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Hormonal control of resilin deposition in the blowfly, Calliphora erythrocephala
J. Insect Physiol., 1974, Vol. 20, pp. 935 to 946. Perganon Press. Printed in Great Britain
HORMONAL CONTROL OF RESILIN DEPOSITION IN THE BLOWFLY, CALLIPHORA ERYTHROCEPHALA M. SABARATNAM” Department of Zoology, West Mains Road, University of Edinburgh, Scotland (Received 3 May
1973 ; revised 27 October 1973)
Abstract-The deposition of the resilin tendon in the blowfly Calliphora crythrocephala was investigated in normal and in various experimental conditions. The results showed that the weight of the protein resilin that is deposited is controlled by diet as well as by the hormone secreted by the medial neurosecretory cells. Endocrinologically abnormal Calliphora adults deposited a tendon with normal ultrastructure but showed signs of premature ageing while Calliphora fed on a sugar diet deposited a tendon with abnormal ultrastructure.
INTRODUCTION
ENDOCUTICLEdeposition in adult insects is known to be controlled by such factors as the availability of food (NEVILLE, 1965; CULLEN, 1969) and the hormone bursicon which is secreted by the medial neurosecretory cells of the brain (FOGAL and FRAENKEL,1969; VINCENT, 1971). A link between diet and hormone secretion was established by THOMSEN(1952) and subsequently various authors have dealt with the importance of diet and hormones in the post imaginal growth of insects (review by WIGGLESWORTH, 1970) but it is still not clear how general these effects are nor what cellular changes are involved. It was therefore decided to examine the way in which the deposition of the elastic cuticular protein resilin (WEISFOGH, 1960) takes place during adult life and how far the mechanism may be related to that involved in other processes of growth and metamorphosis. MATERIALS
AND METHODS
Calliphora erythrocephala were bred in a 12 hr light 12 hr dark photoperiod at 20°C and were normally fed on a diet which consisted of meat, sugar, and water. The pleurotergal muscle with its resilin tendon or the tendon alone were dissected out from the various groups of experimental flies and weighed in a Cahn Gram Electrobalance after drying to a constant weight (SABARATNAM, 1973). Similarly the weights of the second abdominal tergite and sternite were also recorded. * Present address: Department of Ceylon, Sri Lanka. 31
of Zoology, Thurstan
935
Road, Colombo 3, University
936
M.
SABARATNAM
Experiments were performed to examine the role of the endocrine systems. The medial neurosecretory cells were located by THOMSEN’S(1952) method and the cells were cauterized with an electric wire loop supplied through a transformer at 4 to 6 V a.c. using HILL’s (1962) method. The corpus allatum-cardiacurn complex was removed by THOMSEN’S (1942) method but in my experiments neck muscles were left in place. The corpus allatum-cardiacurn complex was transplanted into the abdomen of flies deprived of their medial neurosecretory cells by THOMSEN’S(1952) method. Fixation for electron microscopy and sectioning of the Araldite blocks were carried out according to SAECARATNAM (1973).
RESULTS E#ect of diet in unoperated plies
Two batches of over five hundred flies were kept respectively- on a meat and sugar diet or on a sugar diet. At 1 day, 7, 14, 21, 28, 35, and 42 days after emergence batches of 50 flies were removed for dissection from the two groups. The mean weights of the resilin tendon with and without muscle and of the sclerotized cuticle of the second abdominal tergite and sternite in the two groups of flies are given in Figs. 1, 2, 3, 4, and 5. As the weights observed at 6 weeks were similar to the weights observed from flies surviving to a 2 to 24 months, flies were only kept for 6 weeks. With both sugar-fed and meat-fed flies, the weights of the sternal plate increased significantly during the first 2 weeks of adult life. Thereafter the weight of the sternal plate remained more or less constant to 5 weeks but fell by the sixth week of post-emergent adult life to a weight that did not differ significantly with either diet from that at 1 week (Fig. 1). On the other hand, the weight of the tergal plate increased during the first week then remained constant to week 4 and then decreased significantly to week 6 in the meat fed and sugar fed flies (Fig. 2). The weight of the sclerotized cuticle in meat-fed flies was significantly greater than that of the sugar-fed flies between weeks 1 and 6. Although the weight of the cuticle fell between the fourth and sixth weeks, the weight remained greater than in a newly emerged fly and the final weight in the meat-fed flies was greater than in the sugar-fed flies. It is noteworthy that the increase in weight in the first 2 weeks of adult life was greater in the meat-fed flies but the decrease in weight between weeks 4 and 6 was similar with both diets. A similar pattern was observed for the resilin tendon (Fig. 4) but the weight of muscle after the initial increase during the first week in sugar-fed flies or during the first 2 weeks in meat-fed flies changed very little (Figs. 3, 5). Observations and/or measurements of the size of the oocytes, corpora allata, and fat body cells were also made from flies 14 days after emergence for purposes
HORMONAL CONTROL OF RBSILIN DEPOSITION IN BLOWFLY
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DAYS ___+ FIG. 1. Graph showing the weight of the second sternal plate against the age of the flies. The points show the means of samples from 5 insects and the bar lines show the 95 per cent confidence limits of the means. Sugar-fed flies, O-0-0 ; meat-fed flies, 0 . . . 0 . . . 0.
180
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DAYS -+ FIG. 2. Graph showing the weight of the second tergal plate against the age of the flies. Sample size and confidence limits as in Fig. 1. Sugar-fed flies, O-O-O; meat-fed flies, 0 . . . 0 . . . 0.
937
938
M. SABARATNAM Resilin + MUSCLE
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FICL 3. Graph showing the weight of five pleurotergal muscles with resilin tendons against the age of the flies. The points show the means for five samples each of five muscles with tendons and the bar lines show 95 per cent confidence limits of these means. Sugar-fed flies, O-O-O ; meat-fed flies, 0 . . . 0 . . . 0 ; operated flies, l ; flies with medial neurosecretory cells cauterized, -mnc; flies with corpus allatum removed, -ca; flies with medial neurosecretory cells cauterized and two corpora allata implanted, - mnc + ca.
Meat
flies
Sugar flies
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42
DAYS -
FIG. 4.
Graph showing the weight of five tendons against the age of the flies. Sample size, confidence limits, and symbols as in Fig. 3.
of comparison in the later experiments. In the meat-fed flies the length of the mature oocytes was from 1.40 to 1.50 mm, while in the case of the sugar-fed flies the range was from 0*128 to O-136 mm. The average diameter of the corpus allatum in the meat-fed flies was 0.126 T 0*012 mm while in sugar-fed flies it was O-092 r 0.006 mm at 2 weeks. In the meat-fed flies the fat body cells showed a cyclic activity which coincided with the development of the egg cells. In the sugar-fed flies, the fat body cells were sparse, and the few cells present were small and transparent, which is due to the quantity of glycogen, fat, and proteins (THOMSEN, 1952).
HORMONAL
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OF RESILIN
DEPOSITION
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22
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FIG. 5. Graph showing the weight of five pleurotergal muscles against the age of the flies. The points are obtained by subtraction of the data from Fig. 4 from that of Fig. 3. Sample size, confidence limits, and symbols as in Fig. 3.
EfJect of extirpation of medial neurosecretorycells The medial neurosecretory cells were cauterized from 80 flies 3 to 4 hr after emergence. A sham-operated control of 30 flies was also set up. Of the 80 experimental flies, 30 died during the 14 days of the experiment and another 15 had fully or partly developed ovaries. In the operated control 25 flies survived, but 5 of these did not have their ovaries fully developed. These flies together with the 15 experimental groups with ovaries present were discarded as in these the operation was thought to be unsuccessful (THOMSEN, 1952). All the operated flies appeared to feed normally as the mid-gut was always found to contain reddish liquid (meat) in the crop. The weight of the muscle plus tendon and of the tendon alone from the experimental and operated control flies is shown in Figs, 3 and 4, and in flies deprived of their medial neurosecretory cells the weight of the tendon and of the muscles was very significantly smaller than in the meat-fed flies of the same age group (PC OmOOl),but the weight is not significantly different from that in the 1 day flies or from the sugar-fed flies of the same age group, i.e. in flies deprived of their medial neurosecretory cells, the resilin tendon and the muscle did not increase in weight. The oocytes of the experimental flies were similar in size to those of the sugarfed flies. The individual oocytes were 0.12 to 0.18 mm long. The operated control flies started to lay eggs by the seventh day after the operation: no further observations were made on the state of their ovaries. In the experimental flies the fat body cells were smaller and somewhat transparent. The operated control flies had fat body cells very similar to those of the normal egg laying females.
940
M.
SABARATNAM
E#ect of allatectomy
The corpus allatum was removed from 60 flies up to 45 min. after emergence and a sham-operated control of 40 flies was set up at the same time. Only 28 of the experimental flies and 25 of the operated flies survived the 14 days of the experiment. Of the surviving experimental flies, 5 had fully or partly developed ovaries, while 2 of the operated controls did not have mature ovaries. These flies were discarded, as in these cases the operation was considered to be unsuccessful by THOMSEN’S(1952) criterion. The gut was examined for meat as in the third section of Results and only flies that had been feeding were used. The weights of the pleurotergal muscle and resilin tendon and of the tendon alone in experimental and operated control flies are shown in Figs. 3, 4, and 5. The weight of the resilin tendon with its muscle in allatectomized flies was significantly less than that in the meat-fed flies in experiment 1 or of the operated control flies in this experiment. However, the weight of the resilin tendon alone in the allatectomized flies was significantly greater than in flies of the same age with their medial neurosecretory cells extirpated and in the sugar-fed flies of the first and second sections of Results and this experiment. The muscle weights in the allatectomized flies did not differ significantly from those of the meat-fed flies (Fig. 5). The oijcytes in the experimental flies were only 0.14 to 0.18 mm long as is expected from the operation. These experimental flies had fat bodies with remarkably hypertrophied cells while those of the operated controls appeared normal. Effect of extirpation of medial neurosecretory cells and implantation of corpora allatacardiaca
The medial neurosecretory cells were extirpated (p. 936) from flies 3 to 4 hr after emergence. Two days later two corpora allata-cardiaca complexes were taken from three to four day female flies and transplanted into the abdomen of each experimental fly. In these double operated flies, the mortality rate was high as in the case of the allatectomized flies. About 20 of the 70 operated flies survived for the 14 days of the experiment. Thirty of the flies deprived of their medial neurosecretory cells died before the second operation. All the surviving experimental flies had mature oocytes and these were used for weight measurements. Figs. 3 and 4 show the weights of resilin tendon and the tendon with its muscle of the 2 weeks double operated flies 12 days after the second operation. The transplanted corpora allata induced significant growth of muscle and tendon. The weight of these tissues was only slightly less than in the normal meat-fed and sham-operated control flies and very much greater than in the flies without medial neurosecretory cells (experiment 2) or in the sugar fed controls of this experiment. Ultrastructural observations
The normal situation in the 7 day fly has been described elsewhere (SABARAT1973). The normal 14 day fly is very similar but the endoplasmic reticulum has largely disappeared (Fig. 6.).
NAM,
FIG. 6. Electron micrograph showing the epithelium (Epi) adjacent to the The fibrous layer (FL) of resilin tendon (RT) from a 14 day meat-fed Calliphora. the tendon interdigitates with the deep invaginations of the epithelium (Inv) and is joined to the epithelium by hemidesmosomes (H Des). Lateral plasma membrane (LPM), mitochondria (Mi), microtubules (MT), nucleus (N), ribosomal granules (RG). FIG. 7. A slightly oblique section of the epithelium (Epi) adjacent to the resilin tendon (RT) from a 14 day sugar-fed Cdliphoru showing chromatin (Ch), fibrous layer (FL), microtubules (MT), nucleus (N), septate desmosomes (S Des). Glycogen granules (Gly) are found at this stage. FIG. 8. A slightly oblique section through the epithelium (Epi) adjacent to the resilin tendon (RT) of a 14 day Calliphora lacking the medial neurosecretory ,__~ . . ,__-I . ,-.I . . .. -.
943
HORMONAL CONTROLOF BESILIN DEPOSITIONIN BLOWFLY
TABLE ~-COMPARISON OF STRUCTURALFEATDBB~OF THE EPIDERMISANDRESILIN TENDONOF Calliphora AT VARIOUS AGES AND IN VARIOUSEXPERIMENTAL CONDITIONS Flies lacking medial neurosecretory cells, 2 weeks after emergence (Fig. 8)
Very old flies, 70 to 80 days after emergence (Fig. 9)
Meat-fed flies, 2 weeks after emergence (Fig. 6)
Sugar-fed flies, 2 weeks after emergence (Fig. 7)
Condition of the nucleus in the epithelium
Heterochromatic with distinct nucleoli. Distinct nuclear membrane
Heterochromatic chromatin material in patches along periphery and within nucleoplasm. Nuclear membrane illdefined and irregular nucleolus not distinct
Heterochromatic chromatin material in large patches along periphery small but distinct nucleolus nuclear membrane illdefined
Heterochromatic chromatin material occupying a larger part of the nucleus. Nucleolus distinct. The nuclear membranes highly irregular
Endoplastic reticulum in epithelium
Folded membranes and narrow cistemae between fascicles of microtubules
Cytoplasm between microtubules appears
Rare. Non-membrane bound spaces between microtubules
Rare
Scattered between microtubules and along cell margin
Rare
Rare
Rare
Ribosomes in epithelium
empty
Mitochondria epithelium
in
Mitochondria between microtubules and along cell margin
Rare or disorganized
Rare or disorganized
Rare or disorganized
Microtubules epithelium
in
Major portion of cytoplasm occupied by microtubules of dense electron contrast
Sparse
Same as in 2 week meat-fed flies
Less distinct overlaid by spaces of very low electron contrast which are not membrane bound
Absent
Concentrated along cuticular end of cell
Absent
Absent
Glycogen
M. SABARATNAM
944
TABLE 1 (co&) Flies lacking medial neurosecretory cells, 2 weeks after emergence (Fig. 8)
Very old flies, 75 to 80 days after emergence (Fig. 9)
Meat-fed flies, 2 weeks after emergence (Fig. 6)
Sugar-fed flies, 2 weeks after emergence (Fig. 7)
Interdigitation between epithelium and tendon
Deep, 4-40 pm
Same as in meatfed flies
Same as in meatfed flies but disorganized
Same as in meatfed flies but disorganized
Region of tendon adjacent to epithelium
Fibrous layer well established
Homogenous and electron dense
Disorganized
Disorganized with spaces which are not bound by membranes
Inner region of tendon
Electron transparent resilin cross-linked
Homogeneous and electron dense
Disorganized, especially the extension of the fibrous layer into the tendon
Disorganized and homogeneous
The fine structure of the epithelium and the tendon were also examined in 14-day sugar-fed flies (Fig. 7), 14 day flies lacking medial neurosecretory cells (Fig. S), and in 80 day flies (Fig. 9). The principal results are shown in Table 1. Attention is drawn to the similarity in appearance of the epithelium in general and of the mitochondria and ribosomal granules in particular in sugar-fed flies, in flies lacking their medial neurosecretory cells, and in 80 day flies (Figs. 7-9). The tendon of the sugar-fed flies resembles that of the 80 day flies while that of the flies lacking their medial neurosecretory cells appears fairly normal. DISCUSSION
The results obtained by weight measurements from normal meat-fed flies shows that the initial deposition of the cuticle is followed by a period of maturity when there is no change in weight followed by a period during which there is a decline in the weight of the cuticle; this has also been observed by BENNET-CLARK (unpublished). S imi 1ar results are obtained from sugar-fed flies which suggests that a similar process takes place but that the small weight increase in the sugar-fed flies results from the different substrate availability during the deposition of the cuticle. Secondly, a hormone control of oocyte and corpus allatum development is known from the work of THOMSEN (1952). FOGAL and FRAENKEL (1969) and
HORMONAL CONTROL OF RESILIN DEPOSITION IN BLOWFLY
945
VINCENT
(1971) found that a hormone bursicon secreted by the medial neurosecretory cells was required for the deposition of the endocuticle. In this study this site of secretion is shown to be necessary for the deposition in the adult of the rubber-like cuticle, resilin. So it is likely that bursicon is involved here also. However, the availability of food and hormones alone do not seem to be sufficient to bring about the deposition of the cuticle. The results from digging flies (SABARATNAM, 1973) suggest that some stimulus operating at the time of adult emergence is necessary to trigger the hormone system without which even the change from the condition in the pharate adult to that in the adult is arrested. Evidence obtained at the ultrastructural level indicates that endocrine abnormality such as that in flies lacking their medial neurosecretory cells leads to an early ageing process as is seen from the appearance of the nucleus, the absence of the ribosomal granules, mitochondria, and the general dissolution of the epithelium. In these flies, however, the tendon appears similar to that of a normal fly which suggests that although cellular ageing may have occurred, those cellular processes which lead to the dissolution of the tendon observed in aged and sugar-fed flies have not taken place. It is thus possible that the medial neurosecretory cells are responsible for controlling both the deposition and the reabsorption of the cuticle. The situation in the sugar-fed flies is different. In these flies the epithelial cytoplasm (Fig. 7) appears like that of the old flies but the tendon structure is different and there is a dense distribution of glycogen-like particles immediately outside the cuticular end of the cell suggesting that metabolism is abnormal. The ultrastructure of the resilin tendon is similar to that in the old flies (Fig. 9) and is unlike that seen in flies lacking their medial neurosecretory cells (Fig. 8) or in the digging flies (SAEZARATNAM, 1973). It seems reasonable to assume that in these flies, after the initial cross-linkage of the protein and chitin, further resilin deposition does not occur. This is borne out by the fact that the ribosomal granules usually found in a fly of this age are not present, It is also possible that in the sugar-fed fly the resilin tendon has been partially reabsorbed which would account for the similarity of its appearance to that in the very old flies. The way in which changes in both the epithelium and the resilin tendon of old flies is brought about is unclear though it is possible that the hormones that are responsible for general growth in early adult life are equally important in bringing about a resorption of the same tissues in very old flies. Initial examinations of this idea were not successful as it is difficult to carry out surgical operations on mature or aged flies. However, this is a fruitful field for further study. Ac?znowZedgeme-nts--It is a real pleasure to thank Dr. H. C. BRNNET-CLARK for his continued help and guidance throughout this work. Thanks are also due to Mr. GEORGE H. DUNCAN for his help with the electron microscope, to Mr. D. F. CREMER for photographic assistance, and to the University of Ceylon (Colombo) for financial support.
REFERENCES CULLEN M. Trinidad.
biology of the giant water bugs (Hemiptera, PTOC.R. ent. Sot. Load. (A) 44, 123-136.
J. (1969) The
Belostomatidae)
in
946
M. SABARATNAM
FOGALW. and FRAENKELG. (1969) The rBle of bursicon in melanization and endocuticle formation in adult fleshfly, Sarcophaga bullata. J. Insect Physiol. 15, 1235-1247. HILL L. (1962) Neurosecretory control of haemolymph protein concentration during ovarian development in the desert locust. r. Insect Physiol. 8, 609-619. NEVILLEA. C. (1965) Circadian organization of chitin in some insect skeletons. Quart. J. nicr. Sn’. 106, 315-325. SABARATNAMM. (1973) The effect of digging on the development of adult characters in the blowfly Calliphora erythrocephala. J. Insect Physiol. 19, 2143-2154. THOMSEN E. (1942) An experimental and anatomical study of the corpus allatum in the blow-fly, Calliphora erythrocephala (Meig.). Vidensk. Medd. Naturh. Foren Kbh. 106, 319-40s. THOMSJZN E. (1952) Functional significance of the neuro-secretory cells and the corpus cardiacum in the female blow-fly, Calliphora erythrocephala (Meig.). J. exp. Biol. 29, 137-172. VINCENT J. F. V. (1971) Effects of bursicon on cuticular properties in Locusta migratoria migratorioides. J. Insect Physiol. 17, 625-636. WEIS-FOGH T. (1960) A rubber-like protein in insect cuticle. J. exp. Biol. 37, 889-907. WIGGLESWORTH V. B. (1970) Insect Hormones (University reviews in Biology). Oliver & Boyd, Edinburgh.
HORMONAL CONTROL OF RESILIN DEPOSITION IN THE BLOWFLY, CALLIPHORA ERYTHROCEPHALA M. SABARATNAM” Department of Zoology, West Mains Road, University of Edinburgh, Scotland (Received 3 May
1973 ; revised 27 October 1973)
Abstract-The deposition of the resilin tendon in the blowfly Calliphora crythrocephala was investigated in normal and in various experimental conditions. The results showed that the weight of the protein resilin that is deposited is controlled by diet as well as by the hormone secreted by the medial neurosecretory cells. Endocrinologically abnormal Calliphora adults deposited a tendon with normal ultrastructure but showed signs of premature ageing while Calliphora fed on a sugar diet deposited a tendon with abnormal ultrastructure.
INTRODUCTION
ENDOCUTICLEdeposition in adult insects is known to be controlled by such factors as the availability of food (NEVILLE, 1965; CULLEN, 1969) and the hormone bursicon which is secreted by the medial neurosecretory cells of the brain (FOGAL and FRAENKEL,1969; VINCENT, 1971). A link between diet and hormone secretion was established by THOMSEN(1952) and subsequently various authors have dealt with the importance of diet and hormones in the post imaginal growth of insects (review by WIGGLESWORTH, 1970) but it is still not clear how general these effects are nor what cellular changes are involved. It was therefore decided to examine the way in which the deposition of the elastic cuticular protein resilin (WEISFOGH, 1960) takes place during adult life and how far the mechanism may be related to that involved in other processes of growth and metamorphosis. MATERIALS
AND METHODS
Calliphora erythrocephala were bred in a 12 hr light 12 hr dark photoperiod at 20°C and were normally fed on a diet which consisted of meat, sugar, and water. The pleurotergal muscle with its resilin tendon or the tendon alone were dissected out from the various groups of experimental flies and weighed in a Cahn Gram Electrobalance after drying to a constant weight (SABARATNAM, 1973). Similarly the weights of the second abdominal tergite and sternite were also recorded. * Present address: Department of Ceylon, Sri Lanka. 31
of Zoology, Thurstan
935
Road, Colombo 3, University
936
M.
SABARATNAM
Experiments were performed to examine the role of the endocrine systems. The medial neurosecretory cells were located by THOMSEN’S(1952) method and the cells were cauterized with an electric wire loop supplied through a transformer at 4 to 6 V a.c. using HILL’s (1962) method. The corpus allatum-cardiacurn complex was removed by THOMSEN’S (1942) method but in my experiments neck muscles were left in place. The corpus allatum-cardiacurn complex was transplanted into the abdomen of flies deprived of their medial neurosecretory cells by THOMSEN’S(1952) method. Fixation for electron microscopy and sectioning of the Araldite blocks were carried out according to SAECARATNAM (1973).
RESULTS E#ect of diet in unoperated plies
Two batches of over five hundred flies were kept respectively- on a meat and sugar diet or on a sugar diet. At 1 day, 7, 14, 21, 28, 35, and 42 days after emergence batches of 50 flies were removed for dissection from the two groups. The mean weights of the resilin tendon with and without muscle and of the sclerotized cuticle of the second abdominal tergite and sternite in the two groups of flies are given in Figs. 1, 2, 3, 4, and 5. As the weights observed at 6 weeks were similar to the weights observed from flies surviving to a 2 to 24 months, flies were only kept for 6 weeks. With both sugar-fed and meat-fed flies, the weights of the sternal plate increased significantly during the first 2 weeks of adult life. Thereafter the weight of the sternal plate remained more or less constant to 5 weeks but fell by the sixth week of post-emergent adult life to a weight that did not differ significantly with either diet from that at 1 week (Fig. 1). On the other hand, the weight of the tergal plate increased during the first week then remained constant to week 4 and then decreased significantly to week 6 in the meat fed and sugar fed flies (Fig. 2). The weight of the sclerotized cuticle in meat-fed flies was significantly greater than that of the sugar-fed flies between weeks 1 and 6. Although the weight of the cuticle fell between the fourth and sixth weeks, the weight remained greater than in a newly emerged fly and the final weight in the meat-fed flies was greater than in the sugar-fed flies. It is noteworthy that the increase in weight in the first 2 weeks of adult life was greater in the meat-fed flies but the decrease in weight between weeks 4 and 6 was similar with both diets. A similar pattern was observed for the resilin tendon (Fig. 4) but the weight of muscle after the initial increase during the first week in sugar-fed flies or during the first 2 weeks in meat-fed flies changed very little (Figs. 3, 5). Observations and/or measurements of the size of the oocytes, corpora allata, and fat body cells were also made from flies 14 days after emergence for purposes
HORMONAL CONTROL OF RBSILIN DEPOSITION IN BLOWFLY
30
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flies
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35
42
DAYS ___+ FIG. 1. Graph showing the weight of the second sternal plate against the age of the flies. The points show the means of samples from 5 insects and the bar lines show the 95 per cent confidence limits of the means. Sugar-fed flies, O-0-0 ; meat-fed flies, 0 . . . 0 . . . 0.
180
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DAYS -+ FIG. 2. Graph showing the weight of the second tergal plate against the age of the flies. Sample size and confidence limits as in Fig. 1. Sugar-fed flies, O-O-O; meat-fed flies, 0 . . . 0 . . . 0.
937
938
M. SABARATNAM Resilin + MUSCLE
t 40
I
flies flies
36
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0.*-o -
32
20
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1
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DAYS -
FICL 3. Graph showing the weight of five pleurotergal muscles with resilin tendons against the age of the flies. The points show the means for five samples each of five muscles with tendons and the bar lines show 95 per cent confidence limits of these means. Sugar-fed flies, O-O-O ; meat-fed flies, 0 . . . 0 . . . 0 ; operated flies, l ; flies with medial neurosecretory cells cauterized, -mnc; flies with corpus allatum removed, -ca; flies with medial neurosecretory cells cauterized and two corpora allata implanted, - mnc + ca.
Meat
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Sugar flies
1
7
14
21
28
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0**#*0 W
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DAYS -
FIG. 4.
Graph showing the weight of five tendons against the age of the flies. Sample size, confidence limits, and symbols as in Fig. 3.
of comparison in the later experiments. In the meat-fed flies the length of the mature oocytes was from 1.40 to 1.50 mm, while in the case of the sugar-fed flies the range was from 0*128 to O-136 mm. The average diameter of the corpus allatum in the meat-fed flies was 0.126 T 0*012 mm while in sugar-fed flies it was O-092 r 0.006 mm at 2 weeks. In the meat-fed flies the fat body cells showed a cyclic activity which coincided with the development of the egg cells. In the sugar-fed flies, the fat body cells were sparse, and the few cells present were small and transparent, which is due to the quantity of glycogen, fat, and proteins (THOMSEN, 1952).
HORMONAL
CONTROL
30
I
OF RESILIN
DEPOSITION
-
26
-
22
-
18
-
14
-
IN
BLOWFLY
Meat
flies
o**.o
Sugar
flies
o--o
939
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.ElD
-
$6
2
MUSCLE
I’ 1
’ 7
’ 14
’
’
’
’
21
28
35
42
DAYS -
FIG. 5. Graph showing the weight of five pleurotergal muscles against the age of the flies. The points are obtained by subtraction of the data from Fig. 4 from that of Fig. 3. Sample size, confidence limits, and symbols as in Fig. 3.
EfJect of extirpation of medial neurosecretorycells The medial neurosecretory cells were cauterized from 80 flies 3 to 4 hr after emergence. A sham-operated control of 30 flies was also set up. Of the 80 experimental flies, 30 died during the 14 days of the experiment and another 15 had fully or partly developed ovaries. In the operated control 25 flies survived, but 5 of these did not have their ovaries fully developed. These flies together with the 15 experimental groups with ovaries present were discarded as in these the operation was thought to be unsuccessful (THOMSEN, 1952). All the operated flies appeared to feed normally as the mid-gut was always found to contain reddish liquid (meat) in the crop. The weight of the muscle plus tendon and of the tendon alone from the experimental and operated control flies is shown in Figs, 3 and 4, and in flies deprived of their medial neurosecretory cells the weight of the tendon and of the muscles was very significantly smaller than in the meat-fed flies of the same age group (PC OmOOl),but the weight is not significantly different from that in the 1 day flies or from the sugar-fed flies of the same age group, i.e. in flies deprived of their medial neurosecretory cells, the resilin tendon and the muscle did not increase in weight. The oocytes of the experimental flies were similar in size to those of the sugarfed flies. The individual oocytes were 0.12 to 0.18 mm long. The operated control flies started to lay eggs by the seventh day after the operation: no further observations were made on the state of their ovaries. In the experimental flies the fat body cells were smaller and somewhat transparent. The operated control flies had fat body cells very similar to those of the normal egg laying females.
940
M.
SABARATNAM
E#ect of allatectomy
The corpus allatum was removed from 60 flies up to 45 min. after emergence and a sham-operated control of 40 flies was set up at the same time. Only 28 of the experimental flies and 25 of the operated flies survived the 14 days of the experiment. Of the surviving experimental flies, 5 had fully or partly developed ovaries, while 2 of the operated controls did not have mature ovaries. These flies were discarded, as in these cases the operation was considered to be unsuccessful by THOMSEN’S(1952) criterion. The gut was examined for meat as in the third section of Results and only flies that had been feeding were used. The weights of the pleurotergal muscle and resilin tendon and of the tendon alone in experimental and operated control flies are shown in Figs. 3, 4, and 5. The weight of the resilin tendon with its muscle in allatectomized flies was significantly less than that in the meat-fed flies in experiment 1 or of the operated control flies in this experiment. However, the weight of the resilin tendon alone in the allatectomized flies was significantly greater than in flies of the same age with their medial neurosecretory cells extirpated and in the sugar-fed flies of the first and second sections of Results and this experiment. The muscle weights in the allatectomized flies did not differ significantly from those of the meat-fed flies (Fig. 5). The oijcytes in the experimental flies were only 0.14 to 0.18 mm long as is expected from the operation. These experimental flies had fat bodies with remarkably hypertrophied cells while those of the operated controls appeared normal. Effect of extirpation of medial neurosecretory cells and implantation of corpora allatacardiaca
The medial neurosecretory cells were extirpated (p. 936) from flies 3 to 4 hr after emergence. Two days later two corpora allata-cardiaca complexes were taken from three to four day female flies and transplanted into the abdomen of each experimental fly. In these double operated flies, the mortality rate was high as in the case of the allatectomized flies. About 20 of the 70 operated flies survived for the 14 days of the experiment. Thirty of the flies deprived of their medial neurosecretory cells died before the second operation. All the surviving experimental flies had mature oocytes and these were used for weight measurements. Figs. 3 and 4 show the weights of resilin tendon and the tendon with its muscle of the 2 weeks double operated flies 12 days after the second operation. The transplanted corpora allata induced significant growth of muscle and tendon. The weight of these tissues was only slightly less than in the normal meat-fed and sham-operated control flies and very much greater than in the flies without medial neurosecretory cells (experiment 2) or in the sugar fed controls of this experiment. Ultrastructural observations
The normal situation in the 7 day fly has been described elsewhere (SABARAT1973). The normal 14 day fly is very similar but the endoplasmic reticulum has largely disappeared (Fig. 6.).
NAM,
FIG. 6. Electron micrograph showing the epithelium (Epi) adjacent to the The fibrous layer (FL) of resilin tendon (RT) from a 14 day meat-fed Calliphora. the tendon interdigitates with the deep invaginations of the epithelium (Inv) and is joined to the epithelium by hemidesmosomes (H Des). Lateral plasma membrane (LPM), mitochondria (Mi), microtubules (MT), nucleus (N), ribosomal granules (RG). FIG. 7. A slightly oblique section of the epithelium (Epi) adjacent to the resilin tendon (RT) from a 14 day sugar-fed Cdliphoru showing chromatin (Ch), fibrous layer (FL), microtubules (MT), nucleus (N), septate desmosomes (S Des). Glycogen granules (Gly) are found at this stage. FIG. 8. A slightly oblique section through the epithelium (Epi) adjacent to the resilin tendon (RT) of a 14 day Calliphora lacking the medial neurosecretory ,__~ . . ,__-I . ,-.I . . .. -.
943
HORMONAL CONTROLOF BESILIN DEPOSITIONIN BLOWFLY
TABLE ~-COMPARISON OF STRUCTURALFEATDBB~OF THE EPIDERMISANDRESILIN TENDONOF Calliphora AT VARIOUS AGES AND IN VARIOUSEXPERIMENTAL CONDITIONS Flies lacking medial neurosecretory cells, 2 weeks after emergence (Fig. 8)
Very old flies, 70 to 80 days after emergence (Fig. 9)
Meat-fed flies, 2 weeks after emergence (Fig. 6)
Sugar-fed flies, 2 weeks after emergence (Fig. 7)
Condition of the nucleus in the epithelium
Heterochromatic with distinct nucleoli. Distinct nuclear membrane
Heterochromatic chromatin material in patches along periphery and within nucleoplasm. Nuclear membrane illdefined and irregular nucleolus not distinct
Heterochromatic chromatin material in large patches along periphery small but distinct nucleolus nuclear membrane illdefined
Heterochromatic chromatin material occupying a larger part of the nucleus. Nucleolus distinct. The nuclear membranes highly irregular
Endoplastic reticulum in epithelium
Folded membranes and narrow cistemae between fascicles of microtubules
Cytoplasm between microtubules appears
Rare. Non-membrane bound spaces between microtubules
Rare
Scattered between microtubules and along cell margin
Rare
Rare
Rare
Ribosomes in epithelium
empty
Mitochondria epithelium
in
Mitochondria between microtubules and along cell margin
Rare or disorganized
Rare or disorganized
Rare or disorganized
Microtubules epithelium
in
Major portion of cytoplasm occupied by microtubules of dense electron contrast
Sparse
Same as in 2 week meat-fed flies
Less distinct overlaid by spaces of very low electron contrast which are not membrane bound
Absent
Concentrated along cuticular end of cell
Absent
Absent
Glycogen
M. SABARATNAM
944
TABLE 1 (co&) Flies lacking medial neurosecretory cells, 2 weeks after emergence (Fig. 8)
Very old flies, 75 to 80 days after emergence (Fig. 9)
Meat-fed flies, 2 weeks after emergence (Fig. 6)
Sugar-fed flies, 2 weeks after emergence (Fig. 7)
Interdigitation between epithelium and tendon
Deep, 4-40 pm
Same as in meatfed flies
Same as in meatfed flies but disorganized
Same as in meatfed flies but disorganized
Region of tendon adjacent to epithelium
Fibrous layer well established
Homogenous and electron dense
Disorganized
Disorganized with spaces which are not bound by membranes
Inner region of tendon
Electron transparent resilin cross-linked
Homogeneous and electron dense
Disorganized, especially the extension of the fibrous layer into the tendon
Disorganized and homogeneous
The fine structure of the epithelium and the tendon were also examined in 14-day sugar-fed flies (Fig. 7), 14 day flies lacking medial neurosecretory cells (Fig. S), and in 80 day flies (Fig. 9). The principal results are shown in Table 1. Attention is drawn to the similarity in appearance of the epithelium in general and of the mitochondria and ribosomal granules in particular in sugar-fed flies, in flies lacking their medial neurosecretory cells, and in 80 day flies (Figs. 7-9). The tendon of the sugar-fed flies resembles that of the 80 day flies while that of the flies lacking their medial neurosecretory cells appears fairly normal. DISCUSSION
The results obtained by weight measurements from normal meat-fed flies shows that the initial deposition of the cuticle is followed by a period of maturity when there is no change in weight followed by a period during which there is a decline in the weight of the cuticle; this has also been observed by BENNET-CLARK (unpublished). S imi 1ar results are obtained from sugar-fed flies which suggests that a similar process takes place but that the small weight increase in the sugar-fed flies results from the different substrate availability during the deposition of the cuticle. Secondly, a hormone control of oocyte and corpus allatum development is known from the work of THOMSEN (1952). FOGAL and FRAENKEL (1969) and
HORMONAL CONTROL OF RESILIN DEPOSITION IN BLOWFLY
945
VINCENT
(1971) found that a hormone bursicon secreted by the medial neurosecretory cells was required for the deposition of the endocuticle. In this study this site of secretion is shown to be necessary for the deposition in the adult of the rubber-like cuticle, resilin. So it is likely that bursicon is involved here also. However, the availability of food and hormones alone do not seem to be sufficient to bring about the deposition of the cuticle. The results from digging flies (SABARATNAM, 1973) suggest that some stimulus operating at the time of adult emergence is necessary to trigger the hormone system without which even the change from the condition in the pharate adult to that in the adult is arrested. Evidence obtained at the ultrastructural level indicates that endocrine abnormality such as that in flies lacking their medial neurosecretory cells leads to an early ageing process as is seen from the appearance of the nucleus, the absence of the ribosomal granules, mitochondria, and the general dissolution of the epithelium. In these flies, however, the tendon appears similar to that of a normal fly which suggests that although cellular ageing may have occurred, those cellular processes which lead to the dissolution of the tendon observed in aged and sugar-fed flies have not taken place. It is thus possible that the medial neurosecretory cells are responsible for controlling both the deposition and the reabsorption of the cuticle. The situation in the sugar-fed flies is different. In these flies the epithelial cytoplasm (Fig. 7) appears like that of the old flies but the tendon structure is different and there is a dense distribution of glycogen-like particles immediately outside the cuticular end of the cell suggesting that metabolism is abnormal. The ultrastructure of the resilin tendon is similar to that in the old flies (Fig. 9) and is unlike that seen in flies lacking their medial neurosecretory cells (Fig. 8) or in the digging flies (SAEZARATNAM, 1973). It seems reasonable to assume that in these flies, after the initial cross-linkage of the protein and chitin, further resilin deposition does not occur. This is borne out by the fact that the ribosomal granules usually found in a fly of this age are not present, It is also possible that in the sugar-fed fly the resilin tendon has been partially reabsorbed which would account for the similarity of its appearance to that in the very old flies. The way in which changes in both the epithelium and the resilin tendon of old flies is brought about is unclear though it is possible that the hormones that are responsible for general growth in early adult life are equally important in bringing about a resorption of the same tissues in very old flies. Initial examinations of this idea were not successful as it is difficult to carry out surgical operations on mature or aged flies. However, this is a fruitful field for further study. Ac?znowZedgeme-nts--It is a real pleasure to thank Dr. H. C. BRNNET-CLARK for his continued help and guidance throughout this work. Thanks are also due to Mr. GEORGE H. DUNCAN for his help with the electron microscope, to Mr. D. F. CREMER for photographic assistance, and to the University of Ceylon (Colombo) for financial support.
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biology of the giant water bugs (Hemiptera, PTOC.R. ent. Sot. Load. (A) 44, 123-136.
J. (1969) The
Belostomatidae)
in
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FOGALW. and FRAENKELG. (1969) The rBle of bursicon in melanization and endocuticle formation in adult fleshfly, Sarcophaga bullata. J. Insect Physiol. 15, 1235-1247. HILL L. (1962) Neurosecretory control of haemolymph protein concentration during ovarian development in the desert locust. r. Insect Physiol. 8, 609-619. NEVILLEA. C. (1965) Circadian organization of chitin in some insect skeletons. Quart. J. nicr. Sn’. 106, 315-325. SABARATNAMM. (1973) The effect of digging on the development of adult characters in the blowfly Calliphora erythrocephala. J. Insect Physiol. 19, 2143-2154. THOMSEN E. (1942) An experimental and anatomical study of the corpus allatum in the blow-fly, Calliphora erythrocephala (Meig.). Vidensk. Medd. Naturh. Foren Kbh. 106, 319-40s. THOMSJZN E. (1952) Functional significance of the neuro-secretory cells and the corpus cardiacum in the female blow-fly, Calliphora erythrocephala (Meig.). J. exp. Biol. 29, 137-172. VINCENT J. F. V. (1971) Effects of bursicon on cuticular properties in Locusta migratoria migratorioides. J. Insect Physiol. 17, 625-636. WEIS-FOGH T. (1960) A rubber-like protein in insect cuticle. J. exp. Biol. 37, 889-907. WIGGLESWORTH V. B. (1970) Insect Hormones (University reviews in Biology). Oliver & Boyd, Edinburgh.