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The effects of various carbohydrate diets on Aedes aegypti infected with Dirofilaria immitis
JO”R”AL
OF INVERTEBRATE
33, 75-80 (19791
PATHOLOGY
The Effects of Various Carbohydrate Diets on Aedes aegypti Infected with Dirofilaria immitis GEORGE L. STEWART, Qepartment
qf Biology.
The
FRED K. SOIFER’, University
of
Texas
AND J. BRECK STEWART
at Arlington.
Arlington,
Texas
76019
Received May 18, 1978 Aedes aegypti infected with Diro$/aria immitis and uninfected mosquitoes were maintained on various carbohydrate diets (glucose, galactose, fructose, sucrose, trehalose, maltose, and melibiose). The value of each of these sugars in supporting survival of adult A. aegypti. and in supporting egg production, viability of eggs, and development of third-stage larvae of D. immitis in A. aegypti was analyzed. Fructose, glucose, maltose, sucrose, and trehalose provided the strongest support for survival of adult male, and infected and uninfected adult female A. aegypti. Galactose and melibiose provided the least support for survival of all groups of mosquitoes. The mean number of eggs laid per uninfected adult female A. cregypti was greatest when mosquitoes were maintained on glucose. mehbiose, maltose, fructose, sucrose, and trehalose. The same was true for female mosquitoes infected with D. immitis; except for melibiose which provided poor support for egg production. In both Dirojilaria-infected and in uninfected mosquitoes, galactose supported the production of low mean numbers of eggs per adult female A. aegypti. High percentages of eggs laid by uninfected and by infected female mosquitoes fed glucose, melibiose, maltose, sucrose, and trehalose hatched. While galactose supported a high percentage of hatching in eggs laid by uninfected A. aegypti. a much lower percentage of eggs laid by infected female mosquitoes maintained on this same carbohydrate hatched. The lowest percentages of eggs that hatched were from among those laid by infected and by uninfected females fed fructose. The highest mean number of D. immitis larvae CL,) were recovered from adult A. aegypti fed glucose, maltose, fructose, and sucrose; the second best sugar in this regard was trehalose. The lowest mean number of D. immitis larvae were isolated from female A. aegypti fed galactose and melibiose. KEY WORDS: Aedes aegypti; Dirofilarim immitis; mosquito, fecundity; mosquito, longevity; nematode: glucose: maltose: sucrose; galactose: trehalose: fructose: melibiose.
INTRODUCTION
It has been clearly demonstrated that the composition of the diet of some invertebrate hosts is an important factor in determining susceptibility to parasites (Tripp, 1969; Weathersby and Noblet, 1973). Dirojilaria immitis is a nematode parasite of canids which can utilize more than 60 species of mosquitoes as intermediate host (Ludlam et al., 1970). Little work has been done on the influence of diet on the biology of the mosquito host infected with D. immitis or on the development of this parasite within the intermediate host. The present study was undertaken to determine the effects of different carbohydrate diets on survival, ’ Bellaire Boulevard Animal Blvd., Houston, Texas 77036.
Clinic. 6213 Bellaire
fecundity, and egg viability in Aedes aegypti infected with D. immitis, and on development of the third-stage larva of this filarial nematode. MATERIALS
AND METHODS
D. immitis used in these experiments was initially obtained in 1973 from a naturally infected dog maintained at Bellaire Boulevard Animal Clinic, Houston, Texas. A Black-eye Liverpool strain of A. aegypti h as b een maintained in our laboratory since 1973 when it was obtained from Dr. Carl Hacker of The University of Texas School of Public Health, Houston, Texas. Mosquitoes were reared by the methods of Crovello and Hacker (1972). Mosquito larvae were hatched from egg papers immersed in deionized water in white, porcelain pans. Each pan contained between 100
75 0022-201 l/79/010075-06$01.00/O Copyright IV’ 1979 by Academic Prrss. Inc All rightc of reproduction in any form vscrved.
76
STEWART,
SOIFER,
and 200 larvae. Pans were maintained at 27°C and liver powder solution was added to pans as required. Following pupation, 100 male and 100 female pupae were placed on moist filter paper in each of 16 glass Petri dishes. One Petri dish was placed in each of 16-24 x 24 x 24 in. aluminum mesh cages for emergence of adults. All cages were maintained in an insectary at 27°C and 80% relative humidity. Cages of mosquitoes were divided into eight groups consisting of two cages. Following the experimental methods of Weathersby and Noblet (1973), one group of mosquitoes (two cages) was maintained on water alone and the remaining seven groups of mosquitoes were maintained on a 5% solution of one of the following carbohydrates: glucose, galactose, melibiose, maltose, sucrose, fructose,. or trehalose. Water or sugar solutions were changed daily and dead mosquitoes were removed from each cage, sexed, and counted. Four days following emergence of adults, one cage of adult mosquitoes from each of the eight groups was allowed to feed for 20 min on a dog infected with D. immitis (peripheral microfilaremia of 4000 mflml of blood; SE = kl93.7); the remaining eight cages of mosquitoes were allowed to feed for 20 min on a dog determined to be uninfected on the basis of chest X-ray analysis, physical examination, and the absence of circulating microfilariae. This dog had been obtained at 10 weeks of age in 1973 and maintained in a mosquito-proof cage since that time. Two days following the blood meal, an oviposition cup (half-pint paper can containing wet paper towels) was placed in each cage. Oviposition cups were changed daily and the total number of eggs laid by females in each cage was determined by direct count. Samples of eggs from each cage of mosquitoes were kept moist for 5 days to allow embryonation. Egg papers were dried at room temperature. One week later, 3-100 egg samples from each of the 14 cages (all mosquitoes maintained on water died before oviposi-
AND
STEWART
tion) were treated as above for hatching. Each day for 5 days, all larvae hatching from eggs were removed and counted (no eggs hatched after 4 days). These data were used to calculate percentage eggs hatching. On the 13th day following the blood meal, each cage was treated separately in the following manner: all adult mosquitoes were removed from the cage by suction, stunned by agitation, sexed, and counted. Females were individually crushed in cold (SC) phosphate-buffered saline (PBS, pH 7.2), and removed to a small-mesh plastic basket immersed in 2 ml of cold PBS in a small glass vial. Mosquito suspensions were maintained at 37°C for 1 hr and the thirdstage larvae (LJ were removed with a fine point pipet and counted. At the end of the initial incubation period each mosquito was removed from its plastic basket, crushed lightly, and processed a second time as above for recovery of any remaining larvae. All uninfected female mosquitoes from the same cage were pooled and treated in this same manner. Because of high mortality among adult mosquitoes supported on melibiose and galactose, experiments involving these sugars were repeated five times for both infected and uninfected mosquitoes. Experiments in which adult mosquitoes were supported on the other five sugars and on water were repeated three times. All data were statistically analyzed by the Student’s t test to determine the significance of differences. RESULTS
The percentage of the original population of infected and uninfected adult female A. aegypti remaining alive on each day between 2 days before the blood meal and the end of the study (13 days following the blood meal) are presented in Figure 1. From Day 0 to the end of the study, galactose supported the survival of significantly lower percentages of infected female mosquitoes than did all other sugars except melibiose. A similar situation was seen with
EFFECTS OF CARBOHYDRATES
ON DIROFILARIA-INFECTED
+c
77
m-m ‘m-mmm
a,
_ & a
AEDES
Uninfected
~................~..............., 2'0'2
3 4 5 6 7 8 9,011,213
2'0'23456
7 8 9 10 11 12 13
Days FIG. 1. Adult female Aedes aegypti infected with DiroJilaria immitis and uninfected adult female A. fed glucose to), galactose ( l ), melibiose (W), trehalose ((I)), fructose (A), sucrose 0, and maltose (A) presented as percentage of initial population surviving on each day between 2 days before the blood meal and 13 days following the blood meal (blood meal provided on Day 0).
aegypri
uninfected female mosquitoes. Melibiose supported the survival of significantly lower percentages of infected adult female mosquitoes (beginning of the study to Day 8 post-blood meal) and of uninfected female mosquitoes (from 1 day before the blood meal to the end of the study) than did galactose. Trehalose supported the survival of significantly lower percentages of uninfected female mosquitoes (Days 8 through 13 post-blood meal) than did maltose, sucrose, glucose, and fructose. With all sugars except melibiose, greater percentages of uninfected than infected female mosquitoes were alive between 2 days following the blood meal and the end of the study. With melibiose, significantly greater percentages of uninfected than infected female A. aegypti were alive between Days 2 and 11 post-blood meal. Similar percentages of adult male mosquitoes supported on each of the seven sugars tested, whether they were caged with infected or uninfected female mosquitoes, were alive on each day during the study.
The percentages of adult female mosquitoes infected with D. immitis, of uninfected adult female mosquitoes and of adult male mosquitoes, surviving to the end of the study (Day 13 post-blood meal) are presented in Figure 2. Fructose supported survival of the highest percentage of male mosquitoes caged with uninfected female mosquitoes. Glucose and sucrose were second in this regard: then maltose and trehalose; followed by galactose and melibiose. Fructose, sucrose, and glucose provided support for survival of the highest percentage of male mosquitoes caged with infected female mosquitoes; then maltose and trehalose; followed by galactose and melibiose. The highest percentages of uninfected female mosquitoes surviving were among those fed glucose, maltose, fructose, and sucrose; then trehalose; followed by galactose and melibiose. Survival of female A. aegypti infected with D. immitis was best supported by glucose, maltose, fructose, sucrose, and trehalose, while galactose and melibiose provided the poorest support for survival of infected female
78
STEWART,
SOIFER, AND STEWART
_female
+ 5 v G a
20. : ih gl
I
ga me ma
fr
su
gl
tr
ga me
ma
fr
su
tr
Sugar FIG. 2. Adult male Aedes aegypti caged with uninfected adult female A. aegypvpti (white bar) and adult males caged with adult female A. aegypti infected with Dirofilaria immitis (black bar); and infected (white bar) and uninfected (black bar) adult female A. aegypti are presented as percentage adult mosquitoes surviving at the end of the study (Day 13 post-blood meal) versus dietary carbohydrate [glucose (gl). galactose (ga), melibiose (me), maltose (ma), fructose (fr). sucrose (su) and trehalose (tr)]. Standard errors are presented for all data. P < 0.05
mosquitoes. With all sugars except melibiose higher percentages of uninfected than infected female mosquitoes were alive at the end of the study. With all sugars except melibiose, the
mean numbers of eggs laid per female A. aegypti infected with D. immitis and per uninfected female A. aegypti were similar (Figure 3A). The mean number of eggs laid per infected female mosquito fed melibiose
TT
ga me
ma
tr
su
tr
gl ga
me
ma
tr
I
tr
S U gar FIG. 3. (A) Mean number of eggs laid per adult female Aedes aegypti infected with DiroJilaria immitis (white bar) and per uninfected adult female A. aegypti (black bar) fed glucose (gl), galactose (ga), melibiose (me), maltose (ma), fructose (fr), sucrose (su), and trehalose (tr). (B) Viability of eggs laid by adult female A. aegypvpri infected with D. immitis (white bar) and by uninfected adult female A. aegypfi (black bar) fed glucose (gl), galactose (pa), melibiose (me), maltose (ma), fructose (fr), sucrose (su), and trehalose (tr). Dataare presented in terms ofpercentage eggs hatching. Standard errors are shown for all data. P < 0.05
EFFECTS
OF
CARBOHYDRATES
ON DIROFILARIA-INFECTED
was significantly lower than that for uninfected female mosquitoes fed this same sugar. The percentage of hatching was similar among eggs laid by infected and uninfected female mosquitoes fed any of the seven sugars except galactose (Figure 3B). When galactose was the supporting sugar, a significantly lower percentage of eggs laid by infected than by uninfected female mosquitoes hatched. With fructose as the supporting sugar, the percentages of eggs laid by infected and uninfected female A. aegypti that hatched were similar. However, these percentages were significantly lower than that observed with all other sugars except galactose (infected female mosquitoes). Similar mean numbers of third-stage larvae (LJ of D. immitis were obtained from infected female mosquitoes fed glucose, maltose, fructose and sucrose (Figure 4). The mean number of L3 of D. immitis isolated from individual female A. aegypti fed trehalose was significantly lower than with the aforementioned four sugars, but signifi7.5 al t-0
I
gl
ga
mema
fr
su
tr
Sugar FIG. 4. Mean number of Dir&aria immitis larvae isolated per infected adult female Aedes aegyppri fed glucose (gl), galactose (ga), melibiose (me), maltose (ma), fructose (fr), sucrose (su), and trehalose (tr). Standard errors are oresented for all data. P < 0.05
AEDES
79
cantly higher than that obtained from female mosquitoes fed galactose and melibiose. No D. immitis third-stage larvae were recovered from uninfected female mosquitoes. DISCUSSION
In agreement with the findings of Weathersby and Noblet (1973) in their study on Plasmodium gallinaceum, the survival of adult A. aegypti, both uninfected and those infected with D. immitis, was greatest on diets consisting of glucose, maltose, fructose, sucrose, and trehalose. The poorest survival of mosquitoes was among those supported on melibiose and galactose. Significantly greater percentages of uninfected than infected female mosquitoes were alive at the end of the study (Day 13 post-blood meal). On the other hand, similar percentages of male mosquitoes from cages containing uninfected females and from cages housing female mosquitoes infected with D. immitis were alive throughout the study (Day 2 before blood meal to Day 13 post-blood meal). A significantly higher mortality rate among infected than uninfected female A. aegypti first became evident on the second day following the blood meal and continued throughout the study. These differences in the death rate between infected and uninfected mosquitoes is attributed to the pathology created by D. immitis larvae developing in the malpighian tubules of the parasitized female mosquitoes (Christensen and Andrews, 1976). Of all the sugars tested, melibiose provided the poorest support for survival of both infected and uninfected mosquitoes. However, only infected female A. aegypti fed melibiose showed a significant decrease in fecundity. It has been shown by others that a parasite can alter the fecundity of its insect host (Walker, 1964; Hacker, 1971). Although the other six sugars supported similar levels of fecundity in infected and uninfected mosquitoes, we suggest that the stress of parasitism combined with the extremely low nutritive value of melibiose
80
STEWART,
SOIFER, AND STEWART
significantly reduced fecundity in infected female mosquitoes fed this sugar. Galactose supported levels of fecundity in infected and uninfected female mosquitoes which were as low as those supported by melibiose in female mosquitoes infected with D. immitis. One possible explanation for this observation may be that galactose cannot be utilized as efficiently by A. aegypti for support of egg production as can the other six sugars. A similar explanation may be offered for the decreased viability of eggs laid by infected and uninfected female mosquitoes fed fructose. In this situation perhaps fructose cannot be used as efficiently by A. aegypti for support of embryonation as can the other sugars tested. Relative to egg viability, a similar situation was seen with galactose. Lower percentages of eggs laid by infected than uninfected female mosquitoes hatched. It may be that the additive effect of pathology of the filarial infection and the low nutritional value of galactose decreased the quality of eggs produced. Those sugars which provided the poorest support for survival of infected adult female A. aegypti, i.e., galactose and melibiose, supported the development of the lowest numbers of D. immitis third-stage larvae. On the other hand, four of the five sugars that provided strong support for the survival of infected adult female mosquitoes, i.e., sucrose, glucose, maltose, and fructose, supported the development of the highest numbers of D. immitis third-stage larvae. Trehalose is the only exception to
this apparent trend. This sugar provided a level of support for survival of infected adult female mosquitoes similar to that observed with the above four sugars, but supported the development of third-stage larvae of D. immitis at a level significantly below that seen with sucrose, glucose, maltose, and fructose. ACKNOWLEDGMENT The authors thank Dr. Carl S. Hacker of The University of Texas School of Public Health for his advice and assistance in this study.
REFERENCES M., AND ANDREWS, W. N. 1976. Natural infection of A&es trivittatus (COQ.) with Dirofilaria immitis in central Iowa. J. Purusitol., 62, 276-280. CROVELLO, T. J., AND HACI~ER, C. S. 1972. Evolutionary strategies in life table characteristics among feral and urban strains of Aedes uegypti. Evolution, 26, 185- 196. HACKER, C. S. 1971. The differential effect of PLUSmodiwn gallinuceum on the fecundity of several strains of Aedes aeg.vpti. J. fnvertebr. Puthoi.. 18, 373-377. LUDLAM, K. W., JACI~OWSM, L. A., AND OTTO. G. F. 1970. Potential vectors of Dirojflaria immitis. J. Amer. Vet. Med. Ass.. 157, 1354-1359. TRIPP, M. R. 1969. General mechanisms and principles of invertebrate immunity. In “Immunity to Parasitic Animals” (G. J. Jackson, R. Herman, and I. Singer, eds.), Vol I, pp. 111-128. Appleton-CenturyCrofts, New York. WEATHERSBY, A. B., AND NOBLET. R. 1973. Plasmodium gallinaceum: Development in Aedes uegypti maintained on various carbohydrate diets., E-VP. Purusito/. , 34, 426-43 1. WALKER, W. 1964. Parasite-induced changes of internal and external sex characteristics in insects. Exp. Parasitol.. 15, 561-597. CHRISTENSEN,
$.
OF INVERTEBRATE
33, 75-80 (19791
PATHOLOGY
The Effects of Various Carbohydrate Diets on Aedes aegypti Infected with Dirofilaria immitis GEORGE L. STEWART, Qepartment
qf Biology.
The
FRED K. SOIFER’, University
of
Texas
AND J. BRECK STEWART
at Arlington.
Arlington,
Texas
76019
Received May 18, 1978 Aedes aegypti infected with Diro$/aria immitis and uninfected mosquitoes were maintained on various carbohydrate diets (glucose, galactose, fructose, sucrose, trehalose, maltose, and melibiose). The value of each of these sugars in supporting survival of adult A. aegypti. and in supporting egg production, viability of eggs, and development of third-stage larvae of D. immitis in A. aegypti was analyzed. Fructose, glucose, maltose, sucrose, and trehalose provided the strongest support for survival of adult male, and infected and uninfected adult female A. aegypti. Galactose and melibiose provided the least support for survival of all groups of mosquitoes. The mean number of eggs laid per uninfected adult female A. cregypti was greatest when mosquitoes were maintained on glucose. mehbiose, maltose, fructose, sucrose, and trehalose. The same was true for female mosquitoes infected with D. immitis; except for melibiose which provided poor support for egg production. In both Dirojilaria-infected and in uninfected mosquitoes, galactose supported the production of low mean numbers of eggs per adult female A. aegypti. High percentages of eggs laid by uninfected and by infected female mosquitoes fed glucose, melibiose, maltose, sucrose, and trehalose hatched. While galactose supported a high percentage of hatching in eggs laid by uninfected A. aegypti. a much lower percentage of eggs laid by infected female mosquitoes maintained on this same carbohydrate hatched. The lowest percentages of eggs that hatched were from among those laid by infected and by uninfected females fed fructose. The highest mean number of D. immitis larvae CL,) were recovered from adult A. aegypti fed glucose, maltose, fructose, and sucrose; the second best sugar in this regard was trehalose. The lowest mean number of D. immitis larvae were isolated from female A. aegypti fed galactose and melibiose. KEY WORDS: Aedes aegypti; Dirofilarim immitis; mosquito, fecundity; mosquito, longevity; nematode: glucose: maltose: sucrose; galactose: trehalose: fructose: melibiose.
INTRODUCTION
It has been clearly demonstrated that the composition of the diet of some invertebrate hosts is an important factor in determining susceptibility to parasites (Tripp, 1969; Weathersby and Noblet, 1973). Dirojilaria immitis is a nematode parasite of canids which can utilize more than 60 species of mosquitoes as intermediate host (Ludlam et al., 1970). Little work has been done on the influence of diet on the biology of the mosquito host infected with D. immitis or on the development of this parasite within the intermediate host. The present study was undertaken to determine the effects of different carbohydrate diets on survival, ’ Bellaire Boulevard Animal Blvd., Houston, Texas 77036.
Clinic. 6213 Bellaire
fecundity, and egg viability in Aedes aegypti infected with D. immitis, and on development of the third-stage larva of this filarial nematode. MATERIALS
AND METHODS
D. immitis used in these experiments was initially obtained in 1973 from a naturally infected dog maintained at Bellaire Boulevard Animal Clinic, Houston, Texas. A Black-eye Liverpool strain of A. aegypti h as b een maintained in our laboratory since 1973 when it was obtained from Dr. Carl Hacker of The University of Texas School of Public Health, Houston, Texas. Mosquitoes were reared by the methods of Crovello and Hacker (1972). Mosquito larvae were hatched from egg papers immersed in deionized water in white, porcelain pans. Each pan contained between 100
75 0022-201 l/79/010075-06$01.00/O Copyright IV’ 1979 by Academic Prrss. Inc All rightc of reproduction in any form vscrved.
76
STEWART,
SOIFER,
and 200 larvae. Pans were maintained at 27°C and liver powder solution was added to pans as required. Following pupation, 100 male and 100 female pupae were placed on moist filter paper in each of 16 glass Petri dishes. One Petri dish was placed in each of 16-24 x 24 x 24 in. aluminum mesh cages for emergence of adults. All cages were maintained in an insectary at 27°C and 80% relative humidity. Cages of mosquitoes were divided into eight groups consisting of two cages. Following the experimental methods of Weathersby and Noblet (1973), one group of mosquitoes (two cages) was maintained on water alone and the remaining seven groups of mosquitoes were maintained on a 5% solution of one of the following carbohydrates: glucose, galactose, melibiose, maltose, sucrose, fructose,. or trehalose. Water or sugar solutions were changed daily and dead mosquitoes were removed from each cage, sexed, and counted. Four days following emergence of adults, one cage of adult mosquitoes from each of the eight groups was allowed to feed for 20 min on a dog infected with D. immitis (peripheral microfilaremia of 4000 mflml of blood; SE = kl93.7); the remaining eight cages of mosquitoes were allowed to feed for 20 min on a dog determined to be uninfected on the basis of chest X-ray analysis, physical examination, and the absence of circulating microfilariae. This dog had been obtained at 10 weeks of age in 1973 and maintained in a mosquito-proof cage since that time. Two days following the blood meal, an oviposition cup (half-pint paper can containing wet paper towels) was placed in each cage. Oviposition cups were changed daily and the total number of eggs laid by females in each cage was determined by direct count. Samples of eggs from each cage of mosquitoes were kept moist for 5 days to allow embryonation. Egg papers were dried at room temperature. One week later, 3-100 egg samples from each of the 14 cages (all mosquitoes maintained on water died before oviposi-
AND
STEWART
tion) were treated as above for hatching. Each day for 5 days, all larvae hatching from eggs were removed and counted (no eggs hatched after 4 days). These data were used to calculate percentage eggs hatching. On the 13th day following the blood meal, each cage was treated separately in the following manner: all adult mosquitoes were removed from the cage by suction, stunned by agitation, sexed, and counted. Females were individually crushed in cold (SC) phosphate-buffered saline (PBS, pH 7.2), and removed to a small-mesh plastic basket immersed in 2 ml of cold PBS in a small glass vial. Mosquito suspensions were maintained at 37°C for 1 hr and the thirdstage larvae (LJ were removed with a fine point pipet and counted. At the end of the initial incubation period each mosquito was removed from its plastic basket, crushed lightly, and processed a second time as above for recovery of any remaining larvae. All uninfected female mosquitoes from the same cage were pooled and treated in this same manner. Because of high mortality among adult mosquitoes supported on melibiose and galactose, experiments involving these sugars were repeated five times for both infected and uninfected mosquitoes. Experiments in which adult mosquitoes were supported on the other five sugars and on water were repeated three times. All data were statistically analyzed by the Student’s t test to determine the significance of differences. RESULTS
The percentage of the original population of infected and uninfected adult female A. aegypti remaining alive on each day between 2 days before the blood meal and the end of the study (13 days following the blood meal) are presented in Figure 1. From Day 0 to the end of the study, galactose supported the survival of significantly lower percentages of infected female mosquitoes than did all other sugars except melibiose. A similar situation was seen with
EFFECTS OF CARBOHYDRATES
ON DIROFILARIA-INFECTED
+c
77
m-m ‘m-mmm
a,
_ & a
AEDES
Uninfected
~................~..............., 2'0'2
3 4 5 6 7 8 9,011,213
2'0'23456
7 8 9 10 11 12 13
Days FIG. 1. Adult female Aedes aegypti infected with DiroJilaria immitis and uninfected adult female A. fed glucose to), galactose ( l ), melibiose (W), trehalose ((I)), fructose (A), sucrose 0, and maltose (A) presented as percentage of initial population surviving on each day between 2 days before the blood meal and 13 days following the blood meal (blood meal provided on Day 0).
aegypri
uninfected female mosquitoes. Melibiose supported the survival of significantly lower percentages of infected adult female mosquitoes (beginning of the study to Day 8 post-blood meal) and of uninfected female mosquitoes (from 1 day before the blood meal to the end of the study) than did galactose. Trehalose supported the survival of significantly lower percentages of uninfected female mosquitoes (Days 8 through 13 post-blood meal) than did maltose, sucrose, glucose, and fructose. With all sugars except melibiose, greater percentages of uninfected than infected female mosquitoes were alive between 2 days following the blood meal and the end of the study. With melibiose, significantly greater percentages of uninfected than infected female A. aegypti were alive between Days 2 and 11 post-blood meal. Similar percentages of adult male mosquitoes supported on each of the seven sugars tested, whether they were caged with infected or uninfected female mosquitoes, were alive on each day during the study.
The percentages of adult female mosquitoes infected with D. immitis, of uninfected adult female mosquitoes and of adult male mosquitoes, surviving to the end of the study (Day 13 post-blood meal) are presented in Figure 2. Fructose supported survival of the highest percentage of male mosquitoes caged with uninfected female mosquitoes. Glucose and sucrose were second in this regard: then maltose and trehalose; followed by galactose and melibiose. Fructose, sucrose, and glucose provided support for survival of the highest percentage of male mosquitoes caged with infected female mosquitoes; then maltose and trehalose; followed by galactose and melibiose. The highest percentages of uninfected female mosquitoes surviving were among those fed glucose, maltose, fructose, and sucrose; then trehalose; followed by galactose and melibiose. Survival of female A. aegypti infected with D. immitis was best supported by glucose, maltose, fructose, sucrose, and trehalose, while galactose and melibiose provided the poorest support for survival of infected female
78
STEWART,
SOIFER, AND STEWART
_female
+ 5 v G a
20. : ih gl
I
ga me ma
fr
su
gl
tr
ga me
ma
fr
su
tr
Sugar FIG. 2. Adult male Aedes aegypti caged with uninfected adult female A. aegypvpti (white bar) and adult males caged with adult female A. aegypti infected with Dirofilaria immitis (black bar); and infected (white bar) and uninfected (black bar) adult female A. aegypti are presented as percentage adult mosquitoes surviving at the end of the study (Day 13 post-blood meal) versus dietary carbohydrate [glucose (gl). galactose (ga), melibiose (me), maltose (ma), fructose (fr). sucrose (su) and trehalose (tr)]. Standard errors are presented for all data. P < 0.05
mosquitoes. With all sugars except melibiose higher percentages of uninfected than infected female mosquitoes were alive at the end of the study. With all sugars except melibiose, the
mean numbers of eggs laid per female A. aegypti infected with D. immitis and per uninfected female A. aegypti were similar (Figure 3A). The mean number of eggs laid per infected female mosquito fed melibiose
TT
ga me
ma
tr
su
tr
gl ga
me
ma
tr
I
tr
S U gar FIG. 3. (A) Mean number of eggs laid per adult female Aedes aegypti infected with DiroJilaria immitis (white bar) and per uninfected adult female A. aegypti (black bar) fed glucose (gl), galactose (ga), melibiose (me), maltose (ma), fructose (fr), sucrose (su), and trehalose (tr). (B) Viability of eggs laid by adult female A. aegypvpri infected with D. immitis (white bar) and by uninfected adult female A. aegypfi (black bar) fed glucose (gl), galactose (pa), melibiose (me), maltose (ma), fructose (fr), sucrose (su), and trehalose (tr). Dataare presented in terms ofpercentage eggs hatching. Standard errors are shown for all data. P < 0.05
EFFECTS
OF
CARBOHYDRATES
ON DIROFILARIA-INFECTED
was significantly lower than that for uninfected female mosquitoes fed this same sugar. The percentage of hatching was similar among eggs laid by infected and uninfected female mosquitoes fed any of the seven sugars except galactose (Figure 3B). When galactose was the supporting sugar, a significantly lower percentage of eggs laid by infected than by uninfected female mosquitoes hatched. With fructose as the supporting sugar, the percentages of eggs laid by infected and uninfected female A. aegypti that hatched were similar. However, these percentages were significantly lower than that observed with all other sugars except galactose (infected female mosquitoes). Similar mean numbers of third-stage larvae (LJ of D. immitis were obtained from infected female mosquitoes fed glucose, maltose, fructose and sucrose (Figure 4). The mean number of L3 of D. immitis isolated from individual female A. aegypti fed trehalose was significantly lower than with the aforementioned four sugars, but signifi7.5 al t-0
I
gl
ga
mema
fr
su
tr
Sugar FIG. 4. Mean number of Dir&aria immitis larvae isolated per infected adult female Aedes aegyppri fed glucose (gl), galactose (ga), melibiose (me), maltose (ma), fructose (fr), sucrose (su), and trehalose (tr). Standard errors are oresented for all data. P < 0.05
AEDES
79
cantly higher than that obtained from female mosquitoes fed galactose and melibiose. No D. immitis third-stage larvae were recovered from uninfected female mosquitoes. DISCUSSION
In agreement with the findings of Weathersby and Noblet (1973) in their study on Plasmodium gallinaceum, the survival of adult A. aegypti, both uninfected and those infected with D. immitis, was greatest on diets consisting of glucose, maltose, fructose, sucrose, and trehalose. The poorest survival of mosquitoes was among those supported on melibiose and galactose. Significantly greater percentages of uninfected than infected female mosquitoes were alive at the end of the study (Day 13 post-blood meal). On the other hand, similar percentages of male mosquitoes from cages containing uninfected females and from cages housing female mosquitoes infected with D. immitis were alive throughout the study (Day 2 before blood meal to Day 13 post-blood meal). A significantly higher mortality rate among infected than uninfected female A. aegypti first became evident on the second day following the blood meal and continued throughout the study. These differences in the death rate between infected and uninfected mosquitoes is attributed to the pathology created by D. immitis larvae developing in the malpighian tubules of the parasitized female mosquitoes (Christensen and Andrews, 1976). Of all the sugars tested, melibiose provided the poorest support for survival of both infected and uninfected mosquitoes. However, only infected female A. aegypti fed melibiose showed a significant decrease in fecundity. It has been shown by others that a parasite can alter the fecundity of its insect host (Walker, 1964; Hacker, 1971). Although the other six sugars supported similar levels of fecundity in infected and uninfected mosquitoes, we suggest that the stress of parasitism combined with the extremely low nutritive value of melibiose
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significantly reduced fecundity in infected female mosquitoes fed this sugar. Galactose supported levels of fecundity in infected and uninfected female mosquitoes which were as low as those supported by melibiose in female mosquitoes infected with D. immitis. One possible explanation for this observation may be that galactose cannot be utilized as efficiently by A. aegypti for support of egg production as can the other six sugars. A similar explanation may be offered for the decreased viability of eggs laid by infected and uninfected female mosquitoes fed fructose. In this situation perhaps fructose cannot be used as efficiently by A. aegypti for support of embryonation as can the other sugars tested. Relative to egg viability, a similar situation was seen with galactose. Lower percentages of eggs laid by infected than uninfected female mosquitoes hatched. It may be that the additive effect of pathology of the filarial infection and the low nutritional value of galactose decreased the quality of eggs produced. Those sugars which provided the poorest support for survival of infected adult female A. aegypti, i.e., galactose and melibiose, supported the development of the lowest numbers of D. immitis third-stage larvae. On the other hand, four of the five sugars that provided strong support for the survival of infected adult female mosquitoes, i.e., sucrose, glucose, maltose, and fructose, supported the development of the highest numbers of D. immitis third-stage larvae. Trehalose is the only exception to
this apparent trend. This sugar provided a level of support for survival of infected adult female mosquitoes similar to that observed with the above four sugars, but supported the development of third-stage larvae of D. immitis at a level significantly below that seen with sucrose, glucose, maltose, and fructose. ACKNOWLEDGMENT The authors thank Dr. Carl S. Hacker of The University of Texas School of Public Health for his advice and assistance in this study.
REFERENCES M., AND ANDREWS, W. N. 1976. Natural infection of A&es trivittatus (COQ.) with Dirofilaria immitis in central Iowa. J. Purusitol., 62, 276-280. CROVELLO, T. J., AND HACI~ER, C. S. 1972. Evolutionary strategies in life table characteristics among feral and urban strains of Aedes uegypti. Evolution, 26, 185- 196. HACKER, C. S. 1971. The differential effect of PLUSmodiwn gallinuceum on the fecundity of several strains of Aedes aeg.vpti. J. fnvertebr. Puthoi.. 18, 373-377. LUDLAM, K. W., JACI~OWSM, L. A., AND OTTO. G. F. 1970. Potential vectors of Dirojflaria immitis. J. Amer. Vet. Med. Ass.. 157, 1354-1359. TRIPP, M. R. 1969. General mechanisms and principles of invertebrate immunity. In “Immunity to Parasitic Animals” (G. J. Jackson, R. Herman, and I. Singer, eds.), Vol I, pp. 111-128. Appleton-CenturyCrofts, New York. WEATHERSBY, A. B., AND NOBLET. R. 1973. Plasmodium gallinaceum: Development in Aedes uegypti maintained on various carbohydrate diets., E-VP. Purusito/. , 34, 426-43 1. WALKER, W. 1964. Parasite-induced changes of internal and external sex characteristics in insects. Exp. Parasitol.. 15, 561-597. CHRISTENSEN,
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