Experimental Gerontology 45 (2010) 685–690

Contents lists available at ScienceDirect

Experimental Gerontology
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / ex p g e r o

The impact of larval and adult dietary restriction on lifespan, reproduction and
growth in the mosquito Aedes aegypti
Teresa K. Joy, Anam J. Arik, Vanessa Corby-Harris, Adiv A. Johnson, Michael A. Riehle ⁎
Department of Entomology, University of Arizona, Tucson, AZ, United States

a r t i c l e i n f o a b s t r a c t

Article history: Dietary restriction extends lifespan in many organisms, but little is known about how it affects
Received 18 February 2010 hematophagous arthropods. We demonstrated that diet restriction during either larval or adult stages
Received in revised form 14 April 2010 extends Aedes aegypti lifespan. A. aegypti females fed either single or no blood meals survived 30–40% longer
Accepted 26 April 2010
than those given weekly blood meals. However, mosquitoes given weekly blood meals produced far more
Available online 6 May 2010
eggs. To minimize reproduction's impact on lifespan, adult mosquitoes were fed artificial blood meals
Keywords:
containing b10% of the protein in normal human blood, minimizing egg production. A. aegypti fed artificial
Dietary restriction blood meals containing 25 mg/ml of BSA had significantly shorter lifespans than those fed either 10 or 5 mg/
Nutrient restriction ml. To assess the impact of larval dietary restriction on adult lifespan, we maintained larval A. aegypti on 2X,
Aging 1X (normal diet), 0.5X or 0.25X diets. Adult mosquitoes fed 0.5X and 0.25X larval diets survived significantly
Senescence longer than those fed the 2X larval diet regardless of adult diet. In summary, dietary restriction during both
Dengue larval and adult stages extends lifespan. This diet-mediated lifespan extension has important consequences
for understanding how dietary restriction regulates lifespan and disease transmission.
© 2010 Elsevier Inc. All rights reserved.

1. Introduction
One of the most robust mechanisms regulating lifespan across a
wide range of organisms is dietary restriction, where nutrient intake is
limited to below what an organism would take ad libitum, but above
the level of starvation. Lifespan extension from dietary restriction has
been observed in a variety of organisms (Mair and Dillin, 2008),
including insects as diverse as the fruit fly, the house fly, a butterfly,
and even a stick insect (Carey et al., 2002, Cooper et al., 2004, Boggs
and Ross, 1993, Roark and Bjorndal, 2009). In Drosophila melanogaster,
for example, dietary restriction in adults can extend lifespan by over
50% (Grandison et al., 2009, Partridge et al., 2005). Protein and lipid
levels, not calories per se, appear to be especially important in
governing this relationship (Mair et al., 2005). Less is known about the
impact of dietary restriction at the larval stage. One study demon-
strated that while yeast deprived larvae take longer to eclose and
adults are smaller, there was no apparent difference in adult mortality
between larvae fed a restricted or normal diet (Tu and Tatar, 2003).
Given their unique ecology, it is useful to extend this widespread
relationship between diet and longevity to blood feeding arthropods
such as the mosquito Aedes aegypti.
Wild mosquitoes often experience periods of nutrient restriction.
During the aquatic larval stages, container breeding Aedes mosquitoes
⁎ Corresponding author. PO Box 210036, Forbes 410, Department of Entomology,
University of Arizona, Tucson, AZ 85721, United States. Tel.: +1 520 626 8500; fax: +1
520 621 1500.
E-mail address: mriehle@ag.arizona.edu (M.A. Riehle).
develop in small transient pools, where they undergo intense inter-
and intraspecific competition (Reiskind and Lounibos, 2009) for
resources such as bacteria, algae, and other organic matter. Intra- and
interspecific competition at the larval stages can impact adult lifespan
(Reiskind and Lounibos, 2009), but it is unclear whether nutrient
restriction causes these changes. As adults, female A. aegypti largely
consume protein-rich blood meals, but will also feed on nectar when a
blood source is not readily available or if nectar sources are abundant
(Edman, 2003, Martinez-Ibarra et al., 1997). If hosts are in short
supply, as is the case in many rural areas, then blood meals and dietary
protein will be restricted. Larval competition and the availability of
vertebrate blood during adulthood could potentially impact the
mosquito's lifespan in nature.
A. aegypti mosquitoes have a feast and famine feeding behavior. In
D. melanogaster, there is conflicting evidence on whether periods of
feeding interspersed with periods of starvation can lead to increased
lifespan. Some studies using an intermittent feeding schedule showed
no signs of lifespan extension in flies intermittently starved (Le Bourg
and Médioni, 1991, Le Bourg and Minois, 1996). Studies in medflies
and houseflies had similar results (Carey et al., 2002, Cooper et al.,
2004). In contrast, a 30% increase in lifespan was observed when D.
melanogaster were provided sugar water ad libitum and yeast every
sixth day (Partridge et al., 1987). Female A. aegypti adults offer a
unique model for examining this effect. After mating, female A. aegypti
consume a large (∼2 µl) blood meal rich in protein, nearly doubling
their body weight. Following this blood meal the female produces a
clutch of approximately 100 eggs which are oviposited 48–72 h later.
This cyclical pattern of feeding and reproduction is characteristic of

0531-5565/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.exger.2010.04.009
686 T.K. Joy et al. / Experimental Gerontology 45 (2010) 685–690
many blood feeding arthropods and likely plays an important role in
determining overall lifespan. Dietary restriction might be achieved
either through limited blood meals due to the lack of a suitable host or
through incomplete blood meals caused by disruption of the blood
feeding process. In urban environments, where potential hosts are
abundant and A. aegypti consume multiple blood meals during a
reproductive cycle (Scott et al., 1993), partial blood meals are likely to
be a more important source of dietary restriction.
In addition to the insights that blood feeding arthropods can
provide regarding dietary restriction and lifespan, life history changes
in A. aegypti have the potential to impact disease transmission. The
capacity of A. aegypti to transmit pathogens such as the four closely
related flaviviruses collectively known as dengue is largely contingent
on lifespan. In fact, the daily probability of survival is the single most
important factor determining the vectorial capacity of a mosquito
(Macdonald, 1957). This is due to the fact that many mosquito-borne
parasites, including dengue, require an extended developmental
period within the mosquito known as the extrinsic incubation period,
which can last up to two weeks. In the wild, A. aegypti daily probability
of survival is between 70 and 90%, meaning that even under ideal
conditions fewer than 20% of wild A. aegypti survive long enough to
transmit dengue (Harrington et al., 2001, Costero et al., 1998).
However, those mosquitoes that do survive the extrinsic incubation
period are infective for life and can potentially infect every
subsequent person they bite. Thus, reducing the lifespan of older
mosquitoes that transmit a disproportionate amount of the disease
could reduce overall dengue transmission. Having a better under-
standing of how dietary restriction impacts lifespan, both during the
aquatic larval stages and blood feeding adult stages, is critical for
understanding the epidemiology of dengue and may provide new
insights into novel control measures.
One confounding variable in many studies of the relationship
between diet and lifespan is that reproduction, a trait often negatively
correlated with survival, is directly correlated with food quality and
intake (Piper and Partridge, 2007). To address this, Mair et al. (2004)
restricted females' access to males, used mutant genotypes, and
eliminated female germlines showed that dietary restriction extended
lifespan independent of reproduction in D. melanogaster. A. aegypti
reproduction can be easily manipulated, too, in order to reduce the
influence of reproduction on lifespan in dietary restriction studies.
Because their reproduction is only initiated after they take a protein
meal, we can easily manipulate female fecundity by reducing the
amount of protein they eat.
In this study we explored the impact of dietary restriction during
both larval and adult stages on the lifespan and reproductive capacity
of female A. aegypti. At the same time we determined the lifetime
fecundity of these dietary restricted mosquitoes when provided with
a similar number of discrete protein meals. We also examined how
effectively aging mosquitoes produced eggs and whether previous
reproductive cycles impact egg production in subsequent gonotrophic
cycles.
2. Experimental procedures
2.1. Mosquito rearing
A. aegypti were maintained at 27 °C and 70% RH with a 16 h light
and 8 h dark photo period. During the larval stage mosquitoes were
reared on a solution containing 125 mg/l of yeast, ground rabbit chow,
and lactalbumin (1:1:1 ratio). The standard larval diet was as follows:
days 1 and 2 post larval emergence — 400 µl diet/pan, days 3 and 4 —
600 µl/pan, day 5 through pupation — 1.2 ml/pan. Most mosquitoes
pupated by day nine. Adult mosquitoes were maintained in 5 l
canisters and allowed to feed on 10% dextrose ad libitum using a wick.
For egg production mosquitoes were membrane fed on porcine blood
containing 1% sodium citrate as an anticoagulant. Mosquitoes given
blood for the diet experiments were allowed to feed for 2 h. Pig blood
is over 50% protein (520 g/l (Hellwing et al., 2007)), with the
remaining fraction containing mostly water.
2.2. Adult blood feeding assay
A. aegypti used in the adult blood feeding assays were fed the
standard diet during larval stages. Thirty pans of larvae (200 larvae/l/
pan) were established per experiment under identical conditions and
pupae were combined to generate a homogenous mixture. Approx-
imately 1000 female mosquitoes and several hundred males were
placed in large cages (21 l) to generate three treatment groups. One
treatment group received weekly blood meals starting 7 days post-
emergence, the second treatment received a single blood meal 7 days
post-emergence, and the third treatment received no blood meals.
Blood fed treatments were provided with oviposition substrates 48 h
after blood feeding and were allowed to oviposit for 72 h. All
treatment groups were given unlimited access to a wick soaked in
10% dextrose. Dead female mosquitoes were counted and removed
from the cages daily. Experiments were replicated three times.
2.3. Adult dietary restriction
A. aegypti used in the adult dietary restriction tests were fed the
standard diet during larval stages. Fifteen pans of larvae (100 larvae/l/
pan) were established per experiment under identical conditions and
pupae were combined to generate a homogenous mixture. Female
mosquitoes emerging on days seven or eight were separated into four
cages of 200 females each. Male mosquitoes (50) were added to each
cage to ensure that females had mated. Each treatment was given an
artificial blood meal consisting of various concentrations of bovine
serum albumin (BSA; 1 mg/ml, 5 mg/ml, 10 mg/ml or 25 mg/ml) in
feeding buffer (150 mM NaCl, 1 mM ATP at 37 °C). A 10% dextrose
solution was provided via wick throughout the assay for all
treatments. The first artificial blood meal was given ∼ 5 days after
adult emergence. Subsequent feedings were provided twice weekly
on Mondays and Thursdays. Oviposition surfaces were provided 48 h
after the artificial blood meal for 24–36 h. Eggs laid on the oviposition
substrate or the wick of the dextrose source were collected and
counted. Dead female mosquitoes were counted and removed from
the cages daily. Experiments were replicated five times.
2.4. Larval dietary restriction
To test what effects dietary restriction during the larval stage had
on the lifespan of adult A. aegypti we modified larval food intake. On
day one, newly hatched larvae were evenly distributed into ten pans
(100 larvae/l/pan). Five pans were fed half the standard larval diet and
the other five pans were fed one quarter the standard diet. Since
larvae fed the 1X and 2X diet developed more quickly these
treatments were established two days later. This resulted in all of
the adult mosquitoes emerging within a 3–4 days window. Three
cages containing ∼ 200 adult mosquitoes were established for each
larval treatment group (12 cages total). One cage was never bloodfed,
one was given a single blood meal 4 days after adult eclosion, and one
was given weekly blood meals starting 4 days after adult eclosion.
Mosquitoes in all of the cages were given ad libitum access to a wick
saturated with 10% dextrose. Dead females were counted and
removed from the cages on a daily basis.
2.5. Egg production analysis
To investigate the reproductive success of A. aegypti during
successive reproductive cycles, a large cage was established with
1000 newly eclosed females and ∼ 250 males. Mosquitoes were
provided with a blood meal 7 days post eclosion and non blood fed
 T.K. Joy et al. / Experimental Gerontology 45 (2010) 685–690 687

females were discarded. Five small cages each containing 20 blood fed feed on 10% dextrose ad libitum, one group was fed a single blood meal
females were established. Oviposition substrates were placed in the and dextrose ad libitum, and the third group was fed weekly blood
five treatment groups and the master cage. Mosquitoes were allowed meals supplemented with dextrose ad libitum throughout their adult
to oviposit for 72 h after which all mosquitoes were returned to the life. Analysis of the full model showed that diet (X2 = 646.39,
master cage. Eggs from the five treatment groups were dried p b 0.0001) was a significant predictor of time to death. The effect of
overnight and counted using ImageJ 1.42 software following the diet was also a significant predictor of time to death in each replicate
protocol described in Mains et al. (2008). This process was repeated treated separately (replicate 1: X2 = 145.16, p b 0.0001, replicate 2:
on days 14, 21, and 28. The average number of eggs was calculated by X2 = 134.00, p b 0.0001, replicate 3: X2 = 581.67, p b 0.0001). Female A.
dividing the total number of eggs laid by the number of ovipositing aegypti provided with weekly blood meals had the shortest lifespan,
mosquitoes each week. This experiment was replicated three times. surviving an average of eight days or ∼ 30% less than mosquitoes given
To determine if age impacted egg production during the first a single blood meal (X2 = 418.40; p b 0.0001, Fig. 1A) and ten days or
reproductive cycle, newly eclosed female (250) and male (100) ∼40% less than mosquitoes fed only sugar (X2 = 550.90; p b 0.0001,
mosquitoes were established in four separate cages. Each week (7, 14, Fig. 1A). There was no significant difference between mosquitoes fed a
21 or 28 days post eclosion), one of the four cages was provided with single blood meal versus those fed only sugar (Fig. 1A).
blood meal. Following blood feeding (48 h), 5 cages of 20 blood fed
females each were established and oviposition substrates provided for 3.2. Dietary restriction in adult female A. aegypti
72 h. Eggs were counted using ImageJ 1.42 software following the
protocol of Mains et al. (2008). The average number of eggs was We analyzed the effect of dietary restriction during the adult stage
calculated by dividing the total number of eggs laid by the number of on the lifespan of adult A. aegypti. Larval mosquitoes were reared
ovipositing mosquitoes each week. under identical conditions and the resulting adults fed various
dilutions of bovine serum albumin (BSA). Human blood contains
2.6. Statistical analyses ∼200–250 mg/ml of protein (Bliss, 1928) and a complete blood meal
leads to the production of ∼ 100 eggs. Similar concentrations of BSA
A Cox proportional hazards (CPH) model was used to analyze the led to similarly sized egg clutches (data not shown). To minimize egg
survivorship data (Cox, 1972, Therneau et al., 2000). We were production, but still observe the effects of dietary restriction, we fed
particularly interested in investigating the effect of diet on time to artificial bloomeals containing 25, 10, 5, or 1 mg/ml of BSA to
death while controlling for the effect of replicate. As such, in the full mosquitoes twice weekly. Dextrose (10%) was also provided ad
model, we analyzed the effect of diet on mortality while including libitum throughout the experiment. Dead females were counted daily
replicate as a strata variable (Therneau et al., 2000, Allison, 1995). to assess their mortality rate and five replicate experiments were
Additionally, to ask whether the effect of diet was consistently
significant over the multiple experimental replicates, we used a CPH
to ask whether diet was a significant predictor of time to death for
each replicate separately. To ask whether two treatments were
significantly different from each other, a full CPH model was analyzed
with all other treatments omitted. Significant p-values were inter-
preted as significant pairwise differences. All survival analyses were
performed using PROC TPHREG in SAS version 9.1 (SAS Institute, Cary,
NC, USA).
All reproductive data were analyzed using an ANOVA. We initially
investigated the effect of multiple reproductive cycles on egg
production by fitting a model where reproductive cycle was the
predictor variable, replicate was a random effect, and egg count per
cage was the response. The number of mosquitoes was included as a
covariate in the model because the number of mosquitoes per cage
was not constant. Because measurements were taken on the same
cage successively (i.e., estimates of the number of eggs per cage per
reproductive cycle were not independent across time), the ideal way
to analyze these data is a repeated measures analysis where
measurements are correlated across reproductive cycles. However,
because reproductive cycle was the only predictor variable in the
model, we could not use such an analysis. A standard ANOVA was the
next best, although not perfect, option. We investigated whether
female age at their first blood meal affected the number of eggs laid
per cage. Age at first blood meal was the predictor variable and the
number of mosquitoes per cage was included as a covariate, as this
number was not constant among cages. All ANOVA analyses were
conducted using JMP Version 7 (SAS Institute, Cary, NC, USA).

3. Results
3.1. Impact of blood feeding on adult lifespan in female A. aegypti
Female A. aegypti are obligate blood feeders that require a blood
meal to produce a clutch of eggs. To examine what impact the
consumption of these large protein meals has on A. aegypti's lifespan
we established three treatment groups. One group was allowed to
Fig. 1. The impact of blood feeding and dietary protein on adult A. aegypti lifespan. A. A
representative survivorship curve comparing mosquitoes fed weekly blood meals (BM),
a single blood meal, or no blood meals. All treatments were allowed access to 10%
dextrose ad libitum. Dead individuals were counted daily. Letters following the figure
legend indicate significant differences, with different letters indicating significant
differences. B. Combined data showing the lifespan of adult female mosquitoes given
access to an artificial blood meal containing 25, 10, 5 or 1 mg/ml of BSA every three
days. All treatments were allowed ad libitum access to 10% dextrose. Survivorship was
assessed daily. Treatments with the same letters were not different from each other;
those with different letters have significant differences.
688 T.K. Joy et al. / Experimental Gerontology 45 (2010) 685–690

performed. During their first reproductive cycle, mosquitoes fed

25 mg/ml produced less than 10% of the eggs achieved with a normal
blood meal, and egg production steadily declined during each
successive reproductive cycle (Fig. S1). Mosquitoes fed 5 or 1 mg/ml
produced virtually no eggs and those fed 10 mg/ml produced an
intermediate amount. Adult mosquitoes fed 5 or 10 mg/ml of BSA
lived significantly longer than those fed 1 or 25 mg/ml of BSA
(Fig. 1B). There was no significant difference between those fed 5 and
10 mg/ml or those fed 25 and 1 mg/ml. Mosquitoes given artificial
blood meals containing 10 mg/ml BSA survived an average of nine
days longer (∼ 20%) than those fed 25 mg/ml. Those fed 5 mg/ml
survived seven days longer. Analysis of the full model showed that
diet (X2 = 145.35, p b 0.0001) was a significant predictor of time to
death. Diet was a significant predictor of time to death for each
replicate analyzed separately (replicate 1: X2 = 44.16, p b 0.0001,
replicate 2: X2 = 65.70, p b 0.0001, replicate 3: X2 = 65.70, p b 0.0001,
replicate 4: X2 = 30.06, b0.0001, replicate 5: X2 = 16.19, p = 0.0010).

3.3. Dietary restriction in A. aegypti larvae leads to the extension of adult

lifespan

Larval A. aegypti face considerable competition for limited

resources in the small pools of water they reside in, and this could
affect adult lifespan and dengue transmission. To determine the
impact of dietary restriction during the larval stages on adult
longevity we established four treatment groups and fed them
different larval diets 2X, 1X (standard larval diet), 0.5X and 0.25X.
Following emergence, adults were provided with sugar ad libitum and
given access to none, one, or weekly blood meals. Each experiment
was replicated three times.
Overall, dietary restriction during the larval stages significantly
extended adult lifespan. Adult mosquitoes provided with sugar only
achieved maximal lifespan when fed half of the normal diet as larvae
(2X diet: 50% survival = 38 days, 10% survival = 58 days; 1X diet: 50%
survival = 41 days, 10% survival = 59 days; 0.5X diet: 50% survi-
val = 43 days, 10% survival = 61 days; 0.25X diet: 50% survival = 41
days, 10% survival = 63 days; Fig. 2A). Analysis of the full model
showed that larval diet (X2 = 27.33, p b 0.0001) was a significant
predictor of time to death. Larval diet was a significant predictor of
time to death for the first two but not the third replicate (replicate
1: X2 = 12.10, p = 0.0070, replicate 2: X2 = 27.84, p b 0.0001, replicate Fig. 2. The effect of dietary restriction during the larval stages. A. Survivorship of
3: X2 = 4.59, p = 0.20). Adult mosquitoes fed a single blood meal lived mosquitoes fed 2X, 1X, 0.5X or 0.25X their normal diet as larvae and strictly sugar as adults.
Letters following the figure legend indicate whether the differences between treatments
longest when fed the 0.25X diet, and had the shortest lifespan when
were significant. Treatments with the same letters were not different from each other,
fed a nutrient rich larval diet (2X diet: 50% survival = 34 days, 10% while those with different letters had significant differences. B. Survivorship of mosquitoes
survival = 57 days; 1X diet: 50% survival = 42 days, 10% survi- fed 2X, 1X, 0.5X or 0.25X their normal diet as larvae and a single blood meal and 10%
val = 61 days; 0.5X diet: 50% survival = 37 days, 10% survival = 62 - dextrose ad libitum as adults. Letters following the figure legend indicate significant
differences, with different letters indicating significant differences. C. Survivorship of
days; 0.25X diet: 50% survival = 43 days, 10% survival = 67d; Fig. 2B).
mosquitoes fed 2X, 1X, 0.5X or 0.25X their normal diet as larvae and multiple blood meals
The full model analysis showed that larval diet (X2 = 29.00, and 10% dextrose as adults. Letters following the figure legend indicate significant
p b 0.0001) was a significant predictor of time to death. Larval diet differences, with different letters indicating significant differences.
was a significant predictor of time to death in two of the three
replicates (replicate 1: X2 = 5.21, p = 0.1572, replicate 2: X2 = 40.69,
p b 0.0001, replicate 3: X2 = 8.10, p = 0.044). Mosquitoes provided 3.4. The effect of multiple reproductive cycles on egg production
with weekly blood meals reached maximal lifespan with the most
restricted larval diet (2X diet: 50% survival = 25 days, 10% survi- We observed that the number of reproductive cycles a mosquito
val = 46 days; 1X diet: 50% survival = 28 days, 10% survival = 49 undergoes impacts the mosquito's lifespan (Fig. 1A). However, we
days; 0.5X diet: 50% survival = 28 days, 10% survival = 53 days; 0.25X were interested in whether the number of reproductive cycles or the
diet: 50% survival = 28 days, 10% survival = 52 days; Fig. 2C). Under a age of the mosquito affected the number of eggs produced during
full model, larval diet (X2 = 80.69, p b 0.0001) was a significant each cycle. To assess this, we provided a weekly blood meal to a large
predictor of time to death. Larval diet was a significant predictor of pool of mosquitoes, discarded those that failed to blood feed and
time to death for all three replicates (replicate 1: X2 = 12.13, determined total egg production from 20 mosquitoes during their first
p = 0.0070, replicate 2: X 2 = 24.46, p b 0.0001, replicate 3: four reproductive cycles. During their first reproductive cycle the pool
X2 = 71.93, p b 0.0001). A slight, but significant, decrease in body of 20 mosquitoes produced ∼ 1100 eggs (Fig. 3A). Egg production
size was observed in both males and females given different diets as decreased by ∼ 30% during each of the next two cycles before
larvae, with those mosquitoes receiving the least food producing the stabilizing during the fourth cycle. The number of successive
smallest adults based on wing length (Fig. S2). reproductive cycles was a significant predictor of the number of
 T.K. Joy et al. / Experimental Gerontology 45 (2010) 685–690
Fig. 3. Egg production during successive reproductive cycles in A. aegypti and during the first reproductive cycles of aged mosquitoes. A. The number of eggs per cage laid by fully-fed
females assayed for reproductive output on successive reproductive cycles. Different letters indicate a significant difference between treatments. B. Cohorts of adult mosquitoes aged
1, 2, 3, or 4 weeks old were given an identical blood meal and the number of eggs per cage was counted. Different letters indicate significant differences between treatments.
eggs laid (F3,34 = 23.31, p b 0.0001). This indicates that A. aegypti
mosquitoes, at least under laboratory conditions, devote a large
portion of their resources to their initial reproductive efforts. To assess
whether this observed reduction in egg production was due to the
mosquitoes' age we provided pools of mosquitoes with their first
blood meal when they were either 1, 2, 3, or 4 weeks of age.
Mosquitoes aged 1 to 3 weeks laid a similar number of eggs and age
only impacted egg production as females reached 4 weeks of age
(Fig. 3B). Age at first blood meal was a significant predictor of the
number of eggs laid per cage (F3,15 = 6.20, p = 0.0060).
4. Discussion
Dietary restriction extends the lifespan of many different organ-
isms. We have demonstrated that dietary restriction during both the
larval and adult stages also impacts the adult lifespan of a
haematophagic insect, the mosquito A. aegypti. Adult female A. aegypti
consume large, nutrient rich meals determined by the availability of
vertebrate hosts. Therefore, limited access to vertebrate hosts and
their blood may actually increase lifespan and extend their ability to
reproduce and transmit diseases such as dengue.
Our studies indicate that mosquitoes fed only sugar survive nearly
as well as those given a single blood meal, and significantly longer
than those given multiple blood meals. Previous studies examining
the impact of blood feeding versus sugar feeding on various species of
Aedes mosquitoes give conflicting results. While some suggest blood
feeding is detrimental, others suggest it is beneficial. For example, A.
aegypti mosquitoes given daily human blood meals with or without
sugar both survived better than those given only sugar (Styer et al.,
2007a). In the case of daily blood meals, the protein that is consumed
while a reproductive cycle is already underway could be devoted
towards somatic maintenance or stored as reserve nutrients,
potentially extending lifespan. In urban areas where dense host
populations exist it has been shown that A. aegypti mosquitoes
routinely feed multiple times during a reproductive cycle. However, in
rural areas where hosts are not as abundant this may not be the case.
A second study by the same group on A. aegypti mosquitoes recently
collected from the field showed that mosquitoes fed both sugar and
blood survived significantly longer than those fed either sugar or
blood alone (Styer et al., 2007b). Interestingly, mosquitoes in the
blood only treatments given access to blood every day survived much
better than those given blood every other day, providing some
evidence that frequent blood feeding between reproductive cycles can
enhance survivorship. Similarly, A. aegypti and Aedes albopictus
mosquitoes given daily blood meals both survived significantly longer
when they had access to sugar, although lifetime fecundity was
similar (Braks et al., 2006). In contrast, Aedes communis maintained on
25% or 50% sucrose or fructose survived significantly longer than those
689
maintained on 10% sucrose or those given a blood meal supplemented
with either water or 10% sucrose (Andersson, 1992). These conflicting
results suggest that the frequency of blood feeding, availability of
nectar sources, and host density can have a large impact on mosquito
survivorship.
All of the dietary restriction studies in mosquitoes to date compare
the impact of blood feeding on the mosquito's lifespan by limiting the
mosquito's ability to consume a blood meal. However, because each
blood meal leads to the production of a complete clutch of eggs,
reproduction itself might impact lifespan. Lifespan and reproduction
are often negatively correlated, and so dietary restriction could extend
lifespan by reducing reproduction. To uncouple reproduction and
lifespan in our assays, we provided mosquitoes with artificial blood
meals containing low protein concentrations that reduced egg
production. Egg production was reduced by more than 85%, but we
could not eliminate egg production altogether or equally among all
treatment groups. We therefore cannot completely rule out that
reproduction influenced lifespan. Nonetheless, reproduction was
significantly reduced in all of the treatments, which strongly suggests
that dietary restriction and not egg production is the major factor
influencing lifespan extension in A. aegypti.
The present study sheds some light on the relationship between
larval diet and adult lifespan. Previous studies in D. melanogaster did
not find a relationship between larval diet and adult lifespan (Tu and
Tatar, 2003). However, we find that in all cases where larval diet was
restricted, individuals with a restricted diet during the larval stages
showed a modest, but significant, increase in adult lifespan compared
to those given twice the normal amount of food. Interestingly,
mosquitoes fed 25% of their normal larval diet and given only sugar
during adulthood had a reduced lifespan, likely due to depletion of
protein reserves. However, if those same mosquitoes received even a
single blood meal during adulthood they survived as well as or better
than any of the other treatment groups. Therefore, although blood
meals can decrease lifespan, they might also be beneficial when
individuals experience restricted diets as larvae, as they often do in
the wild. To our knowledge, this is the first study to show that dietary
restriction at the larval stages can extend lifespan and is especially
relevant given the ecology of these insects.
Mosquitoes are important vectors of disease parasites, including
malaria, filariasis, dengue, and West Nile virus. Lifespan is the most
important factor affecting how efficiently a mosquito can transmit
these parasites and even modest changes can greatly impact disease
spread (Macdonald, 1957). Our work suggests that dietary restriction
during both the aquatic larval stages and adulthood might increase
the opportunity for disease transmission. This important link between
diet, lifespan, and disease transmission might be used to identify areas
where A. aegypti transmitted diseases might increase. For example, if
inter- and intraspecific competition for food at the larval stages is high
690 T.K. Joy et al. / Experimental Gerontology 45 (2010) 685–690
where pools of water are especially small, dry climates might be more
disease-prone than originally thought. Similarly, if mosquitoes with
restricted access to protein-rich blood meals live longer, A. aegypti
transmitted disease might increase in rural areas where hosts are
limited.
As in other organisms, the lifespan of adult A. aegypti mosquitoes
can be extended through dietary restriction. In contrast to other
invertebrate models of aging, such as D. melanogaster and C. elegans, A.
aegypti's feeding and reproductive behavior are characterized by
bursts of nutrient intake directly linked to the production of a clutch of
eggs. By limiting the amount of protein in the adult females' diet, we
were able to reduce the impact of reproduction in the dietary
restriction assays. Overall, our data suggest that we might find new
opportunities for controlling mosquito-borne diseases by under-
standing the interplay between larval nutrition, adult blood feeding,
reproduction and lifespan. This work opens intriguing avenues into
controlling mosquito and other vector borne disease transmission
through dietary restriction.
Acknowledgments
We would like to thank Frank Ramberg for helping with mosquito
rearing. This work was supported by the Ellison Medical Foundation
and the BIO5 Institute.
Appendix A. Supplemental data
Supplementary data associated with this article can be found, in
the online version, at doi: 10.1016/j.exger.2010.04.009.
References
Allison, P.D., 1995. Survival Analysis Using the SAS System: a Practical Guide. SAS
Institute Inc, Cary, NC.
Andersson, I.H. 1992. The effect of sugar meals and body size on fecundity and longevity
of female Aedes communis (Diptera: Culicidae). Physiol Entomol 17, 203-203-207.
Bliss, S., 1928. The amide nitrogen of blood. Science 67, 515–516.
Boggs, C.L. Ross, C.L. 1993. The effect of adult food limitation on life history traits in
Speyeria mormonia. Ecology 74, 433-433-441.
Braks, M.A., Juliano, S.A., Lounibos, L.P., 2006. Superior reproductive success on human
blood without sugar is not limited to highly anthropophilic mosquito species. Med.
Vet. Entomol. 20, 53–59.
Carey, J.R., Liedo, P., Harshman, L., Zhang, Y., Muller, H.G., Partridge, L., Wang, J.L., 2002.
Life history response of Mediterranean fruit flies to dietary restriction. Aging Cell 1,
140–148.
Cooper, T.M., Mockett, R.J., Sohal, B.H., Sohal, R.S., Orr, W.C., 2004. Effect of caloric
restriction on life span of the housefly, Musca domestica. FASEB J. 18, 1591–1593.
Costero, A., Edman, J.D., Clark, G.G., Scott, T.W., 1998. Life table study of Aedes aegypti
(Diptera: Culicidae) in Puerto Rico fed only human blood versus blood plus sugar.
J. Med. Entomol. 35, 809–813.
Edman, J.D., 2003. Fitness advantages in multiple blood-feeding: the Aedes aegypti
example. In: Takken, W., Scott, T.W. (Eds.), Ecological Aspects for Application of
Genetically Modified Mosquitoes. Kluwer Academic Publishers, Dordrecht; Boston,
p. 63.
Grandison, R.C., Wong, R., Bass, T.M., Partridge, L., Piper, M.D., 2009. Effect of a
standardised dietary restriction protocol on multiple laboratory strains of
Drosophila melanogaster. PLoS One 4, e4067.
Harrington, L.C., Buonaccorsi, J.P., Edman, J.D., Costero, A., Kittayapong, P., Clark, G.G.,
Scott, T.W., 2001. Analysis of survival of young and old Aedes aegypti (Diptera:
Culicidac) from Puerto Rico and Thailand. J. Med. Entomol. 38, 537–547.
Hellwing, A.L.F., Tauson, A.-H., Skrede, A., 2007. Blood parameters in growing pigs fed
increasing levels of bacterial protein meal. Acta Vet. Scand. 49, 33.
Klowden, M.J., Lea, A.O., 1978. Blood meal size as a factor affecting continued host-
seeking by Aedes aegypti (L.). Am. J. Trop. Med. Hyg. 27, 827–831.
Le Bourg, E., Minois, N., 1996. Failure to confirm increased longevity in Drosophila
melanogaster submitted to a food restriction procedure. J. Gerontol. A Biol. Sci. Med.
Sci. 51, B280–B283.
Macdonald, G., 1957. The Epidemiology and Control of Malaria. Oxford University Press,
London.
Mains, J.W., Mercer, D.R., Dobson, S.L., 2008. Digital image analysis to estimate numbers
of Aedes eggs oviposited in containers. J. Am. Mosq. Control. Assoc. 24, 496–501.
Mair, W., Dillin, A., 2008. Aging and survival: the genetics of life span extension by
dietary restriction. Annu. Rev. Biochem. 77, 727–754.
Mair, W., Sgro, C.M., Johnson, A.P., Chapman, T., Partridge, L., 2004. Lifespan extension
by dietary restriction in female Drosophila melanogaster is not caused by a
reduction in vitellogenesis or ovarian activity. Exp. Gerontol. 39, 1011–1019.
Mair, W., Piper, M.D.W., Partridge, L., 2005. Calories do not explain extension of life span
by dietary restriction in Drosophila. PLoS Biol. 3, e223.
Martinez-Ibarra, J.A., Rodriguez, M.H., Arredondo-Jimenez, J.I., Yuval, B., 1997. Influence
of plant abundance on nectar feeding by Aedes aegypti (Diptera: Culicidae) in
southern Mexico. J. Med. Entomol. 34, 589–593.
Partridge, L., Green, A. Fowler, K. 1987. Effects of egg-production and exposure to males
on female survival in Drosophila melanogaster. J Insect Physiol 33, 745-745-749.
Partridge, L., Piper, M.D., Mair, W., 2005. Dietary restriction in Drosophila. Mech. Ageing
Dev. 126, 938–950.
Piper, M.D., Partridge, L., 2007. Dietary restriction in Drosophila: delayed aging or
experimental artifact? PLoS Genet. 3, e57.
Reiskind, M.H., Lounibos, L.P., 2009. Effects of intraspecific larval competition on adult
longevity in the mosquitoes Aedes aegypti and Aedes albopictus. Med. Vet. Entomol.
23, 62–68.
Roark, A.M., Bjorndal, K.A., 2009. Metabolic rate depression is induced by caloric
restriction and correlates with rate of development and lifespan in a parthenoge-
netic insect. Exp. Gerontol. 44, 413–419.
Scott, T.W., Chow, E., Strickman, D., Kittayapong, P., Wirtz, R.A., Lorenz, L.H., Edman, J.D.,
1993. Blood-feeding patterns of Aedes aegypti (Diptera: Culicidae) collected in a
rural Thai village. J. Med. Entomol. 30, 922–927.
Styer, L.M., Carey, J.R., Wang, J.L., Scott, T.W., 2007a. Mosquitoes do senesce: departure
from the paradigm of constant mortality. Am. J. Trop. Med. Hyg. 76, 111–117.
Styer, L.M., Minnick, S.L., Sun, A.K., Scott, T.W., 2007b. Mortality and reproductive
dynamics of Aedes aegypti (Diptera: Culicidae) fed human blood. Vector Borne
Zoonotic Dis. 7, 86–98.
Therneau, T.M., Grambsch, P., Grambsch, P.M., 2000. Modeling Survival Data: Extending
the Cox Model. Springer-Verlag, New York, NY.
Tu, M.P., Tatar, M., 2003. Juvenile diet restriction and the aging and reproduction of
adult Drosophila melanogaster. Aging Cell 2, 327–333.