Response of Anopheles gambiae s.l. (Diptera: Culicidae) to larval habitat age in western Kenya highlands.

0.2 MB·English

by  Munga, Stephen

Mungaetal.Parasites&Vectors2013,6:13
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RESEARCH Open Access
Response of Anopheles gambiae s.l. (Diptera:
Culicidae) to larval habitat age in western Kenya
highlands
Stephen Munga1, John Vulule1 and Eliningaya J Kweka2*
Abstract
Background: Larval control is of paramount importance inthe reduction of vector populations. Previous
observations have suggestedthat, larvae of Anopheles gambiae s.l occur more often in small temporary habitats
whileotherstudies showed thatlong-lasting stable habitats are more productive than unstable habitats.In
addition, thephysicaland biological conditions and stability of larval habitats can change rapidly innatural
conditions. Therefore, weexamined theeffect of larval habitat age on productivity, larval survival and oviposition
preference ofAnopheles gambiae.
Methods: We sampled the threedifferent habitat ages (10,20 and 30 days) on a daily basis for a period of six
months to determine mosquitolarval abundance. Inaddition, we tested theeffect of age of water (habitat age) on
theovipositionchoice preference of An. gambiae,larval developmenttime and survivorship,and wing lengths of
emerging adults. Additionally, chlorophylla and abundance of mosquito larval predators inthese habitats were
monitored.
Results: Anopheles gambiae s.l. larvae were significantly more abundant (P=0.0002) in habitats that were cleared
every 10 days compared to theotherhabitats.In particular, there were 1.7 times more larvae in this habitat age
compared to theones that were cleared every 30 days. There were significantly (P<0.001) more mosquito larval
predators in the‘30 day’habitats comparedto the other habitats. Oviposition experiments revealedthat
significantly more eggs (P<0.05) were laid in fresh water and water that was 5days old compared to water that
was 10 and 15 days old. However, pupation rate, development times and wing lengths ofmale and female An.
gambiae inthedifferent habitat ages was statisticallyinsignificant (P>0.05).
Conclusion: The current study confirmed that age of the habitat significantly influencesthe productivity of malaria
vectors in western Kenya highlands. Given that malaria vectors were found in allhabitats withvarying ages of
water, simpleenvironmentalmethods of maintaining the drainage ditches in the valley bottoms can helpreduce
larval abundance ofmalaria vectors. Such inexpensive methodsof controllingmosquito breeding could be
promoted to supplementothervector control methods, especially in areas where scarce resources are available for
intensive mosquito control.
*Correspondence:[email protected]
2TropicalPesticidesResearchInstitute,DivisionofLivestockandHuman
DiseasesVectorControl,MosquitoSection,P.O.Box3024,Arusha,Tanzania
Fulllistofauthorinformationisavailableattheendofthearticle
©2013Mungaetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
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Background abovesea level and theYala River transects the study site.
Larvae and pupae of Anopheles gambiae Giles, the prin- The area is characterized by undulating topography, with
cipal vector for Plasmodium falciparum malaria in sub- both steep and gently sloping hills. Although most of the
Saharan Africa, tends to occur in small, temporary sunlit study area has been cultivated, small patches of indigen-
pools. An. gambiae mosquito is an r-strategist and is ous forest still remain along the rivers and streams. The
able to colonize suitable aquatic habitats within a few hill is mostly dotted with maize plots and patches of
days after they are created [1]. In small temporary habi- tea plantation, while several wetlands are located along
tats,predators maybeless prevalent andlarvalfood may theYala River valley. The average annual rainfall is about
be more abundant than it is in long-lasting habitats [2]. 1,950 mm, while peak rainfall generally occurs between
However, other studies have demonstrated that stable March and June followed by a short rainy season in
habitats are more productive than temporary unstable October and November.
habitats [3,4]. Further, An. gambiae larval habitats usu-
ally occur near human habitation, and are often subject
EffectsofhabitatageonAn.gambiaes.l.abundance
to anthropogenic activities [5]. For example, cow foot-
We determined the effects of habitat age on mosquito
prints, cultivated swamps and drainage ditches form the
larval abundance in western Kenya highlands for a
majorbreedinghabitatsforAn.gambiaeinwesternKenyan
period of six months from September 2005 to February
highlands[6,7].However,littleisknownabouttheeffectof
2006. Thirty (30) larval habitats in a low lying area near
habitatdisturbanceorhabitatageonmosquitolarvaldevel-
a stream at Iguhu and each habitat measured 7 × 0.5 m
opmentandsurvivorship.
in length and width respectively. The three treatments
In the recent years, western Kenya highlands, where
hadatotaloftenreplicateseach:(1)habitatswerecleared
this study was conducted, have experienced epidemics of
of grass and water was replenished from the local stream
malaria transmission [8]. These highland sites have also
every 10 days (frequent disturbance); (2) habitats were
experienced dramatic land use changes during the same
cleared of grass and water was replenished from the local
period [9]. For example, most of the natural swamps
stream every 20 days (intermediate disturbance); and (3)
have been cultivated for growing agricultural crops, and
habitats were not disturbed during the 30 day period (no
rain forests have been cleared for crop planting, cattle
disturbance). This experiment was conducted in one
grazing, commercial logging, firewood and housing con-
month (30 days) after which the no-disturbance habitats
struction [10]. The drastic land-use alteration can pro-
were also cleared of water and grass and the experiment
mote vector-borne disease transmission in several ways
repeated again. The 30 larval habitats were randomly
[11,12]. First, more human made aquatic habitats be-
assigned the treatments (frequent, intermediate and no
come available for the An. gambiae complex. Second,
disturbance)atthebeginningoftheexperimentsandkept
the physical and chemical properties of mosquito larval
thesameforthestudyperiod.Larvalmosquitoabundance
habitats may change. Third, the micro-climatic condi-
in the habitats was determined daily by making 50 dips
tions of mosquito larval habitats may change, favoring
per habitat using 350 ml standard dippers. Larvae of An.
rapid development of the malaria vector. Since An. gam-
gambiae s.l. occurring in each habitat were counted and
biae prefers to utilize transient freshwater habitats, thus
scoredintoinstars[1,15].Further,wecharacterizedhabitat
habitat age may influence mosquito larvae abundance,
size(length,width,anddepth).
development and survivorship through its effect on pre-
dation and food availability. In this study, we investi-
gated the effects of habitat age on An. gambiae s.l. larval Effectsofhabitatageonlarvaldevelopment
abundance and predator abundance. We also character- In addition, we determined the effect of larval habitat
ized the abundance of green algae, which are considered age on larval development of An. gambiae s.l. using arti-
as an important larval food for An. gambiae [13-15]. ficial habitats created in plastic washbasins (35 cm in
Further, we investigated the influence of habitat age on diameter and 15 cm deep). The artificial habitats were
oviposition preference and development of An. gambiae created by mixing 2 kilograms of dry soil and 2.5 liters
larvae. The information on An. gambiae larval popula- of water in each washbasin. The dry soil was collected
tion regulation is critical for the development of rational from a farmland within the study area. Two holes (3 cm
andcost-effectivelarvalcontrol methods. in diameter) were drilled 1 cm from the top edge of the
washbasin and covered with a screen (mesh size =
Methods 200 μ) to maintain a constant water level during periods
Studysite of rain while preventing larvae from being washed away.
0 0
ThestudysiteislocatedinIguhuvillage(34°45 E,0°10 N), On the first day of the experiment, 6 replicates were
Kakamega district, a highland area in western Kenya. The created and covered with mosquito netting to prevent
altitude of the study site ranges from 1,420 to 1,580 m wild mosquitoes from laying eggs in the experimental
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washbasins. These washbasins were left in the open area also tested if the differences in the predator density be-
tomimicthenaturalhabitats. tween the habitat ages were statistically significant, using
Six other treatments were created on day 5, 10, or 15 the analysis of variance with repeated measures. When
and treated the same way as artificial habitats above. The the effects were significant,Tukey-Kramer multiple com-
experiment was conducted from January to February parison tests were used to compare mean values among
2006. A cohort of 20 first-instar An. gambiae s.s. larvae the habitats. One way analysis of variance (ANOVA) was
(1–3 hrs old) from a laboratory-reared colony that origi- used to test whether the differences between pupation
nated from the study area were introduced to these habi- rate, development time and wing length of resulting An.
tats. The habitats were covered with mosquito netting gambiaeamongwatertreatmentswerestatisticallysignifi-
materials to prevent egg laying by other mosquitoes, and cant. Pupation rate was defined as the proportion of
randomlyplacedinnaturalhabitatsinanopenareawithin first-instarlarvaethatdevelopedtothepupalstage.Devel-
thestudysite.Onadailybasis,wemonitoredthenumber opment time is defined as the length of time that a first-
of mosquito larvae and pupae in these artificial habitats instar larva requires to emerge as an adult. Finally, we
and the larvae were scored into developmental stages. usedone-wayANOVAtotestiftherewereanysignificant
Water lost through evaporation from the habitats was differences in the number of eggs that were oviposited in
replaced by adding water of the same age kept in same the different ages of water. The Tukey-Kramer multiple
conditionas that usedin the experiment. Oncethelarvae comparison test was used to determine the treatments
started pupating, the washbasins were examined twice thatweresignificantlydifferentfromeachother.
dailyfrom0900hrto1200hrsandfrom1500hrsto19
00hrsforthepresenceofpupae,andpupaewereremoved Results
and held in paper cups to allow them to emerge into Effectsofhabitatageonlarvalabundance
adults. The emerged adults were then separated by sex The species composition and larval abundance of mos-
and wing lengths measured to the nearest 0.01 mm with quitoes sampled during the study period are presented
an ocular micrometer from the distal end of the alula to in Table 1. A total of 50,896 mosquito immature stages
thewingtipexcludingthefringescales[16]. were sampled over the study period. Of these, An. gam-
biae s.l. comprised 74.5%, An. funestus 3.9%, An. cous-
HabitatageandovipositionsiteselectionofAn.gambiae tani 0.5% and An. implexus 0.5% and the rest were
s.s culicines. The mean number of larval and pupal stage of
We used 4 oviposition substrates in this experiment: An. gambiae s.l. mosquitoes was significantly influenced
water of the following ages 0 days (fresh rainwater, 5, 10 by habitat age (χ2 = 17.05, df = 2, P = 0.0002; Table 1).
and 15 days old. One hundred ml substrate water was Comparison between the 10 and 30 day treatments
added to a 300 ml transparent plastic cup (the mouth of revealed a significant 1.7 fold increase (P<0.001) in im-
the cup was 6.4 cm in diameter). A piece of white filter mature An. gambiae in the 10 day treatment (Table 1).
paperwasplacedtightlyineachcupbyfoldingthepaper Similarly, habitats cleared after 20 days were 1.3 times
edge upward. This way, all eggs were laid onto the filter more productive (P<0.05) than the non-disturbed habi-
paper making it easy to count. The four cups were tats(Table1).
placed randomly at the corners of the cage. Twenty An. Thenumberofpredators occurringinahabitat signifi-
gambiae s.s. mosquitoes, 36 hr post blood-feeding, were cantly influenced the number of larvae present (χ2 =
introduced to the oviposition cage (30 × 30 × 30 cm in 59.37, df = 3, P<0.0001). Habitats that had fewer preda-
size) that contained the four substrates, and 2% sugar tors were 2 times more productive compared to habitats
solution was provided daily. The adult mosquitoes used
in this experiment were from a laboratory-reared colony
Table1Abundanceofimmaturestagesofanopheline
that originated from the study area. The experiment was
mosquitospeciesandunidentifiedculicinescollected
set up at 18 00 h, and eggs deposited in each oviposition duringthestudyperiodunderthedifferenthabitatages
substrate were counted under the microscope at 10 00 h
Species 10day 20day 30day
on the following day. This experiment was conducted in
Anophelesgambiaes.l. 15589a 12670b 9637c
a room at the study site and thus there were no controls
Anophelesfunestuss.l. 351a 524a 1105b
for lighting, humidity and temperature. The experiment
wasrepeated 5times,with 10replicates pertrial. Anophelescoustani 47 30 195
Anophelesimplexus 70 85 104
Statisticalanalysis Culicines 3185 3525 3779
We used Poisson regression with repeated measures to
Total 19242 16834 14820
testforthedifferencesinthenumberofimmaturestages
*Valueswithdifferentlettersinarowwerestatisticallysignificantdifferent
of An. gambiae s.l. among the three habitat ages. We fromeachother.
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Table2Larval,predatorandchlorophyllaabundance(mean±SE)indifferentwaterhabitats
Habitatage Numberoflarvaeperdip Numberofpredatorsperdip Chlorophylla(ppm)
10day 0.191±0.006a 0.14±0.06a 73.3±1.16a
20days 0.155±0.005b 0.64±0.17b 67.4±1.06b
30days 0.118±0.004c 1.10±0.32c 87.1±1.79c
*Valuesabovearethemeanvalues(±SE)andthelettersfollowingthenumericalvaluesindicatetheresultsofmultiplecomparisontests.Thevalueswith
differentlettersinacolumnweresignificantlydifferentfromeachother.
with more predators. Predator density in the 30 day occurring in water of different ages. We found signifi-
habitatswassignificantly higher(F =8.43, P<0.001) cantlyhighernumbersofmosquitolarvaeinwaterreplen-
2,181
compared to the other habitat ages. Tukey HSD test ished frequently compared to the water replenished after
revealed that there were more predators in 30 day habi- 30 days. Several factors may regulate the population dy-
tats than in the 10 day (1.10 vs. 0.14 predators per dip) namics ofmosquitolarvae in a habitat.One suchimport-
and 20 day habitats (1.10 vs. 0.64 predators per dip) ant factor is the predation effect in relatively stable
(Table 2). Chlorophyll a level in water significantly influ- habitats [17-19]. We found abundant predators such as
enced (χ2 = 59.37, df = 3, P<0.0001) larval and pupal coleopteransandhemipteransinallthehabitats,withsig-
abundance. In general, habitats that had a higher chloro- nificantly higher predator abundance in habitats with
phyllavalueweremoreproductive comparedtohabitats older water (replenished after 30 days). It has been sug-
with low values. Tukey HSD test revealed that there gested that larval mortality is lower in smaller temporary
were significant differences in the levels of chlorophyll a habitats than it is in large, permanent habitats where the
amongthe habitats(Table2). predationrateishigh[2],thustheeffectofpredationmay
have significantly reduced the number of mosquito larvae
Effectsofhabitatageonlarvalsurvivorshipand in these older habitats. Although, predatory effect may be
ovipositionsiteselectionofAn.gambiaes.s higher in such habitats, productivity may be regulated by
Pupation rate did not vary significantly among the water other factors such as the availability of algae, as demon-
of the different ages (F = 0.62, P = 0.42). The high- stratedbythisstudy.Thusolderhabitatsmaystillprovide
3, 23
est mean pupation rate (61%) occurred in freshwater suitable breeding habitats for immature stages of malaria
(day 0) while the lowest pupation rate (50%) occurred in vector.Forexample,inurbanMalinditownattheKenyan
water of day 5 (Table 3). Female and male development coast, unused swimming pools were found to play a
times and wing length measurements were statistically significant role in the breeding of Anopheles immature
insignificant (P>0.05) among habitat ages (Table 3). stages[20].OtherstudiesinwesternKenyahighlandshave
However, age of water significantly influenced the num- demonstrated that, drainage ditches had significantly
ber of eggs laid (F = 9.42, P<0.001). In particular, higherproductivityinallseasonscomparedtootherhabi-
3, 199
significantly more eggs were laid in freshwater and 5 day tat types [6,7], thus productivity of malaria vectors from
old water (mean= 213.9 and 181.5 respectively) com- drainage ditches can play a role in increasing the risk of
pared to 10 and 15 day water (mean= 112.7 and 115.8 malaria transmission. Greater abundance of mosquito
respectively) (Figure 1). The number of eggs laid in larvae in habitats with higher chlorophyll a values also
water of day 0 and 5 did not differ statistically. Similarly, confirmed the importance of chlorophyll a in the diet of
thenumberofeggslaidinwaterthatwas10and15days larvae. Previous studies have demonstrated the import-
oldwere statistically insignificant(P>0.05). ance of chlorophyll a as a dietary content of An. gambiae
larvae [13,14]; consequently food resources may be a
Discussion limiting factor to production of adult mosquitoes. More
This study demonstrated that habitat age significantly stable habitats may have abundant food resources, how-
influenced the abundance of An. gambiae s.l. larvae ever, habitat productivity may be influenced by other
Table3Mean±SEpupationrates,developmenttimesandwinglengthsfromthelarvalsurvivalexperiment
Variable Day0 Day5 Day10 Day15
Pupationrate 0.61±0.06 0.50±0.06 0.53±0.06 0.55±0.06
Maledevelopmenttime(days) 10.21±0.19 10.59±0.20 10.71±0.22 10.90±0.24
Femaledevelopmenttime(days) 10.75±0.24 11.01±0.23 10.39±0.22 10.81±01.6
Femalewinglength(mm) 2.80±0.02 2.83±0.02 2.83±0.02 2.80±0.04
Malewinglength(mm) 2.70±0.02 2.75±0.02 2.76±0.02 2.70±0.02
Allcomparisonswerestatisticallyinsignificant.
Mungaetal.Parasites&Vectors2013,6:13 Page5of6
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vectors.However,ifthesehabitatsareclearedquiteoften
250
and water is left to flow all the times, this would make it
less suitable for breeding of these vectors. Since mos-
200
gs quito larvae were found in habitats of all ages, this may
g
of e 150 suggest that making the water in these drainage ditches
er to flow is likely to reduce the abundance of mosquito
mb 100 larvae. Other studies have demonstrated that the likeli-
u
N
hoodoffindingAnophelesmosquitoesishigherindrains
50
compared to water flowing at normal velocity [24]. Al-
0 though An. gambiae prefers to breed in clear sunlit
0 day 5 days 10 days 15 days
pools of water, ensuring that the water in the drainage
Age of water
ditches is flowing would reduce oviposition in those
Figure1Thenumberofeggs(mean±SE)ovipositedinwater
habitats. Similarly, moving water is likely to have little
fromthefourdifferenthabitatagetypes.
amount of algae, which is food to the mosquito larvae,
thus these habitats would be less suitable for breeding.
factors rather food resources. For example, we found Older habitats were also found to have malaria vectors.
high levels of chlorophyll a in habitats that were older These habitats are likely to be organically polluted; how-
compared to 10 and 20 days old. Though the levels of ever Anopheles species have been demonstrated to breed
chlorophyll a were higher in 30 day old habitats, larval even in highly organically pollutedhabitats[25]. Thus all
abundance was significantly lower compared to 10 and habitatsneedtobeconsideredashabitatsformalariavec-
20dayoldhabitats.Thiscanbeattributedtohigherpre- torsinanycontrolprogram.Apreviousstudyinthisarea
dation pressure since we found higher predator density demonstratedthatpredationisoneofthemostimportant
in30dayhabitats. factorsregulatingtheproductivityofmalariavectorsfrom
The results from the present oviposition experiments relatively stable habitats [6]. The importance of pre-
demonstrated that water conditions additionally affected dation has also been illustrated by other studies [2,18,19].
oviposition preference of An. gambiae s.l. In particular, Additionally, the reduction in eggs oviposited in older
An. gambiae mosquitoes deposited significantly more habitsresultedinalowerproductivityfromsuchhabitats.
eggs in freshwater (day 0) compared to other ages of
water. We also observed that the number of eggs depos- Conclusion
ited in water of day 0 and 5 days were statistically insig- Since malaria vectors were found in all habitats with
nificant. Immature stages of this mosquito occur in varying ages of water, these results suggest that simple
small, sunlit pools such as borrow pits, hoof prints, tire environmental methods of maintaining the drainage
tracks, drainage ditches, and small puddles [1,4,19,21], ditches in the valley bottoms can help reduce productiv-
which may be very temporary in nature. We have also ity of these vectors. Such inexpensive and simple meth-
previously demonstrated that An. gambiae prefers to lay ods to control the breeding of malaria vectors could be
eggs in small pools filled with rainwater [22]. It is inter- promoted to supplement other vector control methods,
esting to notethat differences inpupation rate, male and especially in areas where resources for vector control are
female development time and male and female wing scarce andhabitatsareaggregated inthe valleybottoms.
length were statistically insignificant among the different
Competinginterests
ages of water. This difference could be due to experi-
Theauthorsdeclarethattheyhavenocompetinginterests.
mental design, as each treatment only differed in age by
a few days. Thus, it is possible that the time intervals Authors’contributions
usedmayhavebeentooshort todetectadifference. SMconceptualizedanddesignedthestudy,collectedthedataanddrafted
themanuscript.JV,EJKwereinvolvedintheconceptionofthestudyand
Taken together, our findings suggest that simple envir-
thedraftingofthemanuscript.Allauthorsreadandapprovedthefinal
onmental methods such as keeping the water in the versionofthemanuscript.
drainage ditches moving could reduce the productivity
Acknowledgement
of malaria vectors in drainage ditches in valley bottoms
WethankM.Okonji,S.Ajuma,W.MihesoandA.Wabwilefortechnical
of western Kenya highlands. A recent study at the same assistance.ThisresearchwassupportedbyWHO/UNDP/WORLDBANK
site demonstrated that cropping cycle (land preparation, SpecialProgrammeforResearchandTraininginTropicalDiseases(TDR)
A50116.ThispaperispublishedwiththepermissionoftheDirectorofKenya
crop weeding and plant flowering) influenced larval
MedicalResearchInstitute.
abundance [23]. As the cropping cycle progresses at this
study site, the farmers havea practice of not clearing the Authordetails
1CentreforGlobalHealthResearch,KenyaMedicalResearchInstitute,Nairobi,
drainage ditches in their farms and small pools of stag-
Kenya.2TropicalPesticidesResearchInstitute,DivisionofLivestockandHuman
nant water become suitable breedinghabitatsfor malaria DiseasesVectorControl,MosquitoSection,P.O.Box3024,Arusha,Tanzania.
Mungaetal.Parasites&Vectors2013,6:13 Page6of6
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Received:24October2012Accepted:9January2013 23. KwekaEJ,ZhouG,MungaS,LeeMC,AtieliH,NyindoM,GithekoAK,YanG:
Published:16January2013 Anophelinelarvalhabitatsseasonalityandspeciesdistribution:A
prerequisiteforeffectivetargetedlarvalhabitatscontrolprogramme.
PLoSOne2012,7:e52084.
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