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The maintenance of Glossina morsitans in England for experimental work
638 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE.
Vol. XXXII. No. 5. February, 1939.
T H E M A I N T E N A N C E OF G L O S S I N A M O R S I T A N S
IN E N G L A N D
FOR E X P E R I M E N T A L WORK. BY
j. C. BROOM, M.D.*
The Wellcome Bureau of Scientific Research, London.
The data presented in this note were accumulated in the course of a study of Trypanosoma brucei throughout its cycle of development in Glossina morsitans. It seemed that the experience gained about the feeding and maintaining of considerable numbers of tsetse flies under laboratory conditions in England might be of interest since no such complete records appear to be available in the literature, though YORV~, MURGATROYDand HAWKING (1933) give certain details of the technique they employed in similar work on a smaller scale. The extensive investigation undertaken by BUXTON and L~ccIs (1935) was carried out in Nigeria with a different end in view, but some of their results have been applicable to the present problem. In this country MELLANBY and MELLANBY (1937) maintained a small population of G. palpalis for a number of generations, but the difficulties they met with were of a different nature from ours. For example : as regards feeding, these observers state that flies which are unwilling to feed should be singled out * We should like to record our thanks to Dr. J. F. COaSON who arranged for the regular supply of pupae ; to Mr. KE~ETH MELLANBYfor showing us points of technique in the handling of tsetse flies ; and to Mr. R. J. Rm~Dand Mr. J. A. HAYNESfor the care and patience they exercised in attending to the flies.
684
MAINTENANCE OF G. MORSIT.ZINS I N ENGLAND.
and " coaxed " to do so by giving them repeated additional opportunities. By this means they found that " it is possible to reduce deaths practically to nil during the first 3 months (at least) of the flies' lives ". In our case a much larger population was necessary and anything up to 1,200 flies were under observation a t one time so that any individual treatment was impossible. As a result the death roll was of course relatively heavy, but a sufficient number survived to allow the experiments to be carried on. After a number of attempts to discover the best conditions, the method that has proved most satisfactory is as follows. Pupae of G. morsitans are sent in batches of about 2,000 at fortnightly intervals by air-mail from Tanganyika. The pupae are enclosed in a bag of mosquito-netting, 5 inches by 3 inches tied with a draw-string, and then placed without packing in a cubical box, of about 3 inches inside measurements, made of pieces of 1/4 inch wood nailed together. Half a dozen 1/8 inch holes are bored in each face of the box to ensure a supply of fresh air during the journey which occupies 5 days. A label bearing the address is stuck on one side and no paper or string is used. A certain number of flies are always found hatched out on arrival but they cannot escape from the netting bag. Most of them are dead but often a few, presumably recently emerged, survive and may be used. When they are received the pupae are distributed into two or three glass jars, 4 inches deep and 4 inches wide ; the tops of the jars are covered with mosquito-netting held in position by a rubber band. Each jar is stood in an 11 inch Petri dish containing a saturated solution of common salt to maintain a humidity of 80 per cent. saturation. The whole is covered with a large bell-jar and kept at a temperature of approximately 24 ° C. It has been pointed out by ROUSAUD (1917), who bred small numbers of G. morsitans in Paris, that tsetse flies will not expand their wings in the absence of light. For that reason the great majority of our flies were hatched in an insectarium lighted by daylight; later, when it was not possible to use that room, it was found by experiment that artificial light is equally effective in causing development of the wings. From the 58,000 pupae received to date about 13,500 flies have emerged, i.e., approximately 23 per cent. The adult flies are collected every second day and placed, four or five together, in glass tubes 4 inches by ~ inch one end of which is covered with mosquito-netting fixed on with adhesive strapping and the other end closed with a cork. These tubes are applied to infected rats and the flies given an opportunity to feed. The fed flies are separated and placed, in tens, in glass cylinders* * These cylinders were made by cutting the bottom off 1 lb. glass jam-jars and grinding the rough edge with carborundum paste on a glass slab. Celluloidcylinders (BuxTON and MELLANBY,1934) of comparable size can be obtained but when large numbers are required the difference in price between jam-jars at 10s. a gross and celluloid cylinders at 2s. 6d. each becomes very marked. These jars are much stronger than, for example, lamp glasses and they do not shatter even if knocked off the bench.
J. C. BROOM.
685
4 inches by 2~ inches, one end covered with mosquito-netting, and a short sleeve of the same material attached to the other end, both being fixed on with adhesive strapping. The edges of the sleeve are fastened together with rubber latex. This is simpler and more rapid than sewing and in addition makes a seam that is not affected by frequent washing. Each cylinder thus contains flies all known to have had an infective feed on the same day. The necessary particulars are written on a piece of strapping which is affixed to the cylinder ; this is better than a paper label because strapping can be removed and transferred to a fresh receptacle as required. Unfed flies are returned to the small tubes and given a further chance to feed. In the early experiments in this series, observations were being made on the trypanosomes at different time intervals after ingestion by the fly, and flies which apparently refused to feed on one day were discarded in case they had taken a small feed unnoticed. Later, this precaution was less necessary and a certain number of flies were retained and allowed to feed on another day. When unfed flies were discarded it was found that about 75 per cent. of those hatched could be expected to feed. The complete developmental cycle of the strain of T. brucei used in these experiments occupied at least 25 days in the flies. During the first 3 weeks after the infective feed therefore the flies were fed thrice a week on clean guineapigs : thereafter they were fed at the same intervals on chickens which are very resistant to infection with T. bruceL The cylinders containing the fed flies were kept in a constant temperature room at 24 ° to 25 ° C. in a humidity of about 80 per cent. saturation as recommended by MELLANBY and MELLANBY (1937). This degree of humidity was obtained in the following way. The room is warmed by tubular electric heaters placed about 1 foot above the floor. A piece of lint was draped over one heater with its lower edge dipping into a shallow tray of water, so providing a large warm surface for evaporation. The room was periodically ventilated by means of an electric fan. When the glass cylinders have become fouled with faeces there is a considerable risk of flies getting stuck to the glass by their wings. If this is noticed in time they may be gently freed with a dissecting needle and often appear to be undamaged ; but if left stuck they die, certainly within 24 hours. To avoid this loss the flies are transferred to clean cylinders as the old ones become dirty and the latter are washed and re-assembled. The change is easily carried out by inserting a test-tube through the sleeve vertically into the cylinder and manoeuvering the flies into the tube. They fall to the bottom and are evidently unable to fly in the confined space so that the whole population can be collected without removing the test-tube and then all transferred to fresh quarters. The old label is affixed to the clean cylinder. It is advisable to change the label before transferring the flies as otherwise the label may easily be forgotten, with the result that a batch of flies has to be destroyed for lack of knowledge of its history.
686
MAINTENANCE OF G. M O R S I T A N S IN ENGLAND.
Our earlier experiments on the study of the developmental cycle of trypanosomes entailed killing the flies a few hours or days after they had taken an infective feed so that no observations could be made on the natural death-rate. More recently the flies have been dissected after 35 days (which gives sufficient time for the complete cycle of development of most species of trypanosomes) and notes were kept of the daily mortality. The data from which the table was prepared were obtained by noting the manner in which a population of between 4,000 and 5,000 G. morsitans diminished in number from the time of the infective feed until the survivors were killed for dissection 5 weeks later. The cylinders of flies were examined daily, all dead flies removed, and the deaths booked against the appropriate day after the infective feed. The records of course are not those of a single population but were collected by degrees, and include flies from a number of batches of pupae received over a period of about 6 months. TABLE. THE SURVIVAL OF A POPULATION' OF 1 0 , 0 0 0
Days after Infective Feed. 0 1
2 3 4 5 6 7 8 9
10 11
Numbers of Survivors. 10,000 9,948 9,833 9,632 9,407 9,133 8,799 8,433 8,090 7,812 7,532 7,263
G. mor$itans DURING THE FIRST FIVE WEEKS OF LIFE.
Days after t Number of Infective Feed. Survivors. 12 13 14 15 16 17 18 19 20 21 22 23
7,024 6,803 6,603 6,414 6,268 6,121 5,950 5,768 5,575 5,405 5,223 5,067
Days after Infective Feed. 24 25 26 27 28 29 30 31 32 33 34 35
Number of Survivors.
i ] i i I
l
4,918 4,795 4,655 4,494 4,324 4,171 4,038 3,916 3,803 3,667 3,542 3,423
The death rates were calculated for each day of life. The irregularities due to casual variation in the daily deaths were smoothed to some extent by taking the central death rate of each 3 days instead of the actual rate found on that day. For example: the death-rate for the 7th day was calculated by adding together the deaths occurring on the 6th, 7th and 8th days and dividing by the number of flies living on these days ; in the same way the rate for the 8th day was calculated from the figures for the 7th, 8th and 9th days, and so on. These daily death-rates were then used to calculate the manner in which an original population of 10,000 G. morsitans would have diminished during the first 5 weeks of life.
J. C. BROOM.
687
The table shows, for each day of life after the infective feed, the number* of survivors of such a theoretical population, maintained in the manner described above. By reference to this table one can obtain an indication, at least, as to the number of flies which may be expected to be available for experimental purposes at various times after the original feed, or, conversely with how many flies one should start in order to have a certain number surviving at any time. 10 9 -~
8
e
7
•~ 6 0
•~ 5 ~0
Z
4
2
I
I
0
5
!
10
I
,|
15 20 Days a f t e r infective feed.
|
25
I
30
l
35
Survival of G. morsitans d u r i n g first five weeks of life. : b e t t e r conditions. L o w e r c u r v e : worse conditions.
Upper c u r v e
In the figure the same data are set forth graphically in the upper curve. For the sake of comparison the survival rate of about 650 flies, kept under less satisfactory conditions, is shown on the same chart. The difference in the relative numbers surviving is very marked throughout, and whereas fully onethird of the flies lived for 5 weeks under the better conditions, less than one-tenth reached that age in the other case. * T h e survivors are given to t h e n e a r e s t whole n u m b e r .
688
MAINTENANCEOF
G. M O R ~ I T , 4 N S
IN ENGLAND.
SUMMARY.
1. A method is described for hatching, feeding and maintaining considerable numbers of G. morsitans for experimental work in England. 2. A modified form of life-table is given showing the survival during the first 5 weeks of life after an original feed. REFERENCES. BUXTON,P. A. & L~wIs, D.J. (1935). Climate and tsetse flies : Laboratory studies upon Glossina submorsitans and tachinoides. Philos. Trans. B., 224, 175. & MELLANBY, K. (1934). The measurement and control of humidity. Bull. ent. Res., 25, 171. ~IELLANBY,H. & M~A.~NBY, K. (1937). Rearing tsetse flies in captivity. Proc. R. ent. Soc. Lond. A., 12, 1. ROUBAUD, E. (1917). Histoire d'un 61evage de Glos$ina morsitar~ h l'Institut Pasteur de Paris. Bull. Soc. Path. exot., 10, 629. YORKE, W., MURGATROYD,F. & HAWKING, F. (1933). Studies in chemotherapy. X. Further observations on the transmissibility of tryparsamide-resistance by Glossina. Ann. trop. Med. ParaMt., 27, 157,
Vol. XXXII. No. 5. February, 1939.
T H E M A I N T E N A N C E OF G L O S S I N A M O R S I T A N S
IN E N G L A N D
FOR E X P E R I M E N T A L WORK. BY
j. C. BROOM, M.D.*
The Wellcome Bureau of Scientific Research, London.
The data presented in this note were accumulated in the course of a study of Trypanosoma brucei throughout its cycle of development in Glossina morsitans. It seemed that the experience gained about the feeding and maintaining of considerable numbers of tsetse flies under laboratory conditions in England might be of interest since no such complete records appear to be available in the literature, though YORV~, MURGATROYDand HAWKING (1933) give certain details of the technique they employed in similar work on a smaller scale. The extensive investigation undertaken by BUXTON and L~ccIs (1935) was carried out in Nigeria with a different end in view, but some of their results have been applicable to the present problem. In this country MELLANBY and MELLANBY (1937) maintained a small population of G. palpalis for a number of generations, but the difficulties they met with were of a different nature from ours. For example : as regards feeding, these observers state that flies which are unwilling to feed should be singled out * We should like to record our thanks to Dr. J. F. COaSON who arranged for the regular supply of pupae ; to Mr. KE~ETH MELLANBYfor showing us points of technique in the handling of tsetse flies ; and to Mr. R. J. Rm~Dand Mr. J. A. HAYNESfor the care and patience they exercised in attending to the flies.
684
MAINTENANCE OF G. MORSIT.ZINS I N ENGLAND.
and " coaxed " to do so by giving them repeated additional opportunities. By this means they found that " it is possible to reduce deaths practically to nil during the first 3 months (at least) of the flies' lives ". In our case a much larger population was necessary and anything up to 1,200 flies were under observation a t one time so that any individual treatment was impossible. As a result the death roll was of course relatively heavy, but a sufficient number survived to allow the experiments to be carried on. After a number of attempts to discover the best conditions, the method that has proved most satisfactory is as follows. Pupae of G. morsitans are sent in batches of about 2,000 at fortnightly intervals by air-mail from Tanganyika. The pupae are enclosed in a bag of mosquito-netting, 5 inches by 3 inches tied with a draw-string, and then placed without packing in a cubical box, of about 3 inches inside measurements, made of pieces of 1/4 inch wood nailed together. Half a dozen 1/8 inch holes are bored in each face of the box to ensure a supply of fresh air during the journey which occupies 5 days. A label bearing the address is stuck on one side and no paper or string is used. A certain number of flies are always found hatched out on arrival but they cannot escape from the netting bag. Most of them are dead but often a few, presumably recently emerged, survive and may be used. When they are received the pupae are distributed into two or three glass jars, 4 inches deep and 4 inches wide ; the tops of the jars are covered with mosquito-netting held in position by a rubber band. Each jar is stood in an 11 inch Petri dish containing a saturated solution of common salt to maintain a humidity of 80 per cent. saturation. The whole is covered with a large bell-jar and kept at a temperature of approximately 24 ° C. It has been pointed out by ROUSAUD (1917), who bred small numbers of G. morsitans in Paris, that tsetse flies will not expand their wings in the absence of light. For that reason the great majority of our flies were hatched in an insectarium lighted by daylight; later, when it was not possible to use that room, it was found by experiment that artificial light is equally effective in causing development of the wings. From the 58,000 pupae received to date about 13,500 flies have emerged, i.e., approximately 23 per cent. The adult flies are collected every second day and placed, four or five together, in glass tubes 4 inches by ~ inch one end of which is covered with mosquito-netting fixed on with adhesive strapping and the other end closed with a cork. These tubes are applied to infected rats and the flies given an opportunity to feed. The fed flies are separated and placed, in tens, in glass cylinders* * These cylinders were made by cutting the bottom off 1 lb. glass jam-jars and grinding the rough edge with carborundum paste on a glass slab. Celluloidcylinders (BuxTON and MELLANBY,1934) of comparable size can be obtained but when large numbers are required the difference in price between jam-jars at 10s. a gross and celluloid cylinders at 2s. 6d. each becomes very marked. These jars are much stronger than, for example, lamp glasses and they do not shatter even if knocked off the bench.
J. C. BROOM.
685
4 inches by 2~ inches, one end covered with mosquito-netting, and a short sleeve of the same material attached to the other end, both being fixed on with adhesive strapping. The edges of the sleeve are fastened together with rubber latex. This is simpler and more rapid than sewing and in addition makes a seam that is not affected by frequent washing. Each cylinder thus contains flies all known to have had an infective feed on the same day. The necessary particulars are written on a piece of strapping which is affixed to the cylinder ; this is better than a paper label because strapping can be removed and transferred to a fresh receptacle as required. Unfed flies are returned to the small tubes and given a further chance to feed. In the early experiments in this series, observations were being made on the trypanosomes at different time intervals after ingestion by the fly, and flies which apparently refused to feed on one day were discarded in case they had taken a small feed unnoticed. Later, this precaution was less necessary and a certain number of flies were retained and allowed to feed on another day. When unfed flies were discarded it was found that about 75 per cent. of those hatched could be expected to feed. The complete developmental cycle of the strain of T. brucei used in these experiments occupied at least 25 days in the flies. During the first 3 weeks after the infective feed therefore the flies were fed thrice a week on clean guineapigs : thereafter they were fed at the same intervals on chickens which are very resistant to infection with T. bruceL The cylinders containing the fed flies were kept in a constant temperature room at 24 ° to 25 ° C. in a humidity of about 80 per cent. saturation as recommended by MELLANBY and MELLANBY (1937). This degree of humidity was obtained in the following way. The room is warmed by tubular electric heaters placed about 1 foot above the floor. A piece of lint was draped over one heater with its lower edge dipping into a shallow tray of water, so providing a large warm surface for evaporation. The room was periodically ventilated by means of an electric fan. When the glass cylinders have become fouled with faeces there is a considerable risk of flies getting stuck to the glass by their wings. If this is noticed in time they may be gently freed with a dissecting needle and often appear to be undamaged ; but if left stuck they die, certainly within 24 hours. To avoid this loss the flies are transferred to clean cylinders as the old ones become dirty and the latter are washed and re-assembled. The change is easily carried out by inserting a test-tube through the sleeve vertically into the cylinder and manoeuvering the flies into the tube. They fall to the bottom and are evidently unable to fly in the confined space so that the whole population can be collected without removing the test-tube and then all transferred to fresh quarters. The old label is affixed to the clean cylinder. It is advisable to change the label before transferring the flies as otherwise the label may easily be forgotten, with the result that a batch of flies has to be destroyed for lack of knowledge of its history.
686
MAINTENANCE OF G. M O R S I T A N S IN ENGLAND.
Our earlier experiments on the study of the developmental cycle of trypanosomes entailed killing the flies a few hours or days after they had taken an infective feed so that no observations could be made on the natural death-rate. More recently the flies have been dissected after 35 days (which gives sufficient time for the complete cycle of development of most species of trypanosomes) and notes were kept of the daily mortality. The data from which the table was prepared were obtained by noting the manner in which a population of between 4,000 and 5,000 G. morsitans diminished in number from the time of the infective feed until the survivors were killed for dissection 5 weeks later. The cylinders of flies were examined daily, all dead flies removed, and the deaths booked against the appropriate day after the infective feed. The records of course are not those of a single population but were collected by degrees, and include flies from a number of batches of pupae received over a period of about 6 months. TABLE. THE SURVIVAL OF A POPULATION' OF 1 0 , 0 0 0
Days after Infective Feed. 0 1
2 3 4 5 6 7 8 9
10 11
Numbers of Survivors. 10,000 9,948 9,833 9,632 9,407 9,133 8,799 8,433 8,090 7,812 7,532 7,263
G. mor$itans DURING THE FIRST FIVE WEEKS OF LIFE.
Days after t Number of Infective Feed. Survivors. 12 13 14 15 16 17 18 19 20 21 22 23
7,024 6,803 6,603 6,414 6,268 6,121 5,950 5,768 5,575 5,405 5,223 5,067
Days after Infective Feed. 24 25 26 27 28 29 30 31 32 33 34 35
Number of Survivors.
i ] i i I
l
4,918 4,795 4,655 4,494 4,324 4,171 4,038 3,916 3,803 3,667 3,542 3,423
The death rates were calculated for each day of life. The irregularities due to casual variation in the daily deaths were smoothed to some extent by taking the central death rate of each 3 days instead of the actual rate found on that day. For example: the death-rate for the 7th day was calculated by adding together the deaths occurring on the 6th, 7th and 8th days and dividing by the number of flies living on these days ; in the same way the rate for the 8th day was calculated from the figures for the 7th, 8th and 9th days, and so on. These daily death-rates were then used to calculate the manner in which an original population of 10,000 G. morsitans would have diminished during the first 5 weeks of life.
J. C. BROOM.
687
The table shows, for each day of life after the infective feed, the number* of survivors of such a theoretical population, maintained in the manner described above. By reference to this table one can obtain an indication, at least, as to the number of flies which may be expected to be available for experimental purposes at various times after the original feed, or, conversely with how many flies one should start in order to have a certain number surviving at any time. 10 9 -~
8
e
7
•~ 6 0
•~ 5 ~0
Z
4
2
I
I
0
5
!
10
I
,|
15 20 Days a f t e r infective feed.
|
25
I
30
l
35
Survival of G. morsitans d u r i n g first five weeks of life. : b e t t e r conditions. L o w e r c u r v e : worse conditions.
Upper c u r v e
In the figure the same data are set forth graphically in the upper curve. For the sake of comparison the survival rate of about 650 flies, kept under less satisfactory conditions, is shown on the same chart. The difference in the relative numbers surviving is very marked throughout, and whereas fully onethird of the flies lived for 5 weeks under the better conditions, less than one-tenth reached that age in the other case. * T h e survivors are given to t h e n e a r e s t whole n u m b e r .
688
MAINTENANCEOF
G. M O R ~ I T , 4 N S
IN ENGLAND.
SUMMARY.
1. A method is described for hatching, feeding and maintaining considerable numbers of G. morsitans for experimental work in England. 2. A modified form of life-table is given showing the survival during the first 5 weeks of life after an original feed. REFERENCES. BUXTON,P. A. & L~wIs, D.J. (1935). Climate and tsetse flies : Laboratory studies upon Glossina submorsitans and tachinoides. Philos. Trans. B., 224, 175. & MELLANBY, K. (1934). The measurement and control of humidity. Bull. ent. Res., 25, 171. ~IELLANBY,H. & M~A.~NBY, K. (1937). Rearing tsetse flies in captivity. Proc. R. ent. Soc. Lond. A., 12, 1. ROUBAUD, E. (1917). Histoire d'un 61evage de Glos$ina morsitar~ h l'Institut Pasteur de Paris. Bull. Soc. Path. exot., 10, 629. YORKE, W., MURGATROYD,F. & HAWKING, F. (1933). Studies in chemotherapy. X. Further observations on the transmissibility of tryparsamide-resistance by Glossina. Ann. trop. Med. ParaMt., 27, 157,