Vol. 30, no. 1
Journal of Vector Ecology
11
Reproductive physiology of Anopheles gambiae and Anopheles atroparvus
Luís Fernandes and Hans Briegel
Institute of Zoology, University of Zürich, CH-8057 Zürich, Switzerland
Received 2 March 2004; Accepted 15 November 2004
ABSTRACT: When exposed to a human host, Anopheles gambiae started probing 4 h post-eclosion, but 95% successfully
blood-fed by 16-20 h with maximal blood volumes of 5-10 µl per female. When fed sugar, the 95% feeding was not observed
until 36-40 h post-eclosion; sugar meals appeared to interfere with blood meals. Similarly in An. atroparvus, maximum
volumes were 10 µl when starved but only 6 µl when fed sugar. This species did not bite before 2 d, and 95% biting was by
4 d. Given single blood meals to water-kept An. gambiae, a threshold body size for oogenesis was detected. With wing
lengths below 2.8 mm, eggs never matured, but when sugar-fed, females of all sizes matured eggs including the synthesis of
maternal deposits. Although sugar feeding interfered with blood feeding, more lipid was transferred to the yolk. In water-kept
An. atroparvus only 5% of the females produced eggs. When sugar-fed for 4 d, all females matured eggs, so in this species
sugar feeding appeared to be essential for oogenesis. An. gambiae always took multiple blood meals, tested at any time after
the first ones, leading to 120 mature eggs/female. Yolk composition was 3.9 mcal protein and 3.8 mcal lipid/oocyte when kept
on water, but 2.8 mcal protein and 4.3 mcal lipid/oocyte with intermittent sugar meals, thus marking a surprising flexibility in
synthesis of yolk protein and lipid that strongly depends on additional carbohydrates sources. Only 80% of water-fed An.
atroparvus re-fed 2 d after a first blood meal with small females taking three blood meals but they still showed reduced
fecundity. Only the large water-fed females matured eggs, with blood volumes higher than 9-12 µl. When fed sugar, the blood
meal input was reduced, but oogenesis was possible, whereas water-fed females required three blood meals to reach the
caloric level comparable to pre-feeding sugar-fed females. Water-fed An. gambiae could survive on daily blood meals alone,
but survival was further extended by intermittent sugar meals. When offered a blood donor daily, there was a behavioral
difference. Females maintained alone showed a more or less regular 3 d feeding and oviposition activity, while females kept
in groups fed daily followed a daily oviposition pattern, suggesting gonotrophic discordance. Journal of Vector Ecology 30:
11-26. 2005.
Keyword Index: An. gambiae, An. atroparvus, ovarian development, multiple blood feeding, survival.
INTRODUCTION
Many species of the genus Anopheles are notorious as
vectors of Plasmodium species that can cause malaria in
vertebrates. Anopheles gambiae Giles sensu stricto, the
African malaria mosquito, causes an extremely heavy burden
for humans on that continent. Yet its reproductive physiology
has not been adequately investigated. The same is true for
An. atroparvus Van Thiel, formerly a malaria vector of the
Northern hemisphere. Therefore, we have studied comparative
aspects of their physiology during the reproductive cycle. In
most mosquitoes, survival depends on plant sugars, which is
the only food source for males. Sugar feeding by female An.
gambiae in the field is uncertain. Although earlier reports
described them feeding on sugar in the field (Laarman 1968,
McCrae et al. 1969), more recent studies point to the opposite
(Beier 1996). In the laboratory, (Briegel 1990b) found An.
gambiae females to complement their nutritional deficiencies
at eclosion by sugar feeding. However, sugar did not increase
fecundity in An. gambiae, which obtained the same energetic
Università degli Studi di Perugia, Dipartimento di Medicina
Sperimentale e Scienze Biochimiche, Sezione Microbiologia,
Via del Giochetto, 06122 Perugia, Italy.
input from more frequent blood feeding (Straif and Beier
1996, Gary and Foster 2001). Feeding exclusively on blood
tends to increase the biting frequency, as also observed in An.
quadrimaculatus and Ae. aegypti (Foster and Eischen 1987,
Canyon et al. 1999).
In contrast to Aedes, blood meal utilization for oogenesis
in Anopheles is characterized by low efficiency (Briegel
1990b). Anopheles females with their limited midgut volume
have evolved the mechanism of blood meal concentration
(Briegel and Rezzonico 1985) in combination with the
ingestion of multiple blood meals, observed under laboratory
conditions in An. albimanus, An. gambiae, and An. stephensi
(Briegel and Hörler 1993). This is accompanied by hostseeking behavior during the gonotrophic cycle (Klowden and
Briegel 1994). Multiple blood meals have also been reported
from the field for An. gambiae s.l. and An. funestus (Gillies
1954, Beier 1996), even though there is an inhibition of hostseeking in large An. gambiae individuals for at least 40 h
after blood feeding (Takken et al. 2001).
The issue of multiple blood meals was addressed by
(Gillies 1954, 1955), whose studies implicate that a first blood
meal might promote the ovarian follicles from Christophers
stage I to the resting stage II (Detinova 1962) before oogenesis
could proceed with a second blood meal. He coined the term
“pre-gravid state” for such field-caught An. gambiae and An.
12
Journal of Vector Ecology
funestus. The need for more than one blood meal to complete
oogenesis was explained by (Briegel 1990a, b) by the fact
that female Anopheles in general have much lower teneral
reserves than culicine mosquitoes.
The significance of body size in the reproductive
physiology of mosquitoes has been recognized and
emphasized by Briegel et al. (2001a), Briegel and
Timmermann (2001), and Briegel et al. (2002). Body size is
determined by environmental conditions during the larval
period (Briegel 1990a, b, Lyimo and Koella 1992,
Timmermann and Briegel 1993, 1999, Briegel et al. 2001a,
Briegel and Timmermann 2001, Briegel et al. 2001b). Small
mosquitoes have several disadvantages compared to large
ones, including decreased survival, smaller teneral reserves,
and reduced fecundities (Takken et al. 1998). A higher
probability of survival was related to larger body size in An.
arabiensis (Ameneshewa and Service 1996) and An. dirus
(Kitthawee et al. 1992), and blood meal size and fecundity
also improved with size (Hogg et al. 1996). Ramasamy et al.
(2000) found a positive effect of body size on teneral reserves
and the number of ovarian follicles in An. tessellatus. Body
size also affected infection rates with Plasmodium falciparum
(Lyimo and Koella 1992). Lyimo and Takken (1993) found
that wild An. gambiae females with wing lengths above 3
mm were able to mature eggs from the first blood meal, while
smaller females required two or three blood meals to mature
the first batch of eggs. These results were later confirmed
under laboratory conditions (Takken et al. 1998).
Our study of nutritional status before a blood meal, the
mobilization of teneral reserves, and the reserve synthesis from
sugar meals may explain the extent of blood consumption
and the efficiency of its utilization for either reserve synthesis
or oogenesis. The “pre-gravid state” described by Gillies
(1954, 1955) will be related to the body size of a female and
its dietary history, both determining the course of oogenesis
and survival. The issue of multiple blood feedings is addressed
in relation to diverse metabolic parameters and compared
between the two vector species with their different
physiotypes.
MATERIALS AND METHODS
Mosquito colonies
Our Anopheles (Cellia) gambiae s.s. colony originated
from Lagos, Nigeria, and has been routinely colonized for 20
years at 27°±1°C and 85±5% R.H. under long day conditions
(14L:10D), with 40 min dimming periods simulating sunrise
and sunset. Routinely, 400 newly-hatched larvae were counted
into 16 x 24 x 5 cm pans and fed standardized volumes of
pulverized TetraMin ® daily. In order to obtain small
mosquitoes, at least 800 larvae were counted per pan, since
intraspecific competition at the larval stage causes a decrease
in adult body size (Timmermann and Briegel 1993). Anopheles
(Anopheles) atroparvus has been colonized continuously for
9 years in our laboratory at room temperature (22±1°C) under
long day conditions (14L:10D). Routinely, 300 newly-hatched
larvae were counted into polystyrol pans and fed a different
amount of TetraMinBaby® daily with the same set of feeding
June 2005
spoons. At least 800 larvae were counted to obtain small
adults. For colony maintenance all blood meals were given
from restrained guinea pigs.
Experimental procedures
Mosquitoes used in experiments originated from both
rearing conditions to create the full range of body sizes, and
females were not mated. Body size of all mosquitoes was
determined by wing length measurements from the alula to
the tip, excluding the fringes. Measurements were converted
to cubic values (WL3), which are more suitable for scaling
physiological data than linear dimensions (Schmidt-Nielsen
1984, Briegel 1990a). Teneral females were sampled not
longer than 1 h after eclosion and fixed immediately for caloric
measurements, or they were kept with access to water or
sucrose concentrations ranging from 0.1% to 20% in round
cages (11.4 cm diameter x 13.0 cm height) for survival and
blood feeding experiments. The different sucrose
concentrations were used to determine the ideal concentration
for mosquito survival. An. gambiae was always studied at
27°C and An. atroparvus at 22°C, but survival experiments
of both species were performed at both temperatures, 27°C
and 22°C. The extent of reserve mobilization under nutritive
stress was measured in females starved to death with access
to water only. These experiments revealed the minimal
irreducible amounts (MIA), necessary for survival (Van
Händel 1984, Briegel 1990a, b). Females kept with access to
10% sucrose were sampled at regular intervals to estimate
lipid and glycogen synthesis. Development of avidity to blood
ingestion was studied in females maintained on water or 10%
sucrose from eclosion and exposed to a host in 0.2 dm3 cages
at 4 h intervals in An. gambiae and daily intervals in An.
atroparvus. At 22°C mosquitoes develop more slowly than at
27°C, so timing of experiments was adapted to the different
development times of the two species. An. atroparvus females
seldom ingested blood from a human arm, so a guinea pig
was offered instead. Either the human arm or a guinea pig
were exposed for 15 min, or until all females ceased probing.
Shortly afterwards, each female was checked for the presence
of blood with transmitted light and kept individually in vials
(13 x 100 mm) with access to water. Feces were collected for
later determination of hematin. After blood meals were
digested (44 - 50 h for An. gambiae and 80 h for An.
atroparvus), the females were dissected for measurements of
follicle and yolk lengths and for oocyte counts. Wings were
kept for the measurement of body size. Mosquitoes with
undigested blood in the midgut were discarded.
The development of ovaries before a blood meal was
also studied at various times after eclosion in females kept
with access to water or 10% sugar. The ovaries were examined
at 300x to determine the Christophers’ stages as described by
(Detinova 1962), and the follicle lengths were measured at
100x. Their caloric content of protein, lipid, and glycogen
was also measured. To reach measurable amounts, the resting
stage ovaries from 30 to 70 An. gambiae females, and 25 An.
atroparvus females had to be pooled for each sample,
depending on the developmental stages.
To study oogenesis, groups of water-fed An. gambiae
Vol. 30, no. 1
Journal of Vector Ecology
were blood fed on a human arm 24 h after eclosion, or after 5
d when fed on sugar, and their ovaries were dissected in 3-12
h intervals after blood ingestion. Due to their slower
development and decreased blood hunger An. atroparvus were
blood fed on a guinea pig 8 d after eclosion, which included
5 d of sugar feeding plus 3 d of starvation. Some had their
ovarioles teased apart and their follicle and yolk lengths
measured, while others had their entire ovaries used for
determinations of protein, lipid, and glycogen contents. In
addition, females were analyzed for their total caloric contents
before and after oviposition to assess the processing of a blood
meal for oogenesis, for energetic demands, or for maternal
reserve synthesis.
The significance of multiple blood meals during one
gonotrophic cycle was studied thoroughly in both species of
all size classes. An. gambiae females were maintained with
access to only water and blood fed 24 h after eclosion. Some
females were then offered a second blood meal 24 or 48 h
later (48 or 72 h after eclosion). An. atroparvus were also
kept with access to water, and offered a guinea pig daily. After
digestion was completed, the females and their ovaries were
analyzed and their oocytes counted.
Survival with daily blood meals from humans was studied
in An. gambiae with access to water or 10% sucrose from
eclosion in square 8 dm3 cages. Between 50 and 100 sugarfed males were added daily to the cages containing 15-30
females (depending on experiment) for the first week to ensure
insemination. The cages had an oviposition cup and were
screened daily for dead mosquitoes, which were removed and
had their wing length measured. The number of mosquitoes
taking a blood meal and their gonotrophic status was recorded
and ovipositions were counted daily. A group of 50 females
eclosed the same day, was kept with 10% sucrose, and used
as a control.
Analytical procedures
For biochemical analyses, mosquitoes were fixed
individually in reacting tubes (10 x 75 mm) with 100 µl of
ethanol and heated to 90°C for 3 min. The Kjeldahl digestion
and subsequent Nesslerization of ammonium sulfate was used
to measure the protein content, as described by Briegel (1986).
Lipid and carbohydrates were measured in the same specimen,
fixed in 13 x 100 mm tubes, following procedures by Van
Handel and Day (1988). Lipid contents were measured on
the basis of the vanillin reaction and carbohydrate by the
anthrone method. All data were converted to calories per insect
to allow direct energetic comparisons, based on the proportion
of 110 mg lipid and 250 mg carbohydrate or protein for 1 cal
(=4.18J); the corresponding conversion factors are 0.009 cal
per mg lipid and 0.004 cal per mg carbohydrates or protein.
To normalize caloric measurements for body size, the calories
were divided by WL3, leading to a size-specific caloric content
(SSCC-values) (Timmermann and Briegel 1993).
Fecal material of individual females was eluted in 1%
lithium carbonate for measurement of hematin (Briegel 1980).
The amount of blood ingested was quantified retrospectively
from the hematin readings for each female based on the
stoichiometric relationship between fecal hematin and
13
hemoglobin ingested. The molar proportions between
vertebrate hemoglobin and fecal hematin allowed us to
calculate the blood consumption of a female, first in terms of
hemoglobin, then in total protein, and finally as µl of host
blood ingested (Briegel 1986). This method accurately reflects
the amount of protein ingested, but the total volume of blood
should be taken as a reference and not as an actual value,
because Anopheles mosquitoes concentrate their blood meal
during feeding (Briegel and Rezzonico 1985), and fecal
hematin reflects the hemoglobin digested by the midgut. Blood
expelled during blood feeding (prediuresis) was discarded.
The hemoglobin titer of the blood donor was monitored by
Drabkin’s solution (Briegel et al. 1979).
RESULTS
Teneral conditions
Body size and total protein, lipid, and glycogen contents
were measured within 1 h of eclosion for all possible body
sizes. In An. gambiae, wing lengths were in the range of 2.33.2 mm; females were slightly larger than males. Their caloric
values were about two-thirds of protein and close to another
third of lipids, while carbohydrates always remained below
one-tenth of the total. Plotting the individual caloric values
of females against their body size (WL3), the following linear
regressions were obtained: protein y=0.037x-0.133 (N=118,
r2=0.925, t=37.91, p<0.0001); lipid y=0.030x-0.357 (N=174,
r2=0.803, t=26.46, p<0.0001); glycogen y=0.004x-0.019
(N=154, r2=0.501, t=12.34, p<0.0001). When normalized for
body size (WL3) and plotted as SSCC-values against body
size, equally linear regressions were revealed for all three
components: protein y=0.0003x+0.024 (N=118, r2=0.236 ,
t=5.99, p<0.0001); lipid y=0.0008x-0.0035 (N=174, r2=0.595,
t=15.90, p<0.0001); glycogen y=0.00005x+0.00214 (N=154,
r2=0.067,t=3.31 p<0.01). Protein and glycogen showed
isometric relationships with body size, while total lipids
revealed a weak positive allometry.
In An. atroparvus, body sizes of females were larger than
males, within the ranges of 3.4 - 4.8 mm and 3.0 – 4.2 mm
wing length respectively. The caloric contents of females
revealed significant linear regressions when plotted against
body size (WL3): protein y=0.024x-0.247 (N=135, r2=0.865,
t=29.23, p<0.0001); lipid y=0.024x-1.107 (N=149, r2=0.80,
t=24.26, p<0.0001); glycogen y=0.004x-0.155 (N=149,
r2=0.572, t=14.02, p<0.0001). Regression formulas for males
were somewhat different (data not shown). When the caloric
values of females were transformed to SSCC-values, the
following regressions were obtained: protein
y=0.00005x+0.017 (N=135, r2=0.139, t=4.641, p<0.0001);
lipid y=0.0002x-0.0063 (N=149, r 2 =0.643, t=16.26,
p<0.0001); glycogen y=0.00003x+0.00001 (N=149,
r2=0.201, t=6.08, p<0.0001). There were no major differences
between protein and glycogen of small and large females, but
lipids were in positive allometry.
Survival and reserve mobilization
Under starvation conditions with access to only water
from eclosion, all An. gambiae died within 4 d and An.
14
Journal of Vector Ecology
atroparvus died within 8 d. Small females died earlier as
previously reported by Takken et al. (1998). When provided
with sugar solutions, however, survival was substantially
extended. Cohorts of 50 large (WL ≥3.0 mm) and 50 small
An. gambiae females (WL ≤2.6 mm) with access to 10%
sucrose showed maximal survival times of 20 and 15 d (50%
survival times 9 and 11 d), respectively. In An. atroparvus,
access to sugar solutions extended survival to 33 and 40 d
(50% survival times 16 and 21 d) for 50 large and 50 small
females, respectively. Survival curves largely overlapped, so
body size had no major consequences on survival times when
sucrose was available as an energy source.
Various sugar concentrations from 0.1% up to 20% in
identical cages were tested for female survival of An. gambiae.
Sucrose concentrations below 0.5% revealed no differences
in survival to water, whereas higher concentrations led to a
gradual increase of the 50% survival times. Maximum survival
times for An. gambiae were obtained with concentrations of
5-10%; 20% solutions increased mortalities.
In An. atroparvus, the effect of different sugar
concentrations (0.1%-20%) was similar. Concentrations
higher than 0.4% gradually increased the 50% survival times,
and maximum survival was also reached with 10% sucrose.
The effect of temperature was compared between An.
atroparvus and An. gambiae with 10% sucrose (Figure 1). In
both species the lower temperature extended their survival.
The caloric protein, lipid, and glycogen contents of
females that died during these survival experiments were
determined. The protein contents of dead females were similar
to teneral values; in An. gambiae mean contents were
Figure 1. Comparison of female survival between An. gambiae
(triangles), and An.atroparvus (squares), and between two
temperatures (open and filled), with access to 10% sucrose. (27°C,
N=609; 22°C, N=98); An. atroparvus (27°C, N=149; 22°C,
N=236).
June 2005
Table 1. Oogenesis in An. atroparvus with access to water or sugar
from eclosion, given a blood meal at successive days (M±SE).
Time of blood
meal (d)
Water
Sugar
N Gravid (%)
eggs
N
Gravid (%)
eggs
2
71
1
(1)
152
14
1
(7)
194
3
103
4
(4)
134±22
21
17
(81)
194±30
4
66
2
(3)
139±6
12
12
(100)
184±26
5
55
0
43
43
(100)
158±26
8
-
-
44
44
(100)
166±31
15
-
-
42
42
(100)
154±43
0.65±0.08 cal protein (N=32, range 0.47-0.83), 0.21±0.15
cal lipids (N= 43, range 0.01-0.86), and 0.05±0.03 cal
glycogen (N=43, range 0.01-0.16). In An. atroparvus, the
corresponding values were 1.90±0.13 cal protein (N=13, range
of 1.73-2.14), also similar to their teneral values (t=1.16,
p>0.2). The caloric lipid content was 1.19±0.97 (N=56, range
0.13-4.59 cal), and glycogen was 0.29±0.31 (N=56, range
0.02-1.37 cal).
Complete starvation, i.e. access to water only, uncovered
the “minimal irreducible amounts” (MIA) required for
survival. The differences to the teneral values indicate maximal
mobilization of reserves. Depending on body size, in An.
gambiae from 5-36% of the teneral protein was catabolized
by smallest and largest females, respectively. At the same time
25-80% of the lipid and roughly 60-80% of the glycogen was
mobilized. Thus, lipid contributed the largest absolute segment
of teneral reserve mobilization, with a 30-fold difference
between the extreme sizes; obviously, the extent of
mobilization of teneral lipid and protein reserves for survival
was strongly affected by body size. The time course of lipid
and glycogen mobilization was followed in 4 h intervals,
requiring 30-40 h to reach the MIA levels. This period
coincided with the aforementioned survival time.
In An. atroparvus large females utilized 5-42% of the
teneral protein content, 37-85% of the teneral lipid, and 4050% of glycogen, always depending on size. Large females
had a clear advantage; they could utilize more teneral reserves,
particularly in absolute amounts, which explains their longer
survival under starvation over An. gambiae. The lipid and
glycogen contents of females which died either early or late
during the survival experiments often varied considerably.
Therefore, death may not always be caused by exhausted
reserves because dead insects sometimes contained large
amounts of sugar.
Reserve synthesis from sugar meals
Beginning at eclosion, female An. gambiae with access
to 10% sucrose were sampled for the first 2 d at 4 h intervals
and from day 3 onwards for another 3 wk at 2 d intervals.
They were screened for body size and analyzed for
accumulation of lipid and glycogen (Figure 2). Glycogen was
steadily synthesized from eclosion for 36 h to its maximal
levels of roughly 0.5 cal in large and 0.25 cal in small females,
Vol. 30, no. 1
Journal of Vector Ecology
Figure 2. Lipid and glycogen content of large female An. gambiae
kept for three weeks with 10% sucrose (M±SE, N=12-32 per point).
The teneral values corresponding to their mean body size (WL3
24.28±3.25, N=465) are indicated by a filled symbol.
representing a 6-7-fold increase over teneral levels for both
size classes. During the next 3 wk the glycogen contents
gradually declined.
Lipogenesis followed a different pattern (Figure 2). In
large females it took about 2 d until teneral levels had roughly
doubled the lipid contents. Thereafter, this remained at this
level for another 2 wk, although with great variability. In small
females the lipid content oscillated irregularly around teneral
levels during the first day and then slowly increased, but rarely
doubled; after 5 d it decreased.
The same experiments with An. atroparvus are
summarized in Figure 3 for large females. Glycogen synthesis
varied between 0.5 and 1.5 cal with an average maximum of
1.2 cal, i.e. a 4-5-fold increase over teneral values. Synthesis
of lipids was clearly delayed; it took about 2 wk to reach the
maximum levels. By day 3 a substantial lipogenesis had
started. Large females attained maxima more than 3-fold the
teneral values and in small females the lipid maximum was
reached 2 d earlier with a 4-fold increase. Compared to An.
gambiae, An. atroparvus is characterized by remarkably high
caloric lipid accumulations, even when normalized for body
size (data not shown).
Development of blood “hunger”
The first successful ingestion of blood was determined
15
Figure 3. Lipid and glycogen content of large female An. atroparvus
kept for four weeks at 22°C with 10% sucrose (M±SE, N=7-23
per point). Teneral values are indicated by filled symbols for an
average size of WL3 94.32±8.33 (N=295).
by offering a human arm every 4 h to inexperienced females
that had permanent access to water or 10% sucrose after
eclosion. With An. gambiae, a few females attempted to bite
at 4 h after eclosion, but none successfully fed until 8 h posteclosion. For both treatments, the 95% threshold was observed
at 16-20 h, and by 24 h 100% of the water-kept females had
fed. In females with previous access to sugar the 95% feeding
threshold was delayed to 36-40 h. In An. atroparvus, females
maintained on water did not feed before day 2 and only by
day 4 was the 95% feeding threshold reached. When kept on
sugar, An. atroparvus females always showed a decreased
tendency (below 30% by day 5) to blood-feed.
The amount of blood ingested was determined for An.
gambiae on the basis of hematin measurements (Figure 4).
Females maintained on water gradually increased their blood
meals until maximum volumes were reached at 24-28 h. The
females kept with sugar initially behaved similarly, but their
blood meals were slightly smaller and at 24 h they showed a
decline in blood meal consumption. This is explained by
voluminous crops due to previous sugar consumption. Indeed,
in such females only half of the abdomen was filled with bright
red blood while the other half appeared translucent,
characteristic of filled crops. From 36-40 h onwards the blood
consumption increased and became similar in both treatments.
The maximal volumes of 5-10 µl per female were possible
16
Journal of Vector Ecology
Table 2. Total caloric increase above teneral values of An. gambiae
after processing one blood meal given after 1 d of access to water.
Table 3. Total caloric increase above teneral values of An. gambiae
after one blood meal given after 5 days of continuous access to 10%
sucrose.
Caloric increase over teneral values
WL
Gravid
*
June 2005
Protein
(%)
Lipid (%)
2.8
0.38
(62)
0.38
(117)
2.9
0.24
(28)
0.45
(122)
3.0
0.29
(32)
0.49
3.1
0.32
(33)
0.50
WL
Caloric increase over teneral values
Protein (%)
Lipid
(%)
2.4
0.10
(25)
0.57
(699)
2.5
0.12
(27)
0.69
(548)
(111)
2.6
0.10
(19)
0.72
(482)
(88)
2.7
0.12
(23)
0.86
(413)
2.8
0.00
(0)
0.85
(260)
Gravid
*
Non2.4
oogenic
0.10
(25)
0.21
(254)
2.9
0.00
(0)
0.92
(248)
2.5
0.11
(26)
0.16
(125)
3.0
0.00
(0)
1.26
(283)
2.6
0.08
(15)
0.29
(191)
3.1
0.01
(1)
1.09
(191)
2.7
0.24
(44)
0.29
(141)
2.8
0.18
(29)
0.16
(50)
Non2.4
oogenic
0.00
(1)
0.51
(623)
2.9
0.05
(5)
0.25
(67)
2.5
0.10
(23)
0.49
(392)
2.6
0.05
(10)
0.49
(326)
3.0
0.09
(10)
0.14
(32)
2.7
0.19
(34)
0.57
(275)
3.1
0.14
(15)
0.30
(53)
*Ovarian values are included.
*Ovarian values are included.
Table 4. Multiple blood meals of An. gambiae. Increase of blood meal size, percentage of gravid females, and fecundity of females kept
with constant access to water. They were fed blood once at 24 h after eclosion, twice at 24 and 48 h, or at 24 and 72 h. Twenty females
even took three blood meals at 24 h intervals. The average body size was 2.75 mm wing length (range 2.3-3.1 mm; M±SE, N in
parentheses).
Time of blood
meal (h)
Size of blood meal
Gravid females
(%)
Eggs
µg hemoglobin
µl blood
24
326±178 (35)
2.2±1.2
40
56±18 (14)
24/48
652±184 (118)
4.4±1.2
97
93±24 (36)
24/72*
446±103 (79)
3.0±0.7
100
120±45 (31)
24/48/72
740±143 (20)
4.8±0.9
100
99±21 (9)
*By 72 h, females had already digested blood taken at 24 h, so the values measured at
72 h were reduced. Therefore, total blood consumption was estimated by adding the
values measured at 24 h and at 72 h, i.e. 772 µg or 5.2 µl.
Vol. 30, no. 1
Journal of Vector Ecology
Figure 4. Development of blood consumption in An. gambiae kept
with water (open circles) or sugar (filled squares) after eclosion.
Amount of blood ingested based on fecal hematin determinations.
The hemoglobin values were converted to approximate volumes
of blood ingested and concentrated during feeding (Mean±SE,
N=13-55 per data point).
because of the concentration mechanism during feeding
(Briegel and Rezzonico 1985).
In An. atroparvus, the maximal blood uptake was about
10 µl, reached within 4 d when kept on water. Providing
females first with sugar had an equally negative effect on blood
consumption; fewer females ingested blood and they always
took smaller meals of roughly 6 µl. This reduced blood feeding
persisted until day 15 but could be improved when sugar
sources were removed before. Up to 80% of the females kept
with sugar for 5 d, and then starved for 3 d took blood,
although the blood volume was not increased (data not shown).
Ovarian development prior to a blood meal
Christophers’ stages and follicle lengths in ovaries of An.
gambiae were compared between water-fed and sugar-fed
females. In An. gambiae, Christophers’ stage I corresponds
to 40 µm of follicle lengths, stage I-II to 40-50 µm, and stage
II to 50-65 µm. There was no significant difference between
Table 5. Multiple blood-feeding in An. atroparvus kept with water.
Percentage of refeeding females, the amount of blood ingested (µl),
the percentage of oogenic females, and fecundity (M±SE, N in
parentheses).
Time of blood
meal (d)
Tested
Blood-fed*
(%)
Blood consumption
(µl)**
Gravid
(%)
Eggs
3+4
131
70±13 (91)
12±4
28 (56)
179±22
3+5
77
80±13 (57)
15±4
37 (74)
183±29
3+4+5
54
58±12 (32)
13±5
13 (45)
163±56
*Percentage based on the number of females that had ingested a
previous blood meal (=100%).
**These values correspond to mean blood volumes of 12-15µl with
a range of 3-26µl per female.
17
water-fed and sugar-fed females, as both had reached stage II
within 2 d after eclosion. With sugar however, follicles were
slightly larger. The maximum follicle length of 60 µm, attained
36 h after eclosion, corresponded to the stable, pre-gravid
state described by Gillies (1954), or the “resting stage,” or
the last stage of the previtellogenic phase, according to
Clements (1992). In both water-fed and sugar-fed An.
atroparvus, the resting stage was reached only by day 5 with
mean follicle lengths of 90 µm. In Christophers’ stage I the
follicles measured 40-50 µm, in stage I-II 50-65 µm, and in
stage II 65-105 µm. Large and small females of both species
were able to reach stage II when given sugar, with no major
differences in oocyte length or caloric content. When starved,
small females were not able to reach stage II because they
died before 36 h in An. gambiae or 5 d in An. atroparvus.
In starved females of An. gambiae, the ovarian caloric
content at 12 h was 8.0±3.9 mcal/female protein (N=3),
1.4±0.2 mcal lipid (N=4), and 0.4±0.2 mcal glycogen (N=5).
While lipid and glycogen levels remained fairly stable between
12 h and 36 h, protein levels fluctuated. In sugar-kept females
the ovarian contents at 108 h after eclosion was 10.0±2.5 mcal/
female protein (N=3), 2.6±0.3 mcal lipid (N=3), and 1.1±0.1
mcal glycogen (N=3). These values were maintained for 4-5
d at the teneral level. Ovarian lipid always remained below 3
mcal/female and glycogen below 1 mcal/female.
In An. atroparvus, the teneral ovarian content was
10.3±0.9 mcal/female protein (N=3), 2.4±0.3 mcal lipid
(N=4), and 0.5±0.1 mcal glycogen (N=4). When kept with
water, the ovarian protein doubled within 1 d and remained
constant until death occurred 3 d later. With access to sugar
for 2 d however, the ovarian contents were 34.6±1.7 mcal/
female for protein (N=2), 3.1±1.4 mcal for lipid (N=3), and
0.7±0.2 mcal for glycogen (N=3). The ovarian protein tripled
within 2 d, whereupon it remained stable, and ovarian lipid
doubled. The ovarian lipid never exceeded 6 mcal, and
glycogen always remained within 2 mcal. Ovarian protein
appeared to be more flexible in both species.
Oogenesis with single blood meals
Water or sugar-fed An. gambiae received single blood
meals at 24 h after eclosion. Their feces were collected during
the period of blood digestion and oogenesis for determining
their blood meal sizes, and the females were either dissected
for egg counts or fixed for quantification of total protein, lipid,
and glycogen. The amount of blood ingested was clearly sizedependent (Figure 5). All non-oogenic females were
significantly smaller than oogenic ones (t=17.6, p<0.0001)
and had ingested significantly smaller blood meals (t=22.2,
p<0.0001). Figure 6 shows the gravidity data for each body
size and a threshold body size for oogenesis. None of the
water-fed females equal to or smaller than 2.7 mm became
gravid, and with body sizes of 2.8 mm only 11% were able to
mature eggs, while larger-sized females were increasingly
successful in developing eggs. Among sugar-fed females,
however, most entered and completed oogenesis, although
with a size-dependent frequency. No size threshold was
encountered.
Among 295 females of An. atroparvus kept on water,
18
Journal of Vector Ecology
June 2005
Table 6. Overview of female and ovarian caloric contents in both Anopheles species given only one or two blood meals. An. atroparvus
without sugar rarely started oogenesis. The values of non-oogenic females are not included, but were similar in protein and glycogen to
teneral females, while lipid was increased over one-third (mean cal/female).
Protein
Lipid
Glycogen
Σ
Teneral
0.75
0.35
0.07
1.17
d 1*
Non-oogenic
Before oviposition
After oviposition
Ovaries
0.80
1.11
0.73
0.34
0.52
0.90
0.34
0.36
0.09
0.14
0.07
0.04
1.41
2.15
1.14
0.74
d 1/2*
Before oviposition
After oviposition
Ovaries
1.08
0.73
0.34
0.92
0.34
0.34
0.17
0.07
0.04
2.17
1.14
0.72
1.77
0.61
0.15
2.53
An. gambiae
An. atroparvus
Teneral
d 3**
Before oviposition
After oviposition
Ovaries
2.56
1.59
0.96
2.27
0.81
1.11
0.27
0.25
0.06
5.10
2.65
2.13
d 3/5*
Before oviposition
Ovaries
2.81
0.90
1.80
0.87
0.35
0.05
4.96
1.82
*No sugar
**Sugar available
Table 7. Overview of ovarian and oocyte development in An. gambiae and An. atroparvus. Follicle length of ovaries at resting stage II,
together with oocyte caloric content after one blood meal, when kept with water or sugar previously and after multiple blood meals.
Resting stage ovaries*
(mcal/female)
Single blood meal
Water-kept
(mcal/oocyte)
Sugar-kept
(mcal/oocyte)
Multiple blood meals
Water-kept
(mcal/oocyte)
An. gambiae
An. atroparvus
Follicle length (µm)
≤65
≤105
Protein**
10.0
35.3
Lipid**
2.0
6.0
Eggs/female
50-100
150-200
Protein
4.0
5.0
Lipid
4.3
4.1
Total
8.3
9.1
Protein
2.8
5.8
Lipid
4.3
6.8
Total
7.1
12.6
50-150
150-200
Protein
Eggs/female
3.9
5.1
Lipid
3.8
4.8
Total
7.7
9.9
*Oocyte numbers not determined.
**Maximum ovarian values of sugar-kept females.
Vol. 30, no. 1
Journal of Vector Ecology
19
Figure 5. Blood meal sizes of non-oogenic (A) and oogenic (B) An. gambiae fed single blood meals 24 h after eclosion with acess to
water. Regression formula for hemoglobin on body size are: A y=31.9x -181.9 (N=241, r2=0.654, t=21.25, p<0.0001); B y=19.6x +
283.0 (N=216, r2=0.133, t=5.73, p<0.0001).
only 7 entered oogenesis after one blood meal with up to 150
eggs (Table 1). Females kept on water died by day 5-6. With
access to sucrose the percentage of oogenic females rapidly
increased between day 2 and 3, and by day 4 all females
became gravid, maturing 150-200 eggs.
The caloric contents of gravid An. gambiae after single
blood meals again correlated significantly with body size. The
following regressions were obtained for previously starved
females: protein y=0.037x + 0.182 (N=42, r2=0.422, t=5.41,
p<0.0001); lipid y=0.046x - 0.296 (N=50, r2=0.215, t=3.62,
p<0.001); glycogen y=0.008x - 0.055 (N=50, r2=0.284,
t=4.36, p<0.0001). The regressions for protein and lipids had
slopes similar to teneral conditions but were increased by
roughly 25% for protein and 33% for lipids. Blood meals
that did not allow oogenesis led to the formation of maternal
deposits that approximated half the values of yolk. This is
demonstrated in Table 2 for each size class of gravid and nonoogenic An. gambiae that had access to water before. In gravid
females, lipid had by far the highest increase with 0.5 cal;
with protein it was 0.2-0.3 cal per female and glycogen
increased only by 0.01-0.07 cal. Non-oogenic females had
smaller values and smaller increases.
These data express the efficiency of blood meal utilization
for the synthesis of yolk in oogenic females and of reserves
in non-oogenic females. Gravid females had utilized 5-11%
of the blood meal as protein, 10-11% as lipid, while glycogen
remained below 2%. Non-oogenic females, however, utilized
a total of 9-24% of the caloric input in an inverse relation
with body size: protein accounted for 1-8%, lipid 4-15%, and
glycogen 2% or less. Together, these results suggest that nonoogenic An. gambiae were able to use similar proportions of
the blood meal as did oogenic females. Almost as much of a
first and single blood meal was converted to the synthesis of
maternal reserves as was transferred by oogenic females to
yolk.
Similar data are compiled in Table 3 for An. gambiae
that had access to sugar before the blood meals. In gravid
females, the regressions of caloric content on body size were
the following: protein y=0.028x + 0.115 (N=55, r2=0.701,
t=11.15, p<0.0001); lipid y=0.072x - 0.361 (N=48, r2=0.826,
t=14.79, p<0.0001); glycogen y=0.013x - 0.109 (N=48,
r2=0.588, t=8.10, p<0.0001). In gravid females, the relative
gain in lipid was 2-7-fold of teneral values, but only up to
27% in protein, both in an inverse relationship to body size.
20
Journal of Vector Ecology
Figure 6. Oogenesis in An. gambiae after a single blood meal
depending on body size. Females had either access to water for 1 d
(A), or to sugar for 5 d (B) before the blood meal. The percentage
of gravid females is given for each size class. With water 457
females were tested and 216 became gravid (47%), with sugar this
was 150 females (81%) out of 185 tested.
However, in absolute terms, large females accumulated
considerably more calories than smaller ones. Non-oogenic
females also gained up to 34% protein and 2-6-fold lipid
(Table 3).
Although sugar-feeding had a deleterious effect on blood
meal volume as shown before (Figure 4), it had a positive
effect on egg production (Table 3). Sugar-kept females had
much lower protein contents that were apparently
compensated by increased lipid contents derived from
previous sugar meals.
In An. atroparvus, the total protein and lipid contents
after digestion of a single blood meal were also in significantly
linear regression with body size: protein y=0.029x+0.046
(N=70, r2=0.717, t=13.12, p<0.001); lipid y=0.031x-0.335
(N=74, r2=0.344, t=6.14, p<0.001) for sugar-fed oogenic
females. For water-fed non-oogenic females regressions were:
protein y=0.018x+0.275 (N=89, r2=0.603, t=11.49, p<0.001);
lipid y=0.011x+0.061 (N=87, r2=0.147, t=3.82, p<0.001).
Oogenic females had roughly doubled their teneral protein
and doubled or tripled their teneral lipid, depending on body
size. In non-oogenic females the teneral protein and lipids
remained at similar levels, thus no maternal deposits were
discerned.
Multiple blood meals, fecundity, and reserve accumulation
Sugar meals reduced the tendency to feed on blood, so
only water-fed females were tested. An. gambiae showed a
continued avidity for blood; after a first blood meal all females
readily took a second meal at any time tested. Up to three
blood meals were ingested on consecutive days after eclosion
(Table 4). With two blood meals in 24-h intervals almost all
females developed eggs, compared to only 40% with one
June 2005
blood meal. With a second blood meal at 48 h the mean
number of mature eggs increased from 56 to 93 per female
and when fed at 24 and 72 h, mean fecundity rose to 120 eggs
(Table 4). Because digestion of a single blood meal took 40
to 44 h, the females with the second meal at 72 h had largely
digested their first meal, but oviposition was denied. In
females fed three-times, fecundity was somewhat lower,
because of a volumetric interference between the second and
third blood meals that limited total ingestion.
We analyzed the caloric content of mature oocytes in
females taking multiple blood meals. Among females that had
no sugar before, the caloric content per mature oocyte was
fairly constant: 3.9±0.4 mcal of protein, 3.8±0.4 mcal of lipid,
and 0.4±0.2 mcal of glycogen (N=17-19). Protein and lipid
as the main constituents of yolk occurred in roughly
equicaloric amounts, while glycogen always remained below
10%. Females that blood fed once with previous access to
sugar ingested smaller blood meals and matured oocytes with
lipid as a dominant component: 2.8±0.7 mcal protein, 4.3±0.6
mcal lipid, and 0.5±0.3 mcal glycogen (N=22-23). The total
was 7.6 mcal, significantly less than in water-kept females
with 8.1 mcal per oocyte (t=8.11, p<0.005).
In a similar approach, An. atroparvus with access to water
were tested for multiple feedings. This species was not as
prone to take multiple blood meals as the previous one. The
results are presented in Figure 7 in relation to body size.
Females were offered their first blood meal on day 3 after
eclosion, a second blood meal on day 4 or 5, or three blood
meals at days 3, 4, and 5. Even though females were starved,
there were always some that did not bite. The large females
ingested sufficient amounts of blood to mature eggs (Figure
7), but there appeared to be a critical blood meal size between
4.5 – 6.0 cal protein (9 - 12 µl blood), above which oogenesis
was possible. The highest average refeeding of 80% was
observed with an interval of 2 d between two blood meals
(Table 5). Correspondingly, blood meal size and gravidity
were maximal. Fewer females took blood on three consecutive
days, and the ones that did were almost all of a smaller size.
Smaller females ingested relatively smaller volumes, which
in turn affected oogenesis. Therefore, even though females
took three blood meals, fecundity appeared reduced (Table
5).
Sugar feeding was advantageous to An. atroparvus
females because it allowed oogenesis with a single blood meal,
while in water-kept females three blood meals were required
to attain similar caloric amounts to females that had access to
sugar before the first blood meal. However, females with
access to sugar took considerably smaller blood meals. The
caloric content per mature oocyte also differed between both
treatments. Sugar-maintained An. atroparvus had oocytes that
contained 5.8±1.0 mcal of protein, 6.8±1.0 mcal of lipid, and
0.3±0.2 mcal of glycogen (N = 39), i.e. a total of 12.9 mcal,
significantly higher than water-kept females with 5.1±0.4 mcal
of protein, 4.8±0.5 mcal of lipid, and 0.3±0.1 mcal of glycogen
(N = 11-14), totaling 10.2 mcal (t=5.84, p<0.005).
An overview of female and ovarian caloric contents is
given for both species in Table 6. Non-oogenic An. gambiae
increased their lipid deposits by an average of 0.2 cal,
Vol. 30, no. 1
Journal of Vector Ecology
21
Figure 7. Blood consumption of An. atroparvus kept with water from eclosion, and blood-fed twice, i.e. day 3 and 4, or day 3 and 5, or
three times, i.e. at day 3, 4 and 5 after eclosion (N=129). The grey area illustrates the blood meal size assumed to be critical for
oogenesis: Oogenic females (filled circles; N=78), were clearly above this amount and non-oogenic ones (open squares; N=51) below.
The broken line indicates the regression (y = 3.821x+253.46; N=183, r2=0.096, t=4.38, p<0.001) for blood consumption with single
blood meals of females fed sugar before and all were oogenic.
Figure 8. Survival of An. gambiae with daily
blood meals. Either they had permanent access
to water (filled circles; N=47) or sucrose (filled
triangles; N=36). For comparison, survival of
females with access to sucrose only (open
squares; N=50).
22
Journal of Vector Ecology
June 2005
Figure 9. Frequencies and pattern of blood feeding and
oviposition in 16 water-kept, inseminated An. gambiae kept
individually in 8 dm3 cages after blood meals. The oviposition
followed a more or less regular 3-d pattern after the peaks of
blood meals.
Figure 10. Feeding and oviposition frequency of An. gambiae
kept in groups of 18 in 8 dm3 cages. A: blood meals were
taken almost daily. B: The oviposition pattern was continuous.
No clear gonothropic concordance was recognized.
Vol. 30, no. 1
Journal of Vector Ecology
23
demonstrating once more that, for them, the blood meal is an
important energy source in addition to providing the raw
material for oogenesis. In Table 7 an outline of ovarian and
oocyte development is presented for both species. The
composition of yolk was flexible and varied with diet, and
providing sugar before a blood meal caused a proportional
increase in lipid content. While An. gambiae females revealed
the highest total contents per oocyte when given blood alone,
in An. atroparvus highest values were observed when given
access to sugar before the blood meals.
daily and oviposited continuously, thus lacking gonotrophic
concordance. Probably, females kept in groups might
experience disturbances in their feeding activity, apparently
disrupting their gonotrophic concordance. Furthermore,
oviposition activity of An. gambiae was affected by cage size:
the smaller the container, the more reluctantly they oviposited.
In small cups (27 cm3), mature oocytes were retained for over
5 d, despite the presence of sperm in the spermathecae.
Survival with only access to blood
We tested the survival of An. gambiae with blood alone
as the dietary substrate. Between the blood meals, females
had access to water. The number of females taking blood,
their gonotrophic status, and the number of eggs oviposited
were recorded daily; females could not be screened for
insemination. Maximal survival time of blood-fed females
was 54 d when they had access to sugar and 42 d with water.
The corresponding 50% survival times were 27 d and 16 d
respectively, clearly extended when compared to sugar alone
(20 d, Figure 8). The survival curves for large and small
females largely overlapped. Sugar-kept females with daily
blood meals matured and oviposited an average of 606 eggs
per female, more than double the average number of 229 eggs
of blood-fed females maintained on water in-between.
Evidently, blood meals as the only food source allow a
considerably extended survival of An. gambiae, and an
appreciable fecundity. However, additional sugar sources
further improved total fecundity.
In these experiments, the intestinal and gonotrophic status
were also evaluated. Females feeding on the host, while dark
blood still was present in their midguts, were judged to refeed. Females containing yellowish yolk material that were
re-feeding were considered to be gonoactive, while a third
group of females approaching the host appeared empty. It
was impossible to determine whether they were at the end or
at the beginning of another gonotrophic cycle, because some
might have had ingested and digested a small blood meal
without producing eggs.
Because this evaluation was not error-free as a result of
small blood meals that might have escaped visual detection,
an attempt was made to follow the oviposition pattern. After
mating, females were either kept individually or in groups in
large cubic cages (8 dm3) with continuous access to water,
and offered blood every day for at least 10 min or until all
had stopped probing.
When kept singly, the majority of the blood meals were
taken in a 3-day pattern, and oviposition followed 3 d later in
almost the same pattern (Figure 9). Thus, these females
showed regular gonotrophic cycles of 3 d, indicating their
concordance. Some females apparently required two blood
meals to complete their gonotrophic cycles. Females with
empty spermathecae were discarded.
However, when kept and blood fed in groups, females
behaved differently. At 7 out of 12 d they regularly took blood
meals, and their oviposition pattern was followed (Figure 10).
Apparently females were capable of taking blood meals almost
Body size is well-established as a crucial parameter of
fecundity and fitness in many mosquitoes and particularly in
several Anopheles species (Briegel 1990b, Kitthawee et al.
1992, Lyimo and Takken 1993, Ameneshewa and Service
1996, Takken et al. 1998, Charlwood et al. 2003). However,
there exist more complex physiological interactions and
consequences than just linear relationships between size,
ingested blood volume, and fecundity, as was generally noted
for Aedes aegypti (Briegel 1990a, Clements 1992). Some farreaching consequences of variable body sizes relate to the
vectorial capacity and to the concept of the gonotrophic cycles
in the Anophelini. In a series of classical field studies, Gillies
(Gillies 1953, 1954, 1955, 1961, Gillies and Wilkes 1965)
reached several important conclusions with respect to An.
gambiae. He observed and introduced the “pre-gravid” status
of females approaching hosts in the field, referring to newly
emerged unmated females, which led him to suggest that two
blood meals were required to mature the first batch of eggs
(Gillies 1954). He further maintained that their first blood
meal could be taken independent of mating, because reaching
the pre-gravid state was considered more important. An.
gambiae females were recently observed taking blood meals
before mating in the field (Charlwood et al. 2003). Laarman
(1968), however, found sugar feeding to be part of the normal
feeding behavior of An. gambiae and An. funestus, and more
recently, Gary and Foster (2001) investigated the effects of
sugar feeding in An. gambiae on its vectorial capacity, as did
Okech et al. (2003), who promoted the crucial significance
of sugar feeding for survival and thus vectorial capacity of
An. gambiae. Recently, Foster and Takken (2004) found that
newly emerged An. gambiae prefer sugar over blood. The
females used in our experiments were able to mature eggs
without being inseminated, even though in other species
mating influences egg development (Marchi et al. 1978,
Klowden and Chambers 1991, Lounibos 1994).
Many of these findings may be explained by our
physiological and quantitative studies on body size and reserve
status of teneral females. We have found a critical body size
(wing length of 2.8 mm) below which the females’ teneral
reserves are insufficient to allow oogenesis with a single blood
meal, unless these females had obtained sugar before. Takken
et al. (1998) recorded no oogenesis in small females after 4 d
of sugar-feeding. Only after 5 d of exclusive sugar-feeding,
some of our smallest females were able to mature eggs with a
single blood meal. Larger females however, had sufficient
lipid and protein reserves available from the larval growth
period, so that ingested blood permitted oogenesis with a
DISCUSSION
24
Journal of Vector Ecology
single meal. Small females apparently first require an increase
their teneral reserves, either from sugar meals or blood meals,
the latest being the most efficient. After one blood meal, their
oocytes progress from the undifferentiated pupal stage to the
resting stage, and only after a second one to maturity (Gillies
1957, Takken et al. 2002), while in the case of previous, early
sugar meals, the oocytes deposit some lipid from one blood
meal (Table 7). A comparable threshold size was also found
in An. albimanus (Briegel 1990b). Therefore, the pre-gravid
status of An. gambiae, besides cytological changes within the
ovaries, is governed by the physiological fate of the first blood
meal, i.e. to synthesize primarily maternal reserves as we had
also shown earlier for An. albimanus and An. gambiae (Briegel
1990b). Apparently, the amount of protein that can be
accumulated is limited, because lipid constituted the largest
deposits. In An. atroparvus, oogenesis also depended on
maternal reserves, but the teneral amounts were insufficient
for oogenesis after a single blood meal. Only when the lipid
was raised through extended sugar feeding did all females
mature eggs with a single meal. When An. atroparvus was
deprived of sugar, however, it succeeded in synthesizing
reserves from three blood meals to a similar extent as if they
had sugar-fed.
In An. gambiae, another question concerns the metabolic
fate of sugar meals versus first blood meals. Whichever is
taken first, it is primarily maternal reserves that are
synthesized. Of course, sugar meals are funneled into
lipogenesis as has been shown many times in many mosquito
species (Van Handel 1965, Briegel 1990a, b, Briegel et al.
2001a, Briegel et al. 2001b). The significance of lipid reserves
for oogenesis has only recently been fully recognized through
the work of Ziegler and Ibrahim (2001) and Briegel et al.
(2002) for Ae. aegypti. In An. atroparvus, the need for
preceding sugar meals, and thus lipid synthesis is more
pronounced than in An. gambiae; it also occurred much slower,
probably due to the lower temperature. Maximal lipid levels
were only gradually attained during two wk of sugar feeding,
i.e. tripling the teneral lipid values. On the behavioral level,
An. atroparvus was also more reluctant to feed on blood before
2-4 d of sugar feeding had passed after eclosion, and the
requirement for double blood meals was minimal. An.
gambiae, on the other hand, readily and successfully fed on
humans at short distances at 16 h post-eclosion, whether sugar
was available or not, in contrast to reports by Takken et al.
(1998), based on host stimuli. Therefore, we believe that An.
gambiae has an opportunistic feeding behavior: whatever
source is encountered first will be taken, blood or sugar. This
may be the main reason for so many confusing observations
reported in the literature from the field. Sugar meals, however,
do interfere with later blood feeding activities, primarily for
volumetric reasons. Large crop volumes, caused by sugar
fillings, limit the volume of later blood meals in both small
and large females. Accordingly, fecundity may be reduced
despite an improved reserve status. In Ae. aegypti, several
works point to a fitness advantage of exclusive blood feeding
over mixed diets of sugar and blood (Naksathit et al. 1999,
Harrington et al. 2001).
Surprisingly, the caloric content of mature oocytes was
June 2005
very flexible in both species, depending on diet. Sugar-fed
females always deposited an increased proportion of lipid,
apparently using their larger maternal lipid reserves to
compensate for the reduced protein ingestion from smaller
blood meals. The metabolic plasticity of Anopheles in general
and An. gambiae in particular, is also indicated by our earlier
findings that only 10-20% of the caloric blood meal input is
utilized for oogenesis (Briegel 1990b), the rest being available
for deamination and thus reserve synthesis, accompanied by
excretion of tremendous amounts of nitrogen catabolites
(Briegel 2003).
Multiple blood meals and gonotrophic discordance
An. gambiae took daily blood meals, and their survival
times were markedly extended over females with access to
sugar, similar to the 34 d reported by Gillies and Wilkes
(1965), and in line with data given by Straif and Beier (1996),
Gary and Foster (2001), and Okech et al. (2003). Furthermore,
the lack of sugar feeding of An. gambiae was compensated
by more frequent blood feeding, again supporting Beier (1996)
and Straif and Beier (1996), which led to the unusual but
correct statement of higher fecundities in water-kept females
(Gary and Foster 2001). It is also possible that the blood meal
might be used not only as a substrate for oogenesis but as an
important energy source. Under such conditions, the vectorial
capacity also was positively affected. Hocking and MacInnes
(1948) had already noted that An. gambiae and An. funestus
could feed daily on blood even when gravid. When such
observations became frequent, and with our observation of
continuous oviposition patterns, the concept of strict
gonotrophic cycles might not always apply to tropical
Anopheles species. Indeed, Briegel and Hörler (1993)
observed ovarioles of An. albimanus with penultimate mature
oocytes on top of the ultimate mature ones, as we have
observed in An. gambiae females, when offered multiple blood
meals and denied an oviposition site (data not shown). Briegel
and Rüfenacht (unpublished data) had observed continuous
ovipositions in multiple blood-fed An. stephensi onto dry
Plexiglas™ surfaces in the absence of a proper oviposition
jar, strongly indicating the possibility of a continuous
oviposition activity independent of any cyclicity. It appears
that gonotrophic cycles may not have been fully established
during the evolution of the Anophelini, as we know them in
the strictly regulated terms described for many Culicini
(Clements 1992).
This led us to suggest a “gonotrophic discordance”
applicable to tropical species of Anopheles, such as gambiae,
albimanus, and stephensi, although in contrast to the term
“gonotrophic dissociation” coined by Swellengrebel (1929)
for hibernating members of the An. maculipennis complex
with their cessation of gonotrophic activities. This interesting
issue definitely requires further investigation on a comparative
basis. Nevertheless, in An. atroparvus, a member of the
maculipennis complex of the temperate regions, different
strategies exist, with sugar meals being essential for oogenesis
with single blood meals. They resemble more the physiotype
that Briegel (2003) encountered in Ae. vexans (Briegel et al.
2001b), depending more on sugar for the first few days of
Vol. 30, no. 1
Journal of Vector Ecology
imaginal life to improve their lipid reserves and setting the
blood meal protein aside primarily for oogenesis.
An additional aspect of their lipid synthesis, either from
sugar or from blood meal, relates to the energy metabolism.
When Kaufmann and Briegel (2004) investigated the flight
potential of these same two species under comparable
nutritional regimes and physiological conditions, they found
evidence that lipid was utilized during flight of sugar-fed as
well as of blood-fed females. The primary flight substrate
was sugar, if and as long as present, and glycogen, but usually
these substrates were insufficient for extended flights, and
subsequently lipid stores were drawn upon. Although the
actual flight ranges for both species in the field remain
unknown, they are probably less than on flight mills.
Acknowledgments
This work was supported by the Swiss National Science
Foundation (to H.B.), while L.F. was on an assistantship from
the Zoological Institute. Technical advice for rearing and
analyses by Mrs. R. Haigis are gratefully acknowledged. An
ethical clearance for using guinea pigs was given by the Swiss
“Bundesamt für Veterinärwesen.”
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