University of Groningen
Olfactory sensitivity of Anopheles mosquitoes with different host preferences
van den Broek, Ingrid Viola Francine
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Publication date:
2000
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van den Broek, I. V. F. (2000). Olfactory sensitivity of Anopheles mosquitoes with different host preferences
Groningen: s.n.
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Summary
Worldwide, mosquitoesof the genusAnophelesare vectorsof malaria.Only female
mosquitoestransmitmalaria parasiteswhen taking a bloodmealin need of a protein
sourceto developtheir eggs.At night, they searchfor appropriateblood-hosts.In the
courseof evolution, female mosquitoesof various specieshave developeddifferent
strategiesto localisetheir hosts.Someare highly specialisedto take blood from one
type of hosts,e.g.humans,cattle,birds or reptiles;othersdirecttheir feedingto several
typesof hostsand show opportunisticbiting behaviour.
Anophelesgambiaes.s.is the main vector of malariain Africa, causingone of
the major public healthproblemsthere.The species'high capacityto transmitmalaria
arisesfrom its selectivepreferenceto bite a human host, i.e. from its anthropophilic
biting behaviour.An. gambiae s.s. is a member of the An. gambiae s./. species
complex, consisting of 6 closely related African species, which can only be
distinguishedfor certain by their geneticcharacteristics.
In contrasÍto An. gambiae
s.s.,the sibling speciesAn. quadriannulatusshowsa definitepreferenceto feed from
animals,i.e. mainly from cattle and otherbovines.This zoophilic specieshardly ever
is a vector of malaria.Another speciesof the complex,An. arabiensis,shows more
opportunistichost preferences.Dependingon the availability of human or bovine
hosts,ln. arabiensisfemaleswill bite one or the other.
The main questionof the researchprojectdealtwith in this thesisis:
How do anthropophilic,zoophilic and opportunisticmosquitospecies
of the An. gambiaes./. complexlocaliseand distinguishthe different
typesof hostsduringtheir nightly searchfor a bloodmeal?
Earlier researchhas shown that temperature,humidity and carbon dioxide as well as
host body odoursplay an importantrole in the attractionof mosquitoesto their hosts.
An increasein temperatureor humidity may be perceived at a few meters from the
host. A concentrationof COz abovebackgroundlevel, due to exhaledbreath of warmbloodedanimals,may be detectedfrom 15-20 meters.Host-odoursare presumably
perceivedat largerdistances,i.e. 50-60meters,if the mosquitoespossessan olfactory
systemsensitiveto theseodourcompounds.
The research described in this thesis aims aÍ comparing the
sensitivities of the olfactory organs of Ánopheles species with
differenthostpreferences.
t08
SUMÀ,IARY
emale
rotein
In the
ïerent
m one
everal
one of
nalaria
ophilic
;pecies
nly be
zmbiae
'd from
Iy ever
's more
bovine
well as
r hosts.
:om the
f warm;umably
lfactory
The first step was a study of the morphology of the antennae,the main olfactory
organs of the mosquito, using ScanningElectron Microscopy. The antennaeof male
and femalemosquitoesof the threeAfrican Ánophelesspecies,Án, gambiaes.s.,An.
arabiensisandAn. quadriannulatus,were compared.
The antennaeof male and femaleAnophelesmosquitoesare2.2 and 1.5 mm
long, respectively.The antennaeare movably connectedwith the head by their scape
and globularpedicel,and furthermoreconsistof 13 flagellar segments.The flagellar
segmentsof the femaleare coveredwith differenttypes of sensoryhairs, which may
serveto perceiveeither heat or humidity, or tactile stimuli or may have an olfactory
function.The first I I flagellarsegmentsof the male bearwhorls of long hairs,which
are brought into maximal vibration by the flight-tone producedby a female of the
samespecies.The vibrationis perceivedby the Johnston'sorganin the pedicel.Hence,
the male antennais first of all designedto detecta potentialmating partnerby sound.
The last two segmentsof the male antennaeshow a variety of sensoryhairs similar to
that of the femaleantennae.
The numbersand distribution of the different types of sensoryhairs on the
antennaeare similar for all 3 Anophelesspecies.The femaleantennaeshowed5 types
of sensoryhairs: sensilla chaetica,sensilla ampullacea,sensilla coeloconica,sensilla
basiconica ('grooved pegs') and sensilla trichodea. Sensilla ampullacea were not
observedon the male antennae.
Sensillatrichodeaand groovedpegshave beenshown
to play a role in olfaction in various other mosquito species.These hairs contain
dendritesof olfactorycells which lie at the baseof the hair. Sensillacoeloconicamay
have an olfactory function as well. These three types of olfactory sensilla are
connectedto 95oÁof the total numberof sensorycells presentin the femaleantennae
(estimatedat 1800 cells) and to 83oÁof thosein the male antennae(800 cells). The
number of olfactory cells of bloodfeedinginsects is thought to be related to the
importanceof odoursin host-orientation
and to the distancethe insecthas to travel to
find a host. The results of the morphologicalstudy showed that the number of
olfactoryhairs,and also the numberof olfactorycells, is equalfor the anthropophilic,
zoophilicand opportunisticAnophelesspecies.Thereforewe may assumethat the role
of the olfactory systemis equally importantfor thesespeciesdespitetheir different
hostpreferences.
Because of the demonstratedolfactory function of sensilla trichodea and
groovedpegs,and the relativelyhigh numbersof thesesensillapresenton the female
antennae,these two types of sensory hairs were the principal candidates for
investigationin our electrophysiological
research.
t09
SUMMARY
The sensitivities of the sensilla trichodea and the grooved pegs of the
Anophelesspecieswere investigatedin threeelectrophysiological
studies.By meansof
electrophysiology,
action potentials('spikes') of the sensorycells at the base of the
sensoryhairs can be recorded.The olfactory cells of insectsusually show a basal
continuousfiring of spikes.When odour substancesare perceivedby the olfactory
cells, the frequency of the spike-signalchangesand either a temporal increase
(excitation)or decrease
(inhibition)ofspikes canbe observed.
Existing electrophysiologicaltechniques were adapted to the size and
characteristics
of the mosquitoantennae.For 'single-cell'electrophysiologya detailed
view of the olfactory hairs was required, which could be obtained under high
magnification (800x) with an inverted microscope. A tungstenelectrodewith a fine
tip (< I pm) was carefullypositionedat the baseof a sensoryhair to catchthe signalof
the sensorycells underlying the cuticula. This signal was thereafterrecorded by
equipmentspecificallydevelopedfor insectsensoryphysiology.
Chapter3 describesa seriesof electrophysiological
on sensilla
measurements
trichodea on the antennaeof the 3 African Anophelesspeciesand the EuropeanAn.
maculipennis atroparvus. The activity of the sensory cells was measured during
stimulationwith severaldosesof a numberof odour substances,
which are emittedby
humanand bovine hosts.Volatile fatty acids are found in relatively high amountsin
sweatand on the human skin and may play a role in the attractionof the anthropophilic
An. gambiaes.s.Five short-chainfatty acidswere testedin thesestudies.Furthermore,
someothersubstances
were chosen,which areknown to attractzoophilicbloodfeeding
insects:two phenols,mainly found in urine,and l-octen-3-ol,a compounddetectedin
the breathof cattle.
The cellsat the baseof sensillatrichodeashowedclearresponses
to theseodour
substances.On stimulation with fatty acids and phenolsboth excitation and inhibition
was found.The cells often respondedto morethanone compoundand the variouscells
showeddifferent response-profiles.
The anthropophilicAn. gambiaes.s. appearedto
have relatively more cells which are excited by fatty acids than the zoophllic An.
quadriannulatus and Án. m. atroparvus. Moreover, the cells of the anthropophilic
speciesrespondedstrongerto the fatty acidsand to lower dosesofthem than cells of
the zoophilic species.In contrastto this, the sensillatrichodeacells of the zoophilic
specieswere more sensitive to 1-octen-3-ol.The cells of the opportunislicAn.
arabiensisshowed sensitivitiesintermediateto those of the other species.The 4
specieswere equallysensitiveto the phenols.The role of phenolsin the behaviourof
behaviourthey may
mosquitoesis not quite clear.Apart from their role in host-seeking
alsobe involvedin the localisationof ovipositionsites.
1t0
of An
the b
comp
founc
found
ol. In
gïoo!
quad
with r
sens
humi
tricho
respo
gamb
host(
with t
whicl
sensi'
chees
magr
anthr
cells
P€rct
hairs
only
com
Cell
The
capí
vici
moÍ
SUMMARY
)gs of the
y meansof
lase of the
)w a basal
e olfactory
al increase
size and
a
I detailed
under high
with a f,rne
hesigral of
ecordedby
on sensilla
ropean An.
rred during
emittedby
amountsin
hropophilic
urthermore,
loodfeeding
detectedin
theseodour
d inhibition
'ariouscells
lppearedto
ophilic An.
hropophilic
hancells of
Lezoophilic
unistic ln.
:ies. The 4
ehaviourof
:r they may
In chapter4 the resultsof electrophysiological
measurements
on groovedpegs
gambiae
quadriannulatus
of An.
s.s. andAn.
aredescribed.The majority of the cells at
the base of the gtooved pegs respondedto the odours of ammonia and amines,
componentsevaporatingfrom excretionproducts(urine and faeces)and to afatty acid
found on the (human)skin; a smallerpart of the cells respondedto acetone,which is
found in breath.Only a few groovedpegswere responsiveto phenolsand l-octen-3ol. In contrast to the results obtained from the sensilla trichodea, the responsesof
grooved peg-cells of the anthropophilicAn. gambíae s.s. and the zoophilic Án.
quadr i annulatus w ere not si grrifi cantly di fferent.
More than 50% of the groovedpegsshowedexcitatoryresponses
to stimulation
with water vapour. On the other hand, dry air causedinhibition of the activity of some
sensilla.This suggeststhat groovedpegsplay a role in the perceptionof changesin
humidity. These humidity-sensitiveresponseswere never observed from sensilla
trichodea cells.
In the third series of electrophysiologicalmeasurements(chapter 5) the
responsesof olfactory cells of both sensilla trichodea and grooved pegs of An.
gambiaes.s. and Án. quadriannulatusto the completeblend of odours emitted by a
host (a cow) and its excretionproducts(urine,manure,skin products)were compared
with the responsesto individual odour components.The odour of Limburger cheese,
which had beenshownto attractAn. gambiae.r.^r..,
was alsotested
Groovedpeg-cellsshowedhigherresponses
to urine,manureand ammoniathan
sensilla trichodea cells. The latter respondedmore strongly to the odours of Limburger
cheese,to l-octen-3-ol and to 3-methyl phenol. No differenceswere found in the
magnitude of responseto natural odour mixtures between olfactory cells of the
anthropophilicand zoophilicspecies.
Thesethreeelectrophysiological
studieshavemadeclearthat both the olfactory
cells underlying sensilla trichodea and grooved pegsof Ánophelesfemalesare able to
perceivevariousodour componentsemittedby humanand bovine hosts.The sensory
hairsand underlyingolfactorycells are not specialisedto detectone odour component
only. The cells also do not seem to respondto a specific combination of odour
components,at leastnot for the dozenof odourswhich were testedin thesestudies.
Cells may respondwith inhibition to one compound,and with excitationto another.
The largevariationof responses
and the broadodour spectrumof the cells enlargesthe
capacityto perceivecomplexodourmixtures,which are likely to be encounteredin the
vicinity of the host. The information of the completecellular network enablesthe
mosquitofemaleto build a detailed'odour-picture'of the potentialhostnearby.
tII
--
SUMMARY
On the antennaeof the anthropophilicas well as the zoophilic Anopheles
femalesaround 1700olfactorycells are found,which altogethershow a large diversity
of responses
to varioushost odours.The anthropophilicand zoophilic speciesshowed
a differentlevel of sensitivityto someodourcomponentswhich are found in the odour
blend emittedby their specifichost,i.e. humansor bovines,but they may have similar
'Human odour' and 'bovine odour' cannot be
sensitivitiesto other components.
characterised
by one or a few odour components,but ratherby a specificcombination
of severalodours.The responsesof anthropophilicand zoophilic speciesto complex
'bovine odours' were similar. Specieswith a specifichost preferencemay not only
respondto the odoursemittedby their specifichost.They alsoperceivethe odour from
other potential hosts, and there are indications that these may have a more or less
repellenteffect on the mosquitoes.In two-choicetestsin the field, it was shown that
the percentageof An. gambiaes.s. femalescaught in a human odour-baitedtrap is
higher when the mosquitoeshave to choosebetweenhuman odour versuscalf odour
(99 vs. l%), than when human odour versus COz is offered (70 vs. 30%). The
anthropophilicmosquitoesapparentlyavoid the calf odour. Similar tests with lt4.
quadriannulalzsshow that femalesof this zoophilic speciesmay avoid the human
odour. Therefore,identificationand localisationof a specific host may not only be
guidedby attractionto odoursofthe host,but also by repellencyby odoursofthe less
preferredhost.
In the last chapterof the thesis,the step from physiologyto behaviouralresearchis
made. 'Actometers',designedfor automaticrecordingof movementof insects,were
used to measurethe behaviouralresponsesof individual mosquitoesto individual
odour componentsand to odour blends. We comparedthree African species,ln.
gambiae s.s., Án. arabiensis and An. quadriannulatus,and the EuropeanAn. m.
atroparvus.
First, the spontaneous
levels of activity during day and night was recordedof
mosquitoesof differentphysiologicalstates.Males and femalesof the African species
showedsimilar activity patterns,with peaksof activity during dawn and dusk. During
the day the mosquitoeswere not active. The Europeanspecieswas mainly active
during the evening,in the hours before midnight. Thesedifferencesare relatedto
climatic differencesin the habitatsof thesespecies.In the tropics the temperature
remainsrelativelyhigh duringthe night and early morning,allowing the insectsto stay
active.In temperatezones,the temperaturemay decreaseduring the night below the
level where insects can stay active. Interestingly, the activity pattems of the
have not changedduring long-term rearing
mosquitoesof theseAnopheles-species
understabletemperatureconditionsin the laboratory.
t12
C
Female
males,a
hungry,
bloodfe
T,
female
during s
stimulat
than 15 r
15 min
stimulat
exist.
Th
significa
opportun
electroph
sensillati
U1
to CO2a
one to 1
bloodfee
enlarged
the beha'
the inhib
may blor
The seri,
provide
compon(
which n
method
mosquitr
Activati,
of the c
mosquit
describe
with ch
hosts.
SUlvíMÁRY
Anopheles
: diversity
esshowed
the odour
rvesimilar
cannotbe
rmbination
o complex
y not onlY
rdourfrom
ore or less
shownthat
ited trap is
calf odour
30%). The
.s with An.
the human
rot only be
; of the less
researchis
lsects,were
r individual
;pecies,An.
eanAn. m.
recordedof
ican species
lusk.During
rainly active
re relatedto
temperature
lsectsto stay
ht below the
;erns of the
term rearing
(10-14days)weremoreactivethanyoungerones(l-4 days).
Oldermosquitoes
Femalemosquitoesshoweda higher activity during the dark part of the night than
males,and inseminatedfemaleswere more activethan virgins. The nightly activity of
hungry, inseminatedmosquito females is mainly directed to host-seekingand
bloodfeeding.One to two daysafterbloodfeedingactivity was stronglydepressed.
To investigatethe effect of odour stimuli on the activity of mosquitoes,only
femalesof 4 to l0 daysof agewere used.An increasein mosquitoactivity was shown
during stimulation with pulses of CO2 and l-octen-3-ol and during continuous
stimulationwith the odour of Limburgercheese.With enduringstimulation,i.e. more
than 15 min, the activity decreased.
Repeatedstimulationwith CO2, after a period of
15 min without stimuli, causeda lower increasein activity than during the first
stimulationperiod.This suggeststhat somekind of peripheralor centralmemory may
exist.
The responsesto l-octen-3-ol of the zoophilic An. quadriannulatus were
significantly higher than those of the anthropophilicAn. gambiae s.s. and the
opportunistic An. arabiensis. These results confirm the results of the
electrophysiological
studies,which showedsimilar differencesin the sensitivityof the
sensillatrichodeato I -octen-3-ol.
Up to least l2 hoursafterbloodfeeding,femalemosquitoeswere not responsive
to COz and l-octen-3-ol,similar to the above-mentioned
decreasein activity during
one to two days after bloodfeeding.It has been shown that immediately after
bloodfeeding
host-seeking
behaviouris inhibitedby 'abdominaldistension',i.e. by the
enlargedbloodfilled abdomen.Thereafter,if the bloodmealtriggers egg production,
the behaviouris inhibited by a hormonalfactor in the haemolymph.In An. gambiae
the inhibitory phasemay be relativelyshort.Field-dataindicatethat thesemosquitoes
may bloodfeedeveryothernight.
The seriesof activity measurements
showedthat the 'actometers'used in this study
provide a sensitivetechniquefor screeningand quantiSing the effects of host-odour
componentson the activity of mosquitoes.In order to identify odour components
which may be used to attractmosquitoesin the field, this may be a more efficient
method than electrophysiology.Actometer-studiesmay quickly determinewhether
mosquitoesperceive an odour and respond to it with increasedactivity levels.
Activationis the first stepin the processof host-seeking.
However,to entanglethe role
of the olfactory system in the recognitionof the odour-blendof a specific host in
mosquitoeswith different host-preferences,
electrophysiologicalresearch such as
describedhere is required.Further researchin this line of work should go together
with chemical studies identi$ing specific odour componentsemitted by different
hosts.
I1.l