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Cytochemical and electrophoretic studies of haemoglobin synthesis in the fat body of a midge, Chironomus thummi
J. Insect Physiol., 1977, Vol. 23. pp. 1233 to 1242. Perganwn Press. Printed in Great Britain.
CYTOCHEMICAL AND ELECTROPHORETIC STUDIES OF HAEMOGLOBIN SYNTHESIS IN THE FAT BODY OF A MIDGE. CHIRONOMUS THUMMI Krssu SCHIN.’ HANS LAUFER~and ERIC CARR’ ‘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, Corm.. 06268, U.S.A. (Received 26 May 1977)
Abstract-The fat body of developing mid- and late fourth instar larvae of a midge, Chirorlomus thummi, has been investigated by means of the benzidine reaction for the localization of haemoglobin within cells. In the subepidermal fat body the reaction deposits of the haemoglobin pseudo-peroxidase activity appear predominantly in the intracisternal cavities of ER and the Golgi. and later, in the pharate pupal stage, in small dense granules (0.5-l pm in. diameter). All the major protein bands of fat body extracts, which are resolved in electrophoresis, give the benzidine reaction and show incorporation of 14C-amino levulinic acids, in this case a specific marker for haemoglobin synthesis. In addition, labelled proteins show identical electrophoretic mobility as the haemoglobins of the haemolymph, suggesting that haemoglobins are synthesized in the fat body. Two types of fat body cells seem to differ with respect to their r8le in haemoglobin metabolism. together with available structural as well as other molecular data on haemoglobins (BRAUNITZERand BRAUN, 1965; AMICONIet al., 1972) suggest that the IN Chironomus haemoglobins are the major protein relationship of the functional ultrastructure of fat constituent of the haemolymph, comprising as much as 90% of the total blood protein (MANWELL,1966). body cells to the production of the haemoglobins should be examined. Haemoglobins serve as a major, yolk-building materDissected fat body and body wall, including epiial (TRAMSand SCHIN,1976, 1977), possibly as a protein source of the salivary gland @CHIN and LAUFER, dermis and appending fat body tissues, have previously been identified as the sites of haemoglobin 1974), and, perhaps most importantly, functions as synthesis in organ culture (BERGTROMet al., 1976; a respiratory pigment for the insect which is often exposed to very low oxygen tension (NEWMANN, LAUFERet al., 1976). However these studies do not 1961). Unlike most other haemoglobin-containing provide specific information on the relationship of the organisms, Chironomus displays a striking haemoultrastructure of the fat body cells to haemoglobin globin polymorphism (MANWELL, 1966). Furthersynthesis. Therefore, the present cytochemical and more, different Chironomus species exhibit species- biochemical studies were undertaken to seek more specific forms of the haemoglobin (THOMPSONand detailed information, especially on the relationship ENGLISH,1966; ENGLISH,1969). Some forms of hae- between fat body cell structures and the biosynthesis moglobins also show ontogenetic changes (WALKER of haemoglobin. et al., 1969), and these changes are believed to be attributable to the activities of several gene loci MATERIALS AND METHODS (MANWELL,1966; TICHY, 1970) which are presumably active in the nuclei of fat body cells, where haemoChironomus thummi were grown by the method deglobins are synthesized (BERGTROMet al., 1976; scribed previously (LAUFERand WILSON, 1971). Fat LAUFERet al., 1976). Changes in the relative concenbody or “body wall” were dissected from developing tration of haemoglobins have been demonstrated to mid- and late fourth instar and pharate pupa and peroxidase activity occur during development (LAUFERand POLUHOWICH, processed for endogeneous according to the modified technique of GRAHAMand 1971), and it has been suggested that these changes result from the activation and inactivation of gene KARNOVSKY(GOLDFISCHER,1967). “Body wall” inloci at specific stages of development (WALKERet al., cludes the epidermis, cuticle, intersegmental muscu1969). Recent studies have suggested that there exists lature, as well as all of the adhering fat body. The a degradative mechanism that appears to control the control medium differed from the complete incubaconcentration of haemoglobins (SCHIN et al., 1974). tion medium for peroxidase activity in that either DAB (=3,3’-diaminobenzidine) or HzOz were omitThe recent studies of the fat body in haemoglobin synthesis (LAUFERet al., 1976; BERCTROM et al., 1976). ted. The reaction of several other tissues such as midINTRODUCTION
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HANSLAUFER,AND ERICCARR
gut, ovaries, and salivary glands were added to the incubation medium for comparison. Other controls involved overnight fixation of tissues with Karnovsky’s fixative (KARNOVSKY, 1965) or by incubation in acidified, alkalized or alcoholized DAB incubation mixture which destroys true peroxidase activity but not the pseudo-peroxidase activity of the haemoglobin (INADAet al., 1961). The following experiment was carried out to determine whether the animals in different developmental stages depend on new synthesis of protein for peroxidase activity. The animals were wrapped in tissuepaper in order to reduce their motility and were kept in a medium containing cycloheximide (1 mg/ml) overnight to inhibit protein synthesis. Animals under this experimental condition always survived but did not show incorporation of radioactive amino acids into the fat body protein. The fat body from these animals was then incubated in the DAB incubation mixture and processed for light and electron microscopy as described previously (TRAVIS and SCHTN, 1976). Thin sections were examined both with and without post-staining (REYNOLDS, 1963). Samples of soluble proteins from homogenates obtained from the fat body, and pooled haemolymph were subjected to standard polyacrylamide electrophoresis in a discontinuous buffer system (DAVIS, 1964; SCHIN et al., 1974). The initial current of 1 mA per tube was applied until the bromphenol blue front entered the separating gel (7.5% acrylamide, pH 8.9). The current was then doubled until the front of the tracking dye reached the end of the gel. One member of each set of duplicate gels was incubated in DMB (= dimethoxy-benzidine) incubation mixture containing 0.2% DMB, 0.3% H,Oz, and 5% acetic acid, to test for haemoglobin (Owner et aI., 1958). The replicate gel was stained for protein in 0.1% Coomassi B.B. overnight and was subsequently destained. The bands of the two sets were then compared in order to establish the identity of the DAB (or DMB) and haemoglobin bands of fat body with those of the haemolymph. In a final experiment subepidermal fat body, and, some body walls, were incubated in a Chironomus culture medium (SCHIN and MOORE, 1977) containing i4C-amino-levulinic acid (= ALA, spec. act.: 25.4 mc/ mole, from New England Nuclear), which had been adjusted to pH 6.7 to an osmolarity of 230 mM with sucrose. Pooled fat body samples were incubated overnight in the radioactive medium as described elsewhere (BERGTROM et al., 1976) and these were then homogenized. The homogenates were run through separating gels as described above. The gels to be used for scintillation counting contained DATD (=N,N’ Diallyltartardiamide) instead of BIS. No effort was made to remove unincorporated aminolevulinic acids since the latter migrate as a single band near the tracking dye. For comparison a duplicate set was stained for haemoglobin peroxidase activity with DMB or DAB. DMB-incubated gels were then
scanned with a Gelman densitometer at the wavelengths of approximately 500 nm (a wide spectrum of the visible wavelength). The other set of unstained gels was sliced into 1 mm segments which were then subjected to scintillation counting.
RESULTS In Chironomus there are at least two types of fat body. One contains green-pigment in the fourth instar, the cells are generally small, and is located below the epidermis and above the intersegmental musculature. The rest of the fat body is white, the cells in this tissue are relatively larger, and these cells are often attached to the viscera or to the intestine. These fat body masses which seem to be scattered suspended within the body cavity may also be attached at various points along the length of the haemocoele. In addition to its smaller size, and characteristic coloration, the subepidermal fat body cells of the developing fourth instar shows a clear cytological difference from cells of the visceral fat body, in that they contain more endoplasmic reticulum (Fig. 1A). Visceral fat body cell on the other hand exhibits a reduction in the organized structures including peripheral cytoplasm, and are occupied largely by structures resembling lipids and lipid droplets (Fig. 1B). The presence of many electron dense granules (0.5-l pm in diameter) is characteristic of the subepidermal fat body cell of the growing fourth instar larva. Most of the granules do not appear to be autophagic vacuoles. On the other hand, the vacuoles that are seen in the visceral fat body cells appear to be mostly autophagic. These cytological distinctions are even more manifest in terms of endogeneous peroxidase activity, as represented by the localization of DAB reaction products. Endogeneous peroxidase activity, which represents either true peroxidase activity and/or pseudo-peroxidase activity of the haemoglobin, is localized mostly in the subepidermal fat body cell of all the developing mid- and late fourth instar larvae (Fig. 2) but is rarely seen in the nonsubepidermal visceral fat body cells of the corresponding larvae. In the subepidermal fat body cells the reaction deposits are confined sharply to the intracistemal cavities of rough ER and Golgi complex (Fig. 2, 3, 4, 5). The nuclear envelope whose outer membrane is studded with ribosomes (therefore representing a derivative of the ER) also displays strong peroxidase activity. The electron density of the structures containing DAB reaction products is not due to the lead deposits of the post-staining, but to the DAB reaction deposits only (compare Fig. 2 with Fig. 3!). Figure 4 shows a portion of the subepidermal fat body of the late fourth instar in which, in the absence of the lead poststaining, the DAB reaction products are seen in the ER regions to have an electron density almost identical to that of cells which had been post-stained.
Fig. 2. A portion of a subepidermal fat body cell of the late fourth deposits. N = nucleus; Sg = small dense granules; E = extracellular stained with lead citrate.
Fig. 3. A portion
instar larva showing DAB reaction space; M = mitochondria.-Post-
of a subepidermal fat body of the late fourth instar larva products in the absence of lead citrate post-staining.
showing
DAB
reaction
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Fig. 4. A high resolution micrograph of a subepidermal fat body cell of the late fourth instar larva. DAB reaction deposits are in the endoplasmic reticulum and perinuclear space (P) of the nuclear envelope.-Not post-stained. Fig. 5. DAB reaction
deposits
in the intracisternal cavities of ER and the Golgi vesicles in the subepiderma1 fat body of the late fourth instar larva.
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Fig. 6. A portion of a subepidermal fat body of the late fourth instar larva. The animal was treated with cycloheximide (1 mg/ml) overnight before incubation into DAB mixture. DAB reaction deposits are present only in the extracellular space (arrows).
Haemoglobin synthesis in chironomus fat body
1 Fig. I. Diagrammatic presentation of two types of fat body cells in the developing mid- or late fourth instar larvae. A: Drawing of sections of typical subepidermal fat body cells. Cy = dense cytoplasm containing endoplasmic reticulum (large dots); N = nucleus; L = lipids or lipid vacuoles; Sg = small granules. B: Drawing of sections of typical non-subepidermal visceral fat body cells with less dense cytoplasm. Most, if not all of, the small dense granules are autophagic vacuoles (Av).
Figure 5 shows that these DAB reaction products are confined exclusively to the intracistemal cavities of the ER and not to the spaces between adjacent ER. While ER is the most prominent structure associated with reaction deposits, the strongest electron density resulting from the DAB reaction is shown by the Golgi complex (Fig. 5). Electron opaque granules (0.5-l pm in diameter) are scattered throughout the cytoplasm. Some of these may have originated from Golgi complexes and they also exhibit some electron density (Figs. 1, 2, 3). However, these structures show some electron opaqueness even in the absence of the DAB reaction in unstained sections. Therefore, the electron density of these granules does not seem to account for the endogeneous peroxidase activity alone. Mitochondria in fat body cells display no DAB reaction deposits. Occasionally DAB reaction deposits appear in the extracellular spaces between two adjacent fat body
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cells. The accumulation of reaction deposits in the extracellular spaces is much more pronounced in the fat body cells of pharate pupa where DAB reaction deposits are no longer visible in the ER cavities (SCHIN et al., unpublished). The presence of the reaction products in the ER cavities of the subepidermal fat body cells is stagespecific. All the mid- and late fourth instar larvae display reaction products, but in the pharate pupa and pharate adult the reaction products are no longer visible in the ER. The appearance of heavy DAB deposits in the extracellular spaces between adjacent cells appears to be specific for certain developmental stages as well. In the pharate pupa the reaction products appear in the extracellular spaces concurrently with the disappearance of these products from the ER. In contrast. in the developing mid- or late fourth instar, when DAB reaction deposits prevail in the ER, there is little or no detectable reaction product in the extracellular spaces. In the pharate pupal stage, the number of intracellular electron dense granules showing DAB reaction has increased significantly. Fat body cells incubated in the incubation mixture without H,Oz or DAB do not show any electron density comparable to that of DAB reaction products. These controls do not however distinguish between true peroxidase and pseudo-peroxidase of haemoglobin. Therefore, as additional controls for the haemoglobin reaction, fat body cells were fixed with Karnovsky’s fixative overnight or fixed for 30min and treated with 70T0 ethanol or 0.1% acetic acid or NaOH before the incubation in the DAB mixture. It has been shown that under these experimental conditions true peroxidase activity is completely inhibited while haem proteins, such as haemoglobin exhibit pseudo-peroxidase activity (INADA et al., 1961: OWEN et al., 1958). Under these conditions, most if not all of the larval subepidermal fat body cells examined displayed DAB reaction products in ER and Golgi complex. The results of the experiments support our contention that the DAB reaction deposits represent haemoglobin pseudo-peroxidase activity and not true peroxidase activity. They do not give us a clear indication of the origin of these proteins, although the presence of the enzyme activity, especially in the ER system strongly supports the idea of de nouo synthesis of this protein during the fourth instar. Therefore, DAB reaction tests were conducted on a group of developing mid- and late fourth instar larvae that were previously incubated for 6 to 8 hours in a medium containing cycloheximide (1 mg/ml) to inhibit protein synthesis. Cells of the subepidermal fat body of mid- and late fourth instar treated in this way show DAB reaction products, but only in the extracellular spaces; none was in the ER system or Golgi complex (Fig. 6). The group of dense granules (0.5 to 1 pm in diameter) whose electron density is not entirely due to the DAB reaction, do not appear significantly affected.
KISSU SCHIN,HANS LAUFER,ANDERIC CARR
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Fig. 7. Densitometric profiles of DMB-positive haemoglobin bands of the haemolymph (-.-.-,) and the subepidermal fat body (------), and the amount (counts/min) of radioactivity of ‘%-ALA incorporated into the haemoglobins (-_te ). Dot-dashed line: optical density of haemolymph proteins from late fourth instar larvae. The gel was incubated in a DMB-mixture for haemoglobin detection. Dashed line: optical density of subepidermal fat body from late fourth instar larvae. The gel was incubated in a DMB-mixture for haemoglobin detection. Part of the haemoglobin pattern is due to the contamination of fat body cells with haemolymph. Solid line = incorporation of radioactivity (14C-ALA) into proteins of subepidermal fat body from late fourth instar larvae. The largest peak of radioactivity represents unincorporated free 14C-ALA. T = tracking dye; Hb = haemoglobin.
Etectrophoretic and radioisotopic experiments
All of the electrophoretically resolvable major protein bands exhibit a positive DAB reaction (Fig. 7). This is shown by extract of either fat body or haemolymph of developing late fourth instar larvae. The haemolymph of the late fourth instar shows in its electrophoreogram at least 9 protein bands, of which 8 are haemoglobins according to their red coloration before fixation. All of these haemoglobins display a positive DAB reactions. The fat body extracts show at least 5 major protein bands, all of which exhibit DMB reaction products. Figure 7 also shows that the positions of DMB-positive protein bands of the fat body strictly correspond to those of the DMB-positive haemoglobin bands of the haemolymph. Combined radioisotopic and electrophoretic experiments show that the fat body cells manufacture most, if not all, of the haemoglobin polypeptides of the haemolymph (see also BERGTROMet al., 1976). All the haemoglobin bands with possible exception of bands 8 and perhaps 9 show peaks of 14C-ALA incorporation. Haemoglobin band 8 and 9 show the largest single peak of radioactivity, but much of the radioactivity in this region is due to the presence of unincorporated 14C-ALA.
DISCUSSION The present cytochemical and electrophoretic studies suggest that sub-epidermal fat body is a major site of endogeneous peroxidase activity, and supports previously stated conclusion that the fat body is an active site of haemoglobin synthesis (BERGTROMef al.. 1976 ; LAUFER et al., 1976). The reaction deposits formed after incubation of tissues with DAB and H,Oz are considered to be reliable indicator of peroxidase activity (GOLDFISCHER,1967; OWEN et al., 1958). They represent true peroxidase activity and/or pseudo-peroxidase activity of haemproteins or haemin compounds with nitrogeneous bases (INADA et al., 1961). Though it is possible that a minor part of DAB or DMB reaction might be due to true peroxidase activity, the reaction deposits in Chironomus fat body in all likelihood represent a predominant pseudo-peroxidase activity of haemoglobin. This conclusion is supported strongly by electrophoretic evidence that the haemoglobin is. the only visible, DAB-(or DMB-)reacting substance among all of the proteins from the fat body (Fig. 7). Some caution has to be exercised in the interpretation of the results of electrophoretic experiments with body wall because the body wall with its attached
1241
Haemoglobin synthesis in chironomus fat body fat body may contain small amounts of haemolymph.
However, this residual haemolymph can be washed away by prolonged rinsing. Nevertheless, reaction products are observed in body wall extracts containing subepidermal fat body after prolonged washing of the tissue prior to homogenization. These positive DAB reactions, are considered to be evidence for the presence of haemoglobin in the homogenate. In the developing mid- and late fourth instar the pseudo-peroxidase activity of the haemoglobin is confined primarily to the intracisternal cavities of the endoplasmic reticulum, as evidenced by the presence of DAB reaction products. Though the reaction deposits in these regions are not as strong as in the Golgi bodies, the cavities of ER, including the perinuclear space of the nuclear envelope, are the most prominent structures associated with this peroxidase activity. The frequent association of ribosomes with its outer surface and the presence of DAB deposits in the perinuclear space of the nuclear envelope could mean that the nuclear envelope represents part of the elaborate ER system. The small dense cytoplasmic granules show strong electron density, but it appears that much of this electron density is not due to the presence of DAB reaction products, as these structures are electron opaque even in ordinary micrographs and in the controls lacking DAB or HzO,. In view of the fact that these structures rapidly assume strong DAB reactivity only at the later, pharate pupal stage, the small granules in the developing fourth instar may not be functioning actively in the storage of haemoglobin. Notably, these granules fail to show structures such as digestive remnants of mitochondria or ER within their membrane boundaries. This might suggest that in the subepidermal fat body of the developing fourth instar the electron density of DAB reaction products. if there is any, such as that seen in the small granules, is not due to the uptake of haemoglobin from the haemolymph. The lack of pinosomes or coated vesicles at this stage sup port the above suggestion. The prominent localization of DAB reaction products in the intracisternal cavities of ER and Golgi vesicles, and their absence in most other structures suggest that these reaction deposits represent de nom synthesis of haemoglobin. This conclusion is sup ported further by the observations that the distribution of DAB reaction products is protein synthesis dependent. The in vivo or in vitro cycloheximide treatment prevents the deposition of reaction products from haemoglobin peroxidase activity coincident with the cessation of protein synthesis. This conclusion receives further support in that the electrophoreograms of fat body extracts. DAB- or DMB-positive haemoglobin display noticeably large peaks of radioactive ALA incorporation. This is an indication of haemoglobin synthesis (see also BERCTROMer al., 1976; LAUFERet al., 1976). The results indicate that if there exist any temporal relationship between various DAB deposit-containing structures in the in-
tracellular localization and distribution of DAB deposits, the sequence would be: synthesis of the haemoglobin on the rough ER+ intracellular deposits in the ER cisternae-+ transport to the Golgi vesicles(?)--+ secretion by means of exocytosis into extracellular spaces from which the haemoglobin exudes into the haemolymph. The first and last of these steps is fairly well substantiated by our data. We have not yet found evidence that the Golgi vesicles carry their contents to the extracellular spaces where DAB reaction deposits have been found. We conclude from our results, that the fat body of Chironomus is actively involved in haemoglobin metabolism in the fourth instar stage. The subepiderma1 fat body cells seem to be more active in de novo synthesis than the visceral fat body. This is supported’ by the finding of differential amounts of peroxidase activity, endoplasmic reticulum, and Golgi vesicles, and the dependence of the peroxidase-positive activity upon protein synthesis. There seem therefore to be at least two different types of fat body function differently in haemoglobin metabolism. The observations, that in the developing mid- and late fourth instar much of the subepidermal fat body displays specific intracellular haemoglobin localization and synthesis, and furthermore, that much of the visceral fat body exhibits little or none, are suggestive of such a specialized function. The fact that a large number of the visceral fat body cells examined contain autophagic vacuoles, and occasionally, pinosomes is an indication that these fat body cells are geared to the uptake and storage rather than to the synthesis of haemoglobin. The findings that ferritin appeared in the autophagic vacuoles of the visceral fat body within 15 min after the injection into the animal fall well into line with above observations (SCHIN and CLEVER, 1968). Taken together, these results tend to indicate that Chironomus has developed effective multiple mechanisms to control the concentration of the blood proteir-through synthesis, storage, utilization, and finally through degradation (SCHINand CLEVER,1968; SCHIN et al., 1974; BERGTROMet al., 1976; LAUFER et al., 1976; TRAVIS and SCHIN, 1976, 1977). These mechanisms must be of fundamental importance to the life of the insect. Acknowledgements-The authors are indebted to Dr R. SUNY college at Plattsburgh, for his kind criti-
CLARK at
cism and advice in the preparation of the manuscript. Thanks are due also to Dr H. Z. Lru, Dr R. ELLSWORW, and Dr W. GRAZIADEIwho allowed us to use their equip ment and materials for this work. This research was in part supported by a grant from the University of Connecticut Research Foundation.
REFERENCES AMICONIG., ANTONINIE., BRUNORIM., FORMANECK H., and HUBERR. (1972) Functional properties of native and
reconstituted thummi.
Eur.
hemoglobins J. Biochem.
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Chironomus thummi
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KISSUSCHIN,HANS LAUFER,AND
BERGTROM G., LAUFERH.. and ROGERSR. (1976) Fat body: a site of hemoglobin synthesis in Chironomus rfiummi (Diptera). J. Ceff &of. 69, 264274. BRAUNITZER G. and BRAUNV. (1965) Zur Phylogenie des Haemoglobin-molektils. Untersuchungen an InsektenHaemoglobinen (Chironomus thummi). Hoppe-Seyfer’s 2. physiol. Chem. 340, 88-91. DAVIS B. J. (1964) Disc electrophoresis-II. Method and applications to human serum proteins. Ann. N.I! Acad. Sci. 121, 404427. ENGLISHD. S. (1969) Ontogenetic changes in hemoglobin synthesis of two strains of Chironomus tentans. J. &nbryol. exp. Morph. 22, 465476. GOLDFI~CHER S. (1967) The cvtochemical localization of myoglobin in striated muscles of man and walrus. J. Cell Biol. 34, 398403. INADAY., KUROZUMIT., and SHIBATAK. (1961) Peroxidase activity of hemoproteins. I. Generation of activity by acid or alkali denaturation of methemoglobin and catalase. Arch. Biochem. Biophys. 93, 30-36. KARNOVSKYM. J. (1965) A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. Cell Biol. 27, 138A. LAUFERH. and POLUHOWICH J. J. (1971) A factor controlling the concentration of hemoglobin in Chironomus during metamorphosis. Limnofogica 8. 1255126. LAUFERH. and WILXIN 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. 1855200. PrenticeHall, N.J. LAUFERH.. BERGTROMG., and ROGERSR. (1976) Stage and tissue-specific hemoglobin synthesis in an invertebrate. In Invertebrate Tissue Culture. Applications in Medicine, Biology, and Agriculture. pp. 227-240. Academic Press, New York. MANWELLC. (1966) Starch gel electrophoresis of the multiple haemoglobins of small and large larval Chironomus-a developmental haemoglobin sequence in an invertebrate. J. Embryo/. exp. Morph. 16, 259-270. I
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NEUMANND. (1961) Der Einfluss des Eisenangebotes auf die Haemoglobinsynthese und die Entwicklung der Clzironomus-Larve. Zeitschrift fiir Natu~orschung 16b, 82tX324. OWEN J. A., SILBERMAN H. J.. and GROT C. (1958) Detection of haemoglobin, haemoglobin-haptoglobin complex and other substances with peroxidase activity after zone electrophoresis. Nature, Land. 182, 1373. REYNOLDS E. S. (1963) The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 27, 208-212. SCHINK. and CLEVERU. (1968) Ferritin-uptake by salivary glands of Chironomus tentans and its intracellular localization. ENI). Cell Res. 49. 208-211. SCHIN K. and LAUFERH. (1974) Uptake of homologous haemolymph protein by salivary glands of Chironomus thummi. J.~Insect
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SCHINK. and MOORER. D. (1977) Cation concentrations in the haemolymph of the fly, Chironomus thummi, during development. J. Insect Physiol. 23, 723-729. SCHIN K.. POLUHOWICHJ. J., GAMO T., and LAUFERH. (1974) Degradation of haemoglobin in Chironomrrs during metamorphosis. J. Insect Physiol. 20, 561-571. THOMP~QNP. E. and ENGLISHD. S. (1966) Multiplicity of hemoglobins in the genus Chironomus (Tendipes). Science,
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TICHY H. (1970) Biochemische und cytogenetische Untersuchungen zur Natur des Haemoglobin-Polymorphismus bei Chironomus tentans und Chironomus pallidiuitatus. Chromosoma (Berl.) 29, 131-188. TRAVISJ. L. and SCHIN K. (1976) Evidence for haemoglobin uptake by oocytes of Chironomus thummi. J. fnsect Physiof.
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TRAVISJ. L. and SCHIN K. (1977) Developmental studies of vitellogenesis in a Dipteran insect, Chironomus thummi. Dij’erentiation 7, 113-125. W~ILKERW., MAIER W.. und BERTAUP. (1969) Untersuchungen tiber die Haemolymph-proteine der Chironomiden (Dipt.). Z. Naturforsch. 24b, 110-I 16.
CYTOCHEMICAL AND ELECTROPHORETIC STUDIES OF HAEMOGLOBIN SYNTHESIS IN THE FAT BODY OF A MIDGE. CHIRONOMUS THUMMI Krssu SCHIN.’ HANS LAUFER~and ERIC CARR’ ‘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, Corm.. 06268, U.S.A. (Received 26 May 1977)
Abstract-The fat body of developing mid- and late fourth instar larvae of a midge, Chirorlomus thummi, has been investigated by means of the benzidine reaction for the localization of haemoglobin within cells. In the subepidermal fat body the reaction deposits of the haemoglobin pseudo-peroxidase activity appear predominantly in the intracisternal cavities of ER and the Golgi. and later, in the pharate pupal stage, in small dense granules (0.5-l pm in. diameter). All the major protein bands of fat body extracts, which are resolved in electrophoresis, give the benzidine reaction and show incorporation of 14C-amino levulinic acids, in this case a specific marker for haemoglobin synthesis. In addition, labelled proteins show identical electrophoretic mobility as the haemoglobins of the haemolymph, suggesting that haemoglobins are synthesized in the fat body. Two types of fat body cells seem to differ with respect to their r8le in haemoglobin metabolism. together with available structural as well as other molecular data on haemoglobins (BRAUNITZERand BRAUN, 1965; AMICONIet al., 1972) suggest that the IN Chironomus haemoglobins are the major protein relationship of the functional ultrastructure of fat constituent of the haemolymph, comprising as much as 90% of the total blood protein (MANWELL,1966). body cells to the production of the haemoglobins should be examined. Haemoglobins serve as a major, yolk-building materDissected fat body and body wall, including epiial (TRAMSand SCHIN,1976, 1977), possibly as a protein source of the salivary gland @CHIN and LAUFER, dermis and appending fat body tissues, have previously been identified as the sites of haemoglobin 1974), and, perhaps most importantly, functions as synthesis in organ culture (BERGTROMet al., 1976; a respiratory pigment for the insect which is often exposed to very low oxygen tension (NEWMANN, LAUFERet al., 1976). However these studies do not 1961). Unlike most other haemoglobin-containing provide specific information on the relationship of the organisms, Chironomus displays a striking haemoultrastructure of the fat body cells to haemoglobin globin polymorphism (MANWELL, 1966). Furthersynthesis. Therefore, the present cytochemical and more, different Chironomus species exhibit species- biochemical studies were undertaken to seek more specific forms of the haemoglobin (THOMPSONand detailed information, especially on the relationship ENGLISH,1966; ENGLISH,1969). Some forms of hae- between fat body cell structures and the biosynthesis moglobins also show ontogenetic changes (WALKER of haemoglobin. et al., 1969), and these changes are believed to be attributable to the activities of several gene loci MATERIALS AND METHODS (MANWELL,1966; TICHY, 1970) which are presumably active in the nuclei of fat body cells, where haemoChironomus thummi were grown by the method deglobins are synthesized (BERGTROMet al., 1976; scribed previously (LAUFERand WILSON, 1971). Fat LAUFERet al., 1976). Changes in the relative concenbody or “body wall” were dissected from developing tration of haemoglobins have been demonstrated to mid- and late fourth instar and pharate pupa and peroxidase activity occur during development (LAUFERand POLUHOWICH, processed for endogeneous according to the modified technique of GRAHAMand 1971), and it has been suggested that these changes result from the activation and inactivation of gene KARNOVSKY(GOLDFISCHER,1967). “Body wall” inloci at specific stages of development (WALKERet al., cludes the epidermis, cuticle, intersegmental muscu1969). Recent studies have suggested that there exists lature, as well as all of the adhering fat body. The a degradative mechanism that appears to control the control medium differed from the complete incubaconcentration of haemoglobins (SCHIN et al., 1974). tion medium for peroxidase activity in that either DAB (=3,3’-diaminobenzidine) or HzOz were omitThe recent studies of the fat body in haemoglobin synthesis (LAUFERet al., 1976; BERCTROM et al., 1976). ted. The reaction of several other tissues such as midINTRODUCTION
I?34
Krssu
SCHIN,
HANSLAUFER,AND ERICCARR
gut, ovaries, and salivary glands were added to the incubation medium for comparison. Other controls involved overnight fixation of tissues with Karnovsky’s fixative (KARNOVSKY, 1965) or by incubation in acidified, alkalized or alcoholized DAB incubation mixture which destroys true peroxidase activity but not the pseudo-peroxidase activity of the haemoglobin (INADAet al., 1961). The following experiment was carried out to determine whether the animals in different developmental stages depend on new synthesis of protein for peroxidase activity. The animals were wrapped in tissuepaper in order to reduce their motility and were kept in a medium containing cycloheximide (1 mg/ml) overnight to inhibit protein synthesis. Animals under this experimental condition always survived but did not show incorporation of radioactive amino acids into the fat body protein. The fat body from these animals was then incubated in the DAB incubation mixture and processed for light and electron microscopy as described previously (TRAVIS and SCHTN, 1976). Thin sections were examined both with and without post-staining (REYNOLDS, 1963). Samples of soluble proteins from homogenates obtained from the fat body, and pooled haemolymph were subjected to standard polyacrylamide electrophoresis in a discontinuous buffer system (DAVIS, 1964; SCHIN et al., 1974). The initial current of 1 mA per tube was applied until the bromphenol blue front entered the separating gel (7.5% acrylamide, pH 8.9). The current was then doubled until the front of the tracking dye reached the end of the gel. One member of each set of duplicate gels was incubated in DMB (= dimethoxy-benzidine) incubation mixture containing 0.2% DMB, 0.3% H,Oz, and 5% acetic acid, to test for haemoglobin (Owner et aI., 1958). The replicate gel was stained for protein in 0.1% Coomassi B.B. overnight and was subsequently destained. The bands of the two sets were then compared in order to establish the identity of the DAB (or DMB) and haemoglobin bands of fat body with those of the haemolymph. In a final experiment subepidermal fat body, and, some body walls, were incubated in a Chironomus culture medium (SCHIN and MOORE, 1977) containing i4C-amino-levulinic acid (= ALA, spec. act.: 25.4 mc/ mole, from New England Nuclear), which had been adjusted to pH 6.7 to an osmolarity of 230 mM with sucrose. Pooled fat body samples were incubated overnight in the radioactive medium as described elsewhere (BERGTROM et al., 1976) and these were then homogenized. The homogenates were run through separating gels as described above. The gels to be used for scintillation counting contained DATD (=N,N’ Diallyltartardiamide) instead of BIS. No effort was made to remove unincorporated aminolevulinic acids since the latter migrate as a single band near the tracking dye. For comparison a duplicate set was stained for haemoglobin peroxidase activity with DMB or DAB. DMB-incubated gels were then
scanned with a Gelman densitometer at the wavelengths of approximately 500 nm (a wide spectrum of the visible wavelength). The other set of unstained gels was sliced into 1 mm segments which were then subjected to scintillation counting.
RESULTS In Chironomus there are at least two types of fat body. One contains green-pigment in the fourth instar, the cells are generally small, and is located below the epidermis and above the intersegmental musculature. The rest of the fat body is white, the cells in this tissue are relatively larger, and these cells are often attached to the viscera or to the intestine. These fat body masses which seem to be scattered suspended within the body cavity may also be attached at various points along the length of the haemocoele. In addition to its smaller size, and characteristic coloration, the subepidermal fat body cells of the developing fourth instar shows a clear cytological difference from cells of the visceral fat body, in that they contain more endoplasmic reticulum (Fig. 1A). Visceral fat body cell on the other hand exhibits a reduction in the organized structures including peripheral cytoplasm, and are occupied largely by structures resembling lipids and lipid droplets (Fig. 1B). The presence of many electron dense granules (0.5-l pm in diameter) is characteristic of the subepidermal fat body cell of the growing fourth instar larva. Most of the granules do not appear to be autophagic vacuoles. On the other hand, the vacuoles that are seen in the visceral fat body cells appear to be mostly autophagic. These cytological distinctions are even more manifest in terms of endogeneous peroxidase activity, as represented by the localization of DAB reaction products. Endogeneous peroxidase activity, which represents either true peroxidase activity and/or pseudo-peroxidase activity of the haemoglobin, is localized mostly in the subepidermal fat body cell of all the developing mid- and late fourth instar larvae (Fig. 2) but is rarely seen in the nonsubepidermal visceral fat body cells of the corresponding larvae. In the subepidermal fat body cells the reaction deposits are confined sharply to the intracistemal cavities of rough ER and Golgi complex (Fig. 2, 3, 4, 5). The nuclear envelope whose outer membrane is studded with ribosomes (therefore representing a derivative of the ER) also displays strong peroxidase activity. The electron density of the structures containing DAB reaction products is not due to the lead deposits of the post-staining, but to the DAB reaction deposits only (compare Fig. 2 with Fig. 3!). Figure 4 shows a portion of the subepidermal fat body of the late fourth instar in which, in the absence of the lead poststaining, the DAB reaction products are seen in the ER regions to have an electron density almost identical to that of cells which had been post-stained.
Fig. 2. A portion of a subepidermal fat body cell of the late fourth deposits. N = nucleus; Sg = small dense granules; E = extracellular stained with lead citrate.
Fig. 3. A portion
instar larva showing DAB reaction space; M = mitochondria.-Post-
of a subepidermal fat body of the late fourth instar larva products in the absence of lead citrate post-staining.
showing
DAB
reaction
1236
Fig. 4. A high resolution micrograph of a subepidermal fat body cell of the late fourth instar larva. DAB reaction deposits are in the endoplasmic reticulum and perinuclear space (P) of the nuclear envelope.-Not post-stained. Fig. 5. DAB reaction
deposits
in the intracisternal cavities of ER and the Golgi vesicles in the subepiderma1 fat body of the late fourth instar larva.
1237
Fig. 6. A portion of a subepidermal fat body of the late fourth instar larva. The animal was treated with cycloheximide (1 mg/ml) overnight before incubation into DAB mixture. DAB reaction deposits are present only in the extracellular space (arrows).
Haemoglobin synthesis in chironomus fat body
1 Fig. I. Diagrammatic presentation of two types of fat body cells in the developing mid- or late fourth instar larvae. A: Drawing of sections of typical subepidermal fat body cells. Cy = dense cytoplasm containing endoplasmic reticulum (large dots); N = nucleus; L = lipids or lipid vacuoles; Sg = small granules. B: Drawing of sections of typical non-subepidermal visceral fat body cells with less dense cytoplasm. Most, if not all of, the small dense granules are autophagic vacuoles (Av).
Figure 5 shows that these DAB reaction products are confined exclusively to the intracistemal cavities of the ER and not to the spaces between adjacent ER. While ER is the most prominent structure associated with reaction deposits, the strongest electron density resulting from the DAB reaction is shown by the Golgi complex (Fig. 5). Electron opaque granules (0.5-l pm in diameter) are scattered throughout the cytoplasm. Some of these may have originated from Golgi complexes and they also exhibit some electron density (Figs. 1, 2, 3). However, these structures show some electron opaqueness even in the absence of the DAB reaction in unstained sections. Therefore, the electron density of these granules does not seem to account for the endogeneous peroxidase activity alone. Mitochondria in fat body cells display no DAB reaction deposits. Occasionally DAB reaction deposits appear in the extracellular spaces between two adjacent fat body
I739
cells. The accumulation of reaction deposits in the extracellular spaces is much more pronounced in the fat body cells of pharate pupa where DAB reaction deposits are no longer visible in the ER cavities (SCHIN et al., unpublished). The presence of the reaction products in the ER cavities of the subepidermal fat body cells is stagespecific. All the mid- and late fourth instar larvae display reaction products, but in the pharate pupa and pharate adult the reaction products are no longer visible in the ER. The appearance of heavy DAB deposits in the extracellular spaces between adjacent cells appears to be specific for certain developmental stages as well. In the pharate pupa the reaction products appear in the extracellular spaces concurrently with the disappearance of these products from the ER. In contrast. in the developing mid- or late fourth instar, when DAB reaction deposits prevail in the ER, there is little or no detectable reaction product in the extracellular spaces. In the pharate pupal stage, the number of intracellular electron dense granules showing DAB reaction has increased significantly. Fat body cells incubated in the incubation mixture without H,Oz or DAB do not show any electron density comparable to that of DAB reaction products. These controls do not however distinguish between true peroxidase and pseudo-peroxidase of haemoglobin. Therefore, as additional controls for the haemoglobin reaction, fat body cells were fixed with Karnovsky’s fixative overnight or fixed for 30min and treated with 70T0 ethanol or 0.1% acetic acid or NaOH before the incubation in the DAB mixture. It has been shown that under these experimental conditions true peroxidase activity is completely inhibited while haem proteins, such as haemoglobin exhibit pseudo-peroxidase activity (INADA et al., 1961: OWEN et al., 1958). Under these conditions, most if not all of the larval subepidermal fat body cells examined displayed DAB reaction products in ER and Golgi complex. The results of the experiments support our contention that the DAB reaction deposits represent haemoglobin pseudo-peroxidase activity and not true peroxidase activity. They do not give us a clear indication of the origin of these proteins, although the presence of the enzyme activity, especially in the ER system strongly supports the idea of de nouo synthesis of this protein during the fourth instar. Therefore, DAB reaction tests were conducted on a group of developing mid- and late fourth instar larvae that were previously incubated for 6 to 8 hours in a medium containing cycloheximide (1 mg/ml) to inhibit protein synthesis. Cells of the subepidermal fat body of mid- and late fourth instar treated in this way show DAB reaction products, but only in the extracellular spaces; none was in the ER system or Golgi complex (Fig. 6). The group of dense granules (0.5 to 1 pm in diameter) whose electron density is not entirely due to the DAB reaction, do not appear significantly affected.
KISSU SCHIN,HANS LAUFER,ANDERIC CARR
I240
Hbl
r
I
I
,
I
I
IO
20
30
40
50
SLICE
I
60
I
70
I
80
NUMBER
Fig. 7. Densitometric profiles of DMB-positive haemoglobin bands of the haemolymph (-.-.-,) and the subepidermal fat body (------), and the amount (counts/min) of radioactivity of ‘%-ALA incorporated into the haemoglobins (-_te ). Dot-dashed line: optical density of haemolymph proteins from late fourth instar larvae. The gel was incubated in a DMB-mixture for haemoglobin detection. Dashed line: optical density of subepidermal fat body from late fourth instar larvae. The gel was incubated in a DMB-mixture for haemoglobin detection. Part of the haemoglobin pattern is due to the contamination of fat body cells with haemolymph. Solid line = incorporation of radioactivity (14C-ALA) into proteins of subepidermal fat body from late fourth instar larvae. The largest peak of radioactivity represents unincorporated free 14C-ALA. T = tracking dye; Hb = haemoglobin.
Etectrophoretic and radioisotopic experiments
All of the electrophoretically resolvable major protein bands exhibit a positive DAB reaction (Fig. 7). This is shown by extract of either fat body or haemolymph of developing late fourth instar larvae. The haemolymph of the late fourth instar shows in its electrophoreogram at least 9 protein bands, of which 8 are haemoglobins according to their red coloration before fixation. All of these haemoglobins display a positive DAB reactions. The fat body extracts show at least 5 major protein bands, all of which exhibit DMB reaction products. Figure 7 also shows that the positions of DMB-positive protein bands of the fat body strictly correspond to those of the DMB-positive haemoglobin bands of the haemolymph. Combined radioisotopic and electrophoretic experiments show that the fat body cells manufacture most, if not all, of the haemoglobin polypeptides of the haemolymph (see also BERGTROMet al., 1976). All the haemoglobin bands with possible exception of bands 8 and perhaps 9 show peaks of 14C-ALA incorporation. Haemoglobin band 8 and 9 show the largest single peak of radioactivity, but much of the radioactivity in this region is due to the presence of unincorporated 14C-ALA.
DISCUSSION The present cytochemical and electrophoretic studies suggest that sub-epidermal fat body is a major site of endogeneous peroxidase activity, and supports previously stated conclusion that the fat body is an active site of haemoglobin synthesis (BERGTROMef al.. 1976 ; LAUFER et al., 1976). The reaction deposits formed after incubation of tissues with DAB and H,Oz are considered to be reliable indicator of peroxidase activity (GOLDFISCHER,1967; OWEN et al., 1958). They represent true peroxidase activity and/or pseudo-peroxidase activity of haemproteins or haemin compounds with nitrogeneous bases (INADA et al., 1961). Though it is possible that a minor part of DAB or DMB reaction might be due to true peroxidase activity, the reaction deposits in Chironomus fat body in all likelihood represent a predominant pseudo-peroxidase activity of haemoglobin. This conclusion is supported strongly by electrophoretic evidence that the haemoglobin is. the only visible, DAB-(or DMB-)reacting substance among all of the proteins from the fat body (Fig. 7). Some caution has to be exercised in the interpretation of the results of electrophoretic experiments with body wall because the body wall with its attached
1241
Haemoglobin synthesis in chironomus fat body fat body may contain small amounts of haemolymph.
However, this residual haemolymph can be washed away by prolonged rinsing. Nevertheless, reaction products are observed in body wall extracts containing subepidermal fat body after prolonged washing of the tissue prior to homogenization. These positive DAB reactions, are considered to be evidence for the presence of haemoglobin in the homogenate. In the developing mid- and late fourth instar the pseudo-peroxidase activity of the haemoglobin is confined primarily to the intracisternal cavities of the endoplasmic reticulum, as evidenced by the presence of DAB reaction products. Though the reaction deposits in these regions are not as strong as in the Golgi bodies, the cavities of ER, including the perinuclear space of the nuclear envelope, are the most prominent structures associated with this peroxidase activity. The frequent association of ribosomes with its outer surface and the presence of DAB deposits in the perinuclear space of the nuclear envelope could mean that the nuclear envelope represents part of the elaborate ER system. The small dense cytoplasmic granules show strong electron density, but it appears that much of this electron density is not due to the presence of DAB reaction products, as these structures are electron opaque even in ordinary micrographs and in the controls lacking DAB or HzO,. In view of the fact that these structures rapidly assume strong DAB reactivity only at the later, pharate pupal stage, the small granules in the developing fourth instar may not be functioning actively in the storage of haemoglobin. Notably, these granules fail to show structures such as digestive remnants of mitochondria or ER within their membrane boundaries. This might suggest that in the subepidermal fat body of the developing fourth instar the electron density of DAB reaction products. if there is any, such as that seen in the small granules, is not due to the uptake of haemoglobin from the haemolymph. The lack of pinosomes or coated vesicles at this stage sup port the above suggestion. The prominent localization of DAB reaction products in the intracisternal cavities of ER and Golgi vesicles, and their absence in most other structures suggest that these reaction deposits represent de nom synthesis of haemoglobin. This conclusion is sup ported further by the observations that the distribution of DAB reaction products is protein synthesis dependent. The in vivo or in vitro cycloheximide treatment prevents the deposition of reaction products from haemoglobin peroxidase activity coincident with the cessation of protein synthesis. This conclusion receives further support in that the electrophoreograms of fat body extracts. DAB- or DMB-positive haemoglobin display noticeably large peaks of radioactive ALA incorporation. This is an indication of haemoglobin synthesis (see also BERCTROMer al., 1976; LAUFERet al., 1976). The results indicate that if there exist any temporal relationship between various DAB deposit-containing structures in the in-
tracellular localization and distribution of DAB deposits, the sequence would be: synthesis of the haemoglobin on the rough ER+ intracellular deposits in the ER cisternae-+ transport to the Golgi vesicles(?)--+ secretion by means of exocytosis into extracellular spaces from which the haemoglobin exudes into the haemolymph. The first and last of these steps is fairly well substantiated by our data. We have not yet found evidence that the Golgi vesicles carry their contents to the extracellular spaces where DAB reaction deposits have been found. We conclude from our results, that the fat body of Chironomus is actively involved in haemoglobin metabolism in the fourth instar stage. The subepiderma1 fat body cells seem to be more active in de novo synthesis than the visceral fat body. This is supported’ by the finding of differential amounts of peroxidase activity, endoplasmic reticulum, and Golgi vesicles, and the dependence of the peroxidase-positive activity upon protein synthesis. There seem therefore to be at least two different types of fat body function differently in haemoglobin metabolism. The observations, that in the developing mid- and late fourth instar much of the subepidermal fat body displays specific intracellular haemoglobin localization and synthesis, and furthermore, that much of the visceral fat body exhibits little or none, are suggestive of such a specialized function. The fact that a large number of the visceral fat body cells examined contain autophagic vacuoles, and occasionally, pinosomes is an indication that these fat body cells are geared to the uptake and storage rather than to the synthesis of haemoglobin. The findings that ferritin appeared in the autophagic vacuoles of the visceral fat body within 15 min after the injection into the animal fall well into line with above observations (SCHIN and CLEVER, 1968). Taken together, these results tend to indicate that Chironomus has developed effective multiple mechanisms to control the concentration of the blood proteir-through synthesis, storage, utilization, and finally through degradation (SCHINand CLEVER,1968; SCHIN et al., 1974; BERGTROMet al., 1976; LAUFER et al., 1976; TRAVIS and SCHIN, 1976, 1977). These mechanisms must be of fundamental importance to the life of the insect. Acknowledgements-The authors are indebted to Dr R. SUNY college at Plattsburgh, for his kind criti-
CLARK at
cism and advice in the preparation of the manuscript. Thanks are due also to Dr H. Z. Lru, Dr R. ELLSWORW, and Dr W. GRAZIADEIwho allowed us to use their equip ment and materials for this work. This research was in part supported by a grant from the University of Connecticut Research Foundation.
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Chironomus thummi
I ‘41 _ _
KISSUSCHIN,HANS LAUFER,AND
BERGTROM G., LAUFERH.. and ROGERSR. (1976) Fat body: a site of hemoglobin synthesis in Chironomus rfiummi (Diptera). J. Ceff &of. 69, 264274. BRAUNITZER G. and BRAUNV. (1965) Zur Phylogenie des Haemoglobin-molektils. Untersuchungen an InsektenHaemoglobinen (Chironomus thummi). Hoppe-Seyfer’s 2. physiol. Chem. 340, 88-91. DAVIS B. J. (1964) Disc electrophoresis-II. Method and applications to human serum proteins. Ann. N.I! Acad. Sci. 121, 404427. ENGLISHD. S. (1969) Ontogenetic changes in hemoglobin synthesis of two strains of Chironomus tentans. J. &nbryol. exp. Morph. 22, 465476. GOLDFI~CHER S. (1967) The cvtochemical localization of myoglobin in striated muscles of man and walrus. J. Cell Biol. 34, 398403. INADAY., KUROZUMIT., and SHIBATAK. (1961) Peroxidase activity of hemoproteins. I. Generation of activity by acid or alkali denaturation of methemoglobin and catalase. Arch. Biochem. Biophys. 93, 30-36. KARNOVSKYM. J. (1965) A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. Cell Biol. 27, 138A. LAUFERH. and POLUHOWICH J. J. (1971) A factor controlling the concentration of hemoglobin in Chironomus during metamorphosis. Limnofogica 8. 1255126. LAUFERH. and WILXIN 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. 1855200. PrenticeHall, N.J. LAUFERH.. BERGTROMG., and ROGERSR. (1976) Stage and tissue-specific hemoglobin synthesis in an invertebrate. In Invertebrate Tissue Culture. Applications in Medicine, Biology, and Agriculture. pp. 227-240. Academic Press, New York. MANWELLC. (1966) Starch gel electrophoresis of the multiple haemoglobins of small and large larval Chironomus-a developmental haemoglobin sequence in an invertebrate. J. Embryo/. exp. Morph. 16, 259-270. I
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