The infectivity of the entomopathogenic fungus
Beauveria bassiana to insecticide-resistant and
susceptible Anopheles arabiensis mosquitoes at
two different temperatures
Christophe K Kikankie1,2,3, Basil D Brooke2,3, Bart GJ Knols3,4, Lizette L Koekemoer2,3, Marit Farenhorst4,
Richard H Hunt1,2, Matthew B Thomas3,5, Maureen Coetzee2,3*
Background: Control of the major African malaria vector species continues to rely extensively on the application
of residual insecticides through indoor house spraying or bed net impregnation. Insecticide resistance is
undermining the sustainability of these control strategies. Alternatives to the currently available conventional
chemical insecticides are, therefore, urgently needed. Use of fungal pathogens as biopesticides is one such
possibility. However, one of the challenges to the approach is the potential influence of varied environmental
conditions and target species that could affect the efficacy of a biological ‘active ingredient’. An initial investigation
into this was carried out to assess the susceptibility of insecticide-susceptible and resistant laboratory strains and
wild-collected Anopheles arabiensis mosquitoes to infection with the fungus Beauveria bassiana under two different
laboratory temperature regimes.
Methods: Insecticide susceptibility to all four classes of insecticides recommended by WHO for vector control was
tested on laboratory and wild-caught An. arabiensis, using standard WHO bioassay protocols. Mosquito
susceptibility to fungus infection was tested using dry spores of B. bassiana under two temperature regimes (21 ±
1°C or 25 ± 2°C) representative of indoor conditions observed in western Kenya. Cox regression analysis was used
to assess the effect of fungal infection on mosquito survival and the effect of insecticide resistance status and
temperature on mortality rates following fungus infection.
Results: Survival data showed no relationship between insecticide susceptibility and susceptibility to B. bassiana.
All tested colonies showed complete susceptibility to fungal infection despite some showing high resistance levels
to chemical insecticides. There was, however, a difference in fungus-induced mortality rates between temperature
treatments with virulence significantly higher at 25°C than 21°C. Even so, because malaria parasite development is
also known to slow as temperatures fall, expected reductions in malaria transmission potential due to fungal
infection under the cooler conditions would still be high.
Conclusions: These results provide evidence that the entomopathogenic fungus B. bassiana has potential for use
as an alternative vector control tool against insecticide-resistant mosquitoes under conditions typical of indoor
resting environments. Nonetheless, the observed variation in effective virulence reveals the need for further study
to optimize selection of isolates, dose and use strategy in different eco-epidemiological settings.
* Correspondence: email@example.com
2Vector Control Reference Unit, National Institute for Communicable
Diseases of the National Health Laboratory Service, Private Bag X4,
Sandringham 2131, Johannesburg, South Africa
Kikankie et al. Malaria Journal 2010, 9:71
© 2010 Kikankie 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/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Malaria vector control relies primarily on the selective
application of residual insecticides through either indoor
residual house spraying (IRS) or insecticide-treated nets
(ITNs). At high coverage, these approaches have proven
highly effective in reducing malaria morbidity and mor-
tality at an affordable cost . However, the ever-
increasing development of resistance to insecticides [2,3]
is of great concern. Insecticide resistance in malaria vec-
tor populations covers all classes of insecticides cur-
rently used in public health and is widespread
geographically [2,4-7]. It is, therefore, not surprising that
interest in alternative non-chemical strategies has
increased over the last decade.
Fungal pathogens commonly infect insects  and
there has been extensive research on numerous species
of Deuteromycete fungi (e.g. Culicinomyces spp., Beau-
veria spp., Metarhizium spp. and Tolypocladium spp.)
for use as biological pest control agents in agriculture
[9-13]. Although such fungi appear to have limited
impact on mosquito populations under natural condi-
tions [8,14], there is increasing evidence supporting the
potential use of isolates of Beauveria bassiana and
Metarhizium anisopliae for control of adult mosquito
Given the emerging problems of insecticide resistance,
one of the key requirements for any new (bio) pesticide
product for mosquito control is to have limited cross-
resistance with existing chemical insecticides [26,27].
Clearly, if resistance to widely used insecticides, such as
permethrin and DDT, confers resistance to fungal
pathogens then potential novel biopesticide products
will have limited utility either as replacements for insec-
ticides, or in integrated strategies for insecticide resis-
tance management . The likelihood of cross-
resistance occurring in mosquitoes, however, appears to
be remote and in fact it would seem that in certain
instances infection with fungi counteracts resistance that
is based on metabolic mechanisms, at least in laboratory
colonies . One aim of the current study, therefore,
was to compare the virulence of a candidate strain of
the entomopathogenic fungus B. bassiana against insec-
ticide-resistant and susceptible Anopheles arabiensis
laboratory colonies, as well as wild collected adult mos-
quitoes to determine fungal susceptibility.
Additionally, because fungal pathogens are living
organisms, the rate at which they penetrate and grow
within an infected host is determined by temperature.
Accordingly, the efficacy of certain fungal biopesticide
products in agriculture has been shown to be strongly
influenced by environmental temperature, in some cases
further mediated by thermal behaviour of the target
insect [28-31]. The effects can be such that under
certain conditions, speed of kill is rapid and overall con-
trol very good, while under other conditions, speed of
kill is very slow and control inadequate [30,31]. In a
recent study, Blanford et al  demonstrated that Ano-
pheles stephensi mosquitoes infected with B. bassiana or
M. anisopliae did not exhibit any change in thermal
behaviour that might affect speed of kill. Nonetheless,
temperature remains an important environmental factor
likely to affect fungal germination and growth rate
inside mosquito hosts. With respect to malaria control,
a critical factor is how the speed of kill (virulence) varies
relative to the extrinsic incubation period (EIP) of the
malaria parasite; if mortality is faster than the rate of
parasite development then impact on transmission will
be greater than if mortality is slower than the parasite
rate of development . Importantly, both the EIP and
pathogen growth vary with temperature [31,32]. The
second aim of the current study, therefore, was to
explore the effect of temperature on virulence of B.
bassiana against insecticide susceptible and resistant An.
arabiensis. The daily average temperature measured
inside traditional African houses between seasons in
western Kenya is 23 ± 1.8°C [33-35]. Therefore, the
impact of B. bassiana was assessed nder temperature
regimes 2°C lower and higher than this average to cap-
ture the range of mean temperatures likely experienced
in indoor resting sites.
Wild mosquito collection
Mosquitoes were collected inside traditional houses in
Karonga (9° 48’51.04” S, 33° 52’.97” E), northern Malawi,
using a mouth aspirator. They were placed into small
polystyrene cups covered with gauze netting and later
identified morphologically using the taxonomic keys of
Gillies and Coetzee . Only female mosquitoes identi-
fied as members of the Anopheles gambiae complex
were retained. A cohort of these mosquitoes was main-
tained on a 10% sucrose solution soaked in cotton pads
for a few hours after which they were subjected to
insecticide susceptibility tests. Another cohort of female
mosquitoes was blood-fed for oviposition. These mos-
quitoes were also maintained on a sucrose solution
before and during transportation to the laboratory for
colony rearing and fungal susceptibility tests.
Colonies of An. arabiensis housed at the Vector Control
Reference Unit of the National Institute for Communic-
able Diseases, Johannesburg, South Africa, were main-
tained under standard insectary conditions of 25 ± 2°C,
80% ± 10% relative humidity (RH) and a 12:12 hour
day/night cycle with 45 minutes dusk/dawn transition
Kikankie et al. Malaria Journal 2010, 9:71
Page 2 of 9
between photo-periods. Field collected mosquitoes were
reared under the same conditions in order to obtain F1
progeny to be used for fungal infection experiments.
Female mosquitoes were offered blood meals two or
three times whereafter each gravid female mosquito was
placed in a glass vial lined with a moistened filter paper
to allow for egg laying. Individual egg batches were
transferred into plastic bowls (250 ml) half filled with
distilled water. F1 larvae were reared in the same bowl
until they pupated. All larvae were fed twice daily with a
mixture of brewer’s yeast and finely ground dog biscuits.
Pupae were transferred daily into plastic vials (50 ml)
half filled with distilled water and covered with gauze
netting. Emerged F1 adult mosquitoes were collected
using an aspirator and kept in small cups (180 ml) cov-
ered with gauze netting or in small plastic cages (2.5
litres). F1 adult progeny were maintained on a 10%
sucrose solution soaked in cotton pads for 2-3 days
before being subjected to fungal susceptibility tests.
The Polymerase Chain Reaction (PCR) method  was
used to test the species integrity of selected laboratory-
reared An. arabiensis colonies as well as to identify
wild-caught material morphologically identified as An.
gambiae complex. Selected laboratory colonies included
SENN and SENN-DDT originating from Sudan, and
MBN and MBN-DDT originating from Kwazulu/Natal,
South Africa. SENN-DDT and MBN-DDT were selected
for resistance to DDT from their respective parent
Insecticide susceptibility tests
Newly emerged adult mosquitoes from each An. ara-
biensis colony were exposed to discriminating dosages
of representative insecticides from all insecticide classes
recommended by WHO for malaria vector control
(Table 1). The standard insecticide susceptibility assays
and test kits were used . Samples of 25 non blood-
fed mosquitoes per cylinder were exposed to insecticide-
treated papers for one hour. After exposure, mosquitoes
were transferred to clean holding tubes and provided
with cotton pads soaked in a 10% sucrose solution.
Knock-down was recorded after 1 h exposure and final
mortality was recorded 24 h post-exposure. Each test
was duplicated for fully insecticide susceptible colonies
and triplicated for insecticide resistant colonies. Tests to
determine the insecticide susceptibility status of field-
collected mosquitoes were limited to DDT (organo-
chlorine) and dieldrin (cyclodiene) because of sample
size limitations. Insecticide susceptibility status was
determined according to WHO criteria, whereby colo-
nies were considered resistant when more than 20% of
individuals survived the diagnostic dose 24 h post-expo-
sure. A final mortality of 98-100% indicates full suscept-
ibility whilst mortality between 80-97% suggests the
possibility of resistance that needs further confirmation
Fungal susceptibility tests
Fungal infection experiments were performed under two
different temperature regimes: 21 ± 1°C and 25 ± 2°C,
both under 70% ± 15% relative humidity. Infection was
carried out using ‘fungal suspensors’ prepared according
to the method described by Scholte et al ; this
approach is not meant to mimic potential operational
delivery of spores but provides a reliable infection
method for controlled laboratory-based assays. For each
trial three suspensors measuring 6.5 cm in height and
2.5 cm in diameter were lined on the inside with a
cylinder of filter paper which also served to hold a plas-
tic vial half filled with a 10% sucrose solution for moist-
ening the filter paper. The sugar solution served as a
mosquito attractant to each suspensor. Approximately
100 mg of B. bassiana (isolate IMI 391510) conidial
powder was weighed and dusted onto treatment suspen-
sors using a small paintbrush. Treated suspensors and
an equal number of untreated, control suspensors were
placed individually into small cages (2.5 litres). Cohorts
of 25-35 sugar-fed 2-3 day old adult female mosquitoes
from each colony were exposed to either treated or
untreated suspensors for 24 h. A minimum of three
trials was conducted per colony, each consisting of three
treatment replicates and three controls. After the 24 h
exposure mosquitoes were transferred into clean holding
cages and fed on a 10% sucrose solution during the
Mortality in treatment and control samples was
recorded daily up until the death of all fungus-infected
mosquitoes. All dead mosquitoes were removed from
their respective cages using dissecting forceps. Each
cadaver was dipped in 70% ethanol in order to eliminate
saprophytic fungi from their cuticles. They were then
placed in Petri dishes lined with moistened filter paper
and sealed with parafilm to maintain high humidity for
Table 1 Insecticides used for WHO insecticide
Kikankie et al. Malaria Journal 2010, 9:71
Page 3 of 9
fungal sporulation. Petri dishes were incubated at 25 ±
2°C for a period of 3-5 days after which cadavers were
screened for external fungal growth under a compound
microscope. Cadavers with visible fungal growth on
their body surface were considered to have died as a
result of fungal infection. Final fungal infection propor-
tion was recorded per sample.
Data recording and analysis
Mosquito survival was recorded daily. Cox’ regression
analysis  using SPSS 15.0 software was used to com-
pare survival between treatments and controls. Compar-
isons of mortality rates were used to assess differences
in the effect of fungus infection on survival between
insecticide susceptible and resistant colonies, laboratory-
reared and wild caught An. arabiensis as well as
between the two temperature regimes. For each factor, i.
e. insecticide susceptibility status, colony and tempera-
ture, two-way interactions (of the factor with fungus
treatment) were included in the Cox Regression model
to test if the effect of fungal infection on mosquito sur-
vival was significantly influenced by the factor.
Species identification and confirmation
A sample of 386 An. gambiae complex mosquitoes was
collected from the field in Malawi. They were all identi-
fied as An. arabiensis by PCR. All samples drawn from
the laboratory-reared colonies were confirmed to be as
Insecticide susceptibility tests
The insecticide susceptibility status of the laboratory
An. arabiensis colonies and the wild-caught sample is
shown in Figure 1. There was no evidence of insecticide
resistance in MBN and field-collected mosquitoes,
although the latter were only exposed to 4% DDT and
4% dieldrin because of limited numbers available for
exposure assays. There was clear evidence of resistance
to 0.75% permethrin in the baseline colony SENN. The
SENN-DDT and MBN-DDT selected colonies showed
measurable resistance to DDT as expected, as well as
resistance to one or more insecticides from each of
the other classes (pyrethroids, carbamates and
Figure 1 Insecticide susceptibility status of four laboratory strains and one field strain of Anopheles arabiensis. Data show the average
mosquito mortality (%) after a 1 hour exposure to insecticide treated papers, recorded 24 hours post-exposure, of two replicates of 25
mosquitoes for the susceptible groups and three replicates for the resistant groups.
Kikankie et al. Malaria Journal 2010, 9:71
Page 4 of 9
Fungal susceptibility tests
Daily survival of the field-collected mosquitoes after fun-
gus exposure is shown in Figure 2. Exposure to dry B.
bassiana spores resulted in significant reductions in
longevity of the wild An. arabiensis mosquitoes (P <
0.05, HR > 1) relative to their respective controls. Com-
parisons of survival following fungal exposure between
wild-caught and baseline laboratory-reared An. arabien-
sis (MBN and SENN, Figure 3) did not reveal any signif-
icant difference in susceptibility to fungal infection (P <
0.05, HR > 1).
Survival curves of the insecticide resistant and sus-
ceptible laboratory colonies are shown in Figure 3.
Exposure to B. bassiana spores resulted in significant
reductions in longevity in all mosquito colonies (P <
0.05, HR > 1) relative to their respective controls,
regardless of their insecticide susceptibility levels and
temperature regimes. Cox-regression analysis revealed
no significant differences in fungus-infected mortality
rates between the insecticide-resistant and baseline
colonies. Interaction analyses indicated no significant
influence of insecticide susceptibility status on fungus-
induced mortality rates at both tested temperatures
(HR = 0.769; P = 0.13 and HR = 1.456; P = 0.61 for
MBN vs MBN-DDT at 21°C and 25°C respectively
and HR = 1.03; P = 0.86 and HR = 0.576; P = 0.27
for SENN vs SENN-DDT respectively at 21°C and 25°
C). Even where fungus mortality rates appeared slower
in insecticide resistant lines, as in MBN-DDT (Figure
3), this is not because of increased fungal resistance
but because the respective untreated control lines also
survived better. Therefore, fungal susceptibility is not
affected by resistance to insecticides. Fungus-induced
mortality rates were relatively rapid at 25°C, with
100% mortality taking 10-12 days post-fungus expo-
sure in the baseline colonies (MBN and SENN) and
field-collected mosquitoes, and 18-21 days in the
DDT-selected colonies (MBN-DDT and SENN-DDT).
At 21°C, equivalent mortalities took 20-28 days and
27-30 days, respectively (Figure 3). The differences in
mortality rate were mirrored in the sporulation data,
with > 90% sporulation of cadavers in the 25°C
regime compared with just over 70% at 21°C. How-
ever, not all infected mosquito cadavers show fungal
sporulation after death because some mosquitoes may
be killed by fungal toxins or secondary infections,
rather than primary fungal growth and invasion of
organs that tends to facilitate sporulation of the cada-
ver. As observed from the hazard ratio values in
Table 2, the fungus had a stronger effect at higher
temperatures than at low temperatures. Cox-regres-
sion interaction analyses revealed a significant differ-
ence (P = 0.045) in the effect of the fungus on
survival between the two temperature regimes. The
hazard ratios (risk of death) were greater at the higher
temperatures of 25 ± 2°C than at the lower tempera-
tures of 21 ± 1°C (Table 2), suggesting that the prob-
ability of a mosquito dying soon after infection was
greater at higher temperatures than at lower tempera-
tures. Thus, quantitative differences were detected
between the two exposure temperatures in all colonies
tested (Figures 2 and 3).
Figure 2 Daily cumulative proportional survival of F1 offspring of An. arabiensis collected in Karonga, Malawi. Data show the average ±
SE survival of nine replicate groups of 25-35 mosquitoes infected with B. bassiana (open symbols) and uninfected control groups (closed
symbols) kept at 21 ± 1°C (red) or 25 ± 2°C (blue).
Kikankie et al. Malaria Journal 2010, 9:71
Page 5 of 9
Mosquitoes caught resting indoors north of Karonga on
Lake Malawi consisted solely of An. arabiensis. Condi-
tions during the collection period were dry and hot,
favouring a preponderance of this member of the An.
gambiae complex, which is generally more tolerant of
such conditions [36,40-42].
There are clear indications of insecticide resistance to
all insecticides and their respective classes tested in one
or more of the An. arabiensis samples used. The con-
trolling mechanisms of these resistance phenotypes are
likely to involve target site mutations such as kdr as well
as metabolic detoxification [2,7,43-47]. Resistance to
insecticides in major malaria vector species, coupled to
the limited number of insecticides available for use in
public health programmes, highlights the need to evalu-
ate the potential efficacy of entomopathogens.
All laboratory-reared and wild-caught An. arabiensis
lines were susceptible to B. bassiana. Though quantita-
tive differences were detected between the two exposure
temperatures in all colonies tested, Beauveria signifi-
cantly reduced mosquito longevity at both temperature
regimes with no evidence for enhanced resistance to
fungal infection due to insecticide resistance. Where
mortality rate was apparently slowed due to DDT resis-
tance, this effect was due to enhanced overall survival in
Figure 3 Daily cumulative proportional mosquito survival of insecticide-susceptible (blue) and insecticide-resistant (red) An. arabiensis
laboratory colonies originating from Sudan (SENN) or Kwazulu/Natal (MBN). Data show the average ± SE survival of nine replicate groups
of 25-35 mosquitoes infected with B. bassiana (open symbols) and uninfected control groups (closed symbols) kept at 21 ± 1°C (top) or 25 ± 2°
Table 2 Mortality P values and hazard ratios of An.
21 ± 1°C25 ± 2°C
Colony P value Hazard ratioP value Hazard ratio
Comparisons were made between fungus-infected and control mosquitoes at
21 ± 1°C and 25 ± 2°C using Cox-regression analysis.
Kikankie et al. Malaria Journal 2010, 9:71
Page 6 of 9
the selected lines relative to the baseline colonies, rather
than any significant reduction in susceptibility to fungus
per se. Why these resistant mosquitoes survived better
than controls in the absence of insecticide exposure is
unclear. The DDT resistance in these lines is linked to
higher levels of expression of glutathione S-transferases
(GST) and esterases . Conceivably these generic
detoxifying enzymes could enhance survival in the
laboratory environment, although trade-offs against
other traits and fitness measures might be expected in
other environments [48-50].
Beauveria killed mosquitoes significantly quicker at
25°C than at 21°C. This result is consistent with the
known temperature-growth profile for this isolate,
which indicates a temperature optimum of around 26°C
(unpublished data). As suggested previously, to interpret
the possible significance of this effect it is important to
consider not just absolute speed of kill, but speed of kill
relative to the length of the EIP. According to the classic
day-degree model of Detinova , the EIP of P. falci-
parum is 12.3 days at 25°C and 22.2 days at 21°C. Thus,
if mosquitoes became infected with malaria and fungus
more or less simultaneously (as would happen if mos-
quitoes contacted fungus on a treated surface following
an infectious blood feed), no mosquitoes would have
survived long enough to transmit malaria in any of the
colonies held at 25°C. At 21°C, fungal infection of the
recently derived Malawi colony would have reduced the
percentage of mosquitoes potentially able to transmit
malaria (i.e. comparing percent alive at day 22 in control
and treated populations) from 64% to 4%, representing a
92% reduction. In the two longer-lived DDT resistant
colonies, the equivalent figures are 64% to 17% and 55%
to 20%, representing reductions in transmission poten-
tial of approximately 70%. Thus, although still contribut-
ing to substantial reductions in transmission potential,
the fungus appears to work less well at 21°C.
Fully extrapolating these results to potential impact in
the field is difficult as mortality schedules could poten-
tially differ markedly between lab and field environ-
ments for a variety of reasons. Moreover, the current
study considers only one dose and it is likely that higher
doses could help compensate for the apparent thermal
constraint at 21°C. Studies exploring higher fungal doses
and different bioassay exposure techniques have shown
the potential for much more rapid mosquito mortality
than observed in the current study [e.g. [15,52]], and
studies with other insect hosts indicate ‘dose x tempera-
ture’ interactions whereby effects of lower doses are
magnified at sub-optimal temperatures . Of course,
selection of a different fungal isolate that is less tem-
perature sensitive, or combining isolates with different
temperature optima could overcome the constraint
completely . Furthermore, the growth rate of the
malaria parasite slows exponentially as temperatures
decrease further towards 18°C , whereas the decline
in fungal growth rate appears more linear over this
range (unpubl. data) so it is likely that at slightly cooler
temperatures still, the relative efficacy of the current iso-
late would recover. In addition, sub-lethal effects of
infection such as impact on malaria parasite develop-
ment [16,18] and reduced feeding propensity [54,55] can
reduce mosquito vectorial capacity irrespective of speed
of kill . Nonetheless, with potential for considerable
variation in both mean conditions and diurnal tempera-
ture ranges across different transmission environments
, understanding the effects of temperature on bio-
pesticide performance is an important area for further
research [see also ].
Overall, the results of the current study demonstrate
that relative susceptibility of Anopheles arabiensis to a
candidate fungal biopesticide strain is not affected by
resistance to insecticides (see also ), that wild-caught
mosquitoes are equally susceptible to fungal infection
and that although there was temperature-dependent var-
iation in fungal virulence, fungal infection led to sub-
stantial reductions in malaria transmission potential in
conditions typical of local African houses (at least in
western Kenya). These empirical data add support to
recent modelling studies suggesting that as long as cov-
erage is high (a goal of most conventional vector control
operations), slow acting biopesticides can deliver sub-
stantial reductions in malaria transmission across a
range of conditions [21,23,58,59]. Of particular relevance
here is the study of Koella et al , who demonstrated
that the level of control (whether biological or conven-
tional) necessary to reduce or even prevent malaria
transmission depends on the background transmission
intensity. In areas of low to moderate transmission,
<50% reduction could provide substantial control,
whereas in areas of very high transmission, even 90%
reduction might not be sufficient to deliver any benefit
due to the strongly saturating relationship between
malaria prevalence and transmission [58,60]. Thus the
significance of the temperature-dependence in biopesti-
cide performance needs to be considered in relation to
the local epidemiology context.
Additionally, the efficacy and impact of a biopesticide
will depend on ultimate use strategy. For example, in a
recent theoretical study, Hancock  demonstrated
that under conditions of intense transmission, high sin-
gle coverage of either ITNs or IRS with a fungal biopes-
ticide might not substantially reduce malaria prevalence
in the human population, whereas intermediate coverage
of both interventions simultaneously could. This conclu-
sion, together with the empirical data demonstrating
that resistance to insecticides does not confer resistance
to fungi, highlights the potential for development of
Kikankie et al. Malaria Journal 2010, 9:71
Page 7 of 9
novel integrated control strategies combining insecticide
and biopesticide interventions. Understanding such
interactions, together with the local environmental con-
text, are important areas for future research to define
possible limits to biopesticide performance and identify
isolates, doses and potential delivery systems to optimise
control strategies across time and space.
We thank Belinda Spillings for assistance with field collections and Joel
Mouatcho for assistance with fungal experiments. This research was funded
by an anonymous charity organisation. BGJK receives financial support from
the Dutch Scientific Organisation (NWO, VIDI grant 864.03.004). MC is
supported by the South African Research Chair Initiative of the Department
of Science and Technology and the National Research Foundation.
1School of Animal, Plant and Environmental Sciences, University of the
Witwatersrand, Private Bag 3, Wits 2050, Johannesburg, South Africa.2Vector
Control Reference Unit, National Institute for Communicable Diseases of the
National Health Laboratory Service, Private Bag X4, Sandringham 2131,
Johannesburg, South Africa.3Malaria Entomology Research Unit, School of
Pathology, University of the Witwatersrand and the National Health
Laboratory Service, Johannesburg, South Africa.4Laboratory of Entomology,
Wageningen University and Research Centre, PO Box 8031, 6700 EH,
Wageningen, the Netherlands.5Centre for Infectious Disease Dynamics and
Department of Entomology, Pennsylvania State University, University Park
16802, PA, USA.
CKK carried out wild mosquito collections, species identification, insecticide
and fungal susceptibility tests, data analysis, interpretation of results, and
drafted the first version of the manuscript. BDB supervised all the laboratory
experiments and contributed to the subsequent writing of the manuscript.
BGJK obtained funding for the project and contributed to the editing of the
manuscript. LLK supervised the insecticide susceptibility assays and species
identification. MF assisted with the statistical analyses and contributed to the
editing of the manuscript. RHH organised the field trip to Malawi and was
involved in wild mosquito collections, identification and rearing. MBT
provided fungal spores, was involved in methodology of fungal experiments
and contributed to editing the manuscript. MC was involved in project
design and contributed to the final editing of the manuscript. All authors
read and approved the final version of the manuscript prior to submission.
The authors declare that they have no competing interests.
Received: 19 November 2009
Accepted: 8 March 2010 Published: 8 March 2010
1. World Health Organization: World malaria report 2008 Geneva, Switzerland:
2. Hemingway J, Ranson H: Insecticide resistance in insect vectors of human
disease. Ann Rev Entomol 2000, 45:371-391.
3. Hargreaves K, Koekemoer LL, Brooke BD, Hunt RH, Mthembu J, Coetzee M:
Anopheles funestus is resistant to pyrethroid insecticides in South Africa.
Med Vet Entomol 2000, 14:181-189.
4. Coetzee M, Horne DWK, Brooke BD, Hunt RH: DDT, dieldrin and pyrethroid
resistance in African malaria vector mosquitoes: an historical review and
implications for future malaria control in southern Africa. South Afr J Sci
5. Etang J, Manga L, Chandre F, Guillet P, Fondjo E, Mimpfoundi R, Toto JC,
Fontenille D: Insecticide susceptibility status of Anopheles gambiae s.l .
(Diptera: Culicidae) in the Republic of Cameroon. J Med Entomol 2003,
6. Coetzee M, Van Wyk P, Booman M, Koekemoer LL, Hunt RH: Insecticide
resistance in malaria vector mosquitoes in a gold mining town in Ghana
and implications for malaria control. Bull Soc Path Exot 2006, 99:400-403.
Abdalla H, Matambo TS, Koekemoer LL, Mnzava AP, Hunt RH, Coetzee M:
Insecticide susceptibility and vector status of natural populations of
Anopheles arabiensis from Sudan. Trans R Soc Trop Med Hyg 2007,
Scholte E-J, Knols BGJ, Samson RA, Takken W: Entomopathogenic fungi for
mosquito control: A review. J Insect Sci 2004, 4:19.
Ferron P: Biological control of insect pests by entomopathogenic fungi.
Ann Rev Entomol 1978, 23:409-422.
Khan HK, Jayaraj S, Gopalan M: Muscardine fungi for the biological
control of agroforestry termite Odontotermes obesus . Insect Sci Appl 1993,
Milner RJ, Prior C: Susceptibility of Australian plague locust, Chortoicetes
terminifera, and wingless grasshopper, Phaulacridium vittatum, to the
fungi Metarhizium spp . Biol Control 1994, 4:132-137.
Thomas MB, Blanford S, Lomer CJ: Reduction of feeding by the variegated
grasshopper, Zonocerus variegates, following infection by the fungal
pathogen, Metarhizium flavoviride . Biocont Sci Technol 1997, 7:327-334.
Shah PA, Pell JK: Entomopathogenic fungi as biological control agents.
Appl Microbiol Biotechnol 2003, 61:413-423.
Clark TB, Kellen W, Fukuda T, Lindegren JE: Field and laboratory studies on
the pathogenicity of the fungus Beauveria bassiana to three genera of
mosquitoes. J Invertebr Pathol 1968, 11:1-7.
Scholte E-J, Njiru BN, Smallegang RC, Takken W, Knols BGJ: Infection of
malaria (Anopheles gambiae s.s.) and filariasis (Culex quinquefasciatus)
vectors with the entomopathogenic fungus Metarhizium anisopliae .
Malar J 2003, 2:29.
Blanford S, Chan BHK, Jenkins N, Sim D, Turner RJ, Read AF, Thomas MB:
Fungal pathogen reduces potential for malaria transmission. Science
Scholte E-J, Ng’habi K, Kihonda J, Takken W, Paaijmans K, Abdulla S,
Killeen GF, Knols BGJ: An entomopathogenic fungus for control of adult
African malaria mosquitoes. Science 2005, 308:1641-1643.
Thomas M, Read AF: Can fungal biopesticides control malaria?. Nat Rev
Farenhorst M, Farina D, Scholte E-J, Takken W, Hunt RH, Coetzee M,
Knols BGJ: African water storage pots for the delivery of the
entomopathogenic fungus Metarhizium anisopliae to the malaria vectors
Anopheles gambiae s.s. and Anopheles funestus . Am J Trop Med Hyg 2008,
Darbro J, Thomas MB: Spore persistence and likely aeroallergenicity of
entomopathogenic fungi used for mosquito control. Am J Trop Med Hyg
Hancock PA, Thomas MB, Godfray HCJ: An age-structured model to
evaluate the potential of novel malaria-control interventions: a case
study of fungal biopesticide sprays. Proc R Soc London 2009, 276:71-80.
Blanford S, Read A, Thomas MB: Thermal behaviour of Anopheles stephensi
in response to infection with malaria and fungal entomopathogens.
Malar J 2009, 8:72.
Read AF, Lynch PA, Thomas MB: How to make evolution-proof
insecticides for malaria control. PLoS Biol 2009, 7:4.
Achonduh OA, Tondje PR: First report of pathogenicity of Beauveria
bassiana RBL 1034 to the malaria vector, Anopheles gambiae s.l. (Diptera;
Culicidae) in Cameroon. Afr J Biotechnol 2008, 7:931-935.
Farenhorst M, Mouatcho JC, Kikankie CK, Brooke BD, Hunt RH, Thomas MB,
Koekemoer LL, Knols BGJ, Coetzee M: Fungal infection counters
insecticide resistance in African malaria mosquitoes. Proc Nat Acad Sci
Nauen R: Insecticide resistance in disease vectors of public health
importance. Pest Manag Sci 2007, 63:628-633.
Kelly-Hope L, Ranson H, Hemingway J: Lessons from the past: managing
insecticide resistance in malaria control and eradication programmes.
Lancet Infect Dis 2008, 8:387-398.
Watson DW, Mullens BA, Petersen JJ: Behavioural response of Musca
domestica (Diptera: Muscidae) to infection by Entomophthora muscae
(Zygomycetes: Entomophtorales). J Invertebr Pathol 1993, 61:10-16.
Kikankie et al. Malaria Journal 2010, 9:71
Page 8 of 9
29. Inglis DG, Johnson DL, Goettel MS: Effects of temperature and Download full-text
thermoregulation on mycosis by Beauveria bassiana in grasshoppers. Biol
Control 1996, 7:131-139.
Klass JI, Blanford S, Thomas MB: Development of a model for evaluating
the effects of environmental temperature and thermal behaviour on
biological control of locusts and grasshoppers using pathogens. Agr
Forest Entomol 2007, 9:189-199.
Klass JI, Blanford S, Thomas MB: Use of geographic information systems
to explore spatial variation in pathogen virulence and the implications
for biological control of locusts and grasshoppers. Agr Forest Entomol
Craig MH, Snow RW, Le Sueur D: A climate-based distribution model of
malaria transmission in sub-Saharan Africa. Parasitol Today 1999,
Knols BGJ, Njiru B, 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,
Okech BA, Gouagna LC, Killeen GF, Knols BGJ, Kabiru EW, Beier JC, Yan G,
Githure JI: Influence of sugar availability and indoor microclimate on
survival of Anopheles gambiae (Diptera: Culicidae) under semifield
conditions in western Kenya. J Med Entomol 2003, 40:657-663.
Okech BA, Gouagna LC, Knols BGJ, Kabiru EW, Killeen GF, Beier JC, Yan G,
Githure JI: Influence of indoor microclimate and diet on survival of
Anopheles gambiae s.s. (Diptera: Culicidae) in village house conditions in
western Kenya. Int J Trop Insect Sci 2004, 24:207-212.
Gillies M, Coetzee M: A Supplement to the Anophelinae of Africa South of the
Sahara The South African Institute for Medical Research, Johannesburg
Scott JA, Brogdon WG, Collins FH: Identification of single specimens of
the Anopheles gambiae complex by the polymerase chain reaction. Am J
World Health Organization: Test procedures for insecticide resistance
monitoring in malaria vectors, Bio efficacy and persistence of
insecticides on treated surfaces. WHO/CDS/CPC/MAL/98.12, Geneva 1998.
Cox DR: Regression models and life tables. J R Stat Soc B 1972,
Gillies M, De Meillon B: The Anophelinae of Africa South of the Sahara
(Ethiopian zoogeographical region) The South African Institute for Medical
Research, Johannesburg 1968.
Lindsay SW, Parson L, Thomas CJ: Mapping the ranges and relative
abundance of the two principal African malaria vectors, Anopheles
gambiae sensu stricto and An. arabiensis, using climate data. Proc Biol Sci
Petrarca V, Nugud AD, Ahmed MA, Harid AM, DiDeco MA, Coluzzi M:
Cytogenetics of the Anopheles gambiae complex in Sudan with special
reference to An. arabiensis : relationships with East and West African
populations. Med Vet Entomol 2000, 14:149-164.
Martinez-Torres D, Chandre F, Williamson MS, Darriet F, Serge JB,
Devonshire AL, Guillet P, Pasteur N, Pauron D: Molecular characterisation
of pyrethroid knockdown resistance (kdr) in the major malaria vector
Anopheles gambiae s.s . Insect Mol Biol 1998, 7:179-184.
Ranson H, Jensen B, Vulule JM, Wang X, Hemingway J, Collins FH:
Identification of a point mutation in the voltage-gated sodium channel
gene of Kenyan Anopheles gambiae associated with resistance to DDT
and pyrethroids. Insect Mol Biol 2000, 9:491-497.
Diabate A, Baldet T, Chandre F, Dabire R, Simard F, Ouedraogo JB, Guillet P,
Hougard JM: The role of agricultural use of insecticides in resistance to
pyrethroids in Anopheles gambiae s.l . in Burkina Faso. Am J Trop Med
Hyg 2002, 67:617-622.
Fanello C, Petrarca V, Della Torre A: The pyrethroid knock-down resistance
gene in the Anopheles gambiae complex in Mali and further indication
of incipient speciation within Anopheles gambiae s.s. Insect Mol Biol 2003,
Matambo TS, Abdalla H, Brooke BD, Koekemoer LL, Mnzava A, Hunt RH,
Coetzee M: Insecticide resistance in the malarial mosquito Anopheles
arabiensis and association with the kdr mutation. Med Vet Entomol 2006,
Agnew P, Berticat C, Bedhomme S, Sidobre C, Michalakis Y: Parasitism
increases and decreases the costs of insecticide resistance in
mosquitoes. Evol 2004, 58:579-586.
49. Tabashnik BE, Dennehy TJ, Carriere Y: Delayed resistance to transgenic
cotton in pink bollworm. Proc Natl Acad Sci USA 2005, 102:15389-15393.
Labbé P, Berticat C, Berthomieu A, Unal S, Bernard C, Weill M, Lenormand T:
Forty years of erratic insecticide resistance evolution in the mosquito
Culex pipiens . PloS Genetics 2007, 3:2190-2199.
Detinova TS: Age-grouping methods in Diptera of medical importance.
World Health Organization, Geneva 1962.
Farenhorst M, Knols BGJ: A novel method for standardized application of
fungal spore coatings for mosquito exposure bioassays. Malar J 2010,
Arthurs SP, Thomas MB: Effect of dose, pre-mortem host incubation
temperature and thermal behaviour on host mortality and mycosis and
sporulation of Metarhizium anisopliae var acridum in Schistocerca
gregaria . Biocontr Sci Technol 2001, 11:411-420.
Scholte E-J, Knols BGJ, Takken W: Infection of the malaria mosquito
Anopheles gambiae with the entomopathogenic fungus Metarhizium
anisopliae reduces blood feeding and fecundity. J Invertebr Pathol 2006,
Ondiaka S, Bukhari T, Farenhorst M, Takken W, Knols BGJ: Effects of fungal
infection on the host-seeking behaviour and fecundity of the malaria
mosquito Anopheles gambiae Giles. Proc Netherlands Entomol Soc Meet
Paaijmans KP, Read AF, Thomas MB: Understanding the link between
malaria risk and climate. Proc Nat Acad Sci 2009, 106:13844-13849.
Blanford S, Read AF, Thomas MB: Thermal behaviour of Anopheles
stephensi in response to infection with malaria and fungal
entomopathogens. Malar J 2009, 8:71.
Koella JC, Lynch PA, Thomas MB, Read AF: Towards evolution-proof
malaria control with insecticides. Evol Appl 2009, ISSN 1752-4571,
Hancock PA: Combining fungal biopesticides and insecticide-treated
bednets to enhance malaria control. PLoS Comput Biol 2009, 5(10):
Beier JC, Killeen GF, Githure JI: Short report: entomologic inoculation rates
and Plasmodium falciparum malaria prevalence in Africa. Am J Trop Med
Hyg 1999, 61:109-113.
Cite this article as: Kikankie et al.: The infectivity of the
entomopathogenic fungus Beauveria bassiana to insecticide-resistant
and susceptible Anopheles arabiensis mosquitoes at two different
temperatures. Malaria Journal 2010 9:71.
Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
Kikankie et al. Malaria Journal 2010, 9:71
Page 9 of 9