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Habitat discrimination by gravid Anopheles
gambiae sensu lato –a push-pull system
Manuela Herrera-Varela
1,2
, Jenny Lindh
3
, Steven W Lindsay
4
and Ulrike Fillinger
1,2*
Abstract
Background: The non-random distribution of anopheline larvae in natural habitats suggests that gravid females
discriminate between habitats of different quality. Whilst physical and chemical cues used by Culex and Aedes
vector mosquitoes for selecting an oviposition site have been extensively studied, those for Anopheles remain
poorly explored. Here the habitat selection by Anopheles gambiae sensu lato (s.l.), the principal African malaria
vector, was investigated when presented with a choice of two infusions made from rabbit food pellets, or soil.
Methods: Natural colonization and larval survival was evaluated in artificial ponds filled randomly with either
infusion. Dual-choice, egg-count bioassays evaluated the responses of caged gravid females to (1) two- to six-day
old infusions versus lake water; (2) autoclaved versus non-autoclaved soil infusions; and assessed (3) the olfactory
memory of gravid females conditioned in pellet infusion as larvae.
Results: Wild Anopheles exclusively colonized ponds with soil infusion and avoided those with pellet infusion.
When the individual infusions were tested in comparison with lake water, caged An. gambiae sensu stricto (s.s.)
showed a dose response: females increasingly avoided the pellet infusion with increasing infusion age (six-day
versus lake water: odds ratio (OR) 0.22; 95% confidence interval (CI) 0.1-0.5) and showed increasing preference to
lay eggs as soil infusion age increased (six-day versus lake water: OR 2.1; 95% CI 1.4-3.3). Larvae survived in soil
infusions equally well as in lake water but died in pellet infusions. Anopheles gambiae s.s. preferred to lay eggs
in the non-autoclaved soil (OR 2.6; 95% CI 1.8-3.7) compared with autoclaved soil. There was no change in the
avoidance of pellet infusion by individuals reared in the infusion compared with those reared in lake water.
Conclusion: Wild and caged An. gambiae s.l. females discriminate between potential aquatic habitats for oviposition.
These choices benefit the survival of the offspring. Although the study was not designed to distinguish between
stimuli that acted over a distance or on contact, it could be demonstrated that the choice of habitat is mediated by
chemical cues based on both preference and avoidance. These cues, if identified, might be developed for ‘push-pull’
strategies to improve malaria vector monitoring and control.
Keywords: Oviposition site selection, Anopheles gambiae, Natural infusion, Olfactory memory
Background
Selection of suitable oviposition sites is a critical step in
the life history of mosquitoes [1]. This is a process
whereby individuals select and occupy a non-random set
of aquatic habitats. Habitat selection is of major import-
ance for the interpretation of spatial and temporal distri-
butions of populations, and for understanding intra and
inter-specific relations that influence the abundance of
individuals [2,3]. Organisms without any parental care
are likely to choose habitats based on a set of innate or
learned cues in order to maximize the survival and fit-
ness of their offspring [2,4].
Mosquitoes utilize a wide range of aquatic niches for
oviposition, including natural ponds, puddles, stream
fringes, marshes, tree-holes and plant axils, man-made
pits, drains, rice fields, and containers [5]. Field studies
have shown that mosquitoes are discriminating in select-
ing sites for egg deposition [6,7] and that oviposition
choices made by gravid females are a key factor in
* Correspondence: ufillinger@mbita.icipe.org
1
Department of Diseases Control, London School of Hygiene and Tropical
Medicine, London, UK
2
International Centre for Insect Physiology and Ecology (icipe)-Thomas
Odhiambo Campus, Mbita, Kenya
Full list of author information is available at the end of the article
© 2014 Herrera-Varela et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the
Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public
Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this
article, unless otherwise stated.
Herrera-Varela et al. Malaria Journal 2014, 13:133
http://www.malariajournal.com/content/13/1/133
determining larval distribution [8-11]. Although different
species are found in the same type of habitat, oviposition
site selectivity is considerably species specific [12]. Im-
mature stages of Anopheles gambiae sensu lato (s.l.), the
major malaria vector in sub-Saharan Africa, are typically
described as inhabiting very small, temporary sunlit
pools and puddles without vegetation [13-16]. However,
reviews of the literature and recent research on larval
ecology have shown that this is a gross oversimplifica-
tion of the wide range of habitats colonized by this spe-
cies [17-19], a fact recognized over half a century ago by
Holstein who reviewed the ‘extraordinary diversity of the
breeding places’of An. gambiae s.l. [19] . Numerous
studies have described how the presence of An. gambiae
s.l. larvae [17,20-23] and the capacity of individual habi-
tats for generating adult mosquitoes [6,8,17,24,25] differs
markedly over space and time, yet these surveys failed to
reveal any risk factors that could consistently predict
sites preferred by An. gambiae s.l. [17,20-23]. This might
lead to the conclusion that this species randomly de-
posits its eggs in a large range of habitats and that the
heterogeneous distribution of larvae results from the
survival of larvae in the aquatic habitat [9,10] rather
than the adults’choice.
Surprisingly, fully gravid malaria vectors looking for
suitable larval habitats have been grossly understudied
[26]. Compared to the wealth of knowledge of the phys-
ical and chemical factors used by gravid culicine for
selecting an oviposition site [27-40] those potentially
used by the world’s most deadly malaria vector remain al-
most unexplored. Whereas many publications recognize
that the distribution of larvae between seemingly suitable
aquatic habitats is probably due to the choice of the gravid
female [6,8,24,41-43] and that this choice probably im-
pacts on the fitness of her offspring, there is little empir-
ical evidence to support these assertions. Most recent
research has evaluated the characteristics of aquatic habi-
tats associated with the presence and absence of larvae
[15,21,44-46] but the understanding of the behaviour of
gravid female An. gambiae s.l. when searching for an ovi-
position site remains, at best, sketchy [8,41,47-53].
Laboratory studies demonstrated that physical condi-
tions of the aquatic habitats influence oviposition site se-
lection in An. gambiae s.l., with females preferring dark
backgrounds to pale ones, muddy water to clear water
and fully hydrated substrates [8,41,50,54]. Turbidity has
been suggested as an important physical cue for ovipos-
ition behaviour in An. gambiae s.l. although the evidence
for this is contradictory [55,56].
Even less is known about the chemical cues and their
interaction with physical factors. Water vapour is itself
an attractant to gravid mosquitoes [57]. It has been
shown that gravid An. gambiae s.l. are sensitive to
bacteria-derived odours [47,53,58] which have been
associated with increased [47,53] and reduced [58] egg
numbers compared to sterile media in cage bioassays.
Whilst over 20 putative oviposition semiochemicals have
been suggested in the literature, based on the analyses
of bacteria- or habitat-derived volatile chemicals and
electro-antennogram studies [53,59], there is only one
report [60] of two chemicals inducing a behavioural re-
sponse in gravid females (one increasing and one de-
creasing the oviposition response).
Here the oviposition behaviour of An. gambiae s.l. was
explored to test the hypotheses that a gravid An. gam-
biae s.l. female evaluates the suitability of a habitat using
chemical cues from water bodies that oviposition choices
made by a gravid female benefit the offspring and that
this choice cannot be modified by experience in one
generation.
Habitat selection by gravid An. gambiae s.l. was tested
by presenting a choice of two infusions; one made with
soil from an area where natural habitats occur fre-
quently, and one made with rabbit food pellets. Rabbit
food pellets are frequently used as diet for mosquito lar-
vae in insectaries [61,62] and infusions made of grass,
hay and other plant material, including rabbit food pel-
lets have shown to be attractive to a range of mosquito
species and have been used in gravid traps [63-66].
The aim was to explore whether Anopheles gambiae
s.l. might also be drawn to this infusion.
Natural colonization and larval survival was evaluated
in artificial ponds filled randomly with either infusion.
As a consequence of the field observations, two-choice,
egg-count bioassays were used to explore pattern of ovi-
position seen in the field. Experiments were designed to
address the following questions: 1) Do gravid An. gam-
biae s.l. females discriminate between different habitats
when searching for an oviposition site? 2) Does the ovi-
position choice benefit the survival of their offspring?
3) Are gravid females guided by preference (attractants/
stimulants) or avoidance (repellents/deterrents)? 4) Are
oviposition choices likely to be based on chemical cues?
and, 5) Is the choice made by a gravid female influenced
by her olfactory memory of her larval habitat?
Methods
Study site
Experiments were carried out at the International Centre
for Insect Physiology and Ecology (icipe), Mbita, on the
shores of Lake Victoria, Western Kenya (geographic co-
ordinates 0°26′06.19″South; 34°12′53.13″East; altitude
1,137 m above sea level). Mbita has a typical tropical cli-
mate; temperatures oscillate between 18-28°C and there
is annual rainfall of 1,436 mm (based on data from icipe
meteorological station for 2010–2012). Two rainy sea-
sons occur annually, the long rainy season between
March and June and the short rainy season between
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October and December. Malaria is endemic in the area
and transmitted by three vectors, which are in order of
their abundance: Anopheles arabiensis,Anopheles gam-
biae sensu stricto (s.s.) and Anopheles funestus [67].
Mosquitoes
Open-field trials were conducted with wild anopheline
and culicine females that oviposited in tubs of water
sunk into the ground. These were colonized within three
days. Laboratory experiments were carried out with
insectary-reared An. gambiae s.s. (Mbita strain) supplied
by icipe’s insectary and reared following standard operat-
ing procedures. Briefly, larvae were reared in round plas-
tic tubs (diameter 0.6 m) filled with water from Lake
Victoria and fed Tetramin® fish food twice daily. Larvae
were collected randomly from several tubs on the day of
experiment. Gravid mosquitoes were prepared by select-
ing 300 female and 300 male mosquitoes, two to three
days old, from their rearing cages at 12.00 and keeping
them in 30 × 30 × 30 cm netting cages at 25-28°C and
68-75% relative humidity. To avoid mosquito desicca-
tion, cotton towels (folded to 25×12 cm) were saturated
with lake water and placed over the cages. Mosquitoes
were starved of sugar for seven hours before blood feed-
ing and allowed to feed on a human arm for 15 min at
19.00 on the same day. After feeding, mosquitoes were
provided with 6% glucose solution ad libitum. This pro-
cedure was repeated 24 hours later. After the first blood
meal unfed female mosquitoes were removed from the
cages. Fed female mosquitoes were kept together with
males for two days after the second blood meal before
using them in an experiment (i e, females four to five
days after first blood meal). In the afternoon (16.30) of
the day of an experiment 45–100 (depending on experi-
ment and availability) visually presumed gravid females,
that is with an enlarged, pale white abdomen, were se-
lected from the holding cage. A small percentage of
these mosquitoes were probably not gravid because most
females needed two blood meals to reach full gravidity
and some never reach full gravidity even after three
feeds [68,69]. Whilst two meals were provided it cannot
be guaranteed that two meals were taken by all females.
This might be the reason that not all mosquitoes ex-
posed to oviposition medium in experiments laid eggs
(responded), therefore the number of responders was
smaller than the number tested. Non-responders were
excluded from the analyses.
Experimental procedures
Do gravid Anopheles gambiae sensu lato females
discriminate between different habitats when searching
for an oviposition site?
To explore natural colonization of habitats by wild mos-
quitoes, 20 artificial habitats were created by implanting
20 plastic tubs (40 cm diameter, 20 cm deep) into an
open-sunlit field during the long rainy season in May
2011. The tubs were placed in four lines of five tubs
each 4 m apart [70]. Two different substrates were ran-
domly offered in the artificial habitats. Half of the tubs
(ten) received 30 g of rabbit food pellets (Scooby® rabbit
and rodent food, Nairobi) containing hay and grains
from maize, wheat, barley, cotton, sunflower, soya bean
meal, and traces of molasses, vitamins and minerals. The
remaining half of the tubs (ten) received 2 kg of dry soil
taken from a field at icipe. Soil texture was characterized
as a silty clay loam according to the US Department of
Agriculture (USDA) texture triangle [71] using the de-
tergent method [72] to separate and quantify soil min-
eral particles of different size. A volume of 15 l of lake
water pumped from Lake Victoria, was added to each
tub and the water level was held constant by adding
water to the 15 l mark daily. The two treatments are
henceforth referred to as pellet and soil infusion. To
study the oviposition response of wild mosquitoes the
tubs were monitored daily between 08.00 and 10.00 by
dipping five times per tub with a standard dipper
(350 ml). Two different dippers were used for the two
treatments to avoid contamination. Four dips were taken
from the edge of the tubs and one from the middle. The
content of each dip was emptied into a white plastic
bowl and all early instars (first and second stage larvae)
counted and recorded for both culicine and anopheline
mosquitoes. All larvae and the water were returned to
the respective tub. The tubs were followed for 16 days.
Ponds were searched for pupae and collected daily to
prevent any emergence of potential disease vectors.
Pupae were allowed to emerge in cages in the laboratory
and any anophelines emerging identified to species using
morphological keys [13,73] and for specimens of the An.
gambiae complex using the ribosomal DNA-polymerase
chain reaction (PCR) method to distinguish between the
two local species of the complex An. gambiae s.s. and
An. arabiensis [74].
Does the oviposition choice benefit the survival of their
offspring?
Larval survival was assessed by introducing individual,
insectary-reared, first instar An. gambiae s.s. larvae in in-
fusions collected from the tubs set up in the field. Infu-
sion samples were taken after one, six, 11, and 16 days.
One-hundred ml of infusion was collected from each of
the ten tubs per treatment and pooled per treatment
(soil or pellet infusion) in a plastic bottle. Lake water
was used as a control. First instars were introduced in
100 ml plastic cups containing 50 ml of pellet infusion,
soil infusion or lake water. Twenty larvae were exposed
individually per treatment and collection day. Larvae
were fed every second day with finely ground Tetramin®
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Baby fish food. Food was provided with a blunt tooth-
pick that was first wetted in lake water and then dipped
quickly, not more than 1 mm deep into the ground food,
and then dipped onto the surface of the test water. Lar-
val development was monitored daily and the time of
death or time to pupation and emergence recorded. This
experiment was implemented under ambient conditions
in a semi-field system (80 sq m) with screened walls and
a glass roof [75].
Are gravid females guided by preferences or avoidance?
Based on the analysis of the field data a series of two-
choice, egg-count bioassays were designed to investigate
if the response of wild gravid females observed in the
field was based on avoidance or preference of an infu-
sion or both.
Gravid females were selected from insectary cages and
transferred individually to 30×30×30 cm cages. In each
cage two glass cups (Pyrex®, 100 ml, 70 mm diameter),
surrounded by tightly fitting aluminium cylinders, so
that mosquitoes could see only the water surface, were
filled with 100 ml of either the control or test medium
and placed in diagonal corners of the cage. Prior to use,
cups and cylinders were cleaned with detergent, then
autoclaved and kept in an oven at 200°C for at least two
hours. The position of oviposition cups containing the
test medium was alternated between adjacent cages to
control for possible position effect. The placement of the
first test cup was randomly allocated for one of the four
cage corners in the first cage. Subsequent test cups were
rotated in the next possible corners in a clockwise direc-
tion relative to the position of the preceding cup. One
control cup was added in each cage diagonal to the test
cup to complete a two-choice set up. The experiments
were carried out in makeshift sheds (Figure 1) that ex-
posed the mosquitoes to ambient light, temperature and
relative humidity but protected the cages from rain.
Two sets of experiments were carried out consecu-
tively (Table 1, Set 1 and 2). In the first set oviposition
choice was evaluated for two-, four- and six-day old pel-
let infusions compared with lake water. In the second
set, the oviposition choice was evaluated for two, four
and six-day old soil infusions compared with lake water.
In both sets of experiments internal controls were used
to validate the two-choice experiment. Here equal num-
bers of cages were set up where both cups in a cage con-
tained lake water and were labelled randomly as control
and test cup, assuming that gravid females lay eggs in
both cups in an equal proportion.
Infusions were prepared in a similar way as for the
field tests. Fifteen l of lake water were either incubated
with 30 g of pellets to prepare a pellet infusion or incu-
bated with 2 kg of soil to prepare a soil infusion. Infu-
sions were prepared in a plastic tub (40 cm diameter
20 cm depth) six days, four days and two days before the
day of experiment in order to test all ages in parallel.
Tubs were covered with mosquito netting and kept in
makeshift sheds at ambient conditions but protected
from rain. Experiments were implemented over three to
nine rounds depending on the availability of gravid fe-
males and the response rate per round (Table 1) with
fresh batches of infusions and different batches of mos-
quitoes for every round. On the day of experiment infu-
sions were sieved through a clean piece of cotton cloth
to remove large debris remaining from the pellets or
soil.
Fifteen to 25 replicate cages per treatment were set up
per round. A single gravid female was introduced per
cage at 17.30. The next morning between 08.00 and
09.00 the absence or presence and the number of eggs
was recorded for the control and test cup in each cage.
Turbidity, conductivity, dissolved oxygen, and pH were
measured in five cups per treatment in four different
batches of pellet, soil infusions and lake water using a
turbidity meter (TURB 355IR, WTW Germany) and a
AB
Figure 1 Location of egg-count bioassays. (A) Sheds (10 m long ×5 m wide ×2.8 m high) with walls made of reed mats and a roof made of
translucent corrugated polycarbonate sheets. (B) Interior of a shed. In each shed two tables hold up to 25 standard cages each, allowing 40 cm
of space between adjacent cages.
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multimeter (Multi 340i, WTW, Germany). In addition
one batch of pellet, soil infusion and lake water was
tested for ammonium (NH
4
+), carbonate hardness, total
hardness, nitrate (NO
3
−
), nitrite (NO
2
−
), and phosphate
(PO
4
3−
) content using Aquamerck® test kits from the
compact laboratory for water testing (Aquamerck®
No.111151, Germany).
Are oviposition choices likely to be based on chemical
cues?
Soil infusions differed strongly in colour and turbidity
from lake water. To assess if the oviposition response
observed was based on visual or chemical cues, a third
set of dual-choice, egg-count bioassays were imple-
mented with six-day old soil infusions (Table 1, Set 3)
comparing the relative attractiveness of autoclaved
and non-autoclaved infusion [47,76]. The experiment
followed the same experimental procedures as described
above. After filtering the infusion through a cloth on the
day of experiment, the infusion was split in two equal
volumes and half autoclaved at 120°C for 20 min to kill
bacteria potentially involved in releasing oviposition se-
miochemicals [47,77] and to reduce the amount of vola-
tile chemicals from the solution whilst maintaining the
colour and turbidity of the infusion. After autoclaving,
the infusion was left to cool to ambient temperature be-
fore setting up the cage bioassays. The oviposition
choice of individual gravid females was evaluated for six-
day old soil infusion versus lake water, autoclaved six-
day old soil infusion versus lake water and for autoclaved
versus non-autoclaved six-day old infusion. An equal
number of cages were set up where both cups in a cage
contained lake water and were randomly labelled as con-
trol and test cup.
In order to confirm that autoclaving sterilized the infu-
sion, samples (1 ml) of both infusions were taken during
each experimental round for bacterial cultures. Samples
were serially diluted (ten-fold) two times in distilled
water. After dilution, 100 μl of each of the ×1 (un-
diluted), ×10
−1
and ×10
−2
dilutions was spread separately
onto the surface of duplicate Lysogeny Broth (LB) agar-
plates (LB Lennox-Fisher Scientific) [78]. Plates were in-
cubated overnight at 30°C and the presence of colonies
recorded.
The same physical and chemical parameters were mea-
sured for the autoclaved infusion as described above for
the non-autoclaved pellet and soil infusions.
Is the choice of the gravid female influenced by her
olfactory memory of her larval habitat?
A fourth set of experiments (Table 1, Set 4) was de-
signed to assess the possibility that a gravid female’s
choice for an oviposition site might be influenced by her
olfactory memory of her larval habitat, as has been sug-
gested for culicine species [4,79].
Table 1 Summary details of dual-choice, egg-count bioassays to evaluate oviposition choices in Anopheles gambiae
sensu stricto
Dual-choice cage,
egg-count bioassays
Treatments No of
rounds
Total no of females responding
for all rounds (total number set up)
Control Test
Set 1: Pellet infusions Lake water Lake water
3
66 (75)
Lake water 2-day old pellet infusion 64 (75)
Lake water 4-day old pellet infusion 67 (75)
Lake water 6-day old pellet infusion 68 (75)
Set 2: Soil infusions Lake water Lake water
9
153 (225)
Lake water 2-day old soil infusion 161 (225)
Lake water 4-day old soil infusion 160 (225)
Lake water 6-day old soil infusion 171 (225)
Set 3: Vision versus olfaction
in soil infusions
Lake water Lake water
12
186 (220)
Lake water 6-day old soil infusion 150 (220)
Lake water Autoclaved 6-day old soil
infusion
157 (220)
Autoclaved 6-day old
soil infusion
6-day old soil infusion 169 (220)
Set 4: Olfactory memory –pellet
infusions
Lake water reared An. gambiae females
Lake water 6 day old pellet infusion 1 31 (45)
Pellet infusion reared An. gambiae females
Lake water 6-day old pellet infusion 1 37 (45)
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To test this, approximately 2,000 An. gambiae s.s. eggs
were dispensed in 1.5 l of two-day old pellet infusion
and another 2,000 eggs in lake water and reared under
the same conditions to the adult stage. The infusion and
lake water in the rearing pans was replaced every two
days with fresh two-day old infusion or lake water until
all surviving larvae pupated. Larvae were fed with Tetra-
min® fish food twice daily following routine insectary
procedures. Pupae were collected in a cup with 100 ml
of rearing water and placed in 30×30×30 cm cages for
emergence. Gravid females for cage bioassays were ob-
tained as described above.
Dual-choice cage bioassays were carried out in paral-
lel with gravid An. gambiae s.s. reared in the infusion
and gravid An. gambiae s.s. reared in lake water. A sin-
gle mosquito was offered a choice between six-day old
pellet infusion or lake water. Forty-five replicates were
set up in parallel for both treatment groups as described
above.
Sample size considerations
The sample size (number of responders) in the four sets
of cage experiments differed for a number of reasons.
Due to adverse climate conditions affecting the mos-
quito supply during the pellet infusion bioassays, the
production of colony-reared mosquitoes was low. Never-
theless, two-sample comparison of proportions power
calculation showed that 66 responders in each arm in
the pellet infusion bioassays (Table 1, Set 1) was suffi-
cient to detect a 23% increase or decrease in the propor-
tion of eggs laid in the treatment compared to the lake
water only experiment with 80% power at the 5% level
of significance. The effect of the pellet infusion observed
on oviposition response was much stronger than 23%. In
the soil infusion experiments (Table 1, Set 2 and Set 3),
a minimum of 150 responders in each arm was analysed.
This was sufficient to detect a 15% increase or decrease
in the proportion of eggs laid in the treatment as com-
pared to the lake water only experiment at the same
power and significance level. This level of accuracy was
deemed appropriate for investigating significant behav-
ioural cues affecting the oviposition choice. The evalu-
ation of olfactory memory required the mosquitoes to be
reared in pellet infusion where larval mortality was
nearly 98%. Therefore, only 45 females could be tested,
out of which only 31 and 37 responded in the two arms
(Table 1, Set 4). The hypothesis for this experiment was
that the preference of gravid females could be changed
and therefore at least double the proportion of eggs laid
in pellet infusion by infusion-reared females as com-
pared to the lake water-reared females. With 31 re-
sponders in each arm the experiment was powered
(80%) to detect a change in the proportion of 35%.
Statistical analyses
All data were analysed in R statistical software version
2.13.1 [80]. The one sample proportion test function was
used to estimate the 95% confidence intervals (CI) for
the proportion of larvae surviving in pellet infusion, soil
infusion and lake water. Pupation time of larvae exposed
to different treatments was calculated using the follow-
ing formula: (A×1) + (B×2) + (C×3)…(G×10)/Total num-
ber of pupae collected, where A, B, C…G are the
number of pupae collected on day 1, 2, 3 to 10. Dual-
choice, egg-count bioassays were analysed using general-
ized linear models (glm-function) with a quasibinomial
distribution fitted to account for the overdispersion. In
the first three sets of experiments the proportion of eggs
laid in test cups in the cages with equal treatments (lake
water in both cups) were compared with the proportion
of eggs laid in test cups in cages with two different treat-
ments. It was hypothesized that gravid females presented
with an identical treatment lay in both cups in an ap-
proximately equal proportion (p = 0.5). The statistical
analysis aimed to reveal if the test treatment of interest
(e g, infusions of different age) received an increased or
decreased proportion of the total number of eggs laid as
compared to the lake water only treatment. Therefore,
the treatment choice (e g, lake water only cages, cages
with infusion versus lake water) and the round of experi-
ment were included as fixed factors to analyse their im-
pact on the outcome (proportion of eggs laid in test
cup). A similar analysis was used for the fourth set of ex-
periments to compare the proportion of eggs laid in test
cups (pellet infusion) by gravid females that were reared
in hay infusion during their larval development, com-
pared to the proportion of eggs laid in test cups by
gravid females that were reared in lake water. The mean
proportion of eggs laid in test cups in different treat-
ments and their 95% CIs were calculated as the expo-
nential of the parameter estimates for models with no
intercept included. Similarly, multiple comparisons of
treatments were calculated based on the model param-
eter estimates.
Results
Gravid Anopheles gambiae sensu lato females discriminate
between habitats when searching for an oviposition site
Mosquitoes oviposited in the artificial ponds shortly
after they had been set up since early instar larvae were
found from day 3 and larvae hatch approximately 24–
48 hours after eggs are laid. Ponds with pellet infusion
were colonized exclusively and in high densities by culi-
cine mosquitoes. Not a single Anopheles larva was de-
tected over the 16-day observation period. In sharp
contrast, early Anopheles instar were consistently found
from day 3 to day 16 in the soil infusion ponds
(Figure 2). Based on the pattern of larval abundance,
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peak oviposition occurred six to ten days after setting up
the ponds. Anopheles nearly always occurred in higher
densities than culicines. Anopheles larval densities are
naturally relatively low in the study area, with approxi-
mately one to three larvae/dip in natural habitats [17].
In the present study an average of ten (95% CI 5–18)
early instar larvae/dip was recorded, indicating that the
soil infusion ponds were a highly favourable habitat.
All pupae collected from the artificial habitats belonged
to the An. gambiae complex. PCR-based species analysis
revealed that nearly all the wild An. gambiae s.l. were An.
arabiensis (98%, 49/50).
The female’s oviposition choice benefits the survival of
her offspring
Anopheles gambiae s.s. larvae survived equally well in
soil infusion and lake water irrespective of the age of the
infusion. In contrast, larvae placed in pellet infusion only
survived in the one-day old infusion in similar numbers
but survival was reduced by over 60% (p < 0.001) in pel-
let infusions six days and older compared to lake water
or soil infusions of the same age (Figure 3). Mean pupa-
tion time for survivors did not significantly differ be-
tween treatments or ages of the infusion and was on
average 7.5 days (95% CI 6.6-8.3).
Gravid female Anopheles gambiae sensu stricto show
avoidances and preferences when selecting an
oviposition site
Figure 4 shows the median response rate of the gravid
females to the test cup in pellet infusion and soil infu-
sion experiments. An approximately equal proportion of
females laid eggs in test and control cups when an equal
choice of lake water was provided. Fewer females laid
their eggs in pellet infusion as it aged, whilst for soil in-
fusion the opposite was the case with an increasing pro-
portion of females laying eggs in soil infusion as it aged.
0
40
80
120
160
200
240
280
320
2345678910111213141516
0
4
8
12
16
20
24
28
32
2345678910111213141516
Average number of larvae
Age of habitats in days
Culicinae Anopheles
AB
Figure 2 Natural colonization of artificial habitats. Daily average of early instar larvae in (A) pellet infusions; (B) soil infusions.
Figure 3 Survival of Anopheles gambiae sensu stricto larvae to the pupal stage kept in different infusions or lake water. Error bars show
95% confidence intervals.
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Similar results were seen for the proportions of eggs
laid although the distribution of eggs between the two
equal choices of lake water was slightly skewed (Figure 5,
Set 1, lake water control cup 0.45 (95% CI 0.34-0.56)
versus lake water test cup 0.55 (95% CI 0.44-0.66))
though not significantly different from 0.5. The distribu-
tion of eggs between lake water and two-day old pellet
infusion did not significantly differ from the distribution
between the two cups with lake water only. However,
pellet water became unattractive from day 4 (Figure 5,
Set 1). It was 6.7 times less likely for an egg to be laid in
the test cup in the treatments that contained six-day old
pellet infusion versus lake water than it was when both
cups contained lake water. During the experiment with
soil infusions a similarly skewed distribution in the pro-
portion of eggs laid in the two cups with lake water was
observed due to chance alone. (Figure 5, Set 2). In con-
trast to the pellet infusion, larger proportions of eggs
were laid in the test cups with increasing age of the
soil infusion. An egg was more than twice as likely to
be laid in the test cup in the treatments that con-
tained six-day old soil infusions compared with lake
water than it was when both cups contained lake
water (Figure 5, Set 2).
AB
Figure 4 Proportion of gravid Anopheles gambiae sensu stricto laying eggs in infusions of different ages compared with control water.
(A) Pellet infusion experiment; (B) soil infusion experiment.
Figure 5 Oviposition response of caged Anopheles gambiae sensu stricto to pellet (Set 1)and soil (Set 2) infusions of different
incubation times and non-autoclaved and autoclaved 6 day soil infusion (Set 3). Multiple comparison of treatments: treatments denoted
with the same letter are not significantly different.
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On average individual females laid 63 eggs (95% CI
60–65) (Figure 5) irrespective of the experiment and
treatment. Notably, 18% (95% CI 11-26%) of gravid fe-
males laid eggs in both cups provided in a cage, a behav-
iour known as skip oviposition in other mosquito
species [81] but rarely reported for An. gambiae s.s. [82].
The average number of eggs laid by skip-ovipositing fe-
males and by females that chose only a single cup was
similar. Whilst the percentage of skip-ovipositing fe-
males was similar in all treatments with two equal lake
water choices and in all soil infusion treatments, this be-
haviour was affected by the pellet infusion. Only a few
An. gambiae s.s. females skip-oviposited in the four- and
six-day old pellet infusion treatments (6%, 95% CI 3-9%).
Pellet and soil infusions differed in key physical and
chemical parameters. All pellet infusions had a strong
smell to the human nose, were more or less transparent
and had a slightly green colour but differed little in ap-
pearance compared with lake water in the oviposition
cups. Correspondingly, turbidity levels were low. In con-
trast, soil infusions did not have any smell to the human
nose, were light brown in colour and turbid, providing a
strong visual contrast to the lake water. Pellet infusions
were also characterized by relatively high conductivity,
low pH and oxygen deprivation. In contrast the soil infu-
sions’conductivity was approximately half that of pellet
infusions, was saturated with dissolved oxygen and had a
higher pH (Table 2). The variability of these measures
between infusions of different incubation times within a
treatment group was relatively low and does not appear
to explain the differences in the behavioural responses.
The only factor that changed over time was turbidity in
the soil infusion and notably the most preferred six-day
old infusion was less turbid than the others.
The increased carbon and total hardness of the pellet
infusion corresponded with the increased conductivity
levels and the high ammonium and phosphate levels
compared with the soil infusion (Table 2).
Chemical cues from the infusions are responsible for the
oviposition choice in cage bioassays
Since soil infusions differed in appearance from the lake
water, an additional set of experiments was carried out
to evaluate whether the attractiveness of this infusion
was due to visual cues. Cage experiments with two equal
choices of lake water confirmed an equal distribution of
eggs between control and test cup. Notably, when gravid
females had a choice between lake water and autoclaved
soil infusion, the lake water was preferred. The ovipos-
ition preference for six-day old soil infusion compared
with lake water was also confirmed in this set of experi-
ments with nearly identical odds ratios as before of 2.2.
The preference for the six-day old infusion was corrobo-
rated when given a choice between autoclaved and non-
autoclaved infusions of similar colour and turbidity
(Figure 5, Set 3).
Results from this set of experiments suggest that
chemical cues are involved in the oviposition responses
observed. If the preference for the six-day old soil infu-
sion over lake water was based on turbidity and/or
colour of the infusion alone, a similar response in the
choice tests with autoclaved versus non-autoclaved infu-
sion should have been seen as in the choice tests with
lake water versus autoclaved infusion. Due to the slight
Table 2 Physical and chemical properties of pellet and soil infusions
Parameter
Oviposition substrates in choice experiments
Lake
water
Pellet infusions Soil infusions
2 days 4 days 6 days 2 days 4 days 6 days Auto-claved
Turbidity (NTU) 1 22 17 25 222 97 73 137
(0.6–1.4) (20–23) (14–19) (20–29) (197–248) (92–102) (61–84) (108–166)
Conductivity (uS/cm) 107 477 553 543 173 207 237 266
(105–110) (462–491) (547–559) (532–555) (171–176) (203–212) (233–242) (258–274)
Dissolved oxygen (mg/l) 4 0.3 0.7 0.3 5.3 6 6.3 4.8
(2.7–5.3) (0.21–1.4) (0.9–1.4) (0.2–0.4) (5.0–5.6) (5.7–6.3) (6.0–6.6) (4.6–5.1)
pH 8.1 6.3 6.7 7.4 7.7 7.9 8 8.9
(7.9–8.1) (6.2–6.3) (6.5–6.9) (7.3–7.5) (7.6–7.7) (7.8–7.9) (7.9–8.0) (8.8–8.9)
Ammonium (mg/l) 0 5 0 0
Nitrate (mg/l) 10 10 10 10
Nitrite (mg/l) 0 0 0.025 0.025
Phosphate (mg/l) 0 3 1.5 1.5
Carbonate hardness (mmol/l) 0.1 3.9 2.7 2.5
Total hardness (mmol/l) 0.1 1.5 0.7 0.7
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avoidance of the autoclaved infusion the odds of finding
an egg in the non-autoclaved six- day old infusion
should have been approximately 1.3 (decreased choice
for autoclaved infusion by 30% or increased choice of
fresh infusion by 30%). Nevertheless, the remaining odds
of 2.2 can only be explained by chemical cues being
either involved in attracting the female from a short dis-
tance or stimulating the female to lay eggs on contact
with water. Physical and chemical water parameters were
similar for autoclaved and non-autoclaved soil infusions.
Bacteria cultures from autoclaved infusions confirmed
that samples did not contain any bacteria that could
grow on LB plates as opposed to the non-autoclaved in-
fusion where colonies of at least three different morph-
ologies were observed.
The oviposition choice of the gravid female is not
influenced by her olfactory memory of her larval habitat
Rearing An. gambiae s.s. in two- to four-day old pellet
infusion did not alter their oviposition response towards
the infusion (p = 0.392). Gravid females reared in lake
water and gravid females reared in pellet infusion show
an equally strong avoidance of the six-day old pellet in-
fusion provided in choice experiments (Figure 6, Set 4).
Discussion
The results confirm that wild and caged An. gambiae s.l.
females discriminate (defined as recognition and under-
standing of the difference between two things [83]) be-
tween potential aquatic habitats for oviposition and
make clear choices when presented with contrasting ovi-
position media. These choices benefit the survival of the
offspring. Although the experimental design does not
allow a conclusion whether the stimuli acted over a dis-
tance (attractants and repellents [84]) or on contact with
the oviposition medium (stimulants and deterrents [85]),
it could be demonstrated that the choice of breeding site
is guided by both avoidance and preference. However,
the exclusive way in which the artificial ponds were
chosen in the field, where they were set up relatively
close to each other, suggest that these characteristics
were detected by both culicines and anophelines from a
distance rather than on contact.
Muddy water has previously been suggested to in-
crease oviposition response of gravid An. gambiae s.s. in
cages when offered together with clear water [41], how-
ever in this study cage experiments could not confirm
this observation. The difference in turbidity between
lake water and infusions cannot explain the avoidance
and preference observed at short range in the cages.
Two-day and six-day old pellet infusions did not differ
in their turbidity but significantly differed in the ovipos-
ition response they received. Similarly, all soil infusions
should have elicited equally strong responses from
gravid females if turbidity was an important oviposition
cue at short range. On the contrary, the six-day old soil
infusion remained equally preferred for oviposition when
tested against a turbid and autoclaved infusion than
when tested against clear lake water. The results suggest
that chemical and not visual cues were responsible for
the responses observed in the cage experiments. The
previously published preference of muddy water [41]
may have been based on chemical cues associated with
the muddy water which was taken from a natural habi-
tat. However the possibility that visual cues played a
role in the selection of oviposition sites by wild mosqui-
toes in field experiments especially when searching
for water bodies from a distance cannot be entirely
excluded.
It is likely that the chemical cues used by mosquitoes
to avoid pellet infusions and to prefer soil infusions were
at least partly of microbial origin, which is supported by
the lack of attraction to the autoclaved soil infusion
compared to clear lake water. It is most likely that the
observed oviposition choices as well as the larval sur-
vival were rooted in the water quality of the habitat and
consequently the associated micro-organisms and che-
micals in the water. The physical and chemical water pa-
rameters measured for the two infusions suggest that
they represented aquatic habitats in different stages of
decay. Contrary to the expectation at the onset of the
experiment, pellet infusions created with increasing age
a habitat type in a severe state of decomposition. High
ammonia and phosphate levels are characteristic of re-
cently inundated organic material [86]. The odour of the
pellet infusion is associated with fermentation of organic
material by facultative and anaerobic bacteria. This leads
to depletion of the oxygen supply and a decrease in pH
as a result of accumulation of organic acids in the water
[87-89]. The soil infusion had the characteristics of a less
nutrient-rich habitat containing relatively little organic
matter. This limits the removal of oxygen by aerobic het-
erotrophic micro-organisms and hence the water col-
umn will stay aerobic [89].
Figure 6 Egg laying responses of Anopheles gambiae sensu
stricto reared in lake water or in pellet infusion to lake water
and pellet infusion (Set 4).
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Anaerobic fermentation products of organic matter
have been previously shown to be highly attractive to a
number of gravid culicine species such as Culex stigma-
tosoma (formerly peus) [87], Culex quinquefasciatus
[33,65,90-92], Culex pipiens [64], Aedes aegypti [77,93]
and Aedes albopictus [94,95]. These infusions have been
associated with a range of bacteria such as Aerobacter
aerogenes, Pseudomona aeruginosa,Bacillus cereus [96-98]
and volatile chemicals produced by them including
4- methylphenol, 3-methylindole, carboxylic acids and me-
thyl esters [33,39,99,100]. It is likely that similar factors
were responsible for the strong repellent/deterrent effect
on gravid An. gambiae s.l. females.
Most stagnant water bodies will show increasing signs
of decomposition over time but the speed and extent
of this will depend largely on habitat quality [101].
Therefore, it is argued that habitat age or permanence
alone is not a good predictor for the oviposition re-
sponse of An. gambiae s.l. as has been suggested [45].
For example, the content and input of organic matter,
source of water and frequency of fresh water inflow will
affect the composition of the biotic community and
chemical and physical characteristics of an aquatic habi-
tat [86,89,101]. This might explain why in some environ-
ments semi-permanent and permanent habitats are just
as well colonized as temporary habitats traditionally
thought to be the preferred An. gambiae s.l. habitats
[13,17,67]. Habitats made of pellet infusion were avoided
by Anopheles from an early habitat age, whilst interest-
ingly, the highest preference of the soil infusion was re-
corded on day 6 in the laboratory and between day 6
and 10 in the field after the habitats were well estab-
lished, contradicting the idea that An. gambiae s.l. is a
pioneer species colonizing temporary habitats immedi-
ately after their occurrence [13].
Typically, it is reported that An. gambiae s.l., although
largely a generalist, is not found in heavily polluted wa-
ters [19,102]. Hancock [103] further observed that An.
gambiae s.l. avoided water with a low pH when it was
also accompanied with high organic matter content.
Addition of freshly cut vegetation (i e, grass cuttings) to
aquatic habitats has also been shown to prevent the lar-
val development of An. gambiae s.l. [16]. The results
from the experiments with pellet infusions support these
observations. On the other hand, there have been recent
reports of An. gambiae s.l. colonizing polluted habitats
especially in urban areas [104-106]. Clearly, the degree
of avoidance or acceptance of a polluted habitat by An.
gambiae s.l. depends on the extent and nature of pollu-
tion [19]. Results show that two-day old pellet infusions
were not rejected by Anopheles and even four-day old
infusions still received a considerable proportion of the
oviposition responses despite their adverse water charac-
teristics. This supports the idea that An. gambiae s.l. has
a very high tolerance level of what they accept as ovipos-
ition sites, especially in the absence of better alternatives
in close vicinity as is often the case in urban environ-
ments and in contrast to the here presented field experi-
ment where good habitats were offered right next to the
unfavoured ones.
Importantly, An. gambiae s.l. appears to have an innate
propensity to avoid specific chemical cues that were
emitted from the pellet infusion. Rearing An. gambiae s.
s. from egg to pupae in this infusion did not alter this
behaviour. Gravid females that had experienced the pel-
let infusion during larval development avoided the infu-
sion for oviposition as much as the females that had no
prior experience of it. This suggests that the environ-
ment in which An. gambiae s.s. develop as larvae does
not determine the preferred oviposition site when they
return to lay eggs. This is in contrast to published work
on Cx. quinquefasciatus where it was demonstrated that
rearing the larvae in an infusion made from guinea-pig fae-
ces cancelled their innate preference for a hay infusion [4].
The cage bioassays with individual gravid females
allowed a number of interesting observations that are
rarely reported since the majority of studies with An.
gambiae s.s. are done with groups of mosquitoes where
the actual number of females laying per cage is unknown
[41]. The occurrence of skip-oviposition in gravid An.
gambiae s.s. and how this is affected by chemical cues
was demonstrated. Furthermore, the design revealed that
the mean number of eggs laid per female in a cage was
similar irrespective of the experiment and treatment; only
the distribution between cups differed when two different
choices were presented. This indicates that gravid females
did not retain their eggs in the presence of an unfavoured
substrate when they were offered a suitable alternative
choice. It also shows that the preferred soil infusion did
not stimulate individual females to lay more eggs than
they would do in lake water. Testing individual females
also excludes potential aggregation effects. Whilst from
the field experiments it might have been possible that
gravid females selected habitats that already received eggs
from conspecific females, cage bioassays with individual
females showed the same avoidance and preference behav-
iour as observed in the field, confirming that conspecifics
alone cannot explain the observed choice.
The potential involvement of microbial activity in break-
ing down organic matter and producing semiochemicals
that impact on the oviposition responses of gravid An.
gambiae s.s. was deduced partly by the lack of attraction
of An. gambiae s.s. to a sterile soil infusion. However, this
must be interpreted with caution since autoclaving the in-
fusion might not only have killed the microbes but af-
fected the chemistry of the resulting infusion, possibly
altering the response of gravid mosquitoes by chemical
changes rather than biological changes [77].
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Batch-to-batch variations were recorded in the re-
sponse of gravid mosquitoes to the infusions, resulting,
for example, in some rounds showing a high preference
and other rounds only a moderate preference for the soil
infusion. This variation can be attributed to differences
in the quality and amounts of odorants released from
the infusions and stochastic events. Fresh infusions were
prepared for every test round with different batches of
pellets and soil. Especially, for the soil it is highly likely
that there were differences in the soil condition as well
as differences in the species composition of the micro-
bial community associated with the natural materials
over time. It has been shown previously that natural infu-
sions can be an inconsistent source of odorants for ovipos-
ition site-seeking mosquitoes and therefore every batch
needs to be verified to be behaviourally active before it can
be used for subsequent experiments [77]. Ideally, if info-
chemicals were to be used for monitoring and/or control-
ling gravid malaria vectors, specific chemically defined
oviposition cues would be preferred over natural infusions
to ensure a consistent response in gravid females either
pushing them away from human population [107,108] or
pulling them towards a gravid trap [38,109].
Whilst the observed avoidance behaviour towards the or-
ganically rich pellet infusion was strong and in the same
range as reported for other species in response to un-
favourable chemical cues [107,108,110], the observed pref-
erence in the cages for the soil infusion was relatively weak
and it is questionable whether it could compete with other
suitable habitats from a larger distance. Nevertheless, con-
sistent response derived from over 150 replicates in two
experiments likely represents a genuine effect. Further in-
vestigations are in progress to characterize the bacteria
communities associated with the infusions and the volatile
chemicals emitted from the infusions and detected by
gravid An. gambiae s.s. using gas-chromatography coupled
to mass-spectrometry and coupled gas chromatography-
electroantennogram detection.
It must be cautioned that not all soils and all rabbit
food pellets will lead to the same physical and chemical
parameters as the infusions presented here. Therefore
the two infusions of this study only serve as specific ex-
amples for two highly contrasting media. Further work
is needed to screen other soil samples to see if the ob-
served response is a response common for all soil infu-
sions prepared under standard conditions and if the
same bacteria and chemical profiles can be detected, or,
which is more likely, that there are significant differences
depending on the source of the soil.
Conclusion
This work illustrates that a gravid An. gambiae s.l. female
selects a suitable habitat for oviposition using chemical
cues from water bodies. It furthermore emphasizes that
natural infusions can be used to manipulate the ovipos-
ition behaviour of An. gambiae s.l.. Soil infusions have the
potential to be used to bait gravid traps for the collection
of An. gambiae s.l., although further work must be imple-
mented to elucidate whether the observed preference was
based on the specific soil type tested or whether similar
responses can be achieved with any soil. The low An. gam-
biae s.l. catching efficacy reported for gravid traps oper-
ationally used for Culex and Aedes monitoring might
partly be explained by the infusions routinely used in these
traps, i e, fermented hay infusions, rabbit food pellet and
cow manure infusions [64-112]. The identification of the
chemicals responsible for the preference of the soil infu-
sion might be exploited to bait gravid traps specifically for
the collection of An. gambiae s.l.
Competing interests
The authors declare that they have no competing interests.
Authors’contributions
UF, JL, MH-V and SWL conceived the idea for this research and developed
the experimental design. MH-V developed all protocols and implemented
the experiments. MH-V and UF analysed the data and wrote the first draft of
the manuscript. All authors contributed to the final draft, read and approved
the manuscript.
Acknowledgements
We thank David Alila, Elisha Obudho and Benard Oyembe for providing
mosquitoes for this study and Paul Ouma, Elizabeth Masinde, Gregory
Masinde and Rose Atieno for their technical support. We also extend our
gratitude to Benedict Orindi for statistical advice. MH-V was supported
by the Colombian Department of Science, Technology and Innovation
(COLCIENCIAS) through the Scholarship programme “Francisco Jose de
Caldas”. This project received funding from the National Institute of Health
(NIH) through grant no. R01AI082537.
Author details
1
Department of Diseases Control, London School of Hygiene and Tropical
Medicine, London, UK.
2
International Centre for Insect Physiology and
Ecology (icipe)-Thomas Odhiambo Campus, Mbita, Kenya.
3
Royal Institute of
Technology, Stockholm, Sweden.
4
School of Biological and Biomedical
Sciences, Durham University, Durham, UK.
Received: 5 February 2014 Accepted: 27 March 2014
Published: 2 April 2014
References
1. Refsnider JM, Janzen FJ: Putting eggs in one basket: ecological and
evolutionary hypotheses for variation in oviposition-site choice. Annu Rev
Ecol Evol Syst 2010, 41:39–57.
2. Rejmankova E, Higashi R, Grieco J, Achee N, Roberts D: Volatile substances
from larval habitats mediate species-specific oviposition in Anopheles
mosquitoes. J Med Entomol 2005, 42:95–103.
3. Morris DW: Toward an ecological synthesis: a case for habitat selection.
Oecologia 2003, 136:1–13.
4. McCall PJ, Eaton G: Olfactory memory in the mosquito Culex
quinquefasciatus.Med Vet Entomol 2001, 15:197–203.
5. Laird M: The natural history of larval mosquito habitats. London: Academic
Press; 1988.
6. Muirhead-Thomson RC: Studies on the breeding places and control of
Anopheles gambiae and A. gambiae var. melas in coastal districts of
Sierra Leone. Bull Entomol Res 1945, 38:527–558.
7. Macan TT: Factors that limit the range of freshwater animals. Biol Rev
Camb Philos Soc 1961, 36:151–198.
8. Bates M: Oviposition experiments with anopheline mosquitoes. Am J Trop
Med Hyg 1940, 20:569–583.
Herrera-Varela et al. Malaria Journal 2014, 13:133 Page 12 of 15
http://www.malariajournal.com/content/13/1/133
9. Muirhead-Thomson RC: Studies on the behaviour of Anopheles minimus.
Part I. The selection of the breeding place and the influence of light and
shade. J Malar Inst India 1940, 3:265–294.
10. Muirhead-Thomson RC: Studies on the behaviour of Anopheles minimus.
Part II. The influence of water movement on the selection of the
breeding place. J Malar Inst India 1940, 3:295–322.
11. Wallis RC: A study of oviposition activity of mosquitoes. Am J Hyg 1954,
60:135–168.
12. Bentley MD, Day JF: Chemical ecology and behavioral aspects of
mosquito oviposition. Annu Rev Entomol 1989, 34:401–421.
13. Gillies MT, De Meillon B: The Anophelinae of Africa South of the Sahara.
Johannesburg: Publications of the South African Institute of Medical
Research; 1968:54.
14. Muirhead Thomson RC: Mosquito Behaviour in Relation to Malaria
Transmission and Control in the Tropics. London: Edward Arnold & Co; 1951.
15. Gimnig JE, Ombok M, Kamau L, Hawley WA: Characteristics of larval
Anopheline (Diptera: Culicidae) habitats in western Kenya. J Med Entomol
2001, 38:282–288.
16. Minakawa N, Sonye G, Yan G: Relationships between occurrence of
Anopheles gambiae s.l. (Diptera: Culicidae) and size and stability of larval
habitats. J Med Entomol 2005, 42:295–300.
17. Fillinger U, Sonye G, Killeen GF, Knols BG, Becker N: The practical
importance of permanent and semipermanent habitats for controlling
aquatic stages of Anopheles gambiae sensu lato mosquitoes: operational
observations from a rural town in western Kenya. Trop Med Int Health
2004, 9:1274–1289.
18. Fillinger U, Lindsay SW: Larval source management for malaria control in
Africa: myths and reality. Malar J 2011, 10:353.
19. Holstein MH: Biology of Anopheles gambiae: Research in French West Africa.
World Health Organization (Geneva); 1954.
20. Majambere S, Fillinger U, Sayer DR, Green C, Lindsay SW: Spatial
distribution of mosquito larvae and the potential for targeted larval
control in The Gambia. Am J Trop Med Hyg 2008, 79:19–27.
21. Fillinger U, Sombroek H, Majambere S, van Loon E, Takken W, Lindsay SW:
Identifying the most productive breeding sites for malaria mosquitoes in
The Gambia. Malar J 2009, 8:62.
22. Munga S, Yakob L, Mushinzimana E, Zhou G, Ouna T, Minakawa N, Githeko
A, Yan G: Land use and land cover changes and spatiotemporal
dynamics of anopheline larval habitats during a four-year period in a
highland community of Africa. Am J Trop Med Hyg 2009, 81:1079–1084.
23. Ndenga BA, Simbauni JA, Mbugi JP, Githeko AK, Fillinger U: Productivity
of malaria vectors from different habitat types in the western Kenya
highlands. PLoS 2011, 6:e19473.
24. Muirhead-Thomson RC: Studies on salt-water and fresh-water
Anopheles gambiae on the east african coast. Bull Entomol Res
1951, 41:487–502.
25. Mutuku FM, Bayoh MN, Gimnig JE, Vulule JM, Kamau L, Walker ED, Kabiru E,
Hawley WA: Pupal habitat productivity of Anopheles gambiae complex
mosquitoes in a rural village in western Kenya. Am J Trop Med Hyg 2006,
74:54–61.
26. Ferguson HM, Dornhaus A, Beeche A, Borgemeister C, Gottlieb M, Mulla MS,
Gimnig JE, Fish D, Killeen GF: Ecology: a prerequisite for malaria
elimination and eradication. PLoS Med 2010, 7:e1000303.
27. Laurence BR, Pickett JA: erythro-6-Acetoxy-5-hexadecanolide, the major
component of a mosquito oviposition attractant pheromone. J Chem Soc
Chem Commun 1982, 0:59–60.
28. Beehler JW, Millar JG, Mulla MS: Protein hydrolysates and associated
bacterial contaminants as oviposition attractants for the mosquito Culex
quinquefasciatus.Med Vet Entomol 1994, 8:381–385.
29. Beehler JW, Millar JG, Mulla MS: Field evaluation of synthetic compounds
mediating oviposition in Culex mosquitoes (Diptera: Culicidae). J Chem
Ecol 1994, 20:281–291.
30. Isoe J, Beehler JW, Millar JG, Mulla MS: Oviposition responses of Culex
tarsalis and Culex quinquefasciatus to aged Bermuda grass infusions.
J Am Mosq Control Assoc 1995, 11:39–44.
31. Isoe J, Millar JG: Characterization of factors mediating oviposition site
choice by Culex tarsalis.J Am Mosq Control Assoc 1995, 11:21–28.
32. Millar JG, Chaney JD, Beehler JW, Mulla MS: Interaction of the Culex
quinquefasciatus egg raft pheromone with a natural chemical
associated with oviposition sites. J Am Mosq Control Assoc 1994,
10:374–379.
33. Millar JG, Chaney JD, Mulla MS: Identification of oviposition attractants
for Culex quinquefasciatus from fermented Bermuda grass infusions.
J Am Mosq Control Assoc 1992, 8:11–17.
34. Bentley MD, McDaniel IN, Yatagai M, Lee HP, Maynard R: p-Cresol:
an oviposition attractant of Aedes triseriatus.Environ Entomol 1979,
8:206–209.
35. Hwang Y, Schultz GW, Axelrod H, Kramer WL, Mulla MS: Ovipositional
repellency of fatty acids and their derivatives against Culex and Aedes
mosquitoes. Environ Entomol 1982, 11:223–226.
36. Mendki MJ, Ganesan K, Prakash S, Suryanarayana MVS, Malhotra RC, Rao KM,
Vaidyanathaswamy R: Heneicosane: an oviposition-attractant pheromone
of larval origin in Aedes aegypti mosquito. Curr Sci 2000, 78:1295–1296.
37. Ganesan K, Mendki MJ, Suryanarayana MVS, Prakash S, Malhotra RC: Studies
of Aedes aegypti (Diptera: Culicidae) ovipositional responses to newly
identified semiochemicals from conspecific eggs. Aust J Entomol 2006,
45:75–80.
38. Seenivasagan T, Sharma KR, Sekhar K, Ganesan K, Prakash S, Vijayaraghavan
R: Electroantennogram, flight orientation, and oviposition responses of
Aedes aegypti to the oviposition pheromone n-heneicosane. Parasitol Res
2009, 104:827–833.
39. Ponnusamy L, Xu N, Nojima S, Wesson DM, Schal C, Apperson CS:
Identification of bacteria and bacteria-associated chemical cues that
mediate oviposition site preferences by Aedes aegypti.Proc Natl Acad Sci
USA 2008, 105:9262–9267.
40. Ponnusamy L, Wesson DM, Arellano C, Schal C, Apperson CS: Species
composition of bacterial communities influences attraction of
mosquitoes to experimental plant infusions. Microb Ecol 2010, 59:158–173.
41. McCrae AW: Oviposition by African malaria vector mosquitoes II. Effects
of site tone, water type and conspecific immatures on target selection
by freshwater Anopheles gambiae Giles, sensu lato. Ann Trop Med Parasitol
1984, 78:307–318.
42. Kennedy J: On water-finding and oviposition by captive mosquitoes.
Bull Entomol Res 1942, 32:279–301.
43. Gu W, Regens JL, Beier JC, Novak RJ: Source reduction of mosquito larval
habitats has unexpected consequences on malaria transmission.
Proc Natl Acad Sci USA 2006, 103:17560–17563.
44. Gouagna LC, Rakotondranary M, Boyer S, Lemperiere G, Dehecq JS,
Fontenille D: Abiotic and biotic factors associated with the presence of
Anopheles arabiensis immatures and their abundance in naturally
occurring and manmade aquatic habitats. Parasit Vectors 2012, 5:96.
45. Munga S, Vulule J, Kweka EJ: Response of Anopheles gambiae s.l. (Diptera:
Culicidae) to larval habitat age in western Kenya highlands. Parasit Vectors
2013, 6:13.
46. Kweka EJ, Zhou G, Munga S, Lee MC, Atieli HE, Nyindo M, Githeko AK, Yan
G: Anopheline larval habitats seasonality and species distribution: a
prerequisite for effective targeted larval habitats control programmes.
PLoS One 2012, 7:e52084.
47. Sumba LA, Guda TO, Deng AL, Hassanali A, Beier JC, Knols BGJ: Mediation
of oviposition site selection in the African malaria mosquito Anopheles
gambiae (Diptera: Culicidae) by semiochemicals of microbial origin.
Int J Trop Insect Sci 2004, 24:260–265.
48. Sumba LA, Ogbunugafor CB, Deng AL, Hassanali A: Regulation of
oviposition in Anopheles gambiae s.s.: role of inter- and intra-specific
signals. J Chem Ecol 2008, 34:1430–1436.
49. Huang J, Walker ED, Vulule J, Miller JR: The influence of darkness and
visual contrast on oviposition by Anopheles gambiae in moist and dry
substrates. Physiol Entomol 2001, 32:34–40.
50. Huang J, Walker ED, Giroux PY, Vulule J, Miller JR: Ovipositional site
selection by Anopheles gambiae: influences of substrate moisture and
texture. Med Vet Entomol 2005, 19:442–450.
51. Huang J, Walker ED, Otienoburu PE, Amimo F, Vulule J, Miller JR: Laboratory
tests of oviposition by the African malaria mosquito, Anopheles gambiae,
on dark soil as influenced by presence or absence of vegetation. Malar J
2006, 5:88.
52. Kweka EJ, Owino EA, Mwang’onde BJ, Mahande AM, Nyindo M, Mosha F:
The role of cow urine in the oviposition site preference of culicine and
Anopheles mosquitoes. Parasit Vectors 2011, 4:184.
53. Lindh JM, Kannaste A, Knols BG, Faye I, Borg-Karlson AK: Oviposition re-
sponses of Anopheles gambiae s.s. (Diptera: Culicidae) and identification
of volatiles from bacteria-containing solutions. J Med Entomol 2008,
45:1039–1049.
Herrera-Varela et al. Malaria Journal 2014, 13:133 Page 13 of 15
http://www.malariajournal.com/content/13/1/133
54. Balestrino F, Soliban SM, Gilles J, Oliva C, Benedict MQ: Ovipositional
behavior in the context of mass rearing of Anopheles arabiensis.
J Am Mosq Control Assoc 2010, 26:365–372.
55. Paaijmans KP, Takken W, Githeko AK, Jacobs AF: The effect of water
turbidity on the near-surface water temperature of larval habitats of the
malaria mosquito Anopheles gambiae.Int J Biometeorol 2008, 52:747–753.
56. Ye-Ebiyo Y, Pollack RJ, Kiszewski A, Spielman A: Enhancement of
development of larval Anopheles arabiensis by proximity to flowering
maize (Zea mays) in turbid water and when crowded. Am J Trop Med Hyg
2003, 68:748–752.
57. Okal MN, Francis B, Herrera-Varela M, Fillinger U, Lindsay SW: Water vapour
is a pre-oviposition attractant for the malaria vector Anopheles gambiae
sensu stricto.Malar J 2013, 12:365.
58. Huang J, Miller JR, Chen SC, Vulule JM, Walker ED: Anopheles gambiae
(Diptera: Culicidae) oviposition in response to agarose media and
cultured bacterial volatiles. J Med Entomol 2006, 43:498–504.
59. Blackwell A, Johnson SN: Electrophysiological investigation of larval water
and potential oviposition chemo-attractants for Anopheles gambiae s.s.
Ann Trop Med Parasitol 2000, 94:389–398.
60. Rinker DC, Pitts RJ, Zhou X, Suh E, Rokas A, Zwiebel LJ: Blood meal-induced
changes to antennal transcriptome profiles reveal shifts in odor sensitiv-
ities in Anopheles gambiae.Proc Natl Acad Sci USA 2013, 110:8260–8265.
61. Haeger J, Provost M: The laboratory colonization of Opifex. Bull World
Health Organ 1964, 31:451–452.
62. Rasgon JL: Wolbachia induces male-specific mortality in the mosquito
Culex pipiens (LIN strain). PLoS One 2012, 7:e30381.
63. Nguyen TT, Su T, Mulla MS: Bacteria and mosquito abundance in
microcosms enriched with organic matter and treated with a Bacillus
thuringiensis subsp. israelensis formulation. J Vector Ecol 1999, 24:191–201.
64. Jackson BT, Paulson SL, Youngman RR, Scheffel SL, Belinda H: Oviposition
preferences of Culex restuans and Culex pipiens (Diptera: Culicidae) for
selected infusions in oviposition traps and gravid traps. J Am Mosq
Control Assoc 2005, 21:360–365.
65. McPhatter LP, Debboun M: Attractiveness of botanical infusions to
ovipositing Culex quinquefasciatus,Cx. nigripalpus, and Cx. erraticus in
San Antonio, Texas. J Am Mosq Control Assoc 2009, 25:508–510.
66. Silver JB: Mosquito Ecology: Field Sampling Methods. London: Springer; 2008.
67. MinakawaN,DidaGO,SonyeGO,FutamiK,NjengaSM:Malaria vectors in Lake
Victoria and adjacent habitats in Western Kenya. PLoS One 2012, 7:e32725.
68. Gillies MT: A modified technique for the age-grading of populations
of Anopheles gambiae.Ann Trop Med Parasitol 1958, 52:261–273.
69. Lyimo EO, Takken W: Effects of adult body size on fecundity and the
pre-gravid rate of Anopheles gambiae females in Tanzania. Med Vet
Entomol 1993, 7:328–332.
70. Fillinger U, Knols BG, Becker N: Efficacy and efficiency of new Bacillus
thuringiensis var israelensis and Bacillus sphaericus formulations against
Afrotropical anophelines in Western Kenya. Trop Med Int Health 2003,
8:37–47.
71. Brown RB: Soil texture. http://edis.ifas.ufl.edu/pdffiles/SS/SS16900.pdf.
72. Whiting D, Card A, Wilson C, Reeder J: Estimating soil texture: sand, silt or
clay? http://www.ext.colostate.edu/mg/gardennotes/214.html.
73. Gillies MT, Coetzee M: A supplement to the anophelinae of Africa South of the
Sahara (Afrotropical Region). Johannesburg: South African Institute for
Medical Research; 1987.
74. Scott JA, Brogdon WG, Collins FH: Identification of single specimens of
the Anopheles gambiae complex by the polymerase chain reaction.
Am J Trop Med Hyg 1993, 49:520–529.
75. Knols BG, Njiru BN, Mathenge EM, Mukabana WR, Beier JC, Killeen GF:
MalariaSphere: a greenhouse-enclosed simulation of a natural Anopheles
gambiae (Diptera: Culicidae) ecosystem in western Kenya. Malar J 2002, 1:19.
76. Ponnusamy L, Boroczky K, Wesson DM, Schal C, Apperson CS: Bacteria stimulate
hatching of yellow fever mosquito eggs. PLoS One 2011, 6:e24409.
77. Ponnusamy L, Xu N, Boroczky K, Wesson DM, Abu Ayyash L, Schal C,
Apperson CS: Oviposition responses of the mosquitoes Aedes aegypti and
Aedes albopictus to experimental plant infusions in laboratory bioassays.
J Chem Ecol 2010, 36:709–719.
78. Bertani G: Lysogeny at mid-twentieth century: P1, P2 and other experi-
mental systems. J Bacteriol 2004, 186:595–600.
79. Kaur JS, Lai YL, Giger AD: Learning and memory in the mosquito Aedes
aegypti shown by conditioning against oviposition deterrence. Med Vet
Entomol 2003, 17:457–460.
80. Development Core Team R: R: A language and enviroment for statistical
computing. pp. http://www.R-project.org/. Vienna, Austria: R Foundation for
statistical Computing; 2011:http://www.R-project.org/.
81. Colton YM, Chadee DD, Severson DW: Natural skip oviposition of the
mosquito Aedes aegypti indicated by codominant genetic markers. Med
Vet Entomol 2003, 17:195–204.
82. Chen H, Fillinger U, Yan G: Oviposition behavior of female Anopheles
gambiae in western Kenya inferred from microsatellite markers. Am J
Trop Med Hyg 2006, 75:246–250.
83. Oxford-Dictionaries: Discriminate [Def.2]. http://oxforddictionaries.com/
definition/english/discrimination.
84. Dethier VG, Barton BL, Smith CN: The designation of chemicals in terms of
the responses they elicit from Insects. J Econ Entomol 1960, 53:134–136.
85. Isoe J, Millar JG, Beehler JW: Bioassays for Culex (Diptera: Culicidae)
mosquito oviposition attractants and stimulants. J Med Entomol 1995,
32:475–483.
86. Palmer R: Classification and ecological status of aquatic ecosystems in the
eastern Caprivi, Namibia. Windhoek: Ministry of Agriculture, Water and Rural
Development; 2002.
87. Gerhardt RW: The influence of soil fermentation on oviposition site
selection by mosquitoes. Mosq News 1959, 19:151–155.
88. Cunha A, Almeida A, Coelho FJRC, Gomes NCM, Oliveira V, Santos AL:
Current Research, Technology and Education Topics in Applied Microbiology
and Microbial Biotechnology. Bajadoz: Formatex Research Center; 2010.
89. Sigee DC: Fresh water microbiology: biodiversity and dynamic interactions of
microorganisms in the aquatic environment. England: John Wiley & Sons Ltd; 2005.
90. Burkett-Cadena ND, Mullen GR: Field comparison of Bermuda-hay infusion
to infusions of emergent aquatic vegetation for collecting female mos-
quitoes. J Am Mosq Control Assoc 2007, 23:117–123.
91. Mboera LEG, Mdira KY, Salum FM, Takken W, Pickett JA: Influence of
synthetic oviposition pheromone and volatiles from soakage pits and
grass infusions upon oviposition site-selection of Culex mosquitoes in
Tanzania. J Chem Ecol 1999, 25:1855–1865.
92. Kramer WL, Mulla MS: Oviposition attractants and repellents of
mosquitoes: oviposition responses of Culex mosquitoes to organic
infusions. Environ Entomol 1979, 8:1111–1117.
93. Santana AL, Roque RA, Eiras AE: Characteristics of grass infusions as
oviposition attractants to Aedes (Stegomyia) (Diptera: Culicidae). J Med
Entomol 2006, 43:214–220.
94. Zhang LY, Lei CL: Evaluation of sticky ovitraps for the surveillance of
Aedes (Stegomyia) albopictus (Skuse) and the screening of oviposition
attractants from organic infusions. Ann Trop Med Parasitol 2008,
102:399–407.
95. Trexler JD, Apperson CS, Schal C: Laboratory and field evaluations of
oviposition responses of Aedes albopictus and Aedes triseriatus (Diptera:
Culicidae) to oak leaf infusions. J Med Entomol 1998, 35:967–976.
96. Hazard EI, Mayer MS, Savage KE: Attraction and oviposition stimulation
of gravid female mosquitoes by bacteria isolated from hay infusions.
Mosq News 1967, 27:133–136.
97. Ikeshoji T, Saito K, Yano A: Bacterial production of the ovipositional
attractants for mosquitoes on fatty acid substrates. Appl Entomol Zool
1975, 10:239–242.
98. Hasselschwert D, Rockett CL: Bacteria as ovipositional attractant for Aedes
Aegypti (Diptera:Culicidae). Great Lakes Entomol 1988, 21:163–168.
99. Allan SA, Kline DL: Evaluation of organic infusions and synthetic
compounds mediating oviposition in Aedes albopictus and Aedes aegypti
(Diptera: Culicidae). J Chem Ecol 1995, 21:1847–1860.
100. Trexler JD, App erson CS, Gemeno C, Perich MJ, Carlson D, Schal C: Field
and laboratory evaluations of potential oviposition attractants for
Aedes albopictus (Diptera: Culicidae). J Am Mosq Control Assoc 2003,
19:228–234.
101. Ruppel RE, Setty KE, Wu M: Decomposition rates of Typha spp. in northern
freshwater wetlands over a stream–marsh–peatland gradient.
Sci Discipulorum 2004, 1:26–37.
102. Symes CB: Malaria in Nairobi. East Afr Med J 1940, 17:332–355.
103. Hancock GLR: Some records of Uganda mosquitoes and oecological
associations of their larvae. Bull Soc Entomol Egypte 1930, 1:38–56.
104. Castro MC, Kanamori S, Kannady K, Mkude S, Killeen GF, Fillinger U: The
importance of drains for the larval development of lymphatic filariasis and
malaria vectors in Dar es Salaam, United Republic of Tanzania. PLoS Negl Trop
Dis 2010, 4:e693.
Herrera-Varela et al. Malaria Journal 2014, 13:133 Page 14 of 15
http://www.malariajournal.com/content/13/1/133
105. Kudom AA, Mensah BA, Agyemang TK: Characterization of mosquito larval
habitats and assessment of insecticide-resistance status of Anopheles
gambiae senso lato in urban areas in southwestern Ghana. J Vector Ecol
2012, 37:77–82.
106. Awolola TS, Oduola AO, Obansa JB, Chukwurar NJ, Unyimadu JP: Anopheles
gambiae s.s. breeding in polluted water bodies in urban Lagos,
southwestern Nigeria. J Vector Borne Dis 2007, 44:241–244.
107. Seenivasagan T, Sharma KR, Ganesan K, Prakash S: Electrophysiological,
flight orientation and oviposition responses of three species of mosquito
vectors to hexadecyl pentanoate: residual oviposition repellent activity.
J Med Entomol 2010, 47:329–337.
108. Siriporn P, Mayura S: The effects of herbal essential oils on the
oviposition-deterrent and ovicidal activities of Aedes aegypti (Linn.),
Anopheles dirus (Peyton and Harrison) and Culex quinquefasciatus (Say).
Trop Biomed 2012, 29:138–150.
109. Seenivasagan T, Sharma KR, Prakash S: Electroantennogram, flight
orientation and oviposition responses of Anopheles stephensi and Aedes
aegypti to a fatty acid ester-propyl octadecanoate. Acta Trop 2012,
124:54–61.
110. Tennyson S, Ravindran KJ, Eapen A, William SJ: Effect of Ageratum
houstonianum Mill. (Asteraceae) leaf extracts on the oviposition activity
of Anopheles stephensi,Aedes aegypti and Culex quinquefasciatus
(Diptera: Culicidae). Parasitol Res 2012, 111:2295–2299.
111. Lewis LF, Clark TB, O’Grady JJ, Christenson DM: Collecting ovigerous Culex
pipiens quinquefasciatus Say near favorable resting sites with louvered
traps baited with infusions of alfalfa pellets. Mosq News 1974, 34:436–439.
112. Lampman RL, Novak RJ: Oviposition preferences of Culex pipiens and
Culex restuans for infusion-baited traps. J Am Mosq Control Assoc 1996,
12:23–32.
doi:10.1186/1475-2875-13-133
Cite this article as: Herrera-Varela et al.:Habitat discrimination by gravid
Anopheles gambiae sensu lato –a push-pull system. Malaria Journal
2014 13:133.
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