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The development of mosquitoes in complete darkness
467 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE.
Vol. XXX.
No. 4.
January, 1937
T H E D E V E L O P M E N T OF M O S Q U I T O E S IN C O M P L E T E DARKNESS. BY
B JOBLING,
Wellcome Entomological Field Laboratories, Wellcome Bureau of Scientific Research, London.
INTRODUCTION.
It has already been mentioned in my previous paper e that the autogenous
Culexpipiens can develop perfectly, mate and lay fertile eggs in complete darkness. Since the publication of that paper I have made the same experiments with the common, nonautogenous Culex pipiens, Culex fatigans and the yellow fever mosquito Aides argenteus. It is the purpose of this paper to give the description of the technique which has been used and the results of these experiments. EXPERIMENTS.
The experiments with the above-mentioned mosquitoes were carried out in an apparatus which was made of thick cardboard (Fig. 1). The walls of this *JOBLING, I~. (1935). The effect of light and darkness on oviposltion in mosquitoes. Trans. R. Soc. trop. Med. & Hyg., xxix, 157.
468
DEVELOPMENT OF MOSQUITOES IN DARKNESS.
apparatus-were 12 inches high and 7 inches wide. The inside dimensions of it were adapted for the accommodation of an enamelled dish, 6 inches in diameter, which was used for the breeding of the mosquitoes. The three walls of this apparatus extended about 2 inches below the bottom, which was provided with a ventilating hole (o.) in each corner. Each ventilating hole was screened below the bottom by a U-shaped piece of cardboard (u.p.) attached to the bottom and to the wall. Inside the apparatus each of these holes was screened by an obliquely fixed, oblong plate (p.). In the middle of the top of the apparatus there was a round ventilating hole, 2 inches in diameter. Inside the apparatus this hole was screened by a disc (d.) which was attached under it to the top by two wooden blocks, {- inch long. On top it was surrounded by an inner cylinder (i.c.) of slightly bigger diameter. This had two semicircular holes (o.) in the upper part and to its rim was attached a disc (d'.) which screened the interior from above. The inner cylinder was surrounded by an outer cylinder (o.c.). This was about 2 inches longer than the inner one and of such a diameter that its wall was separated by ~ inch from the edge of the upper disc. To prevent the escape of the mosquitoes through the ventilators, they were covered inside the apparatus with mosquito netting (m.n.). The door (do.) was made of a wooden board to which was fixed a wooden frame (w.f.). Between this frame and the edge of the door the board was lined with a thick woollen material. When the apparatus was closed, the frame of the door rested in the wooden frame of the apparatus and was in contact with a narrow screen (s.). For aerating the infusion in the dish, the apparatus was provided with an aerating pipe (a.p.). This pipe was inserted through a cork which was fixed in the wall and was connected by rubber tubing with an air pump. In order to exclude completely the passage of light into the pipe, between the end of the rubber tubing and the cork, the latter had an excavation round the pipe to receive the end of the rubber tubing. The temperature of the air and water was measured by two thermometers. The projecting part of the air thermometer was screened by a light-proof case inside the apparatus, whereas the water thermometer was screened from outside. All the internal parts of the apparatus were painted black, while externally it was given two coats of white enamel. The light-proof construction of the apparatus was tested by the following method : - - I n a dark room a photographic plate was cut in two halves. One half was immediately developed and fixed, whereas the other half was put inside the apparatus, which was closed and placed in front of a window. After a week the apparatus was brought into the dark room, the plate was taken out, developed and fixed and then compared with the other half. On comparison of these two halves it was found that there was no difference in their colouration, both being quite clear. This indicated the absence of light in the apparatus when exposed to daylight.
B. J O B L I N G .
469
The liquid for the breeding of the mosquitoes was dog biscuit infusion in all the experiments, because it has been found that the conditions in this remain constant for a much longer period of time than in hay infusion. The infusion was prepared a day before and then strained into two dishes of the same size. Into each dish was introduced a raft of eggs. These were always of the same age and contained the same number of eggs. In the experiments with the nonautogenous Culex pipiens and C. fatigans one big raft was divided into two equal parts. After the introduction of the eggs, one dish was placed in the apparatus, while the other was left exposed to light.
-d:
~----
--
7
-
.
.
.
.
.
FIG. 1. APPARATUS FOR BREEDING MOSQUITOES IN ABSENCE OF LIGHT.
470
DEVELOPMENT OF MOSQUITOES IN DARKNESS.
The breeding in the exposed dish has not been considered as a control in these experiments, because it is impossible to maintain exactly the same conditions in the two separate dishes with the same infusion. It has often happened during the breeding of the mosquitoes that in one dish all the eggs develop to the adult stage, whereas in the other dish, with the same infusion, the eggs from the same raft develop normally only to the first or the second stage larvae, and then they begin to die. In this case the mortality greatly increases when the remaining larvae reach the fourth stage, so that none of them develop to adults. This mortality of the larvae is produced by the action of the bacteria in the infusion. Sometimes the sudden dying of the larvae begins together with a change of colour of the infusion and a slight change of the pH. The dog biscuit and the bread infusions change from milky to brownish colour and become much more transparent. But sometimes there are no obvious changes in the infusion when the larvae begin to die, and the pH remains the same from the moment of the introduction of the eggs up to the appearance of the fourth stage larvae. The breeding in the open dish was used as an indicator of the end of the experiment, and the apparatus was always opened and examined 1 or 2 days after the hatching of the last mosquito from the open dish. With each species of mosquito the experiment was repeated. As it was impossible to avoid the introduction of diffused light into the apparatus when the pH and the temperature of the infusion were taken, the second experiment was performed without the reading of these factors. In all the experiments the pH of the infusion in both dishes was always the same, and its Changes occurred simultaneously in both dishes, with the exception of one experiment, where for 2 days the difference was 0.5. The pH of two of the experiments is shown in the chart together with the temperature of the infusions and the air temperature (Fig. 2). As regards the development of the mosquitoes in the two dishes, the temperature of the infusion was the most important factor. Since in all the experiments the temperature of one dish never varied more than 1° C. from that of the other, and the mean temperature was practically the same in both, this factor will not be given for each experiment separately. THE DEVELOPMENT OF THE MOSQUITOES.
The Common Nonautogenous Culex pipiens. In the experiments with this mosquito the infusion in both dishes was equally aerated and an equal number of eggs from the same raft was introduced into each dish. The first experiment, when the temperature and the pH were taken, was not successful. Out of 80 eggs only 11 developed to the adult stage in the dish exposed to light. The greatest mortality in this dish occurred amongst the fourth stage larvae and the pupae. In the dish in the apparatus all the larvae died in the third or the fourth stage.
B. JOBLING.
471
The experiment was repeated without taking the temperature and the samples of infusion for the pH. At the end of the experiment 36 adults emerged from the dish exposed to light and 23 dead pupae and 14 dead fourth stage larvae were found in the infusion. In the apparatus 24 adults, 6 dead pupae and 14 dead fourth stage larvae were found. The result of the last experiment indicates that the nonautogenous C. pipiens can develop in complete darkness. The small percentage of development (30 per cent.) was not due to the absence of light, but to the unfavourable conditions in the infusion. The larvae of this mosquito are more sensitive to changes in their environment than are those of the other mosquitoes used in these experiments.
~,~st--T-F-P-
I
1
~3.J F .q t I 2~?.o t '. l ; '.
i . :
2 ~..s-I
I
I
I ,~.,VI
I
I ",L
,
j
¢.~,a i i~qvi "~0
I¥ I o"j I
..7o,0
~
I I
i IIA~ I ! ~ "...,~
/s'oi i i l
/8..S
/,~.ol
i
i
i
/d..J ~
i
i
i
:
FiG. 2. GRAPHS
SHOWING
VARIATION
OF
pH, IN
TEMPERATURE TWO
OF
INFUSION
AND
AIR
TEMPERATURE
EXPERIMENTS.
The Autogenous Culex pipiens. In the experiment with this mosquito the infusion was equally aerated in both dishes, and a separate raft of eggs was introduced into each. These rafts had the same number of eggs and were laid at the same time. During the first two experiments the pH, the temperature of the infusion and the air temperature were taken every day. In one of these experiments 51 eggs produced 46 adults in the dish exposed to light, while from the same number of eggs there were
479'
DEVELOPMENT
OF MOSQUITOES IN DARKNESS.
found 47 adults and 2 live pupae in the apparatus. In the other experiment out of 53 eggs in the open dish, 45 developed to the adult, whereas in the apparatus the same number of eggs produced 47 adults, and three new rafts of eggs were found from which numerous first stage larvae had already hatched. In order to exclude the light completely the experiment was repeated without taking the temperature and the pH. In this experiment 36 eggs were introduced into each dish. Twenty-four days after the introduction of the rafts, the breeding in the open dish was finished, and out of 36 eggs, 29 developed to the adult. The apparatus was opened 16 days after the emergence of the last mosquito from the open dish, and in it were found 15 dead adults, 10 live and 9 dead fourth stage larvae and 22 pupa cases. As regards the adults and the pupa cases, they were undoubtedly of the first generation, but it is impossible to know definitely the exact number of the former, because their dead bodies and the empty pupa cases usually disintegrate very quickly in the infusion. All the fourth stage larvae must have belonged to the second generation, because at the mean temperature 19.4 ° C. in the room during the experiment, each stage of the larva lasts only 3 or 4 days. A few larvae may prolong their development, but this retardation indicates their unhealthy condition. Such larvae rarely develop to the adult and generally die during pupation. The presence of only a small number of larvae of the second generation, was due to the deficiency of food and the unfavourable conditions in the infusion, which was found to be clear and brownish in colour. The results of these experiments show quite conclusively that this mosquito can develop, mate and produce a second generation in complete darkness.
Culex fatigans. In the experiments with this mosquito the infusion was not aerated and eggs of the same raft were used for both dishes. The first experiment, when the temperature and the pH were taken, gave the following result. Out of 70 eggs placed in the dish exposed to light, 57 produced mosquitoes, while in the apparatus the same number of eggs produced 67 mosquitoes. In the other experiment, when the pH and the temperature of the infusion were not taken, the result was practically the same. Out of 75 eggs placed in the dish exposed to light, 58 developed to the adult, while in the complete darkness of the apparatus, the same number of eggs produced 63 adults. The following experiment was made in order to see whether this mosquito can lay eggs in complete darkness. Six females were put into the apparatus after they had been fed. Four days later the apparatus was opened and in the dish were found six rafts of eggs from which numerous first stage larvae had already hatched. The above described experiments show quite conclusively that Culexfatigans can lay eggs and breed perfectly in complete darkness.
B. JOBLINC..
4:78
Aides argenteus. In the experiments with this mosquito the infusion in the dishes was not aerated. As the females of this mosquito rarely produce a large number of eggs at one laying, in the first experiment, when the temperature of the infusion and the pH were taken, each dish received 70 eggs which were laid at the same time, but by different females. In the dish exposed to light, out of 70 eggs, 47 produced adults and there were present several unhatched eggs, 3 dead fourth stage larvae and 5 dead pupae. In the apparatus the same number of eggs produced 38 adults and 6 dead third stage larvae, 2 dead fourth stage larvae and several unhatched eggs were found in the infusion. During the second experiment neither the temperature of the infusion nor the pH were taken. From 50 eggs laid by one female in the dish exposed to light, 45 developed to the adult, while in the apparatus, from the same number of eggs, which were laid by another female, 45 developed to the adult and in the infusion were found 3 live fourth stage larvae and 2 dead pupae. The results of these experiments show that Aides argenteus can develop perfectly in complete darkness. The following experiment shows that this mosquito can also lay eggs in complete darkness. Six fed females were put into the apparatus. When 4 days later it was opened 450 eggs were found in the infusion. That all the six females took part in the laying of these eggs was shown by the examination of their abdomens. CONCLUSION.
The results of the experiments show quite conclusively that the common, nonautogenous Culex pipiens, the autogenous Culex pipiens, Culex fatigans and Aides argenteus can develop perfectly in complete absence of light. The ability of the larvae of these mosquitoes to exist under this condition is probably due to their feeding habit. They are omnivorous and are not affected by the absence of algae, such as Cyanophyceae and Chlorophyceae and the high aquatic plants, which cannot exist without light. In complete darkness the food of these larvae consists of small particles of organic matter, and of such organisms as bacteria, some Mastigophora and Ciliata. The existence of these organisms depends also upon the presence of organic matter in the water and they also are not affected by the absence of light. The experiments also show that Culex fatigans, Aides argenteus and the autogenous Culex pipiens can lay eggs in complete darkness and that the last named mosquito can produce a second generation. As regards the oviposition, it has already been pointed out in my previous paper (JoBLI~G, 1935) that in complete darkness it is governed either by the chemical condition of the water, or by the water vapour, or by the combined action of these two factors, because in complete darkness there is no stimulus to develop the phototactic reaction in the mosquito. The same can be said about the mating. The male cannot Fe
474
DEVELOPMENT OF MOSQUITOES IN DARKNESS.
see the female in complete darkness; therefore, it is attracted to it either by the scent, or the sound which the female may produce, or by both of these factors. The results of these experiments may have some practical interest when we remember that suitable breeding places may be present in completely dark situations. The larvae of some of these mosquitoes have been found in dark cellars, in dark interiors of cisterns and in tanks built inside walls. Such places represent more dangerous mosquito breeding grounds than those in the open, especially as regards the urban species, because breeding in these places is not always easily detected. SUMMARY°
The experiments were made with two races of Culex pipiens, Culexfatigans and Aides argenteus in a specially constructed apparatus. T h e results of these experiments show quite conclusively that all these mosquitoes can develop perfectly in complete darkness. Aides argenteus, Culex fatigans and the autogenous Culex pipiens can lay eggs in complete darkness and the last named mosquito can mate and produce a second generation. T h e ability of the larvae of these mosquitoes to develop perfectly in absence of light is probably due to their feeding habit. They are omnivorous and can exist upon organic matter, and upon such organisms as can also live in complete absence of light if organic matter is present in the water.
Vol. XXX.
No. 4.
January, 1937
T H E D E V E L O P M E N T OF M O S Q U I T O E S IN C O M P L E T E DARKNESS. BY
B JOBLING,
Wellcome Entomological Field Laboratories, Wellcome Bureau of Scientific Research, London.
INTRODUCTION.
It has already been mentioned in my previous paper e that the autogenous
Culexpipiens can develop perfectly, mate and lay fertile eggs in complete darkness. Since the publication of that paper I have made the same experiments with the common, nonautogenous Culex pipiens, Culex fatigans and the yellow fever mosquito Aides argenteus. It is the purpose of this paper to give the description of the technique which has been used and the results of these experiments. EXPERIMENTS.
The experiments with the above-mentioned mosquitoes were carried out in an apparatus which was made of thick cardboard (Fig. 1). The walls of this *JOBLING, I~. (1935). The effect of light and darkness on oviposltion in mosquitoes. Trans. R. Soc. trop. Med. & Hyg., xxix, 157.
468
DEVELOPMENT OF MOSQUITOES IN DARKNESS.
apparatus-were 12 inches high and 7 inches wide. The inside dimensions of it were adapted for the accommodation of an enamelled dish, 6 inches in diameter, which was used for the breeding of the mosquitoes. The three walls of this apparatus extended about 2 inches below the bottom, which was provided with a ventilating hole (o.) in each corner. Each ventilating hole was screened below the bottom by a U-shaped piece of cardboard (u.p.) attached to the bottom and to the wall. Inside the apparatus each of these holes was screened by an obliquely fixed, oblong plate (p.). In the middle of the top of the apparatus there was a round ventilating hole, 2 inches in diameter. Inside the apparatus this hole was screened by a disc (d.) which was attached under it to the top by two wooden blocks, {- inch long. On top it was surrounded by an inner cylinder (i.c.) of slightly bigger diameter. This had two semicircular holes (o.) in the upper part and to its rim was attached a disc (d'.) which screened the interior from above. The inner cylinder was surrounded by an outer cylinder (o.c.). This was about 2 inches longer than the inner one and of such a diameter that its wall was separated by ~ inch from the edge of the upper disc. To prevent the escape of the mosquitoes through the ventilators, they were covered inside the apparatus with mosquito netting (m.n.). The door (do.) was made of a wooden board to which was fixed a wooden frame (w.f.). Between this frame and the edge of the door the board was lined with a thick woollen material. When the apparatus was closed, the frame of the door rested in the wooden frame of the apparatus and was in contact with a narrow screen (s.). For aerating the infusion in the dish, the apparatus was provided with an aerating pipe (a.p.). This pipe was inserted through a cork which was fixed in the wall and was connected by rubber tubing with an air pump. In order to exclude completely the passage of light into the pipe, between the end of the rubber tubing and the cork, the latter had an excavation round the pipe to receive the end of the rubber tubing. The temperature of the air and water was measured by two thermometers. The projecting part of the air thermometer was screened by a light-proof case inside the apparatus, whereas the water thermometer was screened from outside. All the internal parts of the apparatus were painted black, while externally it was given two coats of white enamel. The light-proof construction of the apparatus was tested by the following method : - - I n a dark room a photographic plate was cut in two halves. One half was immediately developed and fixed, whereas the other half was put inside the apparatus, which was closed and placed in front of a window. After a week the apparatus was brought into the dark room, the plate was taken out, developed and fixed and then compared with the other half. On comparison of these two halves it was found that there was no difference in their colouration, both being quite clear. This indicated the absence of light in the apparatus when exposed to daylight.
B. J O B L I N G .
469
The liquid for the breeding of the mosquitoes was dog biscuit infusion in all the experiments, because it has been found that the conditions in this remain constant for a much longer period of time than in hay infusion. The infusion was prepared a day before and then strained into two dishes of the same size. Into each dish was introduced a raft of eggs. These were always of the same age and contained the same number of eggs. In the experiments with the nonautogenous Culex pipiens and C. fatigans one big raft was divided into two equal parts. After the introduction of the eggs, one dish was placed in the apparatus, while the other was left exposed to light.
-d:
~----
--
7
-
.
.
.
.
.
FIG. 1. APPARATUS FOR BREEDING MOSQUITOES IN ABSENCE OF LIGHT.
470
DEVELOPMENT OF MOSQUITOES IN DARKNESS.
The breeding in the exposed dish has not been considered as a control in these experiments, because it is impossible to maintain exactly the same conditions in the two separate dishes with the same infusion. It has often happened during the breeding of the mosquitoes that in one dish all the eggs develop to the adult stage, whereas in the other dish, with the same infusion, the eggs from the same raft develop normally only to the first or the second stage larvae, and then they begin to die. In this case the mortality greatly increases when the remaining larvae reach the fourth stage, so that none of them develop to adults. This mortality of the larvae is produced by the action of the bacteria in the infusion. Sometimes the sudden dying of the larvae begins together with a change of colour of the infusion and a slight change of the pH. The dog biscuit and the bread infusions change from milky to brownish colour and become much more transparent. But sometimes there are no obvious changes in the infusion when the larvae begin to die, and the pH remains the same from the moment of the introduction of the eggs up to the appearance of the fourth stage larvae. The breeding in the open dish was used as an indicator of the end of the experiment, and the apparatus was always opened and examined 1 or 2 days after the hatching of the last mosquito from the open dish. With each species of mosquito the experiment was repeated. As it was impossible to avoid the introduction of diffused light into the apparatus when the pH and the temperature of the infusion were taken, the second experiment was performed without the reading of these factors. In all the experiments the pH of the infusion in both dishes was always the same, and its Changes occurred simultaneously in both dishes, with the exception of one experiment, where for 2 days the difference was 0.5. The pH of two of the experiments is shown in the chart together with the temperature of the infusions and the air temperature (Fig. 2). As regards the development of the mosquitoes in the two dishes, the temperature of the infusion was the most important factor. Since in all the experiments the temperature of one dish never varied more than 1° C. from that of the other, and the mean temperature was practically the same in both, this factor will not be given for each experiment separately. THE DEVELOPMENT OF THE MOSQUITOES.
The Common Nonautogenous Culex pipiens. In the experiments with this mosquito the infusion in both dishes was equally aerated and an equal number of eggs from the same raft was introduced into each dish. The first experiment, when the temperature and the pH were taken, was not successful. Out of 80 eggs only 11 developed to the adult stage in the dish exposed to light. The greatest mortality in this dish occurred amongst the fourth stage larvae and the pupae. In the dish in the apparatus all the larvae died in the third or the fourth stage.
B. JOBLING.
471
The experiment was repeated without taking the temperature and the samples of infusion for the pH. At the end of the experiment 36 adults emerged from the dish exposed to light and 23 dead pupae and 14 dead fourth stage larvae were found in the infusion. In the apparatus 24 adults, 6 dead pupae and 14 dead fourth stage larvae were found. The result of the last experiment indicates that the nonautogenous C. pipiens can develop in complete darkness. The small percentage of development (30 per cent.) was not due to the absence of light, but to the unfavourable conditions in the infusion. The larvae of this mosquito are more sensitive to changes in their environment than are those of the other mosquitoes used in these experiments.
~,~st--T-F-P-
I
1
~3.J F .q t I 2~?.o t '. l ; '.
i . :
2 ~..s-I
I
I
I ,~.,VI
I
I ",L
,
j
¢.~,a i i~qvi "~0
I¥ I o"j I
..7o,0
~
I I
i IIA~ I ! ~ "...,~
/s'oi i i l
/8..S
/,~.ol
i
i
i
/d..J ~
i
i
i
:
FiG. 2. GRAPHS
SHOWING
VARIATION
OF
pH, IN
TEMPERATURE TWO
OF
INFUSION
AND
AIR
TEMPERATURE
EXPERIMENTS.
The Autogenous Culex pipiens. In the experiment with this mosquito the infusion was equally aerated in both dishes, and a separate raft of eggs was introduced into each. These rafts had the same number of eggs and were laid at the same time. During the first two experiments the pH, the temperature of the infusion and the air temperature were taken every day. In one of these experiments 51 eggs produced 46 adults in the dish exposed to light, while from the same number of eggs there were
479'
DEVELOPMENT
OF MOSQUITOES IN DARKNESS.
found 47 adults and 2 live pupae in the apparatus. In the other experiment out of 53 eggs in the open dish, 45 developed to the adult, whereas in the apparatus the same number of eggs produced 47 adults, and three new rafts of eggs were found from which numerous first stage larvae had already hatched. In order to exclude the light completely the experiment was repeated without taking the temperature and the pH. In this experiment 36 eggs were introduced into each dish. Twenty-four days after the introduction of the rafts, the breeding in the open dish was finished, and out of 36 eggs, 29 developed to the adult. The apparatus was opened 16 days after the emergence of the last mosquito from the open dish, and in it were found 15 dead adults, 10 live and 9 dead fourth stage larvae and 22 pupa cases. As regards the adults and the pupa cases, they were undoubtedly of the first generation, but it is impossible to know definitely the exact number of the former, because their dead bodies and the empty pupa cases usually disintegrate very quickly in the infusion. All the fourth stage larvae must have belonged to the second generation, because at the mean temperature 19.4 ° C. in the room during the experiment, each stage of the larva lasts only 3 or 4 days. A few larvae may prolong their development, but this retardation indicates their unhealthy condition. Such larvae rarely develop to the adult and generally die during pupation. The presence of only a small number of larvae of the second generation, was due to the deficiency of food and the unfavourable conditions in the infusion, which was found to be clear and brownish in colour. The results of these experiments show quite conclusively that this mosquito can develop, mate and produce a second generation in complete darkness.
Culex fatigans. In the experiments with this mosquito the infusion was not aerated and eggs of the same raft were used for both dishes. The first experiment, when the temperature and the pH were taken, gave the following result. Out of 70 eggs placed in the dish exposed to light, 57 produced mosquitoes, while in the apparatus the same number of eggs produced 67 mosquitoes. In the other experiment, when the pH and the temperature of the infusion were not taken, the result was practically the same. Out of 75 eggs placed in the dish exposed to light, 58 developed to the adult, while in the complete darkness of the apparatus, the same number of eggs produced 63 adults. The following experiment was made in order to see whether this mosquito can lay eggs in complete darkness. Six females were put into the apparatus after they had been fed. Four days later the apparatus was opened and in the dish were found six rafts of eggs from which numerous first stage larvae had already hatched. The above described experiments show quite conclusively that Culexfatigans can lay eggs and breed perfectly in complete darkness.
B. JOBLINC..
4:78
Aides argenteus. In the experiments with this mosquito the infusion in the dishes was not aerated. As the females of this mosquito rarely produce a large number of eggs at one laying, in the first experiment, when the temperature of the infusion and the pH were taken, each dish received 70 eggs which were laid at the same time, but by different females. In the dish exposed to light, out of 70 eggs, 47 produced adults and there were present several unhatched eggs, 3 dead fourth stage larvae and 5 dead pupae. In the apparatus the same number of eggs produced 38 adults and 6 dead third stage larvae, 2 dead fourth stage larvae and several unhatched eggs were found in the infusion. During the second experiment neither the temperature of the infusion nor the pH were taken. From 50 eggs laid by one female in the dish exposed to light, 45 developed to the adult, while in the apparatus, from the same number of eggs, which were laid by another female, 45 developed to the adult and in the infusion were found 3 live fourth stage larvae and 2 dead pupae. The results of these experiments show that Aides argenteus can develop perfectly in complete darkness. The following experiment shows that this mosquito can also lay eggs in complete darkness. Six fed females were put into the apparatus. When 4 days later it was opened 450 eggs were found in the infusion. That all the six females took part in the laying of these eggs was shown by the examination of their abdomens. CONCLUSION.
The results of the experiments show quite conclusively that the common, nonautogenous Culex pipiens, the autogenous Culex pipiens, Culex fatigans and Aides argenteus can develop perfectly in complete absence of light. The ability of the larvae of these mosquitoes to exist under this condition is probably due to their feeding habit. They are omnivorous and are not affected by the absence of algae, such as Cyanophyceae and Chlorophyceae and the high aquatic plants, which cannot exist without light. In complete darkness the food of these larvae consists of small particles of organic matter, and of such organisms as bacteria, some Mastigophora and Ciliata. The existence of these organisms depends also upon the presence of organic matter in the water and they also are not affected by the absence of light. The experiments also show that Culex fatigans, Aides argenteus and the autogenous Culex pipiens can lay eggs in complete darkness and that the last named mosquito can produce a second generation. As regards the oviposition, it has already been pointed out in my previous paper (JoBLI~G, 1935) that in complete darkness it is governed either by the chemical condition of the water, or by the water vapour, or by the combined action of these two factors, because in complete darkness there is no stimulus to develop the phototactic reaction in the mosquito. The same can be said about the mating. The male cannot Fe
474
DEVELOPMENT OF MOSQUITOES IN DARKNESS.
see the female in complete darkness; therefore, it is attracted to it either by the scent, or the sound which the female may produce, or by both of these factors. The results of these experiments may have some practical interest when we remember that suitable breeding places may be present in completely dark situations. The larvae of some of these mosquitoes have been found in dark cellars, in dark interiors of cisterns and in tanks built inside walls. Such places represent more dangerous mosquito breeding grounds than those in the open, especially as regards the urban species, because breeding in these places is not always easily detected. SUMMARY°
The experiments were made with two races of Culex pipiens, Culexfatigans and Aides argenteus in a specially constructed apparatus. T h e results of these experiments show quite conclusively that all these mosquitoes can develop perfectly in complete darkness. Aides argenteus, Culex fatigans and the autogenous Culex pipiens can lay eggs in complete darkness and the last named mosquito can mate and produce a second generation. T h e ability of the larvae of these mosquitoes to develop perfectly in absence of light is probably due to their feeding habit. They are omnivorous and can exist upon organic matter, and upon such organisms as can also live in complete absence of light if organic matter is present in the water.