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Observations on the breeing of Phlebotomus papatash
452
OBSERVATIONS
ON
THE BREEDING PAPA TASII.
OF P H L E B O T O M U S
BY
M. ASHNER,* Department of Hygiene, Hebrew University, Jerusalem. In connection with investigations on sandfly fever started in our laboratory during the summer of 1926, we attempted to rear Phlebotomus papatasiL We tested the technique employed by CHRISTOPHERSand his co-workers for rearing P. argentipes as well as that of Dr. CH. W. YouNG of the Peking Union Medical College, recommended by him to Dr. ADLER during his visit to Jerusalem. In CHRISTOPHERS'method petri dishes are used containing several layers of damp filter paper to keep the food material moist. In YOUNG'S method a porous earthenware pot, standing in a dish partly filled with water, serves the same purpose ; in this respect this method resembles closely the one described by McCoMBIE YOUNG and his co-workers. CHRISTOPHERS' method did not yield good results in our hands. There were dishes from which only two or three adults emerged, although at the beginning larv~ had been observed in large numbers. With YOUNa'S method the results were better but extremely variable, and from the best pots we never got more than a three-fold number of offspring. Owing to the failure to obtain uniform and satisfactory results, we undertook a detailed study of the factors influencing the development of P. papatasii. This showed that good results depend mainly on three conditions: (1) A mould-free food; (2) Optimal temperature conditions; (3) A minimum of humidity, particularly at the time the larvae pupate. f~:~, With the food recommended by CHRISTOPHERS, that is sterile rabbit f~ces mixed with dried blood, an exuberant growth of moulds had already appeared during the first week, so that the normal development of the Phlebotomus was quite impossible. Death occurred specially among the very young larvae and the defenceless pupae; but even the older larvae suffered, many of them failing to pupate even if brought into better conditions. After various trials it was found that the moulds are easily suppressed by using a food consisting of fresh rabbit faeces mixed with soil. To obtain a homogenous medium for comparative observations, one part of sifted garden soil was mixed with two parts of the crushed f~eces, water added to make a thick paste, and the mixture dried in the air with repeated stirring. Of this * Presented by I. J. KLIGLER.
OBSERVATIONS ON THE BREEDING OF
P.P.4.PATASII.
d~8
mixture the larva~ were allowed just as much as could easily be worked over and kept spongy by them ; the newly hatched larva~ were given a few particles only, and the older ones larger amounts according to their number and size, until the bottom of the vessel was covered with a layer of about 1 to 2 cm. e"
/
i
-
i
Earthenware pots for breeding Phlebotomus.
In our first experiments the breeding pots were kept in an incubator at a temperature of 23 ° to 25 ° C., which corresponded to the average outdoor temperature at that time. The results, however, were not satisfactory, and it was soon found that a temperature of 30 ° to 32 ° C. is much more favourable to the development of the larva~. This observation was surprising, since the average summer temperature in Jerusalem is never as high as this. The full development at 23 ° to 25 ° C. extended over a period of about 60 to 65 days, the egg stage lasting 12 to 15, the larval stage 30 to 35, and the pupal period 13 to 15 days. At a temperature of 30 ° to 32 ° C., however, the corresponding times are--for the egg 6, the larva 20, and the pupa 6 days. The process of development at the higher temperature not only is thus reduced by about half the time, but the larvae, because of their greater activity and quicker feeding, prevent entirely the growth of the injurious moulds. As a result of this the development of the larvae and the hatching of the pupae progress with more regularity and certainty than at lower temperatures. The period of development of different batches of flies at the higher temperature, the other factors being favourable, revealed a striking uniformity. This rendered it possible in most cases to predict the exact day when the hatching of the Phlebotomus would begin. The third important point is to avoid excessive humidity. A large number of pupa~ fail to hatch, and die, whenever the larvm are forced to pupate in a too moist environment. The necessary degree of humidity can hardly be stated in exact terms. At any rate, as far as we observed, the proper degree of moisture is already surpassed if under the binocular microscope water is seen in the form of tiny droplets or, as suggested by WHITTINGHAM, as a thin film on the surface of the particles. Normal conditions of moisture are present, if the grownup larvm do not emigrate, but pupate in the food substrate. In the petri dishes
~54
OBSERVATIONS ON THE BREEDING OF P. PAPATASH.
the moisture cannot be regulated as well as in the earthenware pots, and so the excess of water seems to have been the reason for our failure to get satisfactory results with CHRISTOPHERS' method. We now employ the following procedure, which gives us absolutely uniform, constant, and predictable results : Twenty to thirty females are brought into an earthenware pot, about 15 cm. in diameter and 5 era. deep. The pot is covered with a piece of cloth and kept in an incubator at a temperature of 30 ° to 32 ° C. The necessary moisture inside the pot is provided by placing it in a dish of water, and the concentration regulated by increasing or decreasing the depth of the water in the dish. In case the pots are too porous we found it more convenient to place them in a dish containing damp soil, the moisture in the pots being regulated by the degree of dampness of the soil. This method saves the trouble of adding water to the pans daily, because the evaporation from the soil is slower. After five to six days most of the females will have laid their eggs and died. The pot is then opened and the number of eggs laid determined. The eggs are found both scattered and in batches on the bottom and the wall of the vessel. As a rule three or four days after opening the pot the first larwe hatch, and now food must be given immediately, because the newly hatched larwe do not live more than a day without food. It is also possible to scatter a few particles of the food mixture into the pot before putting in the females, so that there is enough food present when the larvae appear. After each examination the pot should be covered again with the cloth, to prevent the larvae escaping and other insects from laying their eggs in the food substrate. When the pupae start hatching the pots are opened daily in a small muslin cage, where the emerged adults are easily caught into test-tubes. Under normal conditions the hatching of the pupae will be finished in two or three weeks ; but, where the conditions are not optimal, one can still find unpupated larwe a month or so after the first adult comes out. As a rule males and females hatch at the same time and in equal numbers ; the females thus become fertilized while still in the breeding pot. The laboratory-bred females feed much more readily than those caught wild. They may feed two or three times during the first week without difficulty. At a temperature of 25 ° C., at which our flies are kept, it takes about nine days after the first blood meal for the eggs to develop and then oviposition will take place if the females are placed in the breeding pots. It is thus possible to rear several generations of P. papatasii in the laboratory without any difficulty. REFERENCES.
WHITTINGHAM, H. E. and ROOK, A. F. (1923). The life-history and bionomics of Phlebotomus papatasii. Brit. M e d . Jl., ii., 1144. CI-mlSTOPHnas, S. R., SHORT, H. E. and BARRAUD, J. P. (1926). Technique employed in breeding Phlebotomus argentipes in Assam. Ind. ffl. Med. Res. Mem. 6 ; February. YOUNG, T . C. McCOMBIE, RICHMOND,A. E., and BRENDISH, G . R . (1926). Sandflies and sandfly fever in the Peshawar district. Ind. ffl. Med. Res., xiii, (4), 961 ; April.
OBSERVATIONS
ON
THE BREEDING PAPA TASII.
OF P H L E B O T O M U S
BY
M. ASHNER,* Department of Hygiene, Hebrew University, Jerusalem. In connection with investigations on sandfly fever started in our laboratory during the summer of 1926, we attempted to rear Phlebotomus papatasiL We tested the technique employed by CHRISTOPHERSand his co-workers for rearing P. argentipes as well as that of Dr. CH. W. YouNG of the Peking Union Medical College, recommended by him to Dr. ADLER during his visit to Jerusalem. In CHRISTOPHERS'method petri dishes are used containing several layers of damp filter paper to keep the food material moist. In YOUNG'S method a porous earthenware pot, standing in a dish partly filled with water, serves the same purpose ; in this respect this method resembles closely the one described by McCoMBIE YOUNG and his co-workers. CHRISTOPHERS' method did not yield good results in our hands. There were dishes from which only two or three adults emerged, although at the beginning larv~ had been observed in large numbers. With YOUNa'S method the results were better but extremely variable, and from the best pots we never got more than a three-fold number of offspring. Owing to the failure to obtain uniform and satisfactory results, we undertook a detailed study of the factors influencing the development of P. papatasii. This showed that good results depend mainly on three conditions: (1) A mould-free food; (2) Optimal temperature conditions; (3) A minimum of humidity, particularly at the time the larvae pupate. f~:~, With the food recommended by CHRISTOPHERS, that is sterile rabbit f~ces mixed with dried blood, an exuberant growth of moulds had already appeared during the first week, so that the normal development of the Phlebotomus was quite impossible. Death occurred specially among the very young larvae and the defenceless pupae; but even the older larvae suffered, many of them failing to pupate even if brought into better conditions. After various trials it was found that the moulds are easily suppressed by using a food consisting of fresh rabbit faeces mixed with soil. To obtain a homogenous medium for comparative observations, one part of sifted garden soil was mixed with two parts of the crushed f~eces, water added to make a thick paste, and the mixture dried in the air with repeated stirring. Of this * Presented by I. J. KLIGLER.
OBSERVATIONS ON THE BREEDING OF
P.P.4.PATASII.
d~8
mixture the larva~ were allowed just as much as could easily be worked over and kept spongy by them ; the newly hatched larva~ were given a few particles only, and the older ones larger amounts according to their number and size, until the bottom of the vessel was covered with a layer of about 1 to 2 cm. e"
/
i
-
i
Earthenware pots for breeding Phlebotomus.
In our first experiments the breeding pots were kept in an incubator at a temperature of 23 ° to 25 ° C., which corresponded to the average outdoor temperature at that time. The results, however, were not satisfactory, and it was soon found that a temperature of 30 ° to 32 ° C. is much more favourable to the development of the larva~. This observation was surprising, since the average summer temperature in Jerusalem is never as high as this. The full development at 23 ° to 25 ° C. extended over a period of about 60 to 65 days, the egg stage lasting 12 to 15, the larval stage 30 to 35, and the pupal period 13 to 15 days. At a temperature of 30 ° to 32 ° C., however, the corresponding times are--for the egg 6, the larva 20, and the pupa 6 days. The process of development at the higher temperature not only is thus reduced by about half the time, but the larvae, because of their greater activity and quicker feeding, prevent entirely the growth of the injurious moulds. As a result of this the development of the larvae and the hatching of the pupae progress with more regularity and certainty than at lower temperatures. The period of development of different batches of flies at the higher temperature, the other factors being favourable, revealed a striking uniformity. This rendered it possible in most cases to predict the exact day when the hatching of the Phlebotomus would begin. The third important point is to avoid excessive humidity. A large number of pupa~ fail to hatch, and die, whenever the larvm are forced to pupate in a too moist environment. The necessary degree of humidity can hardly be stated in exact terms. At any rate, as far as we observed, the proper degree of moisture is already surpassed if under the binocular microscope water is seen in the form of tiny droplets or, as suggested by WHITTINGHAM, as a thin film on the surface of the particles. Normal conditions of moisture are present, if the grownup larvm do not emigrate, but pupate in the food substrate. In the petri dishes
~54
OBSERVATIONS ON THE BREEDING OF P. PAPATASH.
the moisture cannot be regulated as well as in the earthenware pots, and so the excess of water seems to have been the reason for our failure to get satisfactory results with CHRISTOPHERS' method. We now employ the following procedure, which gives us absolutely uniform, constant, and predictable results : Twenty to thirty females are brought into an earthenware pot, about 15 cm. in diameter and 5 era. deep. The pot is covered with a piece of cloth and kept in an incubator at a temperature of 30 ° to 32 ° C. The necessary moisture inside the pot is provided by placing it in a dish of water, and the concentration regulated by increasing or decreasing the depth of the water in the dish. In case the pots are too porous we found it more convenient to place them in a dish containing damp soil, the moisture in the pots being regulated by the degree of dampness of the soil. This method saves the trouble of adding water to the pans daily, because the evaporation from the soil is slower. After five to six days most of the females will have laid their eggs and died. The pot is then opened and the number of eggs laid determined. The eggs are found both scattered and in batches on the bottom and the wall of the vessel. As a rule three or four days after opening the pot the first larwe hatch, and now food must be given immediately, because the newly hatched larwe do not live more than a day without food. It is also possible to scatter a few particles of the food mixture into the pot before putting in the females, so that there is enough food present when the larvae appear. After each examination the pot should be covered again with the cloth, to prevent the larvae escaping and other insects from laying their eggs in the food substrate. When the pupae start hatching the pots are opened daily in a small muslin cage, where the emerged adults are easily caught into test-tubes. Under normal conditions the hatching of the pupae will be finished in two or three weeks ; but, where the conditions are not optimal, one can still find unpupated larwe a month or so after the first adult comes out. As a rule males and females hatch at the same time and in equal numbers ; the females thus become fertilized while still in the breeding pot. The laboratory-bred females feed much more readily than those caught wild. They may feed two or three times during the first week without difficulty. At a temperature of 25 ° C., at which our flies are kept, it takes about nine days after the first blood meal for the eggs to develop and then oviposition will take place if the females are placed in the breeding pots. It is thus possible to rear several generations of P. papatasii in the laboratory without any difficulty. REFERENCES.
WHITTINGHAM, H. E. and ROOK, A. F. (1923). The life-history and bionomics of Phlebotomus papatasii. Brit. M e d . Jl., ii., 1144. CI-mlSTOPHnas, S. R., SHORT, H. E. and BARRAUD, J. P. (1926). Technique employed in breeding Phlebotomus argentipes in Assam. Ind. ffl. Med. Res. Mem. 6 ; February. YOUNG, T . C. McCOMBIE, RICHMOND,A. E., and BRENDISH, G . R . (1926). Sandflies and sandfly fever in the Peshawar district. Ind. ffl. Med. Res., xiii, (4), 961 ; April.