Malaria mosquitoes use host odours to find their blood sources, but little is known about the semiochemicals that mediate this behaviour. A combined study is undertaken to identify the volatile human-specific compounds that are used in the host-seeking behaviour of the females of Anopheles gambia

Beek, T.A., van

Galimard, A.M.S.

Loon, J.A., van

Posthumus, M.A.

Qiu, Y.T.

Smallegange, R.C.

Takken, W.

Name not available

Why humans are attractive to malaria mosquitoes

Wageningen Yield 

50 Entomologische Berichten 63(3) 2003
Key-words: Anopheles gambiae s.s., malaria mosquito,
odours, behaviour, electrophysiology, chemical analysis
Worldwide between 300 and 500 million people suffer from
malaria and over one million people, especially children un-
der the age of five and pregnant women, die as a result of
this disease each year. Most of the malaria cases occur in
sub-Saharan Africa and these result in about 90% of global
mortality due to malaria. In this region, the females of the
mosquito Anopheles gambiae Giles sensu stricto (Diptera:
Culicidae; figure 1) are the principle vectors of the parasite
Plasmodium falciparum, which is the main cause of severe
clinical malaria and death. Treatment and control of the dis-
ease have become difficult due to the spread of drug-resis-
tant strains of the parasites and of strains of mosquitoes
that became resistant to insecticides or changed their biting
behaviour. Social and economical factors also play an im-
portant role. New, more effective and inexpensive control
measures are needed (Greenwood & Mutabingwa 2002).
Only female mosquitoes take blood meals; blood proteins
are required for egg development. Blood is taken from a wide
range of vertebrate hosts. Several studies showed that host
odours play a major role in the orientation and host-finding
of blood-seeking (nocturnal) female mosquitoes (Takken
1991, Takken & Knols 1999). In some species a definite host
preference is present, which is determined genetically. Some
species feed on one kind of host only. Since host-seeking is
mediated by an olfactory stimulus, it is possible that the
odour complex of the host contains host-specific chemicals
to which only certain mosquito species respond. Specificity
can also be due to quantitative differences in abundance of
generally occurring host-derived chemicals.
In Africa the main vectors of human malaria are species
of A. gambiae sensu lato. This is a group of seven mosquito
species, including vectors and non-vectors. Most members
are opportunistic in their feeding behaviour: they may bite
humans and animals, depending on host availability. The fe-
males of A. gambiae s.s., however,are highly anthropopha-
Why humans are attractive to
malaria mosquitoes
Renate C. Smallegange1, Yu Tong Qiu1, Agnès
M.S. Galimard2, Maarten A. Posthumus2, Teris
A. van Beek2, Joop J.A. van Loon1 & Willem Tak-
ken1
1 Laboratory of Entomology
Wageningen University and Research Centre
PO Box 8031
6700 EH Wageningen
Renate.Smallegange@wur.nl
2 Laboratory of Organic Chemistry
Phytochemical Section
Wageningen University and Research Centre
Dreijenplein 8
6703 HB Wageningen
Malaria poses serious problems to human 
h e a l t h , especially in sub-Saharan Africa. Blood-
feeding female mosquitoes are the vectors of
the malaria parasites. The mosquitoes use host
odours to find their blood sources, but we know
little about the semiochemicals that mediate
this behaviour. A combined study including be-
havioural experiments, electrophysiological
techniques and chemical analysis is undertaken
to identify the volatile human-specific comp-
ounds that are used in the host-seeking
behaviour of the females of the malaria mosqui-
to Anopheles gambiae sensu stricto. Traps
baited with these chemicals may assist in moni-
toring populations and reduce contact between
the vector and human host.
Entomologische Berichten 63(3): 50-53
Malaria
Host odours
Figure 1. A blood-feeding Anopheles gambiae s.s.female on a human
arm. Photo: B. Mans
Een bloedzuigend vrouwtje van Anopheles gambiae s.s.
EBjuni2003-JB  09-03-2009  17:56  Page 50
gic: they have a preference for human blood (White 1974,
Pates et al. 2001a), which makes them the most important
vectors of human malaria. Morphology, physiology and be-
haviour of the females are entirely adapted to feeding from
their preferred host. Therefore, it is possible that they use
human-specific odour blends to find their blood hosts. This
knowledge may be used to improve the effectiveness of (od-
orous) mosquito traps that can be deployed to interrupt the
contact between vector and human host.
It has been shown that anthropophilic mosquito females
use chemical compounds present in the human breath or
that evaporate from the human skin to locate their blood
hosts from a distance. For example, in the field, females of
A. gambiae s.s. are caught in traps baited with carbon dioxi-
de (CO
2
) (Gillies 1980, Costantini et al. 1996). However, CO
2
is exhaled by all warm-blooded vertebrates and it is unlikely
that a mosquito species like A. gambiae s.s.,which has a se-
lective host preference, will be guided to
its host by this compound alone. CO
2
seems to be more important for opportu-
nistic and zoophilic species (Mboera et al.
1997, Mboera & Takken 1997, Dekker &
Takken 1998, Takken & Knols 1999). Re-
search in laboratories showed that human
sweat and human skin residues are highly
attractive for A. gambiae s.s. (Braks et al.
1997; Braks & Takken 1999; Healy & Co-
pland 2000). Also chemicals, like short-
chain carboxylic acids, ammonia, and L-
lactic acid, which are present in human
sweat and/or in human skin emanations,
were found to be attractive (e.g. Knols et
al. 1997, Braks et al. 2001, Costantini et
al. 2001, Dekker et al. 2002, Smallegange
et al. 2002). To date however, it is still not
clear which combination of volatiles the
A. gambiae s.s.-females use to find hu-
mans. The (combination of) attractants
identified thus far are less attractive than
the total odour of the natural host. In our
laboratory research is undertaken to un-
ravel this mystery.
The majority of the compounds present in human sweat and
skin emanations have been identified (Cork & Park 1996,
Bernier et al. 2000, Meijerink et al. 2000). Theoretically, we
can use this knowledge to test which compounds play a role
in the host-seeking behaviour of the malaria mosquitoes by
testing them one by one and in all possible combinations in
behavioural tests. However, this is not feasible as it concerns
more than 300 components! It will take years to test all the-
se chemicals for their behavioural relevance. Therefore, at
Wageningen University we are using another inter- discipli-
nary approach.
Some people are bitten by mosquitoes more often than
others (Curtis 1986). We used this phenomenon in our re-
search and compared the odours of highly attractive indivi-
Entomologische Berichten 63(3) 2003 51
Figure 2. Attractiveness of glass beads handled by six volunteers to females of the malaria mos-
quito A. gambiae s.s., when tested individually in a dual-choice olfactometer (treated beads
versus clean beads + ammonia). The attractiveness is given as the number of mosquitoes attract-
ed to the treated beads over the number of mosquitoes attracted to the control stimulus. Beads
of persons on the right attracted high numbers of mosquitoes compared to clean beads. Arrows
show the individuals tested directly against each other (see figure 3).
Aantrekkelijkheid van knikkers die in contact zijn geweest met handen van zes vrijwilligers voor
vrouwtjes van de malariamug A. gambiae s.s., individueel getest in een tweekeuze-olfactometer
(‘reukmeter’; behandelde knikkers versus schone knikkers + ammoniak). De aantrekkelijkheid is
weergegeven als het aantal tot de knikkers aangetrokken muggen gedeeld door het aantal tot de
schone knikkers aangetrokken muggen. Knikkers van personen rechts in de grafiek trokken veel
muggen aan in vergelijking tot schone knikkers. De pijlen geven de individuen aan die tevens als
tweetal zijn getest (zie figuur 3).
Figure 3. Attractiveness of handled glass beads of
a highly and a less attractive volunteer (see figure
2) when tested simultaneously. The mean of four
experiments is shown. In each experiment 30 fe-
male mosquitoes were released (Chi-square-test:
*** P<0.0001; error bars of mean are shown).
Aantrekkelijkheid van knikkers van een zeer aan-
trekkelijke en van een minder aantrekkelijke vrijwil-
liger (zie figuur 2) die rechtstreeks tegen elkaar zijn
getest. De grafiek laat het gemiddelde van vier ex-
perimenten zien. In elk experiment werden 30 vrou-
welijke muggen losgelaten (Chi-kwadraattoets: ***
P<0,0001; standaardfout van het gemiddelde is
eveneens weergegeven).
Interdisciplinary research
EBjuni2003-JB  09-03-2009  17:56  Page 51
duals with those of less attractive ones. A group of 28 volun-
teers was asked to rub glass beads in their hands during a
few minutes. This method can be used to transfer attractive
emanations from the human hand to the glass surface (Ber-
nier et al. 1999). The handled glass beads of each person
were tested six times for their attractiveness in behavioural
tests with A. gambiae s.s.-females. During these experiments
the beads of one person were tested against clean beads and
a concentration of ammonia shown to be
attractive as the standard control odour.
Indeed, these bio-assays revealed that
human individuals can be ranked based
on their attractiveness to female mosquit-
oes (figure 2). To be certain, we tested
beads handled by a highly attractive pers-
on versus beads handled by a less
attractive person. These experiments con-
firmed our first results (figure 3).
Thermal desorption combined with
gas chromatography (GC) and mass spec-
trometry (MS) (see Bernier et al. 1999) are
used to find the differences in the odour
profiles of most and least attractive vol-
unteers, and to identify the compounds
involved. These differences may be quali-
tative and/or quantitative. In addition, it is
possible that not only attractive but also
repellent compounds cause the differen-
ces in attractiveness between individuals.
The attractiveness of the handled beads
was found to decrease quickly and disap-
peared within five hours after handling
(figure 4). This suggests that at least some
of the attractive compounds are very vola-
tile. This knowledge can be used in our
attempt to identify the chemicals respon-
sible for the attractiveness of humans to
female mosquitoes. To narrow down our
search we are looking for human-specific comp-
ounds.
Based on the differences in human odour pro-
files we may be able to identify chemicals involv-
ed in the host-location by A. gambiae s.s.-fema-
les. A first possible step is to use electrophysiolo-
gical methods: identified chemicals can be tested
for their biological activity with a so-called Elec-
troAntennoGram (EAG) and Single Sensillum
Recording techniques (see Meijerink et al. 2000).
These techniques show whether the olfactory
sensilla on the antennae of the female mosquito
respond to these odorous components. Or in ot-
her words, whether the mosquito can ‘smell’ a
certain single chemical or a combination of
compounds. EAG in combination with GC and MS
(EAG-GC-MS) can also be used as an intermedia-
te step (Smid et al. 2002). It will show to which
identified compounds in the human odour profile
the olfactory sensilla of the mosquito are respon-
ding. These compounds will be worth studying.
In the end, it is necessary to return to the be-
havioural tests. A mosquito may be able to smell
a specific odour, but this information does not
tell us if the compound is attractive, repellent or
neutral to the host-seeking females. In dual-choice olfacto-
meters (Braks et al. 1997; figure 5) in the laboratory, single
chemicals and combinations of chemicals can be screened
for their attractiveness. Finally, field experiments in Africa
with wild A. gambiae s.s.-mosquitoes have to reveal the use-
fulness of traps baited with the promising chemicals under
natural circumstances when they have to compete with hu-
man baits.
52 Entomologische Berichten 63(3) 2003
Figure 4. Attractiveness of handled beads of one person immediately and five hours af-
ter handling. The attractiveness is given as the number of mosquitoes attracted to the
treated beads over the number of mosquitoes attracted to clean beads.
De aantrekkelijkheid van vastgehouden knikkers direct en vijf uur na behandeling. De
aantrekkelijkheid is weergegeven als het aantal tot behandelde knikkers aangetrokken
muggen gedeeld door het aantal aangetrokken tot schone knikkers.
Figure 5. Diagram of the dual-port olfactometer (A) and a mosquito release cage (B) (after Pates
et al. 2001b).
Schematische weergave van een tweekeuze-olfactometer (A) en een loslaatkooitje (B) (naar Pates
et al. 2001b).
EBjuni2003-JB  09-03-2009  17:56  Page 52
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Geaccepteerd 18 maart 2003.
Entomologische Berichten 63(3) 2003 53
Samenvatting
Waarom mensen aantrekkelijk zijn voor malariamuggen
Elk jaar raken 300 tot 500 miljoen mensen besmet met malariapa-
rasieten. Met name in Afrika ten zuiden van de Sahara vormt de
ziekte malaria een groot probleem. Het is daar doodsoorzaak num-
mer één onder kinderen tot vijf jaar oud. De ziekte wordt overge-
bracht door bloedzuigende vrouwelijke muggen. In Afrika zijn de
vrouwtjes van de muggensoort Anopheles gambiae sensu stricto de
belangrijkste vector van de malariaparasiet Plasmodium falciparu m.
De bestrijding van malaria wordt onder andere bemoeilijkt door de
ontwikkeling van resistentie van de parasiet tegen medicijnen en
van de mug tegen insecticiden. Nieuwe, effectievere en goedkopere
bestrijdingsmethoden zullen ontwikkeld moeten worden.
Onderzoek heeft uitgewezen dat muggenvrouwtjes geuren ge-
bruiken om hun bloeddonoren te vinden. Aangezien de vrouwtjes
van A. gambiae s.s. hoofdzakelijk mensen steken is het mogelijk
dat een aantal van deze geuren mensspecifiek zijn. Door gebruik te
maken van de combinatie van gedragsonderzoek, elektrofysiologis-
che en analytisch-chemische technieken proberen we te achter-
halen welke geurstoffen dat zijn. Een vangsysteem gecombineerd
met deze lokstoffen kan gebruikt worden om aantalsfluctuaties in
muggenpopulaties te volgen en om het contact tussen mug en
mens te onderbreken om zo verdere overdracht van malariaparasi-
eten te voorkomen.
References
EBjuni2003-JB  09-03-2009  17:56  Page 53


Why humans are attractive to malaria mosquitoes

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