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Tolerance to dehydration of the blood of Sialis lutaria L.
7. Ins. Physiol., 1960, Vol. 6, pp. 81 to 83. Pergamon Press Ltd., London. Printed in Great Britain
TOLERANCE TO DEHYDRATION SIALIS LUTARIA
OF THE BLOOD OF L.
B. J. SELMAN Department
of Zoology, University (Received 4 October
of Bristol
1960)
Abstract-Blood and epidermal cells of larvae of Sialis Zutaria are competent to form tanned wound plugs when hydrated after a prolonged period of dehydration. The appearance and behaviour of the coagulocyte blood cells do not appear to be altered
by prolonged dehydration; when re-hydrated they clot and fornx pseudopodia as they do before dehydration. In the dehydrated state they tolerate heating to 100°C for 30 min. INTRODUCTION
IT is known that the larva of the Chironomid, Polypedilum vanderplunki Hint., will complete its metamorphosis after it has been dried to a moisture content of 15 per cent or less for several years (HINTON, 1953). HINTON found that the larvae of Polypedilum vanderpZanki grew normally after being dried and then exposed to temperatures of 102-104°C for 1 min, or placed in liquid helium (-270°C) for 5 min (HINTON, 1960a, b). Thus in the dry state this species tolerates a range of 372°C. Many insects that do not survive an appreciable fall in moisture content nevertheless possess tissues that seem to be able to tolerate complete dehydration. For instance, in the Tipulid fly, Taphrophila vitripennis Meig., tissue isolated in the spiracular gills at the pupal-adult moult is able to produce tanned wound plugs after prolonged periods of dehydration (HINTON, 1957). The formation of tanned wound plugs has been considered to be evidence that the tissue was alive after dehydration, but, as HINTON (1960b) points out, evidence of this kind that the tissue is alive is open to objection: it may, for instance, be claimed that the tissue did not survive dehydration and that damage to it after re-hydration simply destroyed a barrier between enzyme and substrate that enabled the tyrosinIn this paper the behaviour of the blood cells of Sialis ase reaction to proceed. Zutariu before and after dehydration is described. The fact that blood cells clot and form pseudopodia after re-hydration as they do before dehydration is considered to be evidence that they are not killed by prolonged and more or less complete dehydration. METHODS
AND OBSERVATIONS
Barly instar Siulis larvae were dried to see if they survived. Using whole larvae, the rates of drying of different parts of the body varied greatly. This resulted in partial cytolysis of some tissues when others were completely dry, causing poisoning of all the tissues. To obtain even drying, only abdomens were used. 81
82
B. J. SELMAN
The abdomens were ligatured, cut off, and placed on glass slides in a small chamber connected through a solid carbon dioxide water trap to a phosphorus pentoxide water trap. The whole system was evacuated by a two-stage ‘High Vat’ oil pump to a pressure of < 6 cm Hg. After dehydration the abdomens were stored over phosphorus pentoxide for long periods. After storing for 17 months the gills could form tanned wound plugs when hydrated. The tanned plugs did not disperse in cold concentrated nitric acid or hot concentrated nitric acid for more than hydrochloric acid, They withstood 5 min. If the plugs were of sclerotin, the presence of a tyrosinase inhibitor should A solution of 0.5 M phenylthiourea in 1% ethyl have prevented darkening. alcohol completely inhibited the formation of tanned plugs. Two gill tips were cut off and placed in 1% ethyl alcohol without phenylthiourea in order to check the toxicity of the alcohol. Tanned plugs were formed. Therefore the wound Serial sections showed the wound plugs were of tanned protein or sclerotin. plugs to be composed mainly of clotted blood cells. Dried abdomens heated to 90°C for 2 hr or to 100°C for 30 min before hydration formed tanned wound plugs. The present results agree closely with those of HINTON (1957) on Tuphrophilu. Immediately after hydration the blood cells were visible within the lumen of the gills. As hydration progressed the blood cells became increasingly easy to see and the different cell types could be identified. Nine minutes after hydration, the blood cells were no longer translucent but slightly opaque. After 80 min, the hyaline blood cells were tanned a light brown. At this stage many of the coagulocytes were attached to the central trachea. In gills wounded immediately after hydration, the hyaline haemocytes formed a tanned wound plug. The epidermal cells at the edge of the wound also tanned. Their nuclei became a much darker brown than the cytoplasm. Gills which had not been dehydrated also were wounded and sectioned. A similar picture was obtained. Tanned plugs seldom formed if the dried gills were cut later than an hour after the start of hydration. The tips were removed from ligatured and dried gills, placed in adrop of air-free saline solution, and surrounded by liquid paraffin. A cover glass was placed on top, and the gills were observed with a phase-contrast microscope. Immediately after hydration the cover glass was pressed to squeeze out the contents of the gills. Granular haemocytes and coagulocytes were obtained, and almost invariably the latter had begun to clot. The coagulocytes immediately attached themselves to the glass surface by their pseudopodia. Granules were seen to pass out along long filamentous processes. On one occasion a single elongated, highly-refractile hyaline haemocyte was squeezed out of a gill under pressure. The cell was indistinguishable from a normal coagulocyte before clotting. While the haemocyte was floating freely, its cytoplasm spread out. Vacuoles formed and emptied into the saline solution. There was a violent movement in the refractile centre of the cell around the nucleus, followed by a spinning movement within the central cytoplasm. Then the refractivity faded disclosing a typically clotted nucleus. This is the normal series of changes observed in a cell during clotting.
TOLERANCE TO DEHYDRATION OF THE BLOOD OF
SIALIS
LLJTARIA
L.
83
When no pressure was applied to the gill, a wound plug was formed. If this was pulled away immediately after formation, many fine cytoplasmic strands were formed, comparable to those formed when a cover glass was moved over a clotting blood film. If the clot was pulled away at a stage when tanning had begun there were no strands. This is further evidence that the wound plugs are formed by the clotting of living hyaline haemocytes which have survived dehydration. DISCUSSION It
has been shown that the gills of the larva of Sialis lutaria can be dried and that after many months of storage in a desiccator they can be re-hydrated and the blood cells will still form a clot and thus produce a tanned wound plug. The process of dehydration followed by re-hydration seems to initiate the clotting behaviour of the coagulocyte blood cells. The dehydrated cells apparently retain their morphological structure and physiological capabilities, i.e. they are alive. Thus for a limited time after hydration the blood cells behave as normal cells when forming a clot. Acknowledgement-My course of this work.
best thanks
are due to Dr. H. E. HINTON for his advice during
the
REFERENCES HINTON H. E. (1953) Some adaptations of insects to environments that are alternately dry and flooded, with notes on the habits of the Stratiomyidae. Trans. Sm. Byit. Ent. 11, 209-227. HINTON H. E. (1957) The structure and function of the spiracular gill of the fly Taphrophila vitripennis. Proc. ray. Sot. (B) 147, 90-120. HINTON H. E. (1960a) A fly larva that tolerates dehydration and temperatures of - 270’ to +102”C. Nature, Lond. 188, 336-337. HINTOX H. E. (1960b) Cryptobiosis in the larva of Polypedilunr vanderplanki Hint. (Chironomidae). J. Ins. Physiol. 5, 286-300.
TOLERANCE TO DEHYDRATION SIALIS LUTARIA
OF THE BLOOD OF L.
B. J. SELMAN Department
of Zoology, University (Received 4 October
of Bristol
1960)
Abstract-Blood and epidermal cells of larvae of Sialis Zutaria are competent to form tanned wound plugs when hydrated after a prolonged period of dehydration. The appearance and behaviour of the coagulocyte blood cells do not appear to be altered
by prolonged dehydration; when re-hydrated they clot and fornx pseudopodia as they do before dehydration. In the dehydrated state they tolerate heating to 100°C for 30 min. INTRODUCTION
IT is known that the larva of the Chironomid, Polypedilum vanderplunki Hint., will complete its metamorphosis after it has been dried to a moisture content of 15 per cent or less for several years (HINTON, 1953). HINTON found that the larvae of Polypedilum vanderpZanki grew normally after being dried and then exposed to temperatures of 102-104°C for 1 min, or placed in liquid helium (-270°C) for 5 min (HINTON, 1960a, b). Thus in the dry state this species tolerates a range of 372°C. Many insects that do not survive an appreciable fall in moisture content nevertheless possess tissues that seem to be able to tolerate complete dehydration. For instance, in the Tipulid fly, Taphrophila vitripennis Meig., tissue isolated in the spiracular gills at the pupal-adult moult is able to produce tanned wound plugs after prolonged periods of dehydration (HINTON, 1957). The formation of tanned wound plugs has been considered to be evidence that the tissue was alive after dehydration, but, as HINTON (1960b) points out, evidence of this kind that the tissue is alive is open to objection: it may, for instance, be claimed that the tissue did not survive dehydration and that damage to it after re-hydration simply destroyed a barrier between enzyme and substrate that enabled the tyrosinIn this paper the behaviour of the blood cells of Sialis ase reaction to proceed. Zutariu before and after dehydration is described. The fact that blood cells clot and form pseudopodia after re-hydration as they do before dehydration is considered to be evidence that they are not killed by prolonged and more or less complete dehydration. METHODS
AND OBSERVATIONS
Barly instar Siulis larvae were dried to see if they survived. Using whole larvae, the rates of drying of different parts of the body varied greatly. This resulted in partial cytolysis of some tissues when others were completely dry, causing poisoning of all the tissues. To obtain even drying, only abdomens were used. 81
82
B. J. SELMAN
The abdomens were ligatured, cut off, and placed on glass slides in a small chamber connected through a solid carbon dioxide water trap to a phosphorus pentoxide water trap. The whole system was evacuated by a two-stage ‘High Vat’ oil pump to a pressure of < 6 cm Hg. After dehydration the abdomens were stored over phosphorus pentoxide for long periods. After storing for 17 months the gills could form tanned wound plugs when hydrated. The tanned plugs did not disperse in cold concentrated nitric acid or hot concentrated nitric acid for more than hydrochloric acid, They withstood 5 min. If the plugs were of sclerotin, the presence of a tyrosinase inhibitor should A solution of 0.5 M phenylthiourea in 1% ethyl have prevented darkening. alcohol completely inhibited the formation of tanned plugs. Two gill tips were cut off and placed in 1% ethyl alcohol without phenylthiourea in order to check the toxicity of the alcohol. Tanned plugs were formed. Therefore the wound Serial sections showed the wound plugs were of tanned protein or sclerotin. plugs to be composed mainly of clotted blood cells. Dried abdomens heated to 90°C for 2 hr or to 100°C for 30 min before hydration formed tanned wound plugs. The present results agree closely with those of HINTON (1957) on Tuphrophilu. Immediately after hydration the blood cells were visible within the lumen of the gills. As hydration progressed the blood cells became increasingly easy to see and the different cell types could be identified. Nine minutes after hydration, the blood cells were no longer translucent but slightly opaque. After 80 min, the hyaline blood cells were tanned a light brown. At this stage many of the coagulocytes were attached to the central trachea. In gills wounded immediately after hydration, the hyaline haemocytes formed a tanned wound plug. The epidermal cells at the edge of the wound also tanned. Their nuclei became a much darker brown than the cytoplasm. Gills which had not been dehydrated also were wounded and sectioned. A similar picture was obtained. Tanned plugs seldom formed if the dried gills were cut later than an hour after the start of hydration. The tips were removed from ligatured and dried gills, placed in adrop of air-free saline solution, and surrounded by liquid paraffin. A cover glass was placed on top, and the gills were observed with a phase-contrast microscope. Immediately after hydration the cover glass was pressed to squeeze out the contents of the gills. Granular haemocytes and coagulocytes were obtained, and almost invariably the latter had begun to clot. The coagulocytes immediately attached themselves to the glass surface by their pseudopodia. Granules were seen to pass out along long filamentous processes. On one occasion a single elongated, highly-refractile hyaline haemocyte was squeezed out of a gill under pressure. The cell was indistinguishable from a normal coagulocyte before clotting. While the haemocyte was floating freely, its cytoplasm spread out. Vacuoles formed and emptied into the saline solution. There was a violent movement in the refractile centre of the cell around the nucleus, followed by a spinning movement within the central cytoplasm. Then the refractivity faded disclosing a typically clotted nucleus. This is the normal series of changes observed in a cell during clotting.
TOLERANCE TO DEHYDRATION OF THE BLOOD OF
SIALIS
LLJTARIA
L.
83
When no pressure was applied to the gill, a wound plug was formed. If this was pulled away immediately after formation, many fine cytoplasmic strands were formed, comparable to those formed when a cover glass was moved over a clotting blood film. If the clot was pulled away at a stage when tanning had begun there were no strands. This is further evidence that the wound plugs are formed by the clotting of living hyaline haemocytes which have survived dehydration. DISCUSSION It
has been shown that the gills of the larva of Sialis lutaria can be dried and that after many months of storage in a desiccator they can be re-hydrated and the blood cells will still form a clot and thus produce a tanned wound plug. The process of dehydration followed by re-hydration seems to initiate the clotting behaviour of the coagulocyte blood cells. The dehydrated cells apparently retain their morphological structure and physiological capabilities, i.e. they are alive. Thus for a limited time after hydration the blood cells behave as normal cells when forming a clot. Acknowledgement-My course of this work.
best thanks
are due to Dr. H. E. HINTON for his advice during
the
REFERENCES HINTON H. E. (1953) Some adaptations of insects to environments that are alternately dry and flooded, with notes on the habits of the Stratiomyidae. Trans. Sm. Byit. Ent. 11, 209-227. HINTON H. E. (1957) The structure and function of the spiracular gill of the fly Taphrophila vitripennis. Proc. ray. Sot. (B) 147, 90-120. HINTON H. E. (1960a) A fly larva that tolerates dehydration and temperatures of - 270’ to +102”C. Nature, Lond. 188, 336-337. HINTOX H. E. (1960b) Cryptobiosis in the larva of Polypedilunr vanderplanki Hint. (Chironomidae). J. Ins. Physiol. 5, 286-300.