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Rate of cocoon production by the larva of the fly Rhynchosciara americana and storage sites of precursors
J. Insect Physiol., 1975, Vol. 21, pp. 1547 to 1550. Pergamon Press. Printed in Great Britain.
RATE OF COCOON PRODUCTION BY THE LARVA OF THE FLY RHYNCHOSCIARA AMERICANA AND STORAGE SITES OF PRECURSORS W. R. TERRA and A. G. DE BIANCHI Departamento
de Bioquimica,
Instituto de Quitnica da Universidade SIo Paula, Brasil
de S?io Paula, C.P. 20780,
(Received 14 January 1975 ; revised 3 March 1975) Abstract-A
chemical analysis of Rhynchosciara americana COCOONS at four stages of spinning was performed and the amount and rate of the cocoon production was measured. These results, together with the amount and distribution of the nutrient reserves in the larva during spinning, were used to identify the possible storage sites of cocoon precursors. The physiology, mechanics, and regulation of spinning by R. americana are discussed.
INTRODUCTION
Cocoon samples and rate of cocoon production
THE LARVAE of Rhynchosciara ameticana pupate inside a silken communal cocoon. This cocoon is composed of a peculiar type of silk, several carbohydrates, and calcium carbonate (TERRA and DE BIANCHI, 1974) During the spinning stage there is a remarkable decrease in the larval tissue reserves (TERRA et al., 1975). This decrease must be a consequence of the cocoon production because spinning begins after the animal stops feeding and the salivary glands have no reservoir for secretions. In this paper the amount and production rate of cocoon components is measured and the possible storage sites of cocoon precursors are identified. MATERIALS
Fractionation tions
AND METI-IODS
Animals R. americana was reared in the laboratory according to LARA et al. (1965). We have used only cocoons
made by females. Table
1. Correspondence
Groups of mature larvae were put into different receptacles on a sheet of a moist filter paper where they spin a communal cocoon. At the end of selected spinning stages the cocoons were dried to constant weight at lOO”C, after the removal of the larvae. The dry weight of the cocoon divided by the number of larvae present inside it corresponds to the mass of cocoon produced by each larva. The correspondence between the stage of spinning, the time of spinning, and the various periods of the fourth larval instar (TERRA et al., 1973) are given in Table 1.
of the cocoon and chemical detewaina-
The fractionation of the cocoon by solvents and the determination of ash, calcium, soluble carbohydrates, and soluble proteins have been done according to TERRA and DE BIANCHI (1974).
between stages of spinning and the physiological age of the larvae of
R. americana Stage of spinning
Approximate time intervals of spinning '(hours)
Larval physiological stage (Periods of fourth instar)
-0-15
Third
Early
15-140
Third
Middle
120-192
Very early
.Late
Fourth
Fifth to pharate PePa
.192-240
1547
W. R. TERRAAND A. G. DE BIANCHI
1.548
Table 2. Cocoon chemical composition at the end of the stages of spinning* Percentage Determination
Very
of cocoon
dry-weight
Early
Middle
Late6
material
8.5
6.3
3.1
8.4
Acid-soluble
proteins2
0.3
1.4
1.6
1.9
Acid-soluble
carbohydrates'
0.4
0.4
0.4
0.8
Water-soluble
3.5
11.0
40.4
43.5
25.8
25.4
24.3
23.6
19.2
14.3
9.9
8.2
25.2
19.1
n.d.
4.3
82.9
77.9
79.7
90.7
CaC033 Insoluble
proteins
Insoluble
neutral
.Insoluble
(N~6.25)~ carbohydrates5
ash4
Total
1.
The figures are averages n.d. = not determined.
of three
2.
Determined washed in
acid
3.
Water-insoluble , acetic-acid-soluble proteins and carbohydrates soluble subtracted.
4.
Determined
6.
Released by acid hydrolysis of cocoon insoluble fraction and and extrapolated to zero time of hydrolysis according.to Terra Bianchi (1974).
6.
According to Terra and Bianchi (1974) with the exception insoluble proteins which were determined by the Kjeldahl in the present work.
in the acetic
water.
in the
cocoon
independent
washings
water
Nitrogen in insoluble proteins About 50 mg of dried samples of cocoon after water and acid washings according to TERRA and DE BIANCHI (1974) were used for the determination of nitrogen by the Kjeldahl method according to ALBANFSEand ORTO (1963). RESULTS
early
AND DISCUSSION
Chemical determinations Table 2 shows the results of a chemical analysis of cocoons at the end of selected stages of spinning. The water-soluble, acetic acid-soluble material was considered to be calcium carbonate (TERRA and DE BIANCHI, 1974) and there is, as expected, a correspondence between the relative amount of that fraction (Table 2) and the calcium present in the cocoon, which is 7.5, 19.5 and 19.0 per cent of the cocoon dry weight at the end of the early, middle, and late spinning stages, respectively. Insoluble proteins determined in the late cocoon by Kjeldahl analysis correspond to 23.6 per cent of the cocoon dry weight (Table 2), whereas the proteins when’ calculated from the amino acids released by acid hydrolysis, accounted for 33.6 per cent of the cocoon dry weight (TERRA and DE BIANCHI, 1974). This difference must be a
and
of the
determinations. cocoon
previously.
material. The mass in these conditions
acetic
acid
insoluble
of was fraction.
of method
consequence of an incomplete recovery of nitrogen in the Kjeldahl method as well as of the peculiar amino acid composition of the cocoon proteins which makes the figure of 6.25 unusable in calculating the protein mass from the nitrogen determinations. As a consequence, all the data for insoluble protein shown in Table 2 must be underestimated. Nevertheless, we may conclude that the relative amount of protein in the cocoon does not change during spinning (see Table 2). The relative amounts of insoluble carbohydrate and insoluble ash decrease steadily during spinning (Table 2). Because the relative amount of insoluble neutral carbohydrates decreases during spinning, while the relative amount of insoluble proteins remains approximately constant, it seems that the composition of the organic part of the cocoon, which is extruded by the salivary glands (see below), changes during spinning. According to Table 3 the carbohydrate-protein ratio changes from the very early to the early spinning stage and remains constant afterwards. The water-insoluble material is made up of variable amounts of proteins and carbohydrates which correspond to less than 30 and 20 per cent of that fraction, respectively. The incomplete recovery of the cocoon components, shown in Table 2, should be a consequence
1549
Rate of cocoon production by larva.of Rhyncho$ciara americana Table 3. The amount and the average rate of extrusion of the cocoon components in each stage d spinning Average rate of production'
Amount produced' (pg per animal)
Component
.(ug per'animal per day)
Very early
Early
Middle
Late
Insoluble proteins
66.6
42.6
272
437
Insoluble neutral carbo hydrates
49.5
12.0
90
133
9.0
38.3
571.
892
Insoluble ash
65.0
17.1
Water-soluble material
21.9
5.2
CaC03
Total cocoon3
258
172
67.1 20.3 1100
Very early 107
1940
Middle
9.7
91
79.2
2.7
30.0
14.4
8.7
104 244
Early
35.0 413
66;5 446
13.4
1.2 39.3
219
190
3:9
.Late
6.8 367
122 970
1.
The figures were calculated from Table 2 and from total cocoon production data show
2.
The figures were calculated from the amount produced in each stage shown in this Table taking into account the duration of each spinning stage reported in Table 1.
3.
The figures are averages of three independent determinations carried out as described under Material and Methods;
of the underestimation of the insoluble proteins (see above) and of the non-determination of insoluble uranic acids and galactosamine which are present in significant amounts, at least in the late cocoon (TERRA and DE BIANCHI, 1974). Origin of cocoon components The mass decrease observed in the Malpighian tubules during spinning (TERRA et al., 1975) follows approximately the increase in the mass of calcium carbonate measured in the cocoon (Table 3). The calcium content of the Malpighian tubules at the beginning of spinning (451 pg/larva according to TERRA et al., 1975) is about 70 per cent of the amount of calcium present in the finished cocoon (659 pg/larva, calculated from Table 3). If we bear in mind the difficulties in obtaining Malpighian tubules without any loss of contents we may conclude that the cocoon calcium carbonate is stored in that organ previously. The insoluble ash must come chiefly from the gut lumen contents since the Malpighian tubules contents are mainly ca.lciurn carbonate and hence acid soluble. The decrease of the mass of the gut lumen contents during spinning (TERRA et al., 1975) supports this hypothesis. Preliminary observations suggest that the ash present in the gut lumen is mainly sand ingested by the larvae with the food. The water-soluble material may be derived from the gut lumen contents because about 5 to 10 per cent of this material is water soluble (unpublished results) and/or from the salivary secretion. Contamination by the larval exuvial fluid and/or haemolymph may also occur during the removal of the larvae from the late cocoons, as suggested by TERRA and DE BIANCHI (1974).
in this Table.
The protein present in the cocoon is derived from the salivary secretion but, because the salivary gland stores only about 10 per cent of the total protein that is extruded during spinning, it must be stored in other places. During the very early spinning stage there is an abrupt fall in protein (78 ,ug/animal, according to DE BIANCHI and TERRA, 1975) present in the salivary gland, which is sufficient to account for the cocoon spinning in this stage (Table 3). In the early spinning stage protein remains constant or increases in the larval tissues except in the gut wall where it decreases about 261 pg/larva (TERRA et al., 1975). The amount of protein decreases in all the tissues during the middle and late spinning stages. The decrease is only significant in the haemolymph (700 /.cg/animal) and fat body (359 ,ug/ animal) in the middle spinning stage and haemolymph (662 ,ug/animal) and skeletal muscle (266 ,ug/ animal) in the late spinning stage, according to TERRA et al. (1975). The carbohydrate present in the salivary glands could be sufficient to account for the cocoon produced in the very early spinning stage if it is linked to stored proteins. In this circumstance the carbohydrate became acid insoluble and hence it would not have been detected by DE BIANCHI and TERRA (1975). During the early, middle, and late spinning stage carbohydrate decreases significantly only in fat body (388, 139, and 203 ~g/animal, respectively) and in the skeletal muscle in the late spinning stage (123 pgjanimal) according to TERRA et al. (1975). Rate of cocoon production The rate of cocoon production as shown in Table 3 changes during spinning. The initial high rate corresponds to the use of salivary secretion
W. R. TERRAANDA. G. DE BIANCHI
1550
stored in the salivary glands. Cocoon protein and carbohydrates follow the same rate pattern as total cocoon-. (Table 3). In this connexion it is interesting to note that the highest rate of salivary gland protein labelling by [14C]-phenylalanine occurs around--the end of the middle spinning stage (DE BIANCHI and TERRA, 1975). The rate of calcium carbonate deposition in the cocoon, contrasting to what occurs with proteins and carbohydrates, increases in the middle spinning stage.
Physiology
of cocoon production
We may summarize all the data we have giving a general picture of the process of spinning by the larvae of R. americana. At the onset of spinning the larvae extrude a great amount of salivary secretion stored in the salivary glands with which they spin a net-like tent made of thick filaments cbvering the whole group of larvae. At the same time there is a partial gut emptying resulting in the deposition in the tent of a mineral material consisting mainly of sand. At the early spinning stage the group of larvae becomes completely surrounded by the thick silk filaments and begin to fill the spaces between the filaments with an amorphous and membranous material made of protein and carbohydrate on which they deposit a small amount of the calcium carbonate derived from the Malpighian tubules. The proteins used in spinning at this stage must come from the gut wall through the haemolymph whereas the carbohydrates must come from the fat body. During the middle spinning stage the communal cocoon becomes massive in size and is strengthened by a large amount of calcium carbonate. The cocoon proteins at this stage seem to be derived from precursors stored in the haemolymph and fat body whereas the cocoon carbohydrates should come from the fat body. During the late spinning stage a great amount of cocoon is produced, resulting in the separation of the larvae into individual cells. At the same time, the gut and Malpighian tubules discharge their contents in the cocoon. The proteins used seem to come from the haemolymph and skeletal muscle. During the whole spinning stage, except for the very early one, the haemolymph proteins seem to be
used as major cocoon protein precursors. This agrees with our previous findings which indicate that haemolymph proteins are used at least in part directly in the formation of the salivary secretion (DE BIANCHI
et al., 1973).
The whole process of spinning seems to be controlled by ecdysterone because an injection of 2 ~1 of a 2 x lop3 M solution of this hormone in a late feeding stage larva induces the extrusion of a great amount of salivary secretion and after 24 hr there is a complete emptying of the Malpighian tubules (LARA, personal communication). Acknowledgements-We are much indebted to Professor F. J. S. LARA for advice, encouragement, and for laboratory facilities. We also wish to thank Mrs. I. C. M. TERRA for the Kjeldahl analysis, Miss Y. TAVARES for calcium atomic absorption spectroscopy, and Dr. M. D. MARQUESfor the determination of the solubilities of the cocoon in different solvents. This work was supported by grants from the FundagE de hparo & Pesquisa do Estado de S”ao Paul0 (F.A.P.E.S.P.).
REFERENCES ALBANE~E A. A. and ORTO L. A. (1963) Proteins and amino acids. In Newer Methods of Nutritional Biochemistry (Ed. by ALBANE% A. A.), 1, l-l 12. Academic Press, New York. DE BIANCHI A. G. and TERRA W. R. (1975) Chemical composition and rate of synthesis of the larval salivary secretion of the fly Rhynchosciura americana. J. Insect Physiol. 21, 643-657. DE BIANCHI A. G., TERRA W. R., and LARA F. J. S. (1973) Formation of salivary secretion in Rhynchosciara americana-I. Kinetics of labeled amino acid incorporation. r. Cell Biol. 58, 470-476. LARA F. J. S., TAMAKI H., and PAVANC. (1965) Laboratory &It& of R. angeiae. Am. Nat. 99, 189-191. TERRA W. R. and DE BIANCHI A. G. (1974) Chemical composition of the cocoon of the fly, Rhynchosciara americana. Insect Biochem. 4, 173-183. TERRA W. R.. DE BIANCHI A. G.. GAMBARINI A. G.. and LARA F. J. S. (1973) Haemolymph amino acids and related compounds during cocoon production by the larvae of the fly, Rkynchosciara americana. J. Insect Physiol. 19, 2097-2106. TERRA W. k., FFRREIRA C., and DE BIANCHI A. G. (1975) Distribution of the nutrient reserves during spinning in tissues of the larva of the 0y Rhynchosciara americana. J. Insect Physiol. 21, in press.
RATE OF COCOON PRODUCTION BY THE LARVA OF THE FLY RHYNCHOSCIARA AMERICANA AND STORAGE SITES OF PRECURSORS W. R. TERRA and A. G. DE BIANCHI Departamento
de Bioquimica,
Instituto de Quitnica da Universidade SIo Paula, Brasil
de S?io Paula, C.P. 20780,
(Received 14 January 1975 ; revised 3 March 1975) Abstract-A
chemical analysis of Rhynchosciara americana COCOONS at four stages of spinning was performed and the amount and rate of the cocoon production was measured. These results, together with the amount and distribution of the nutrient reserves in the larva during spinning, were used to identify the possible storage sites of cocoon precursors. The physiology, mechanics, and regulation of spinning by R. americana are discussed.
INTRODUCTION
Cocoon samples and rate of cocoon production
THE LARVAE of Rhynchosciara ameticana pupate inside a silken communal cocoon. This cocoon is composed of a peculiar type of silk, several carbohydrates, and calcium carbonate (TERRA and DE BIANCHI, 1974) During the spinning stage there is a remarkable decrease in the larval tissue reserves (TERRA et al., 1975). This decrease must be a consequence of the cocoon production because spinning begins after the animal stops feeding and the salivary glands have no reservoir for secretions. In this paper the amount and production rate of cocoon components is measured and the possible storage sites of cocoon precursors are identified. MATERIALS
Fractionation tions
AND METI-IODS
Animals R. americana was reared in the laboratory according to LARA et al. (1965). We have used only cocoons
made by females. Table
1. Correspondence
Groups of mature larvae were put into different receptacles on a sheet of a moist filter paper where they spin a communal cocoon. At the end of selected spinning stages the cocoons were dried to constant weight at lOO”C, after the removal of the larvae. The dry weight of the cocoon divided by the number of larvae present inside it corresponds to the mass of cocoon produced by each larva. The correspondence between the stage of spinning, the time of spinning, and the various periods of the fourth larval instar (TERRA et al., 1973) are given in Table 1.
of the cocoon and chemical detewaina-
The fractionation of the cocoon by solvents and the determination of ash, calcium, soluble carbohydrates, and soluble proteins have been done according to TERRA and DE BIANCHI (1974).
between stages of spinning and the physiological age of the larvae of
R. americana Stage of spinning
Approximate time intervals of spinning '(hours)
Larval physiological stage (Periods of fourth instar)
-0-15
Third
Early
15-140
Third
Middle
120-192
Very early
.Late
Fourth
Fifth to pharate PePa
.192-240
1547
W. R. TERRAAND A. G. DE BIANCHI
1.548
Table 2. Cocoon chemical composition at the end of the stages of spinning* Percentage Determination
Very
of cocoon
dry-weight
Early
Middle
Late6
material
8.5
6.3
3.1
8.4
Acid-soluble
proteins2
0.3
1.4
1.6
1.9
Acid-soluble
carbohydrates'
0.4
0.4
0.4
0.8
Water-soluble
3.5
11.0
40.4
43.5
25.8
25.4
24.3
23.6
19.2
14.3
9.9
8.2
25.2
19.1
n.d.
4.3
82.9
77.9
79.7
90.7
CaC033 Insoluble
proteins
Insoluble
neutral
.Insoluble
(N~6.25)~ carbohydrates5
ash4
Total
1.
The figures are averages n.d. = not determined.
of three
2.
Determined washed in
acid
3.
Water-insoluble , acetic-acid-soluble proteins and carbohydrates soluble subtracted.
4.
Determined
6.
Released by acid hydrolysis of cocoon insoluble fraction and and extrapolated to zero time of hydrolysis according.to Terra Bianchi (1974).
6.
According to Terra and Bianchi (1974) with the exception insoluble proteins which were determined by the Kjeldahl in the present work.
in the acetic
water.
in the
cocoon
independent
washings
water
Nitrogen in insoluble proteins About 50 mg of dried samples of cocoon after water and acid washings according to TERRA and DE BIANCHI (1974) were used for the determination of nitrogen by the Kjeldahl method according to ALBANFSEand ORTO (1963). RESULTS
early
AND DISCUSSION
Chemical determinations Table 2 shows the results of a chemical analysis of cocoons at the end of selected stages of spinning. The water-soluble, acetic acid-soluble material was considered to be calcium carbonate (TERRA and DE BIANCHI, 1974) and there is, as expected, a correspondence between the relative amount of that fraction (Table 2) and the calcium present in the cocoon, which is 7.5, 19.5 and 19.0 per cent of the cocoon dry weight at the end of the early, middle, and late spinning stages, respectively. Insoluble proteins determined in the late cocoon by Kjeldahl analysis correspond to 23.6 per cent of the cocoon dry weight (Table 2), whereas the proteins when’ calculated from the amino acids released by acid hydrolysis, accounted for 33.6 per cent of the cocoon dry weight (TERRA and DE BIANCHI, 1974). This difference must be a
and
of the
determinations. cocoon
previously.
material. The mass in these conditions
acetic
acid
insoluble
of was fraction.
of method
consequence of an incomplete recovery of nitrogen in the Kjeldahl method as well as of the peculiar amino acid composition of the cocoon proteins which makes the figure of 6.25 unusable in calculating the protein mass from the nitrogen determinations. As a consequence, all the data for insoluble protein shown in Table 2 must be underestimated. Nevertheless, we may conclude that the relative amount of protein in the cocoon does not change during spinning (see Table 2). The relative amounts of insoluble carbohydrate and insoluble ash decrease steadily during spinning (Table 2). Because the relative amount of insoluble neutral carbohydrates decreases during spinning, while the relative amount of insoluble proteins remains approximately constant, it seems that the composition of the organic part of the cocoon, which is extruded by the salivary glands (see below), changes during spinning. According to Table 3 the carbohydrate-protein ratio changes from the very early to the early spinning stage and remains constant afterwards. The water-insoluble material is made up of variable amounts of proteins and carbohydrates which correspond to less than 30 and 20 per cent of that fraction, respectively. The incomplete recovery of the cocoon components, shown in Table 2, should be a consequence
1549
Rate of cocoon production by larva.of Rhyncho$ciara americana Table 3. The amount and the average rate of extrusion of the cocoon components in each stage d spinning Average rate of production'
Amount produced' (pg per animal)
Component
.(ug per'animal per day)
Very early
Early
Middle
Late
Insoluble proteins
66.6
42.6
272
437
Insoluble neutral carbo hydrates
49.5
12.0
90
133
9.0
38.3
571.
892
Insoluble ash
65.0
17.1
Water-soluble material
21.9
5.2
CaC03
Total cocoon3
258
172
67.1 20.3 1100
Very early 107
1940
Middle
9.7
91
79.2
2.7
30.0
14.4
8.7
104 244
Early
35.0 413
66;5 446
13.4
1.2 39.3
219
190
3:9
.Late
6.8 367
122 970
1.
The figures were calculated from Table 2 and from total cocoon production data show
2.
The figures were calculated from the amount produced in each stage shown in this Table taking into account the duration of each spinning stage reported in Table 1.
3.
The figures are averages of three independent determinations carried out as described under Material and Methods;
of the underestimation of the insoluble proteins (see above) and of the non-determination of insoluble uranic acids and galactosamine which are present in significant amounts, at least in the late cocoon (TERRA and DE BIANCHI, 1974). Origin of cocoon components The mass decrease observed in the Malpighian tubules during spinning (TERRA et al., 1975) follows approximately the increase in the mass of calcium carbonate measured in the cocoon (Table 3). The calcium content of the Malpighian tubules at the beginning of spinning (451 pg/larva according to TERRA et al., 1975) is about 70 per cent of the amount of calcium present in the finished cocoon (659 pg/larva, calculated from Table 3). If we bear in mind the difficulties in obtaining Malpighian tubules without any loss of contents we may conclude that the cocoon calcium carbonate is stored in that organ previously. The insoluble ash must come chiefly from the gut lumen contents since the Malpighian tubules contents are mainly ca.lciurn carbonate and hence acid soluble. The decrease of the mass of the gut lumen contents during spinning (TERRA et al., 1975) supports this hypothesis. Preliminary observations suggest that the ash present in the gut lumen is mainly sand ingested by the larvae with the food. The water-soluble material may be derived from the gut lumen contents because about 5 to 10 per cent of this material is water soluble (unpublished results) and/or from the salivary secretion. Contamination by the larval exuvial fluid and/or haemolymph may also occur during the removal of the larvae from the late cocoons, as suggested by TERRA and DE BIANCHI (1974).
in this Table.
The protein present in the cocoon is derived from the salivary secretion but, because the salivary gland stores only about 10 per cent of the total protein that is extruded during spinning, it must be stored in other places. During the very early spinning stage there is an abrupt fall in protein (78 ,ug/animal, according to DE BIANCHI and TERRA, 1975) present in the salivary gland, which is sufficient to account for the cocoon spinning in this stage (Table 3). In the early spinning stage protein remains constant or increases in the larval tissues except in the gut wall where it decreases about 261 pg/larva (TERRA et al., 1975). The amount of protein decreases in all the tissues during the middle and late spinning stages. The decrease is only significant in the haemolymph (700 /.cg/animal) and fat body (359 ,ug/ animal) in the middle spinning stage and haemolymph (662 ,ug/animal) and skeletal muscle (266 ,ug/ animal) in the late spinning stage, according to TERRA et al. (1975). The carbohydrate present in the salivary glands could be sufficient to account for the cocoon produced in the very early spinning stage if it is linked to stored proteins. In this circumstance the carbohydrate became acid insoluble and hence it would not have been detected by DE BIANCHI and TERRA (1975). During the early, middle, and late spinning stage carbohydrate decreases significantly only in fat body (388, 139, and 203 ~g/animal, respectively) and in the skeletal muscle in the late spinning stage (123 pgjanimal) according to TERRA et al. (1975). Rate of cocoon production The rate of cocoon production as shown in Table 3 changes during spinning. The initial high rate corresponds to the use of salivary secretion
W. R. TERRAANDA. G. DE BIANCHI
1550
stored in the salivary glands. Cocoon protein and carbohydrates follow the same rate pattern as total cocoon-. (Table 3). In this connexion it is interesting to note that the highest rate of salivary gland protein labelling by [14C]-phenylalanine occurs around--the end of the middle spinning stage (DE BIANCHI and TERRA, 1975). The rate of calcium carbonate deposition in the cocoon, contrasting to what occurs with proteins and carbohydrates, increases in the middle spinning stage.
Physiology
of cocoon production
We may summarize all the data we have giving a general picture of the process of spinning by the larvae of R. americana. At the onset of spinning the larvae extrude a great amount of salivary secretion stored in the salivary glands with which they spin a net-like tent made of thick filaments cbvering the whole group of larvae. At the same time there is a partial gut emptying resulting in the deposition in the tent of a mineral material consisting mainly of sand. At the early spinning stage the group of larvae becomes completely surrounded by the thick silk filaments and begin to fill the spaces between the filaments with an amorphous and membranous material made of protein and carbohydrate on which they deposit a small amount of the calcium carbonate derived from the Malpighian tubules. The proteins used in spinning at this stage must come from the gut wall through the haemolymph whereas the carbohydrates must come from the fat body. During the middle spinning stage the communal cocoon becomes massive in size and is strengthened by a large amount of calcium carbonate. The cocoon proteins at this stage seem to be derived from precursors stored in the haemolymph and fat body whereas the cocoon carbohydrates should come from the fat body. During the late spinning stage a great amount of cocoon is produced, resulting in the separation of the larvae into individual cells. At the same time, the gut and Malpighian tubules discharge their contents in the cocoon. The proteins used seem to come from the haemolymph and skeletal muscle. During the whole spinning stage, except for the very early one, the haemolymph proteins seem to be
used as major cocoon protein precursors. This agrees with our previous findings which indicate that haemolymph proteins are used at least in part directly in the formation of the salivary secretion (DE BIANCHI
et al., 1973).
The whole process of spinning seems to be controlled by ecdysterone because an injection of 2 ~1 of a 2 x lop3 M solution of this hormone in a late feeding stage larva induces the extrusion of a great amount of salivary secretion and after 24 hr there is a complete emptying of the Malpighian tubules (LARA, personal communication). Acknowledgements-We are much indebted to Professor F. J. S. LARA for advice, encouragement, and for laboratory facilities. We also wish to thank Mrs. I. C. M. TERRA for the Kjeldahl analysis, Miss Y. TAVARES for calcium atomic absorption spectroscopy, and Dr. M. D. MARQUESfor the determination of the solubilities of the cocoon in different solvents. This work was supported by grants from the FundagE de hparo & Pesquisa do Estado de S”ao Paul0 (F.A.P.E.S.P.).
REFERENCES ALBANE~E A. A. and ORTO L. A. (1963) Proteins and amino acids. In Newer Methods of Nutritional Biochemistry (Ed. by ALBANE% A. A.), 1, l-l 12. Academic Press, New York. DE BIANCHI A. G. and TERRA W. R. (1975) Chemical composition and rate of synthesis of the larval salivary secretion of the fly Rhynchosciura americana. J. Insect Physiol. 21, 643-657. DE BIANCHI A. G., TERRA W. R., and LARA F. J. S. (1973) Formation of salivary secretion in Rhynchosciara americana-I. Kinetics of labeled amino acid incorporation. r. Cell Biol. 58, 470-476. LARA F. J. S., TAMAKI H., and PAVANC. (1965) Laboratory &It& of R. angeiae. Am. Nat. 99, 189-191. TERRA W. R. and DE BIANCHI A. G. (1974) Chemical composition of the cocoon of the fly, Rhynchosciara americana. Insect Biochem. 4, 173-183. TERRA W. R.. DE BIANCHI A. G.. GAMBARINI A. G.. and LARA F. J. S. (1973) Haemolymph amino acids and related compounds during cocoon production by the larvae of the fly, Rkynchosciara americana. J. Insect Physiol. 19, 2097-2106. TERRA W. k., FFRREIRA C., and DE BIANCHI A. G. (1975) Distribution of the nutrient reserves during spinning in tissues of the larva of the 0y Rhynchosciara americana. J. Insect Physiol. 21, in press.