J.
Meci. EDt.
Vol. 12.
DO.
30 DeceJnber 1975
5: 547-550
BUOYANCY AND VENTILATION IN AEDES AEGYPTI PUPAE (DIPTERA: CULICIDAE) I
(L.)
By William. S. Rom.oser2 Abstract: Various aspects of buoyancy in Aedes aegypti (L.) pupal' were studied. Gas in the ventral air space is necessary for a pupa to be buoyant and maintain its normal position at the air-water interface. Pupae, under conditions of forced mbmergence, and commonly spontaneously, lose their buoyancy and reach 01' exceed the density of water. Contact with the atmosphere usually results in an immediate recovery of buoyancy. These changes in buoyancy are considered to be caused by changes in volume and/or gas pressure in the tracheal system. An hypothetical explanation for the observed changes in buoyancy is presented and aspects of the functional and adaptive siKnilicanceof buoyancy and changes in buoyancy are discussed.
'This project W:iS supported in part by grant no. 453 from the Ohio University Research Committee and in part by Public Health Rl'search Grant AI-12641. 'Dl'partment of Zoology & Microbiology, Ohio University, Athens, Ohio 45701, U.S.A.
AND METHODS
The species studied was Aedes aegypti (L.), Rockefeller strain. The eggs were hatched in tap water and larvae, in groups of 50, were reared at 27°C on a diet consisting of ground lab chow, liver powder and yeast. All experiments were carried out at room temperature (23 ± 2°C). At all other times specimens were maintained at 27 :1.: 1°C. Behavioral observations were made on single pupae introduced into a SOO-mlgraduated cylinder. Stimulation to cause diving was accomplished by sharply tapping the side of the cylinder. Pale, recently emerged specimens (0-1 hr old); darker, older specimens (5 hr+); and black, old (3S hr·j·) specimens were observed in response to a single stimulus (i.e., 1 sharp tap) and in response to continuous stimulation (i.e., continuous sharp tapping). Conditions m which pupae were forcibly submerged were required for experimental purposes. These conditions were produced by a tight-fitting plunger in the 500-ml cylinder or by using plastic pill vials (diam. = 2.9 em, height = 5.3 em) with snap
caps
and
small
test
tubes
(diam.
= 7 mm,
length = 120 mm). Holes of a diameter slightly larger than those of the test tubes were drilled in the snap caps. To effect forced submergence of a pupa in a tube, the plastic vial was 1/3 filled with water, and the test tube containing the specimen was inverted, placed through the hole in the snap cap and brought to rest against the bottom of the vial. Due to the small diameter of each tube, water did not run out. In the righted position, the vial and cap served as a holder for the test tube. RESULTS
Pupal behavior The following information is based on the observation of 50 individuals. Undisturbed pupae characteristically rest quietly at the air-water interface. The distal ends of the ventilatory trumpets penetrate the surface film and provide the pupa with continuity between its tracheal system and the air, facilitating gaseous exchange. In response to an appropriate stimulus, a pupa resting at the air-water interface responds by break-
Downloaded from http://jme.oxfordjournals.org/ by guest on April 24, 2016
The fact that mosquito pupae are less dense than water, and hence float, has been well known to mosquito investigators for many years. As early as 1890, Hurst, studying Culex sp., noted that this buoyancy was due to the presence of gas in the cavity formed by the developing wings, mouthparts and legs, which remain cemented together throughout the pupal period. Hurst further recognized that the "air cavity," in addition to providing buoyancy, played a role in the hydrostatic balance of the pupa, " ... serving not only to make the pupa float when at rest, but to make it float in a definite position, with the thorax uppermost and the apertures of the siphons at the surface of the water." Hurst also recognized that the pair of spiracles on the 1st abdominal segment are functional during the pupal period and open into the air cavity, placing it " ... in direct communication with the tracheal system." Christophers (1960) briefly reviewed Hurst's observations and added: " ... pressure in the sac is apparently under control. Looking down upon a young (white) pupa the presence of the bubble ... in the region of the halteres is very evident. From time to time as this area is watched something very like a wink occurs. At first this was thought to be due to some movement of the halteres, but was afterwards seen to be due to the opening and closing of the large first abdominal spiracles." Christophers called the "air cavity" of Hurst the "ventral air space." Christophers' term will be llsed in this paper. The purpose of this investigation was to examine buoyancy and possible changes in buoyancy during the pupal period of a mosquito.
MATERIALS
J. Merl.
548
ing contact with the surface film and actively descending against the buoying force of the gas in the ventral air space. Active descent to a given depth is generally followed by an alternation of short intervals of active swimming and passive rising toward the airwater interface. If undisturbed, the pupa eventually penetrates the surface film and comes to rest. In response to continuous stimulation or a long period of forced submergence, an individual, unless it is nearly ready to emerge as an adult, eventually reaches a state in which its density is equal to or greater than that of water at a given depth. In this state the pupa may remain stationary at some depth or passively sink. During the course of all the observations
made
in this investigation,
passive
Relationship between passive sink and passive ascent During the observations of pupal behavior, nearly every specimen induced to become stationary beneath the water's surface or to begin to passively sink regained its buoyancy upon contacting the air-water interface. In order to determine how rapidly buoyancy was regained and the extent of variation in the time required for a pupa to lose its buoyancy, i.e., reach the state of passive sink, the following experiment was carried out: ( 1) a specimen was taken from the rearing container and placed in a test tube; (2) the tube was then inverted and the time required for the pupa to reach the state of passive sink was measured to the nearest 5 min.; (3) when passive sink was reached, the tube
Vol. 12, no. 5
TABLE 1.
Buoyancy of mosquito pupae allowed brief (approximately I sec.) exposure to the atmosphere after the onset of passive sink.
TIME TO PASSIVE SINK (MIN.) NUMBER Standard No. REAGE OFSPECdevia- GAINING (HR) IMENS Mean Range tion BUOYANCY 0-5 21 22 5-70 20 21 20 II 29 10-45 13 II 35+*· ** II 38 25-90 22 7 *Two specimens failed to reach passive sink and drowned at the tops of the inverted tubes. **Two specimens reached passive sink, but upon righting of the tubes they were unable to regain contact with the airwater interface.
was righted and the pupa allowed to swim to the air-water interface; (4) the moment the trumpets made contact with the atmosphere, the specimen was immediately stimulated to dive and its state of buoyancy (buoyant or nonbuoyant) was observed. This experiment was carried out on young (0-5 hr), intermediate aged (approximately 20 hI') and old, dark (35 hr+) pupae. All of the specimens which lost their buoyancy (TABLE I) and were able to regain contact with the air-water interface regained buoyancy immediately (within about I sec.). The mean time to passive sink appears to increase in pupae between the age of 0-5 hr and 35 hr+. Among the 35-hr+-old specimens, 2 individuals were unable to reach the air-water interface and drowned after passively sinking to the bottoms of their respective tubes. Two specimens in the 35-hr+ group drowned at the tops of their tubes while still buoyant. These specimens had apparently attained at least part of the increase in buoyancy which was previously noted to occur some time prior to adult emergence. The increase in buoyancy late in the pupal period may also account for the apparent increase in mean time to passive sink.
State of buoyancy as a function of time The occurrence of passive sink under conditions of forced submergence suggested that pupae may spontaneously lose their buoyancy. In order to determine if, in fact, such spontaneous loss of buoyancy occurs under "normal" conditions, 10 pupae were placed individually in small vials with a water column approximately 2.5 em long. The state of buoyancy (buoyant or nonbuoyant or stationary just beneath the water's surface) was checked at 5- or 10-min. intervals for 2 or more hours duration. Between checks, the pupae usually rested quietly at the air-water interface. FIG. 1 shows the results of these observations. Eight of
Downloaded from http://jme.oxfordjournals.org/ by guest on April 24, 2016
sinking was followed by active ascent to the water's surface. In a few cases a pupa that had been stationary near the bottom of the container became slightly buoyant as it neared the air-water interface. Specimens held under conditions of forced submergence after reaching the state of passive sink eventually lose their ability to reach the air-water interface and finally sink to the bottom and drown. Pupae which have been forced to drown and which have ceased movement have little or no gas in the ventral air space. A few dark, old specimens (35 hr+), when forced to submerge, did not reach the state of passive sink, even after the passage of several hours. Examination and dissection of several other old pupae revealed the presence of gas in the posterior midgut and between the pharate adult and pupal cuticles. This additional gas caused a substantial increase in buoyancy. After a single tap, pupae were often observed to descend to the bottom of the cylinder and remain motionless for a minute or more before actively ascending to the surface.
Ent.
549
Aedes aegypti pupae buoyancy
Romoser:
1975
hand, a pupa has just regained its lost buoyancy. then the time to passive sink will be longer.
A B
CONCLUSIONS
c o E
F G
100
200
CD
300
o
100
[)
FIG. I. Spontaneous loss of buoyancy in mosquito pupae as indicated by ability of pupae to remain stationary bellt'ath the water's surface or to passively sink. Ordinate: specimens A-J; Abcissa: time in minutes. Circled numbers indicate day I and day 2. The state of buoyancy of each sped men was checked at 10-min. intervals for the first 170 min. 011 day I and at 5-min. intervals for the remainder of day I alld all of day 2. A vertical line indicates a spontant'Ous loss of buoyancy. Otherwise, a pupa was obsl'f\'ed to be buoyant.
the 10 specimens were observed to be stationary beneath the water's surface at least once. The number of times individuals were observed to be stationary varied from I to 31. Two specimens remained buoyant, at a maximum depth of 2.5 em, during the entire period of observation. From these results it is clear that pupae commonly undergo periodic, spontaneous fluctuations in buoyancy. Obviously, since the pupae were not observed continuously, occurrences of these spontaneous changes in buoyancy could easily have been missed. Also, since the occurrence of a pupa being stationary or slightly more dense than water (passive sink) were the only criteria used to determine changes in buoyancy, pupae could have undergone undetected decreases or increases in buoyancy. No regularity of changes in buoyancy is evident from the data presented. Variation in the time to passive sink under conditions of forced submergence is accounted for, at least in part, by spontaneous changes in buoyancy. If the density of a pupa approaches the density of water when forced to submerge, then the time to passive sink will be very short. If, on the other
DISCUSSION
J\1echanism of decrease and increase in buoyancy A plausible explanation for the changes in buoyancy might be as follows. The partial pressure of oxygen in the tracheal system decreases as this gas is utilized in respiration. Most or all of the carbon dioxide produced as a respiratory waste product does not enter the tracheal system, but remains in solution in the hemolymph or diffuses out of the pupa. The combination of loss of oxygen from the tracheal system without the compensatory addition of carbon dioxide would result in a net decrease in volume and/or pressure in the tracheal system. Development of the negative pressure (relative to atmospheric) in the tracheal system possibly results from elastic properties of the tracheae which oppose collapse. The development of negative pressure within the tracheal system would then afford a
Downloaded from http://jme.oxfordjournals.org/ by guest on April 24, 2016
o
(I) Since pupae which are initially buoyant eventually lose their buoyancy under conditions of forced submergence, there must be an increase in their density. (2) Since the ventilatory trumpets provide the only contact with the atmosphere, the increase in buoyancy after a pupa has reached or exceeded the density of water must be provided via the trumpets. Thus the changes in buoyancy must be due to changes within the tracheal system. (3) Since a decrease in density (increase in buoyancy) sufficient to restore lost buoyancy occurs immediately upon regaining contact with the atmosphere at the air-water interface, a negative pressure must develop in the tracheal system during forced submergence. (4) Since pupae undergo spontaneous changes in buoyancy, there must be spontaneous changes in volume and/or gas pressure within the tracheal system. (5) It appears that the ventral air space is filled with gas to a point such that when the decrease in volume and/or gas pressure within the pupa's tracheal system is maximum, the pupa's density is equal to or slightly less than that of water when measured just below the surface. (6) Since drowned pupae have little or no gas in the ventral air space, it is clear that the gas in the ventral air space can move into the tracheal system via the 1st abdominal spiracles.
550
J. Med. Ent.
mechanism for the immediate intake of air when the tracheal system is opened to the atmosphere.
Buoyan0' Since removal of the gas from the ventral air space causes a pupa to be more dense than water, this gas is obviously necessary for the pupa to be buoyant. Since pupae are often observed to be at the same density as water, it may be concluded that even near the water's surface their density alternates between that of water and slightly below. This situation is somewhat analogous with that found in many teleost fish which possess swimbladders in their body cavities. Alexander (1968) explains: "Many teleost fish have densities very close to the density of the water they live in, and can hover in mid-water with only very gende fin movements. This is because they have a bag of gas called the swimbladder in the body cavity. Without it, their densities would lie between about 1.06 and 1.09 g/cm3• They would sink whenever they stopped swimming and they would need more energy to swim at any given speed. It has been estimated that a fish which swims all the time is likely to use 8% less energy in its metabolism if it has a swimbladder, than if it has not." The fact that the density of mosquito pupae is very close to that of water explains the bottom-
resting behavior described in the behavior section. When a pupa resting at the air-water interface is stimulated to dive, it must do so against a buoying force. However, as the pupa swims downward and since its density is near that of water, the increasing pressure of the surrounding water apparently causes the density of the pupa to reach that of water by causing compression of the gases in the tracheal system and ventral air space. If the dive is deep enough, the pupa reaches passive sink and can then continue to sink passively to the bottom. The depth at which a pupa reaches the density of water and finally passive sink depends, of course, upon how dense (or buoyant) the pupa was at the beginning of the dive. Thus as a pupa actively dives, the buoyant force acting against it decreases as the depth of dive increases, and eventually it can literally hover in the water or finally passively sink and remain on the bottom. Some pupae which were observed to be stationary in the water at the bottom of the cylinder were slightly buoyant near the top. Obviously the effect of the density of the surrounding water is reversed when a pupa is in the process of ascending. Thus the effort required for the pupa to ascend decreases as the pupa approaches the water's surface. Both increase in body density while diving and decrease in density while ascending would be energy conserving. Since pupal mosquitoes do not feed, the time spent in this stage constitutes a considerable drain on the energy stores accumulated during the larval stage. In addition to the energy expended in maintenance of the life processes, energy must be expended during the profound tissue reorganization taking place. Further, mosquito pupae, unlike the pupae of most other holometabolous insects, are highly motile and, depending upon circumstances, may be quite active. Given these categories of energy drain and realizing that the newly emerged adult mosquito must fly to a sugar or blood source before there can be any renewal of energy stores, any energy-conserving mechanism during the pupal period would have survival value. Acknowledgments: I wish to express my appreciation for the invaluable assistance of Clifford R. Arko and Paul T. Zielinski during the course of this project. LITERATURE CITED
Alexander, R. M. 1968. Animal mechanics. Sidgwick & Jackson, London. xi + 346 p. Christophers, S. R. 1960. Aedes aegypti (L.). The yellow fever mosquito. Cambridge University Press, Cambridge, England. xii 739 p. Hurst, C. H. 1890. On the life history and development of a gnat (Culex). p. 7. Guardian Press, Manchester, England.
+
Downloaded from http://jme.oxfordjournals.org/ by guest on April 24, 2016
Ventilation Pupae, which have free access to the atmosphere via the air-water interface, spontaneously reach the density of water. It appears that the volume of gas in the ventral air space is such that when the volume and/or gas pressure in the tracheal system is at its minimum, the pupa is at or just slightly less than the density of water just beneath the surface. Thus the ventral air space gas allows the spontaneous decreases in buoyancy without buoyancy being lost to the extent that the pupa becomes unable to swim to the air-water interface, and hence sinks to the bottom and drowns. The occurrence of a negative pressure within the tracheal system and the consequent ability of a pupa to rapidly take air into the tracheal system ensures that the pupa can obtain a fresh supply of oxygen regardless of the brevity of contact with the atmosphere. Under natural circumstances, this ability would enable a pupa to remain submerged for long periods of time, requiring only momentary contact with the air-water interface. This would appear to be of survival value if a pupa was induced to dive in response to the presence of a potential predator. Since, in pupae under conditions of forced submergence, gas is eventually drawn from the ventral air space, this space may well provide an air store.
Vol. 12, no. 5
Meci. EDt.
Vol. 12.
DO.
30 DeceJnber 1975
5: 547-550
BUOYANCY AND VENTILATION IN AEDES AEGYPTI PUPAE (DIPTERA: CULICIDAE) I
(L.)
By William. S. Rom.oser2 Abstract: Various aspects of buoyancy in Aedes aegypti (L.) pupal' were studied. Gas in the ventral air space is necessary for a pupa to be buoyant and maintain its normal position at the air-water interface. Pupae, under conditions of forced mbmergence, and commonly spontaneously, lose their buoyancy and reach 01' exceed the density of water. Contact with the atmosphere usually results in an immediate recovery of buoyancy. These changes in buoyancy are considered to be caused by changes in volume and/or gas pressure in the tracheal system. An hypothetical explanation for the observed changes in buoyancy is presented and aspects of the functional and adaptive siKnilicanceof buoyancy and changes in buoyancy are discussed.
'This project W:iS supported in part by grant no. 453 from the Ohio University Research Committee and in part by Public Health Rl'search Grant AI-12641. 'Dl'partment of Zoology & Microbiology, Ohio University, Athens, Ohio 45701, U.S.A.
AND METHODS
The species studied was Aedes aegypti (L.), Rockefeller strain. The eggs were hatched in tap water and larvae, in groups of 50, were reared at 27°C on a diet consisting of ground lab chow, liver powder and yeast. All experiments were carried out at room temperature (23 ± 2°C). At all other times specimens were maintained at 27 :1.: 1°C. Behavioral observations were made on single pupae introduced into a SOO-mlgraduated cylinder. Stimulation to cause diving was accomplished by sharply tapping the side of the cylinder. Pale, recently emerged specimens (0-1 hr old); darker, older specimens (5 hr+); and black, old (3S hr·j·) specimens were observed in response to a single stimulus (i.e., 1 sharp tap) and in response to continuous stimulation (i.e., continuous sharp tapping). Conditions m which pupae were forcibly submerged were required for experimental purposes. These conditions were produced by a tight-fitting plunger in the 500-ml cylinder or by using plastic pill vials (diam. = 2.9 em, height = 5.3 em) with snap
caps
and
small
test
tubes
(diam.
= 7 mm,
length = 120 mm). Holes of a diameter slightly larger than those of the test tubes were drilled in the snap caps. To effect forced submergence of a pupa in a tube, the plastic vial was 1/3 filled with water, and the test tube containing the specimen was inverted, placed through the hole in the snap cap and brought to rest against the bottom of the vial. Due to the small diameter of each tube, water did not run out. In the righted position, the vial and cap served as a holder for the test tube. RESULTS
Pupal behavior The following information is based on the observation of 50 individuals. Undisturbed pupae characteristically rest quietly at the air-water interface. The distal ends of the ventilatory trumpets penetrate the surface film and provide the pupa with continuity between its tracheal system and the air, facilitating gaseous exchange. In response to an appropriate stimulus, a pupa resting at the air-water interface responds by break-
Downloaded from http://jme.oxfordjournals.org/ by guest on April 24, 2016
The fact that mosquito pupae are less dense than water, and hence float, has been well known to mosquito investigators for many years. As early as 1890, Hurst, studying Culex sp., noted that this buoyancy was due to the presence of gas in the cavity formed by the developing wings, mouthparts and legs, which remain cemented together throughout the pupal period. Hurst further recognized that the "air cavity," in addition to providing buoyancy, played a role in the hydrostatic balance of the pupa, " ... serving not only to make the pupa float when at rest, but to make it float in a definite position, with the thorax uppermost and the apertures of the siphons at the surface of the water." Hurst also recognized that the pair of spiracles on the 1st abdominal segment are functional during the pupal period and open into the air cavity, placing it " ... in direct communication with the tracheal system." Christophers (1960) briefly reviewed Hurst's observations and added: " ... pressure in the sac is apparently under control. Looking down upon a young (white) pupa the presence of the bubble ... in the region of the halteres is very evident. From time to time as this area is watched something very like a wink occurs. At first this was thought to be due to some movement of the halteres, but was afterwards seen to be due to the opening and closing of the large first abdominal spiracles." Christophers called the "air cavity" of Hurst the "ventral air space." Christophers' term will be llsed in this paper. The purpose of this investigation was to examine buoyancy and possible changes in buoyancy during the pupal period of a mosquito.
MATERIALS
J. Merl.
548
ing contact with the surface film and actively descending against the buoying force of the gas in the ventral air space. Active descent to a given depth is generally followed by an alternation of short intervals of active swimming and passive rising toward the airwater interface. If undisturbed, the pupa eventually penetrates the surface film and comes to rest. In response to continuous stimulation or a long period of forced submergence, an individual, unless it is nearly ready to emerge as an adult, eventually reaches a state in which its density is equal to or greater than that of water at a given depth. In this state the pupa may remain stationary at some depth or passively sink. During the course of all the observations
made
in this investigation,
passive
Relationship between passive sink and passive ascent During the observations of pupal behavior, nearly every specimen induced to become stationary beneath the water's surface or to begin to passively sink regained its buoyancy upon contacting the air-water interface. In order to determine how rapidly buoyancy was regained and the extent of variation in the time required for a pupa to lose its buoyancy, i.e., reach the state of passive sink, the following experiment was carried out: ( 1) a specimen was taken from the rearing container and placed in a test tube; (2) the tube was then inverted and the time required for the pupa to reach the state of passive sink was measured to the nearest 5 min.; (3) when passive sink was reached, the tube
Vol. 12, no. 5
TABLE 1.
Buoyancy of mosquito pupae allowed brief (approximately I sec.) exposure to the atmosphere after the onset of passive sink.
TIME TO PASSIVE SINK (MIN.) NUMBER Standard No. REAGE OFSPECdevia- GAINING (HR) IMENS Mean Range tion BUOYANCY 0-5 21 22 5-70 20 21 20 II 29 10-45 13 II 35+*· ** II 38 25-90 22 7 *Two specimens failed to reach passive sink and drowned at the tops of the inverted tubes. **Two specimens reached passive sink, but upon righting of the tubes they were unable to regain contact with the airwater interface.
was righted and the pupa allowed to swim to the air-water interface; (4) the moment the trumpets made contact with the atmosphere, the specimen was immediately stimulated to dive and its state of buoyancy (buoyant or nonbuoyant) was observed. This experiment was carried out on young (0-5 hr), intermediate aged (approximately 20 hI') and old, dark (35 hr+) pupae. All of the specimens which lost their buoyancy (TABLE I) and were able to regain contact with the air-water interface regained buoyancy immediately (within about I sec.). The mean time to passive sink appears to increase in pupae between the age of 0-5 hr and 35 hr+. Among the 35-hr+-old specimens, 2 individuals were unable to reach the air-water interface and drowned after passively sinking to the bottoms of their respective tubes. Two specimens in the 35-hr+ group drowned at the tops of their tubes while still buoyant. These specimens had apparently attained at least part of the increase in buoyancy which was previously noted to occur some time prior to adult emergence. The increase in buoyancy late in the pupal period may also account for the apparent increase in mean time to passive sink.
State of buoyancy as a function of time The occurrence of passive sink under conditions of forced submergence suggested that pupae may spontaneously lose their buoyancy. In order to determine if, in fact, such spontaneous loss of buoyancy occurs under "normal" conditions, 10 pupae were placed individually in small vials with a water column approximately 2.5 em long. The state of buoyancy (buoyant or nonbuoyant or stationary just beneath the water's surface) was checked at 5- or 10-min. intervals for 2 or more hours duration. Between checks, the pupae usually rested quietly at the air-water interface. FIG. 1 shows the results of these observations. Eight of
Downloaded from http://jme.oxfordjournals.org/ by guest on April 24, 2016
sinking was followed by active ascent to the water's surface. In a few cases a pupa that had been stationary near the bottom of the container became slightly buoyant as it neared the air-water interface. Specimens held under conditions of forced submergence after reaching the state of passive sink eventually lose their ability to reach the air-water interface and finally sink to the bottom and drown. Pupae which have been forced to drown and which have ceased movement have little or no gas in the ventral air space. A few dark, old specimens (35 hr+), when forced to submerge, did not reach the state of passive sink, even after the passage of several hours. Examination and dissection of several other old pupae revealed the presence of gas in the posterior midgut and between the pharate adult and pupal cuticles. This additional gas caused a substantial increase in buoyancy. After a single tap, pupae were often observed to descend to the bottom of the cylinder and remain motionless for a minute or more before actively ascending to the surface.
Ent.
549
Aedes aegypti pupae buoyancy
Romoser:
1975
hand, a pupa has just regained its lost buoyancy. then the time to passive sink will be longer.
A B
CONCLUSIONS
c o E
F G
100
200
CD
300
o
100
[)
FIG. I. Spontaneous loss of buoyancy in mosquito pupae as indicated by ability of pupae to remain stationary bellt'ath the water's surface or to passively sink. Ordinate: specimens A-J; Abcissa: time in minutes. Circled numbers indicate day I and day 2. The state of buoyancy of each sped men was checked at 10-min. intervals for the first 170 min. 011 day I and at 5-min. intervals for the remainder of day I alld all of day 2. A vertical line indicates a spontant'Ous loss of buoyancy. Otherwise, a pupa was obsl'f\'ed to be buoyant.
the 10 specimens were observed to be stationary beneath the water's surface at least once. The number of times individuals were observed to be stationary varied from I to 31. Two specimens remained buoyant, at a maximum depth of 2.5 em, during the entire period of observation. From these results it is clear that pupae commonly undergo periodic, spontaneous fluctuations in buoyancy. Obviously, since the pupae were not observed continuously, occurrences of these spontaneous changes in buoyancy could easily have been missed. Also, since the occurrence of a pupa being stationary or slightly more dense than water (passive sink) were the only criteria used to determine changes in buoyancy, pupae could have undergone undetected decreases or increases in buoyancy. No regularity of changes in buoyancy is evident from the data presented. Variation in the time to passive sink under conditions of forced submergence is accounted for, at least in part, by spontaneous changes in buoyancy. If the density of a pupa approaches the density of water when forced to submerge, then the time to passive sink will be very short. If, on the other
DISCUSSION
J\1echanism of decrease and increase in buoyancy A plausible explanation for the changes in buoyancy might be as follows. The partial pressure of oxygen in the tracheal system decreases as this gas is utilized in respiration. Most or all of the carbon dioxide produced as a respiratory waste product does not enter the tracheal system, but remains in solution in the hemolymph or diffuses out of the pupa. The combination of loss of oxygen from the tracheal system without the compensatory addition of carbon dioxide would result in a net decrease in volume and/or pressure in the tracheal system. Development of the negative pressure (relative to atmospheric) in the tracheal system possibly results from elastic properties of the tracheae which oppose collapse. The development of negative pressure within the tracheal system would then afford a
Downloaded from http://jme.oxfordjournals.org/ by guest on April 24, 2016
o
(I) Since pupae which are initially buoyant eventually lose their buoyancy under conditions of forced submergence, there must be an increase in their density. (2) Since the ventilatory trumpets provide the only contact with the atmosphere, the increase in buoyancy after a pupa has reached or exceeded the density of water must be provided via the trumpets. Thus the changes in buoyancy must be due to changes within the tracheal system. (3) Since a decrease in density (increase in buoyancy) sufficient to restore lost buoyancy occurs immediately upon regaining contact with the atmosphere at the air-water interface, a negative pressure must develop in the tracheal system during forced submergence. (4) Since pupae undergo spontaneous changes in buoyancy, there must be spontaneous changes in volume and/or gas pressure within the tracheal system. (5) It appears that the ventral air space is filled with gas to a point such that when the decrease in volume and/or gas pressure within the pupa's tracheal system is maximum, the pupa's density is equal to or slightly less than that of water when measured just below the surface. (6) Since drowned pupae have little or no gas in the ventral air space, it is clear that the gas in the ventral air space can move into the tracheal system via the 1st abdominal spiracles.
550
J. Med. Ent.
mechanism for the immediate intake of air when the tracheal system is opened to the atmosphere.
Buoyan0' Since removal of the gas from the ventral air space causes a pupa to be more dense than water, this gas is obviously necessary for the pupa to be buoyant. Since pupae are often observed to be at the same density as water, it may be concluded that even near the water's surface their density alternates between that of water and slightly below. This situation is somewhat analogous with that found in many teleost fish which possess swimbladders in their body cavities. Alexander (1968) explains: "Many teleost fish have densities very close to the density of the water they live in, and can hover in mid-water with only very gende fin movements. This is because they have a bag of gas called the swimbladder in the body cavity. Without it, their densities would lie between about 1.06 and 1.09 g/cm3• They would sink whenever they stopped swimming and they would need more energy to swim at any given speed. It has been estimated that a fish which swims all the time is likely to use 8% less energy in its metabolism if it has a swimbladder, than if it has not." The fact that the density of mosquito pupae is very close to that of water explains the bottom-
resting behavior described in the behavior section. When a pupa resting at the air-water interface is stimulated to dive, it must do so against a buoying force. However, as the pupa swims downward and since its density is near that of water, the increasing pressure of the surrounding water apparently causes the density of the pupa to reach that of water by causing compression of the gases in the tracheal system and ventral air space. If the dive is deep enough, the pupa reaches passive sink and can then continue to sink passively to the bottom. The depth at which a pupa reaches the density of water and finally passive sink depends, of course, upon how dense (or buoyant) the pupa was at the beginning of the dive. Thus as a pupa actively dives, the buoyant force acting against it decreases as the depth of dive increases, and eventually it can literally hover in the water or finally passively sink and remain on the bottom. Some pupae which were observed to be stationary in the water at the bottom of the cylinder were slightly buoyant near the top. Obviously the effect of the density of the surrounding water is reversed when a pupa is in the process of ascending. Thus the effort required for the pupa to ascend decreases as the pupa approaches the water's surface. Both increase in body density while diving and decrease in density while ascending would be energy conserving. Since pupal mosquitoes do not feed, the time spent in this stage constitutes a considerable drain on the energy stores accumulated during the larval stage. In addition to the energy expended in maintenance of the life processes, energy must be expended during the profound tissue reorganization taking place. Further, mosquito pupae, unlike the pupae of most other holometabolous insects, are highly motile and, depending upon circumstances, may be quite active. Given these categories of energy drain and realizing that the newly emerged adult mosquito must fly to a sugar or blood source before there can be any renewal of energy stores, any energy-conserving mechanism during the pupal period would have survival value. Acknowledgments: I wish to express my appreciation for the invaluable assistance of Clifford R. Arko and Paul T. Zielinski during the course of this project. LITERATURE CITED
Alexander, R. M. 1968. Animal mechanics. Sidgwick & Jackson, London. xi + 346 p. Christophers, S. R. 1960. Aedes aegypti (L.). The yellow fever mosquito. Cambridge University Press, Cambridge, England. xii 739 p. Hurst, C. H. 1890. On the life history and development of a gnat (Culex). p. 7. Guardian Press, Manchester, England.
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Ventilation Pupae, which have free access to the atmosphere via the air-water interface, spontaneously reach the density of water. It appears that the volume of gas in the ventral air space is such that when the volume and/or gas pressure in the tracheal system is at its minimum, the pupa is at or just slightly less than the density of water just beneath the surface. Thus the ventral air space gas allows the spontaneous decreases in buoyancy without buoyancy being lost to the extent that the pupa becomes unable to swim to the air-water interface, and hence sinks to the bottom and drowns. The occurrence of a negative pressure within the tracheal system and the consequent ability of a pupa to rapidly take air into the tracheal system ensures that the pupa can obtain a fresh supply of oxygen regardless of the brevity of contact with the atmosphere. Under natural circumstances, this ability would enable a pupa to remain submerged for long periods of time, requiring only momentary contact with the air-water interface. This would appear to be of survival value if a pupa was induced to dive in response to the presence of a potential predator. Since, in pupae under conditions of forced submergence, gas is eventually drawn from the ventral air space, this space may well provide an air store.
Vol. 12, no. 5
