishable from some haemolymph proteins (DOYLE and LAUFER,1969a, b; LAUFERand NAKASE, 1965) which suggested that among the functions of the gland cells was the transport of proteins from the haemolymph to the lumen of the gland. In experiments involving injection of the exogenous protein, ferritin, we found that injected protein rapidly entered the lysosomal compartment of glandular cells (SCHIN and CLEVER,1968). Using this procedure we were not able to observe any transport activity of the gland. Both KLOJZTZEL and LAUFER(1970) and DOYLEand LAUFER(1969a, b) used isotopic labels either in conjunction with or without the EM and found evidence in support of transport of blood protein into the gland lumen. In the studies reported here we used fractions of Chironomus blood proteins, labelled with Is11 or lzsI or 3H, to determine whether gland cells are involved in the uptake and transport of specific fractions of homologous proteins. The
406
KISSUSCHINANDHANSLAUFER
experimental conditions used were as close to physiological as possible, in order not to initiate novel processes. The experiments determined whether the transport function was directed toward their own blood components. MATERIALS AND METHODS Animals Chironomus thummi larvae were reared in plastic tanks at 20°C in aerated water on a diet of nettle powder (LAUFERand WILSON, 1970). They were staged according to their external appearance as follows: mid-fourth instar larvae, late fifth instar larvae, early and late pharate pupae (LAUFERand NAKASE,1965).
Procedure for radioiodination of larval blood protein Haemolymph collected from mid- and late fourth instar larvae was centrifuged at 100,000 g. The supernatant was decanted and passed through a S-29 x 42 column of Sephadex G-100 medium (Pharmacia, Uppsala, Sweden) which had been preequilibrated with borate buffer (pH 8). The fractions were collected in 40 to 50 5 ml test-tubes. Two major protein fractions and two less prominent fractions were among those proteins which were individually subjected to the radioiodination. The approximate molecular weight of these protein fractions was determined by the use of Squire’s formula (SQUIRE, 1964), or with the aid of protein markers (Manns Res. Lab., Inc.). The protein samples were designated by the following labels on the basis of molecular weight and the colour of the solutions as follows: I, yellow proteins (mol. wt.: > 67,000); II, haemoglobin containing mixture (mol. wt.: > 25,000- < 45,000); III, unpigmented proteins of moderate size (mol. wt.: <25,000); and IV, unpigmented low molecular size proteins (mol. wt.: ca. 12,400). These proteins were iodinated with carrier-free r311 in the presence of iodine monochloride (HELMKAMPet al., 1960). The amount of i311used was 50 PCi for 0.25 mg of fraction I, 50 $Zi for O-4 mg of fraction II, 100 $Zi for 0.75 mg of fraction III, and 100 &i for 1.0 mg of fraction IV. Radioactivity was measured in an aliquot taken from each preparation and the remainder was passed through Sephadex G-25 to remove free iodine. Protein samples emerging from the gel contained only labelled proteins which could be precipitated with 10% TCA. The protein solutions were rapidly lyophilized and dissolved in 0.154 M NaCl to a desired volume. The radioactivity of each sample was: I, 4.9 x lo4 counts/min per ~1; II, 7.5 x lo4 counts/min per ~1; III, 3.7 x lo4 counts/min per ~1; and IV, 2.7 x lo3 counts/min per ~1. The major protein fraction II, containing haemoglobin, was subjected to acrylamide gel electrophoresis to determine whether or not these proteins were denatured. The haemoglobin which was eluted through Sephadex gel in the presence of borate buffer (pH 8.0) sh owed all major bands which were indistinguishable from those of fresh Chironomus haemoglobin (SCHIN et al., in press, 1973).
UPTAKE OF BLOOD PROTEIN BY INSECT SALIVARY GLANDS
407
Tritiation of larval blood protein 3H-amino acids mixture (1~1) (Schwartz Biochem. Inc., sp. act.: 05 mCi/ml) was injected into early fourth instar larvae. After 18 hr or longer haemolymph was collected from each larva, pooled, and centrifuged at 100,000 g. The supernatant was filtered through a Sephadex gel-25 column in the presence of ammonium bicarbonate buffer (pH 8.2) and then lyophilized. Lyophilized protein, which had a molecular weight greater than 5000 and was freed of amino acids, was resuspended in 0.3 ml of 0.154 M NaCl. Th e activity of the protein fraction after dissolving in the NaCl solution was 6.1 x lo5 counts/min per ml. Injection of labelled homologousprotein 3H-labelled whole blood protein solution (1~1) or 1~1 of 1311-blood protein fraction or 1251-blood protein (for autoradiography) was injected into the haemolymph through the proleg of late fourth instar larvae and of mid- and late pharate pupae of C. thummi. At intervals of 15 min, 30 min, 1 hr, 4 hr, and 18 hr after injection the salivary glands were dissected out and thoroughly rinsed in 0,154 M NaCl solution. They were then prepared for either autoradiography or for scintillation counting. For determination of radioactivity glands were washed with 10% TCA containing an excess of non-radioactive amino acids and measured for the radioactivity using Bray’s solution or Liquiflour in a Packard liquid scintillation counter. For autoradiography, glands were fixed with 1 per cent OsO,-collidine buffer (pH 7.2) for 1 h r and dehydrated in an alcohol series. After embedding in D.E.R. embedding mixture (WINBORN, 1965), 2 pm thick sections covered with NTB-3 (Kodak) were processed for photography after a 1 week exposure. RESULTS
Radioactivity in the salivary glands The loss of radioactivity from the haemolymph after injection of homologous blood protein-aH was characteristically accompanied by an increase in glandular radioactivity. During the first 15 min the haemolymph retained most of the injected radioactivity and few counts/min were recorded from salivary glands (Table 1). Glands dissected out 1 hr after the injection showed increased uptake, and radioactivity in the gland continued to increase during successive periods (4 hr) when the labelling leveled off. Salivary glands after injection of homologous blood protein-1311 1~1 of each of the four different classes of iaiI-blood protein was injected into the old fourth instar larvae, mid- and late pharate pupae. As shown in Table 2 the salivary glands were almost impermeable to proteins I, III, and IV. The small amount of radioactivity detected was not much more than background in many instances (background was about 20-25 counts/min). The length of time following the injection had little or no detectable effect on the uptake of these proteins by the gland. In contrast, glands exposed to iaiI-fraction II displayed significant amounts
KIWJ
408 TABLE
Specimen collected after :
~--RADIOACTIVITY
SCHIN
AND
HANS LAUFER
RECOWRED IN SALIVARY GLANDS AFTER INJECTION
Radioactivity recovered in blood (counts/mm per ml of blood protein)
Radioactivity recovered in salivary glands (counts/min per mg of salivary gland protein)
No. of determinations
Mean
S.E.
No. of determinations
Mean
SE.
9 10 10 10
5289 3268 2680 2670
317.8 170.8 224.1 136.9
10 5 15 9
44 400 692 654
6.7 24.4 41.2 22.3
1.5 min 1 hr 4l-u 18hr
Each larva obtained 1~1 of homologous haemolymph SH-protein (sp. act.: 6.1 x lo6 counts/n-&r per ml). Age of animal : late fourth instar and young pharate pupa. For each determination at least 5 larvae were used. TABLJZ 2-~1o~cmvIT~
(counts/m.in)
lSII-Haemo-
Radioactivity recovered in salivary glands (counts/n& pairs of salivary glands) after :
lymph
protein fractions injected Fraction Fraction Fraction Fraction
I II III IV
No. of 15min determinations Mean SE. 12 10 12 12
39.2 30.0 25.0 19.2
14.3 13.0 10.0 3.3
RECOVERED IN SALIVARY GLANDS AF-IXR INJECTION
1 hr Mean 25.8 165.7 35.0 45.0
S.E. 7.9 14.2 11.5 13.7
4 hr Mean 66.7 415.8 78.7 35.6
S.E. 30.0 67.4 29.7 14.4
per 10
18 hr Mean
270.8
S.E.
19.0
Each larva was injected with 1 ~1 of the following protein fractions : fraction 1: mol. wt. >67,000, sp. act., 4.9 x lo* counts/min per ~1; fraction 2: mol. wt. >25,000-~45,000, sp. act., 7.5 x lo4 counts/mm per ~1; fraction 3: mol. wt. <25,000, sp. act., 3.7 x lo4 counts/mm per ~1; fraction 4: mol. wt. cu. 12,000, sp. act., 2.7 x lOa counts/min per ~1.
of radioactivity (eight times background on the average), though the radioactivity in individual salivary glands varied considerably. The highest radioactivity values were obtained from glands removed at 4 hr after injection of fraction II. Autoradiography of glands dissected out 15 min after the injection of la51labelled fraction II revealed little uptake of protein; however, after 1 hr the number of grains in the cytoplasm had increased. Little radioactivity was noted in the glandular lumen. Glands dissected out 4 hr after injection of fraction II displayed more grains in the lumen of the gland than in their cells. These glands did not show the haemoglobin pigmentation of the fraction from which the radioactivity was derived, despite the accumulation of radioactivity. In contrast to the results obtained with fraction II, none of the glands exposed to radioiodinated protein
UPTAKE OFBLOOD
PROTEINBY
INSECTSALIVARY
409
GLANDS
fractions I, III, or IV showed grains in their cells or glandular lumen. These autoradiographic experiments are consistent with the observations obtained with the scintillation counter, above. The data suggest to us that haemoglobin or a derivative, or another constituent of fraction II, are the only blood constituent which are taken up by the gland specifically and preferentially, and released as part of the secretion into the glandular lumen. Control experiments involving the other fractions or the injection of pepsin-treated fraction IIJslI or free 1311showed that these fragments do not gain entry into the salivary gland cell (Table 3). TABLE%--RADIOACTIVITYRBCOVBREDINSALIVARYGLANDS 4hr AFTERINJECTION
(counts/minperlOpairsofglands),
Radioactivity recovered in salivary glands (counts/mm per 10 pairs of glands 4 hr after injection of: Pepsin-treated fraction IIJ311 (activity: 4.4 x lo4 counts/min per
Free iodinenl (activity: 9.1 x 104/$)
P)
No. of determinations
Mean
S.E.
Mean
10
47.0
15.9
56.0
S.E. 29.9
Each larva was injected either with 1 ~1 of pepsin-treated fraction IIJ311 or free iodine131. Prior to injection, 0.04 ml of 0.154 M NaCl solution containing 0.01 mg of pepsin (Sigma) was added to 0.1 ml (0.5 mg) of fraction II-13r (activity: approximately 6.2 x lo4 counts/mm per ~1). The mixture was then kept at 37°C for 30 min before it was injected into the larva.
Radioactivity in the secretion
Determinations of the amount of radioactivity in the culture medium and in the secretion released from larvae show that both the culture medium and glandular secretion contain radioactivity. In attempts to determine the amount of radioactivity in secretory products, injected larvae were transferred to fresh scintillation vials at intervals of 10 min for the first hour following the injection. Intestinal excretion of injected material no longer appeared after approximately 40 min from the time of injection. At 4 and 8 hr after this action the secretions were collected, thoroughly rinsed in 0.154 M NaCl solution, and measured for radioactivity. The radioactivity in the salivary gland secretions after 8 hr was approximately twice as much as after 4 hr. Despite the low counts/min in the salivary secretion, we found the experiments repeatable and consistent and thus we feel that they are reliable. DISCUSSION It is clear that foreign horse radish peroxidase gland cell (SCHIN 14
and
proteins
such as human
serum
albumin,
ferritin,
and
can enter the salivary gland and gain access to the salivary CLEVER,
1968). Our present data test whether constituents
410
K~ssu SCHIN
AND
HANSLAUFES
of the organism’s own haemolymph proteins enter the salivary gland cell. The small amount of radioactivity detected was not above background for fractions I, III, and IV tested. The data do show that one or more constituents of fraction II which contains the blood pigment, haemoglobin is the major, and probably the only fraction, from which components enter the gland cell and are secreted into the gland lumen. This was demonstrable by two independent assay procedures, autoradiography and scintillation spectrometry. The results were consistent with both procedures. The size-class of constituents of fraction II may play a role in determining which components are transported by the gland. It has been reported that DNase and other proteins found in salivary gland cells and secretions have a striking similarity to those of blood in their immunological properties. This was interpreted as a transport of these proteins from the blood into the glandular lumen (LAUFER,1965 ; LAUFERand NAKASE,1965). Subsequent experiments involving the use of an exogeneous protein, ferritin, showed that this protein entered lysosomes of salivary gland cells (SCHIN and CLEVER,1968). The rapid appearance of exogenous protein within the lysosomes was interpreted to mean that some proteins may be degraded within the lysosomes rather than transported intact into the glandular lumen. Our results showed that despite the occurrence of radioactivity in the cell and secretion products, the majority of the glands investigated did not reveal blood pigmentation in the secretion. In the light of the fact that the salivary gland possesses at least two types of haemoglobin proteases (pH 3.5 and S-S), it is suggested that if the proteins taken up by the glandular cell are haemoglobins then they may be digested before being secreted into the glandular lumen (RODEMSet al., 1969; HENRIKSONand CLEVER,1972). However the alternative, that other proteins of fraction II or part of haemoglobin such as globin without haeme, may be transported to the glandular lumen seems likely in the light of our present results. The salivary gland cells synthesize much of their proteins under the control of their own genome (BEERMANN,1961; DOYLE and LAUFER,1969a, b). The appearance of chromosomal puffing in these cells has been considered to reflect differential synthesis of RNA, possibly m-RNA, which in turn may affect the constituents of glandular proteins. This view has been amplified and expanded in accord with the experimental observations made by LAUFERand NAKA~E(1965) because some of the secretory proteins show immunological properties similar to those of blood proteins, and the suggestion was made that the gland may be functioning sa a transport organ (LAUFER, 1965; LAUFERand NAKASE,1965). Our experiments in which Chironomus’ own blood proteins were used, indicate that the glands are indeed involved in the rather selective uptake of blood proteins, constituents of fraction II, but they do not indicate what proportion of the protein taken up by the cell is transported intact and how much is in part degraded in its passage into the gland lumen. The occurrence of the radioactivity in the gland lumen is most likely to be the results of uptake of constituents of fraction II by the gland, an activity which the cell must regulate ultimately with active genes, and perhaps ‘puff’ loci still to be identified.
UPTAKEOF BLOODPROTEINBY INSECTSALIVARY GLANDS
411
Acknowle&ements-The authors are indebted to Dr. RALPH CLARK at SUNY, Plattsburgh, for his kind criticism and advice in the preparation of the manuscript. Thanks are due also to Dr. T. HOLT for his helpful discussion and laboratory assistance. This work was supported by grants from the NSF, the University of Connecticut Research Foundation, and SUNY Research Foundation. REFERENCES BEERMAN W. (1961) Ein Balbiani-Ring als Locus einer Speicheldruesen-Mutation. Chromosoma 12, l-25. DOYLE D. and LAUFJXRH. (1969a) Sources of larval salivary gland secretion in the dipteran Chironomus tentans. J. Cell Biol. 40, 61-78. DOYLE D. and LAUFER H. (1969b) Requirements of ribonucleic acid synthesis for the formation of salivary gland specific proteins in the larvae Chironomus tentans. Exp. Cell Res. 57, 205-210. GROSSBACH U. (1969) Chromosomen-Aktivitaet und biologische Zelldifferenzierung in den Speicheldruesen von Campto chironomus. Chromosome 28,136-187. HELMKAMPR. W., GOODLAND R. L., BALE W. F., SPAR I. L., and MUTSCHLERL. E. (1960) High specific activity iodination of -globulin with iodine-131 monochloride. Cancer Res. 20,149.5-l 500. HENRIKSONP. A. and CLEVERU. (1972) Protease activity and cell death during metamorphosis in the salivary gland of Chironomus tentans. J. Insect Physiol. 18, 1981-2004. KLOETZELJ. and LAUFERH. (1970) Developmental changes in fme structure associated with secretion in larval salivary gland of Chironomus. Exp. Cell Res. 60,327-337. LAUFERH. (1965) Developmental studies of the dipteran salivary gland-III. Relationships between chromosomal puffing and cellular function during development. In Developmental and Metabolic Control Mechanisms and Neoplasia, 19th Ann. M. D. Anderson Symp. on Fundamental Cancer Res., University of Texas, M. D. Anderson Hospital and Tumor Institute, pp. 237-250. LAUFER H. and NAKASEY. (1965) Salivary gland secretion and its relation to chromosomal puffing in the dipteran, Chironomus thummi. Proc. nut. Acad. Sci. U.S.A. 53, 511-516. LAUFERH. and WILSON M. (1970) Hormonal control of gene activity as revealed by puffing of salivary gland chromosomes in dipteran larvae, in laboratory experiments. In General and Comparative Endocrinology (Ed. by PETER R. E. and GORBMANA.), pp. 185-200. Prentice-Hall, Englewood Clifts, N. J. MECHELKEF. (1953) Reversible Strukturmodificationen der Speicheldruesen-Chromosomen von Acricotopus lucidus. Chromosoma 5, 511-543. RODEMSA. E., HENRIKSONP. A., and CLEVERU. (1969) Proteolytic enzymes in the salivary gland of Chironomus tentans. Experientia 25, 686-687. SCHIN K. S. and CLEVER U. (1968) Ultrastructural and cytochemical studies of salivary gland regression in Chironomus tentans. Z. Zellforsch. micr. Anat. 86, 262-279. SCHIN K., POLUHOWICH J. J., GAMO T., and LAUFERH. (1973) Degradation of haemoglobin in Chironomus during metamorphosis. J. Insect Physiol. In press. SQUIREP. G. (1964) A relationship between the molecular weights of macromolecules and their elution volumes based on a model for Sephadex gel filtration. Archs Biochem. Biophys. 107, 471-478. WINBORN W. (1965) Dow epoxy resin with triallyl cyanurate, and similarly modified Araldite and Maraglas mixtures, as embedding media for electron microscopy. Stain Tech. 40,227-231.