3. 3
Abstract
Background
Insecticide killing methods cause high fitness cost and selective pressure against female Anopheles
mosquitoes for malaria control. Environmentally safe and sustainable methods are needed for
mitigating the problem of current interventions. Past studied of Entomopathogenic Fungi (EPF) against
Anopheles gambiae s.s proved effective biological control. For more efficient application of EPF, “heat
and odour” could be considered for inclusion in the delivery of spores for fungal infection. Previous
studied showed that using cues, “heat and odour” evoked the landing response of An. gambiae towards
“heat and odour” release sources. We also assumed that inclusion of “heat and odour” during delivery
of Beauveria bassiana spores on black cotton cloth increase the landing response of Anopheles coluzzii
and subsequently infect and kill them.
Methods
Two free flight experiments were performed with An. coluzzii with spores from B. bassiana isolates
ARSEF 502 and 5641. The isolate ARSEF 502 was more virulent than 5641. Isolate 5641 was used in first
experiment where as 502 in second experiment. In free flight experiment, An. coluzzii were released for
24hrs in a free flight cage of a flight room. A black cotton cloth impregnated with B. bassiana spores
hanged in one side with or without “heat and odour” (see details in materials and methods parts).The
effect of “heat” or “heat and odour” on infection and survival of An. coluzzii was studied during delivery
of spores on black cotton cloth. The survival was recorded for ten days and an infection assay confirmed
the cause of death of An. coluzzii. To observe the negative effect of experimental conditions on An.
coluzzii after fungal exposure a forced exposure experiment was done.
Results
The rate of infection was almost the same in both experiment with inclusion of “heat and odour”.
However the rate of infection did not explain the shortest survival time for An. coluzzii than negative
control. With isolate 5641 there was a non-significant correlation between median survival time (days)
and infection rate (%). On the other hand a significant negative correlation was observed with 502. The
result of the effect of stress (sugar water deprivation) on An. coluzzii after fungal exposure showed that
unavailability of sugar water had impact on the survival of An. coluzzii.
Conclusions
From both experiments using “heat and odour” cues during delivery of B. bassiana spores increased the
infection. But only with isolate 502, the rate of infection was explained the survival time of An. coluzzii.
On the other hand there was less infection observed with more virulent isolate 502. In addition, spores
might be an effect on infection and consequently survival of An. coluzzii. The possible explanation could
be production of volatile compounds that attract An. coluzzii. It was first time to see the added effect of
“heat” or “heat and odour” during delivery of spores in a free-flight set-up. More research should
confirm the effects of “heat” or “heat and odour” on spores delivery in the field.
Keywords: Entomopathogenic fungi, Beauveria bassiana, Heat and odour, Vector control
4. 4
Table of Contents
1. Introduction........................................................................................................................... 6
1.1. Entomopathogenic fungi (EPF): Option for malaria control .................................................. 7
1.2. Mechanism of EPF to kill insect host .................................................................................... 8
1.3. Behavioural correlation of malaria vector and EPF............................................................... 9
1.4. Aim of the study .................................................................................................................10
1.5. Research Question..............................................................................................................10
2. Materials and methods.........................................................................................................11
2.1. Mosquito rearing................................................................................................................11
2.2. Selection of fungal isolates B.bassiana................................................................................11
2.3. Production of selected B. bassiana fungal isolates ..............................................................12
2.4. Preparation of spores suspension .......................................................................................13
2.5. Observing spores viability...................................................................................................13
2.6. Impregnation of black cotton cloth with spores ..................................................................14
2.7. Preparation of MB-5 odour blend .......................................................................................14
2.8. Free flight room exposure experiment................................................................................15
2.9. Effect of stress on survival after fungal exposure................................................................18
2.10. Preparation of forced exposure experiment......................................................................19
2.11. Statistical analysis for free flight and forced exposure experiments...................................19
3. Results ..................................................................................................................................20
3.1. Effect of heat and odour cues on infection of An. coluzzi i with B. bassiana isolate 5641.....20
3.2. Effect of infection by B. bassiana isolate 5641 on survival of An. coluzzii.............................21
3.3. Correlation of infection rate (%) with median survival time (days) ......................................23
3.4. Effect of heat and odour cues on infection of A. coluzzii with B. bassiana isolate 502..........23
3.5. Effect of infection by B. bassiana isolate 502 on survival of An. coluzzii................................24
3.6. Correlation of infection rate (%) with median survival time (days) ......................................26
3.7. Effect of stress on survival after fungal exposure................................................................27
4. Discussion .............................................................................................................................29
4.1. Effect of heat and odour cues for the Infection of An. coluzzii by B. bassiana isolates .........29
4.2. Effect of infection by B. bassiana isolate on survival of An. coluzzii .....................................30
4.3. Correlation between infection rate (%) and median survival time (days).............................31
4.4. Optimization of B. bassiana production ..............................................................................31
6. 6
1. Introduction
Malaria is a devastating disease to third world countries especially Africa as well as few areas in Asia due
to an increase in insecticide resistance. About 438.000 people died due to malaria infection in 2015 and
90% occurred in African countries (WHO, 2015). It is caused by Plasmodium parasites that are vectored
by female Anopheles mosquitoes. The parasite needs an arthropod host to complete its sexual
development, which takes about 7-14 days in female Anopheles mosquitoes (Pumpuni and Beier, 1995).
After successful completion, further transmission occurs via Anopheles mosquito upon biting a human
for taking blood meal (Beier, 1998 and Service, 1996). In general symptoms appear one week later after
biting and disease may cause death if no curative measure is taken. There are five parasites responsible
for malaria and among them Plasmodium falciparum and P. vivax are most common whereas P.
falciparum is most deadly for humans (Malaria report WHO, 2015).
Fortunately, deaths due to malaria significantly reduced from 2000 to 2015 (Bhatt et al.,
2015).Interventions against malaria over the last 15 years have reduced malaria from large parts of
Africa. These efforts of malaria control reduced the incidence of P. falciparum by about 40% and averted
663 million clinical cases in 2015 (Bhatt et al., 2015). The current practices for malaria control is
Insecticide Treated bed Nets (ITNs), Artemisinin-based Combination Therapy (ACT) and Indoor Residual
Spraying (IRS). Among the malaria control interventions, ITNs reduced 68%, whereas 22% and 10% of
cases were averted by ACT and IRS, respectively (figure 1).
Figure 1: The effects of interventions from 2000 to 2015 on averted malaria cases from large parts of
Africa (Bhatt et al., 2015).
7. 7
Meanwhile, there is also concerning news that malaria mosquitoes become resistant against insecticides
e.g.pyrethroids (Yewhalaw et al., 2011, and Chanda et al., 2011).The application of insecticide by
treating bed nets as well as indoor spraying kills mosquitoes within a day after exposure, which
eventually causes a high selection pressure against malaria mosquitoes for malaria control (Knols et al.,
2010). In the long run, insecticide based methods develop high fitness advantages for the resistant
mosquitoes that can be passed to the next generation of malaria vectors.
1.1. Entomopathogenic fungi (EPF): Option for malaria control
Due to the insecticide resistance, scientists are looking for smart options to permanently eradicate the
malaria disease from the earth. Hence they are searching alternative option called biological control
(use of a living organism to kill another living organism) to eliminate the malaria vector. The malaria
vector that showed the resistance against insecticide was found susceptible to EPF. As a result only
biological controls with EPF reduce the insecticide resistant mosquitoes (Blanford et al., 2011).It kills the
mosquitoes very slowly and able to mitigate the problem with insecticide based control tactics. For
example, an isolate with high virulence level allows survival of malaria mosquito up to 7-9 days.
Thereafter EPF cause high mortality of malaria mosquito and reduce the transmission of malaria
parasites (figure 2) (Thomas and Read, 2007).In addition, EPF not only kill malaria vector, but also reduce
the proportion of surviving female mosquitoes carrying sporozoites in their salivary glands. Moreover,
the tendency of infecting mosquitoes by EPF for taking a blood meal is also reduced substantially
compared to uninfected mosquitoes (Blanford et al., 2005). Not only reduced the taking blood meal but
an also killing older female mosquito called late-life action (LLA) is also possible by EPF (Thomas and
Read, 2009).
The success of EPF for reducing crop pests already proved in the field of biological control (Kanzok and
Jacobs-Lorana, 2006). Not only for crop pest but also human disease like malaria EPF remain success.
Beauveria bassiana & Metarhizium anisopliae both are EPF able to invade malaria vectors within 3-14
days based on exposure dose and fungal isolate (Moreira et al., 2009).According to Mnyone et al.,
(2012) the survival of the malaria vector was reduced by 39-57% which was estimated to reduce 75-80%
of malaria transmission after the exposure of spores around the surface of bed nets in a rural village of
Tanzania.
8. 8
Figure 2: Sustainability of chemical and biological interventions against malaria mosquitoes. Female
mosquito transmit malaria parasite if no intervention and become infecticious (A).They die rapidly
when insecticide is used and are unable to transmit malaria diseases.High speed killing may cause
fitness cost as well as selection pressure called insecticide resistant (B).Slow speed fungus kill the
mosquito before transmission of parasite and mitigate selection pressure as well as fitness cost (C)
(Thomas and Read, 2007).
In a nutshell, only chemical control seems inappropriate to eliminate malaria vectors in the future.
Combination of biological and chemical control called Integrated Vector Management (IVM) could be
the best solution to control malaria diseases.
For the evaluation of B. bassiana isolates, Valero-Jimenez et al., (2014) used 29 fungal isolates and
classified the fungal virulence based on the hazard ratio with isolate IMI 391510 as a reference. The
results classified all fungal isolates according to the relative virulence of B. bassiana. The virulence,
viability, infectivity as well as persistence of the spores in the field are crucial for the success of future
biological interventions against malaria vectors. Hence, the development of a long lasting formulation as
well as improving the delivery platform of B. bassiana spores needs to be explored (Knols et al., 2010).
1.2. Mechanism of EPF to kill insect host
The infection mechanism of EPF involves multiple steps, which ends with sporulation of fungus on an
infected cadaver (Figure 3). At the beginning of infection, conidia (spores) first adhere to the host
cuticle. Afterwards they germinate and develop a germ tube as well as a penetration structure called
appressorium. Then, the germ tube enters into the host body by degrading the host cuticle. The
combination of mechanical pressure as well cuticle degrading enzymes is involved here. When the fungi
enter into the host body, they grow vegetatively in the host haemocoel and infection is confirmed by the
growth of conidia on infected cadaver (Gillespie et al., 2000 and Roberts and Legar, 2004).
A B C
9. 9
Figure 3: The mechanism of infection of EPF (Valero-Jimenez et al., 2016).
1.3. Behavioural correlation of malaria vector and EPF
The host seeking behaviour of female malaria mosquitoes is studied because they have a strong
olfactory system to detect the odour released by the host. The antennae as well maxillary palps are
responsible for an olfactory response towards a potential host. The olfactory receptor neuron (ORNs) of
maxillary palp of Aedes aegypti and An.gambiae is strongly sensitive to CO2 as well 1-octen-3-ol. These
cues help them to find the human host for blood meal (George et al., 2011).George et al., 2013 stated
that B. bassiana spores can attract Anopheles stephensi. The reason behind the attraction of An.
stephensi towards B. bassiana spores may be the production of volatiles that mimic human odour. It was
the first report that stated B. bassiana spores can attract An. stephensi. Not only CO2 ,1-octen-3-ol and
human odour but also heat can influence the attraction of An. gambiae (Spitzen, et al., 2013).
The delivery efficacy of B. bassiana spores increased when black cotton cloth and heat was included in a
small mosquito cage experiment (Kaasschieter, 2015). It was supposed that An. coluzzii was forced to
contact the spores in this small mosquito cage experiment (Kaasschieter report, 2015).He concluded
that the infection rate was increased due to inclusion of heat during exposure of mosquitoes in small
bug-dorm cages. He also mentioned that it could be beneficial for field application when it would work
in larger spaces as he did this experiment in small bug-dorm cages. Hence to scale up the results of small
cage experiments, the use of a free flight experimental set-up may provide confirmation of the effect of
heat on infection by B. bassiana spores and consequently survival of An. coluzzii.
10. 10
1.4. Aim of the study
The main goal of my study was to improve the delivery efficacy of B. bassiana spores by exploiting the
natural behaviour of An. coluzzii with the inclusion of “heat and odour” in a free flight room.
1.5. Research Question
The flight behaviour of An. gambiae was tracked with 3-D analysis with the inclusion of the effects of
heat and human odour (Spitzen, et al., 2013). They concluded that addition of heat and human odour
increases the flight speed as well landing response of An. gambiae on the sources. The rate of infection
and survival of An. coluzzii with different fungal isolates of B. bassiana with the inclusion of heat and
odour is thus interesting to further explore. Hence the proposed research question of my thesis was:
RQ: How could the delivery method of B. bassiana be improved by exploiting the natural behaviour of
An. coluzzii with the inclusion of heat and odour in a free-flight experimental set-up?
Main hypothesis
It is assumed that the exposure of An. coluzzii to the spores of B. bassiana with heat and odour will
increase the attraction and landing response of An. coluzzii on fungus-infected black cotton cloth in a
free-flight experiment. Hence, the more An. coluzzii will be infected by spores, the higher proportion of
An. coluzzii will die as a result of the infection.
To answer my main research question I formulated two sub-research questions
SRQ1: Does the inclusion of heat and/or odour result in increased fungal infection rates in An. coluzzii?
H1: Inclusion of heat and odour will increase attraction and landing of An. coluzzii on B. bassiana spores
treated black cotton cloth and then infect and kill them. The most virulent isolate will show a higher
infection than the least virulent isolate.
SRQ2: Does the inclusion of heat and/or odour result in decreased survival of An. coluzzii?
H2: Higher infection rates with more virulent isolate result in shorter survival of An. coluzzii than least
one.
Beside test of above two hypotheses, we also did an additional experiment to answer the following
research question
SRQ3: How experimental conditions could negatively affect the survival of An. coluzzii?
H3: It is assumed that 24hrs deprivation from sugar water might be caused quick mortality of An. coluzzii
than not deprived mosquitoes.
11. 11
2. Materials and methods
2.1. Mosquito rearing
The African malaria mosquito An. coluzzii was used in this study. This mosquito has been cultured in the
Laboratory of Entomology since 1988 and was collected from Suakoko, Liberia (Courtesy of Prof. M.
Coluzzi) in 1987. In my study system An. coluzzii cultures were used to test with B. bassiana. Mosquitoes
were reared at 27±1o
C, 70±5% R.H., and a day-night cycle of 12:12 L: D. Mosquitoes were fed with a 6%
glucose solution ad libitum and offered a blood meal twice a week for 10 minutes. Females laid eggs on
wet filter paper and these were transferred to water trays before hatching. Larvae were fed with
TetraminH (Tetrawerke, Melle, Germany) fish food daily (Spitzen et al., 2013).
2.2. Selection of fungal isolates B.bassiana
Valero-Jimenez et al., (2014) tested 29 fungal isolates and classified these based on their different
virulence level. All isolates were able to kill An. coluzzii after exposure. He concluded that the daily
chance of death with the most virulent isolate of B. bassiana was ten times higher than with the least
virulent one. The B. bassiana isolates were obtained from a reference collection of fungal isolates
(Agricultural Research Services Entomopathogenic Fungi (ARSEF), USDA). The isolates originated from
different countries of the world. In my study system isolates ARSEF 502 and 5641 were used for testing
An. coluzzii in a free flight experimental set-up (see paragraph 2.9). The isolates ARSEF 502 is more
virulent (Figure 4) than 5641 based on hazard ratio. The two isolates used for free flight exposure
experiments described in table 1.
Figure 4: The figure shows different fungal isolates of B. bassiana grouped as highly virulent (triangle),
medium virulent (circle) and least virulent (square). Hazard ratio of fungal isolates is compared in
reference to isolate IMI 391510.The isolate marked in red was used in the experimental bioassay. The
other selected isolate ARSEF 5641 was not in the list but belongs to the least virulence category
(Valero-Jimenez et al., 2014).
12. 12
Table 1. Selected B. bassiana isolate with their original host and place of origin
Isolate Host Place of origin Reference
ARSEF 5641 Orthoptera; Acrididae;
Schistocerca gregaria
Ethiopia: Eritrea,
Shelsela
Valero Jimenez et
al., 2014
ARSEF 502 Lepidoptera; Pyralidae;
Ostrinia nubilalis
China Valero Jimenez et
al., 2014
2.3. Production of selected B. bassiana fungal isolates
We performed three spores production trials of selected fungal isolates in the ecology lab of
entomology with the collaboration of phyto-pathology lab. The main goal of these three trials for mass
production of selected B. bassiana isolates to use in free flight experiment. Unfortunately, we could not
produce the selected fungal isolates due to infection (Table 2).Almost 80% infection was found in mass
production of fungal isolates among the trials. During mass production we followed few steps that
performed in different lab due to lack of complete facility in entomology lab. It could be result of
infection among the trials.
For mass production, fungi were first grown with on petri dish using as a medium Sabouraud Dextrose
Agar (SDAY) with 1% yeast extract for 14 days at 27°C. Afterwards spores were harvested with a 0.05%
Tween 80 solution to make spores suspension. Then the spores suspension was kept at −80°C un l mass
production on the 0.2L glass tube system (Valero-Jimenez et al., 2014).The detailed protocol of mass
production of selected isolate is described in appendix 1 and complete set-up was found in figure-5.
Due to the infection of mass production of B. bassiana isolates we collected the selected fungal isolates
that were produced previously by bioprocess engineering at Wageningen University and Research
Centre (WUR), The Netherlands.
Table 2. Results of mass spores production of selected fungal isolates in laboratory of entomology
Trial Isolate Tube number
(0.2L glass tube)
Amount in
Grams (gm)
Comments
1 ARSEF 8028 1 ------- Almost no spores only mycelia, lots of
hemp
ARSEF 8028 2 ------- Infected
2 ARSEF 8028 1 0.28 A lot of spores with little hemp
ARSEF 8028 2 ------- Fungi were grown but I observed the
growth of other fungi
ARSEF 5641 3 ------- Infected
ARSEF 5641 4 ------- Infected
3 ARSEF 220 1 ------- Infected
ARSEF 4135 2 ------- Infected
13. 13
Figure 5: Complete set-up of mass production of fungal isolate placed in climate cabinet (Temp. 250
C)
in ecology lab. A: 0.2L glass tube with hemp and fungus, B: red-capped bottles for humidity, C:
thermometer (Photo; Kamrul).
2.4. Preparation of spores suspension
The infection rate of mosquito by EPF depends on dose, but we used the same concentration of spores
for each experiment. For all three studies the concentration 1*109
spores/ml of spores suspension was
sprayed on black cotton cloth. It makes 1010
spores/m2
on the black cotton cloth. The suspension for
each isolate was made by mixing approximately 50mg of the isolate’s spores with 1.66ml Shellsol® T
mineral oil (G.J. Arkenbout B.V; Rotterdam, Netherlands). This suspension was mixed vigorously on a
vortex. To get a better estimation of the exact concentration, a 1:20 dilution was made and put on a
0.01mm depth Bürker-Türk haemocytometer to count the number of spores (W. Schreck; Hofheim,
Germany). Spores do not survive long in the Shellsol T suspension, so it was essential to prepare a new
suspension with the same concentration, every time mosquitoes were infected (Keizer, 2014). After
counting the spores, it was calculated how much Shellsoll T needed to be added to get 3ml of a 1*109
spores/ml suspension. The prepared 3ml spore suspension was used for spraying a single black cotton
cloth (30×30cm).
2.5. Observing spores viability
To know the viability of spores the percentage of spores that germinated was investigated. From the
1:20 dilution, a 20μl of spore suspension was put on a Sabouraud Dextrose Agar (SDA) plate with 1%
yeast extract. The viability of ARSEF 502 and 5641 was checked before free flight exposure experiments.
Three plates were made per infection, creating three replicates (Keizer, 2014).
C
B
A
14. 14
Figure 6: Germination of spores on an
SDA plate (Keizer, 2014)
Plates were sealed with Parafilm® and incubated at 27°C. After twenty hours the plates were put under
a microscope. Approximate 100 spores were counted from each plate on three randomly selected spots.
The germination of spore was identified when the hyphae was 1.5 times higher than non germinated
spore (figure 6). Afterwards, the percentage of germinated spores was calculated and used as an
indicator for spore viability.
2.6. Impregnation of black cotton cloth with spores
For free-flight experiments a 30×30 cm black cotton cloth was used. A spore suspension was prepared as
described in paragraph 2.4. For spraying black cotton cloth, it was attached to the back side of rearing
tray (58×38cm) with masking tape. An Airbrush ‘Basic’ with a glass vial (Conrad Electronic) was attached
to a Baby AC-55 compressor (Ding Hwa Co Ltd) to spray the spore suspension. The spore suspension was
applied on one side of the black cotton cloth using a zigzag motion. Then the black cotton cloth was
dried overnight in the ecology lab before start of the experiments.
2.7. Preparation of MB-5 odour blend
Keizer (2014) concluded that mixing of odour blend chemicals with spores had a negative effect on
spore viability. Therefore, the odour blend was prepared separately for the experiment. The MB5 odour
blend (Mukabana et al., 2012; Table 3) was used in the first two experiments. During preparation of
odour blend, 1ml of each component was put in a glass vial. Afterwards, the vial was slightly warmed
with heat from the hands to mix the components properly. A clean 26.5×5cm strip of 15 denier nylon
ladies thigh stocking was added to the odour mix in the glass vial. Tweezers were used to prevent
contamination. The vial was closed immediately and placed at room temperature in a fume hood for
thirty minutes. The strip was air dried under a fume hood for 5 hours. Then strips were wrapped in
aluminium foil individually and stored at 40
C in the freezer. For each replicate a new strip was made.
15. 15
Table 3. MB5 odour blend with their concentration and solvent (Mukabana et al., 2012)
2.8. Free flight room exposure experiment
The first experiment was done in the free flight room that was equipped with a large mosquito cage to
scale up the results of small cage (30x30x30 cm) experiments (Kaasschieter, 2015). The size of the flight
room was 3.8×3.7×3.2m and a large netted chamber 3×2.5×2.5m (see figure 7) (Hiscox et al.,2014). In
free flight room a black cotton cloth (30×30cm) that was received either spores or Shellsol T spraying
hanged into a one side of free flight cage (see figure 8A). The following five treatments were evaluated:
Shellsol T (negative control)
Spores suspended in Shellsol T (spores only)
Spores suspended in Shellsol T and inclusion of heat (spores and heat)
Spores suspended in Shellsol T and MB5 odour blend, excluding heat (spores and odour)
Spores suspended in Shellsol T, including MB5 odour blend and heat (spores, heat and odour)
Figure 7: Layout of a free flight room (Kamrul)
Component Concentration Solvent
Ammonia 2.5% Water
L-(+)-lactic acid 88-92% Water
Tetradecanoic acid 0.00025g/l Ethanol
3-Methyl-1-buthanol 0.000001% Parafin oil
Butyl-1-amine 0.001% Parafin oil
Legend
Black cloth
Release point
Humidifier
Heater
CO
2
source
2.5m
3m
16. 16
8A 8B
Figure 8: A black cotton cloth of 30x30 cm (8A) placed in the flight room and heat cable (8B) that was
attached behind the cloth panel (Photo; Kamrul).
Each of the five treatments was replicated three times. The nylon strip, impregnated with the MB5
odour blend, was attached to the black cotton cloth with a small piece of tape at the top.CO2 (5%) was
provided in all treatments through a small pipe from a container, as this can stimulate host-seeking
behaviour. Heat was included with the use of a 4.5m 25W Rep Tech heat cable (Reptile Technologies;
Gorinchem, Netherlands). It was put in a zigzag line with eight turns behind the cloth, using one side of a
bug-dorm cage (Figure 8B). It was reported by Spitzen et al., (2013) that a landing response could be
evoked at 34°C.
Approximately 40-45 female An. coluzzii aged 5-9 days were released in the free flight cage for each of
the replicates. Afterwards thirty female An. coluzzii were collected from inside the free-flight cage. A
larger number of mosquitoes than needed for follow-up were released, because of mortality noticed
during the 24h of the experiments, probably as a result of dehydration or a lack of sugar water in the
free-flight cage. It was assumed that selected female An. coluzzii were mated before and actively
searching for a blood meal when they were exposed to heat and odour cues. An. coluzzii were released
for 24 hrs and they were carefully collected from the free-flight cage with an automatic aspirator.
17. 17
Figure 9: Holding buckets used for keeping mosquitoes in climate cabinet (Photo; Kamrul)
The 30 collected mosquitoes were put in a holding bucket by replicate treatment, with a piece of cotton
wool moistened with 6% sugar water on top (see figure 9). The holding buckets were then placed in the
climate cabinet at 270
C. Each day the number of dead mosquitoes was recorded. The dead mosquitoes
were removed and put into petri dishes for sporulation test. Mosquitoes that showed signs of
sporulation (approximately 3-8 days) were noted as infected (see figure 9). The survival of mosquito was
recorded for ten days. Live mosquitoes after day 10 were ‘censored’ within the data analyses (see
paragraph 2.11).
Figure 10: Sporulating mosquitoes in a Petri dish. (Keizer, 2014)
18. 18
The experiment described above was repeated with isolate ARSEF 502. This isolate was more virulent
than ARSEF 5641.
2.9. Effect of stress on survival after fungal exposure
We assumed that in the free-flight room a significant number of An. coluzzii died due to ‘sugar water
deprivation’ rather than contact with pathogenic spores. The temperature and humidity were not
optimal for free flight experiments. Moreover, we did not provide access to sugar water for the 24hrs of
the experiment in the free-flight room. Therefore, a forced exposure experiment was done with the
following four treatments that simultaneously tested the effects of sugar water deprivation and fungal
exposure on survival.
Stress + infected (T1)
No stress + Infected (T2)
Stress + Uninfected (T3)
No stress + Uninfected (T4)
Figure 11: Forced exposure experiment using PVC tubes (Photo; Kamrul)
Each of the treatments was replicated three times. Mosquitoes for both stress treatments were put in a
bug-dorm cage without sugar water beforehand and then kept in the rearing room for 24hrs.Thirty
female An. coluzzii mosquitoes aged 5-9 days were released per PVC tube for 2hrs (see figure 11).After
that mosquitoes were collected with aspirator and put in holding buckets, with a piece of cotton wool
moistened with sugar water on top. Then, the holding buckets were placed in the climate cabinet with
270
C temperature. Each day the number of dead mosquitoes was recorded up to ten days. Live
mosquitoes after day 10 were ‘censored’ within the data analyses (see paragraph 2.11).
19. 19
2.10. Preparation of forced exposure experiment
During the experiment mosquitoes were exposed in the set-up developed by Farenhorst and Knols
(2010). The inside of the PVC tube (h=15cm, Ø=8cm) was covered with a glossy proofing paper of
30×8cm in size. The spores from isolates 502 were used in this experiment. After vigorously vortexing
the spores solution of 1*109
spores/ml, 0.9 ml of spore suspension was pipetted in a straight horizontal
line at 3cm from the top of the glossy side of the paper. The spores were equally distributed along the
paper with the use of an automatic K-Control Coater with a K-bar of 24μm on speed setting 4 (RK Print
Coat Instruments Ltd; Herts, UK). The control treatment was prepared by using the same set-up, but
with 0.9 ml of Shellsol T only (i.e. without spores). The papers were left to dry overnight. The next day
the papers were cut to fit the tube and afterwards put into the PVC tube. The papers were fixed to the
tube with two paperclips and the tube was closed off on both sides with plastic microwave foil with
some small holes in it.
2.11. Statistical analysis for free flight and forced exposure experiments
Data were analysed with IBM SPSS Vol. 20.1. A chi-square test was performed for comparing infection
rates from the first two experiments. Afterwards, Binary logistic regression analysis was done to see the
difference between the treatments for infection due to inclusion of “heat or “heat and odour”. The
Kaplan Meier log rank (Mantel-Cox) test was done for survival analysis of second experiment. Pair wise
comparison was done for all treatments of each experiment to see the significant differences between
the treatments. A linear regression analysis was done to correlate median survival time (days) and
infection rate (%) for the first two experiments. Finally, a Cox regression analysis was done for the
experiment that evaluated the main effects of stress (sugar water deprivation) and fungal infection, as
well as their interaction.
20. 20
3. Results
3.1. Effect of heat and odour cues on infection of An. coluzzi i with B. bassiana isolate 5641
The goal of the experiments was to investigate whether the “heat” or combination of “heat and odour”
would increase the infection rate of An. coluzzii with the selected isolate of B. bassiana. It was expected
that mosquitoes should be attracted more to “heat” or “heat and odour” treated black cotton cloth and
consequently become infected and then killed. It was found that infection rate was less in all the
treatments of two bio-assay experiments. The infection was also observed in the negative control due to
contamination when no spore suspension was applied.
On the other hand, infection was slightly higher in “spores only” treatment about 17% compared to
either heat (10%) or odour (5%) separately. It was clearly observed that infection rate with the
combination of “heat and odour” increased 13% in compare to “spores only” treatment. There was a
significant difference found among treatments (Binary logistic regression, χ2
= 12.774, df= 3, p<0.005).
The “spores only” treatment was not significantly different to “spore and heat” treatments (Binary
logistic regression, χ2
= 1.650, df= 1, p= 0.199) whereas significantly differ from “spore and odour” and
“spore, heat and odour” treatments (Binary logistic regression, χ2
= 5.238, df= 1, p<0.05 and χ2
= 9.558,
df= 1, p<0.005) respectively. The % of infection was found in figure 12.
Figure 12: Percentage of infection of An. coluzzii exposed to negative control (black cotton cloth with
Shellsol T), black cotton cloth with, respectively, B. bassiana “spores only”, B. bassiana “spores and
heat”, B. bassiana “spores and odour”, and B. bassiana “spores, heat and odour”. Isolate ARSEF 5641
was used in this experiment.* = significantly differ from “spores only” treatments (Binary logistic
regression, p<0.05). NS= non significant from “spore only” treatments (p>0.05). Error bars: +/-
standard error.
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
Negative
Control
Spore only Spore and heat Spore and
odour
Spore, heat and
odour
%Infection
Treatments
NS
*
*
21. 21
3.2. Effect of infection by B. bassiana isolate 5641 on survival of An. coluzzii
The next objective of this study was to investigate how infection would influence the survival of An.
coluzzii females by B. bassiana isolates. It was expected that high infection rate result in shorter survival
of An. coluzzii. All three replicates of each treatment were analysed individually before being pooled
together to see the effects of individual replicates. All three replicates of each treatment had significant
variation (Log-rank Mantel-Cox; p<0.05) except negative control (Log-rank Mantel-Cox; p>0.05).Due to
this variation we did not pooled the data together. The survival curves of experiment-1 can be found in
Figure-13A. To see the variation among the replicates we showed a survival curve for “spore and heat”
treatments (see figure 13B) and for rest of the treatments (see the appendix-2A).
Figure 13A: Cumulative daily survival of female An. coluzzii exposed to negative control (black cotton
cloth with Shellsol), black cotton cloth with, respectively, B. bassiana “spores only”, B. bassiana
“spores and heat”, B. bassiana “spores and odour”, and B. bassiana “spores, heat and odour”. Isolate
ARSEF 5641 was used in this experiment. Error bars: +/- standard error.
0
0.2
0.4
0.6
0.8
1
0 1 2 3 4 5 6 7 8 9 10
Days after exposure
Cumulativesurvival(±SEM)
Negative Control
Only Spore
Spore and heat
Spore and odour
Spore, heat and odour
Survival of An. coluzzii in free flight room
22. 22
Figure 13B: Variation in cumulative survival of female An. coluzzii among three replicates of “spore
and heat” treatments.
The p values of each treatment for the pairwise comparison were found in Table-4. It was found that
mosquitoes survived longest time in negative control in comparison to other treatments and
significantly differing with all other treatments (Table-4). Apart from negative control all the spores
treatment did not differ significantly from each other. In this experiment nearly all mosquitoes died
within 10 days except negative control where 52% mosquitoes were died within the same number of
days.
Table 4. Pairwise comparisons between the treatments by using Log-rank (Mantel- Cox) tests for
experiment-1. Each of the treatment consists of three replicate. The Values with * significantly
different after the Bonferroni correction (p<0.005) was applied.
0
0.2
0.4
0.6
0.8
1
0 1 2 3 4 5 6 7 8 9 10
Days after exposure
Cumulativesurvival
Replicate-1
Replicate-2
Replicate-3
Survival of An. coluzzii for "spore and heat" treatment
Treatment
Spore only Spore and heat Spore and odour Spore, heat and odour
Chi-
Square
Sig. Chi-
Square
Sig. Chi-
Square
Sig. Chi-
Square
Sig.
Negative
Control
67.556 0.000* 56.464 0.000* 80.734 0.000* 60.696 0.000*
Spore only 1.507 .220 .344 .557 1.625 .202
Spore and heat 4.233 .040 .108 .742
Spore and odour 4.321 .038
23. 23
3.3. Correlation of infection rate (%) with median survival time (days)
To investigate the correlation between infections rates (%) with median survival time (days) a linear
regression analysis was done. It was expected that the rate of infection was negatively correlated with
median survival time. The results were showed in figure 14.Each dot in the figure represents the survival
and infection of a single replicate. The analysis indicated that there was a non-significant correlation
between infection rate and median survival time (df=1, F= 2.144, p>0.05).
Figure 14: The rate of infection plotted against median survival time (days). All replicate of
experiment-1 was used as separate data points. Negative control was excluded as it had received only
shellsol T.
3.4. Effect of heat and odour cues on infection of A. coluzzii with B. bassiana isolate 502
The next experiment repeated the first experiment, but this time we used the more virulent isolate
ARSEF 502. It was expected that infection should be higher in the more virulent isolate than the least
virulent one and should result in more infections in mosquitoes than the previous experiment. The
infection was about 26% in “spore, heat and odour” treatment which was almost double than “spore
only” (17% + 9% = 26%, so 13% on average over two experiments) treatments when we combined the
two experiments ( see figure 12 & 15). Approximately 1 in 4 host-seeking mosquitoes becomes infected
within a 24h period when attracted to a smelly and warm surface infected with B. bassiana spores
where as “spores only” treatment it is about 1 in 8 mosquitoes.
There was a significant difference among the treatments (Binary logistic regression χ2
=19.972, df=3,
p<0.01). Binary logistic regression was carried out to see the significant differences among the individual
treatments. The “spore only” treatment was significantly different from “spore and heat” (χ2
=8.655,
y = 0.070x + 4.065
R² = 0.176
0
1
2
3
4
5
6
7
8
9
0.0 5.0 10.0 15.0 20.0 25.0 30.0
Mediansurvivaltime(days)
Infection rate (%)
Effect of infection rate on median survival of An. coluzzii
Median Survival
Time(days)
Linear (Median Survival
Time(days))
24. 24
df=1, p<0.005) and “spore heat and odour” treatment (χ2
=9.876, df=1, p<0.005) but not significantly
different from “spore and odour” treatment (χ2
=0.083, df=1, p=0.774). It could be concluded from
experiment-2 that either “heat” or the combination of “heat and odour” increased the infection rate.
Figure 15: Percentage of infection of An. coluzzii exposed to negative control (black cotton cloth with
Shellsol T), black cotton cloth with, respectively, B. bassiana “spores only”, B. bassiana “spores and
heat”, B. bassiana “spores and odour”, and B. bassiana “spores, heat and odour”. Isolate ARSEF 502
was used in this experiment.* = significantly differ from “spores only” treatments (Binary logistic
regression, p<0.005). NS= non significant from “spore only” treatments (p>0.05). Error bars: +/-
standard error.
3.5. Effect of infection by B. bassiana isolate 502 on survival of An. coluzzii
To investigate how infection could influence the survival of An. coluzzii more virulence B. bassiana
isolate 502 was used in second experiment. It was also expected that high infection rate would result in
shorter survival of An. coluzzii. All three replicate of each treatment were analysed individually before
being pooled together to see the effects of individual replicates. No significant difference between the
replicates was found (Log-rank Mantel-Cox; p>0.05). Afterwards all three replicates were pooled
together to see the differences between the treatments. The Log-rank (Mantel-Cox) test showed a
significant difference among the treatments (χ2
=172.452, df=4, p<0.01). The survival curves of
experiment-2 can be found in figure 16.
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
Negative Control Spore only Spore and heat Spore and odour Spore, heat and
odour
%Infection
Treatments
*
*
NS
25. 25
Figure 16: Cumulative daily survival of female An. coluzzii exposed to negative control (black cotton
cloth with Shellsol), black cotton cloth with, respectively, B. bassiana “spores only”, B. bassiana
“spores and heat”, B. bassiana “spores and odour”, and B. bassiana “spores, heat and odour”. Isolate
ARSEF 502 was used in this experiment. Letters behind the treatments based on original p-value from
log-rank test when the Bonferroni correction (p<0.005) was applied.
Pairwise comparisons of each of the treatments are found in Table-5. Among the treatments, negative
control had highest survival time. The negative control was significantly different from “spore only”
(χ2
=88.759,df=1,p<0.01), “spore and heat” (χ2
=105.445, df=1, p<0.01) “spore and odour” (χ2
=91.536,
df=1, p<0.01) and “spore, heat and odour” treatments (χ2
=106.315, df=1, p<0.01) respectively. Apart
from negative control all the B. bassiana spore treatments did not differ significantly from each other.
Similar to the first experiment with ARSEF 5641, 49% of mosquitoes from the negative control were alive
after 10 days. On the other hand mosquitoes died within 8 days for rest of the treatments. It was
observed that there was little difference in the survival curve of experiment-1 vs. experiment-2.
0
0.2
0.4
0.6
0.8
1
0 1 2 3 4 5 6 7 8 9 10
Days after exposure
Cumulativesurvival(±SEM)
Negative Control
Spore only
Spore and heat
Spore and odour
Spore, heat and odour
Survival of An. coluzzii in free flight room
a
b
b
b
b
26. 26
Table 5. Pairwise comparisons for all treatments using Log-rank (Mantel-Cox) tests for experiment-2.
Each of the treatment consists of three replicate. Values with * significantly different when the
Bonferroni correction (p<0.005) was applied.
3.6. Correlation of infection rate (%) with median survival time (days)
A linear regression analysis was also done in experiment-2 to see the correlation between infection rate
(%) and median survival time (days). As we used a more virulent isolate, % infection should be negatively
correlated with median survival time (days). There should be also a shorter survival time in experiment-2
than experiment-1. The regression curve is found in figure-17. Each dot in the figure represents the
survival and infection of a single replicate. A linear regression analysis showed that infection rate
explained 68% of the variation in median survival time (days). The result showed that there is a
significant negative correlation between infection rate and median survival time (df=1, F= 20.847,
p<0.01).
Treatment
Spore only Spore and heat Spore and odour Spore, heat and odour
Chi-
Square
Sig. Chi-
Square
Sig. Chi-
Square
Sig. Chi-
Square
Sig.
Negative
Control
88.759 0.000* 105.445 0.000* 91.536 0.000* 106.315 0.000*
Spore only 1.666 .197 1.841 .175 2.381 .123
Spore and heat 5.122 .024 .035 .851
Spore and odour 4.056 .044
27. 27
Figure 17: The rate of infection plotted against median survival time (days). Each replicate of
experiment-2 was used as separate data point. Negative control was excluded as it had received only
shellsol T.
3.7. Effect of stress on survival after fungal exposure
The objective of this experiment is to see the effect of stress on survival of An. coluzzii when they are
forcedly exposed in PVC tube. It was observed in the flight room, the infection was lower than expected
in relation to survival curve and a significant number of An. coluzzii might be died due to temperature,
and humidity variation as well as unavailability of sugar water. It was assumed that mosquitoes with
stress treatments died earlier than mosquitoes without stress treatments. The survival curves are found
in figure 18 and pairwise comparison for each treatment was found in Table-6.
The survival curve referred us, there was an impact for stress (sugar water deprivation) on infected
group of mosquitoes VS uninfected group of mosquitoes. Then we did Cox Regression analysis to
evaluate the interaction between stress and infection. A significant interaction was found between
stress and infection (Cox Regression, p<0.05). Afterwards we run this model again to see how large
stress (sugar water deprivation) effect was for the infected and uninfected group of An. coluzzii
separately. The uninfected mosquitoes had a relatively higher chance of death due to stress (Hazard
ratio; 2.21, p<0.001) than infected group for which the hazard was actually not significantly different
between sugar water deprived and normal mosquitoes (Hazard ratio; 1.36, p>0.05).
y = -0.062*x + 4.136
R² = 0.676
0.0
1.0
2.0
3.0
4.0
5.0
6.0
0.0 10.0 20.0 30.0 40.0 50.0
Mediansurvivaltime(days)
Infection rate (%)
Effectof infection on median survival of An. coluzzii
Median Survival
Time(days)
Linear (Median Survival
Time(days))
28. 28
Figure 18: Cumulative daily survival of female An. coluzzii exposed to stress+infected, not stress
infected, stress+uninfected and not stress+uninfected in PVC tube. The isolate ARSEF 502 was used in
this experiment. Error bars: +/- standard error.
Table 6. Pairwise comparisons for all treatments using Log-rank (Mantel-Cox) tests for effect of stress
after fungal exposure in PVC tube. Each of the treatment consists of three replicates. Values with *
indicate significant differences after Bonferroni correction (p<0.008).
0
0.2
0.4
0.6
0.8
1
0 1 2 3 4 5 6 7 8 9 10
Days after exposure
CumulativeSurvival(±SEM)
Stress+Infected
Not stress+Infected
Stress+Uninfected
Not stress+Uninfected
Effect of stress on survival of An. coluzzii
Treatment Stress +Uninfected Stress +infected Not stress +infected
Chi-
Square
Sig. Chi-
Square
Sig. Chi-
Square
Sig.
Not stress + Uninfected 23.152 .000* 98.127 .000* 91.063 .000*
Stress+Uninfected 39.431 .000* 29.597 .000*
Stress+infected 5.591 .018
29. 29
4. Discussion
The main goal of my study was to improve the delivery platform of B. bassiana spores for infecting An.
coluzzii by adding “heat and odour” cues to the surface that was impregnated with entomopathogenic
spores of the fungus. It was assumed that inclusion of “heat” or “heat and odour” would attract more
An. Coluzzii towards B. bassiana treated black cotton cloth, and hence infect and kill them. In my study,
the black cotton cloth was treated with two different B. bassiana isolates with the addition of “heat and
odour”. Two experiments were conducted in the free flight room to answer my research questions.
Isolates ARSEF 5641 and 502 were used in all experiments. Isolate 502 was more virulent than 5641. The
logic for using different virulence levels of isolate was to see the variation in infection as well survival in
both experiments. It was also assumed that there should be a higher infection and shorter survival in
isolate 502 than 5641.
4.1. Effect of heat and odour cues for the Infection of An. coluzzii by B. bassiana isolates
As inclusion of “heat and odour” increases the attraction of An. gambiae (Spitzen et al., 2013), there
should be a higher infection rate. The infection was lower either adding “heat” or “odour” separately
than “spore only” treatments in the first experiment. On the other hand infection was higher when
“heat and odour” used combinedly. The result of isolate 502 was different from 5641. Inclusion of heat
increased the infection than without heat. However in both experiment, infection was highest in “heat
and odour” treatments. The possible explanation could be “heat and odour” combination increased
landing of An. coluzzii towards spores impregnated black cotton cloth (Spitzen et al., 2013). Hence, An.
coluzzii was infected and subsequently killed by spores. The other explanation might be a synergistic
effect between “heat and odour”. According to the report of McMeniman et al., (2014) “heat and
odour” cues had synergistic effect, that was increased the attraction of Ae. aegypti to human. For
revealing such synergistic effect they developed a multi-model integration of heat, odour and CO2 to
attract Ae. aegypti to human.In addition negative control had also infection in only experiment-1 due to
contamination during handling of B. bassiana spores and An. coluzzii. In conclusion, there seems an
influence of “heat” or “heat and odour” on infection rate in both two experiments.
According to Keizer (2014) report, exposure of malaria vectors in the free flight room with black cotton
cloth sprayed with “spore and odour”, the infection never exceeded 5%. Interestingly, in my study,
infection was found maximum 40% (replicate-3, experiment-1) when “heat and odour” were included.
In her studies she never used heat and sprayed odour with spores on black cotton cloth. On the other
hand, in my study odour did not mix together with spores as like Keizer studied. It could be the possible
explanation for higher infection than Keizer studied (2014). Another studied of Kaasschieter, (2015)
“heat and odour” increased the infection in small bug-dorm cages and he concluded that using heat in a
larger area could be the best way to reach this additional effect. My study continued on the findings of
both two previous researches and find out the possibility of field application of “heat and odour” effect
in Africa. In all three studies, isolate 5641 were used, so the results can be compared although there was
a difference for experimental set-up.
30. 30
During field application of EPF using black cotton cloth might be a suitable substrate for spores delivery
than spraying (Darbro et al., 2011). For this reason we also used a black cotton cloth for spores delivery
with addition of “heat and odour”. Mnyone et al., (2012), reported that fungi treated black cotton strips
gave 75% infection when these strips were placed around bed nets. In their study a man inside the bed
nets acts as “heat and odour” source to lure female malaria mosquitoes. In our study we attached a heat
cable behind the fungi treated black cotton cloth to lure female malaria mosquitoes. If the fungi treated
black cotton cloth hanging outside of bed nets then a man sleeping inside bed nets as heat source may
be work better than our experimental set-up. Previous studied Darbro et al., (2011) concluded that the
delivery of spores on cotton substrate to infect Ae. aegypti found more effective than spraying.
However, black cotton cloth with rough surfaces might be difficult for picking spores upon short landing
of female malaria mosquitoes (Personal idea). Then using suitable delivery substrate with smooth
surface would be nice to take the benefit from “heat and odour” effect in the field.
Instead of using black cotton cloth, clay pots also acts as a good substrate for spores delivery. According
to Farenhorst et al., (2008) the delivery of Metarhizium anisopliae spores on African water storage pots
reduced the survival of An. gambiae s.s. and An. funestus. They concluded that clay pots might be
attractive for male and female An. gambiae s.s. for distinct resting site. As a result spores delivery inside
the clay pots have more chance to death by EPF. Moreover, there was not any repellent effect for EPF
on malaria vector during surface treated application (Mnyone et al., 2012 and Farenhorst et al., 2008).
In addition using heat during spores delivery might be a problem with spores viability. In my study, black
cotton cloth was heated with about 340
C, but at this temperature spores could be unviable for couple of
days (Hegedus et al., 1992; Lane et al., 1991).
4.2. Effect of infection by B. bassiana isolate on survival of An. coluzzii
The second research question of my study was how fungal infection influenced the survival of An.
coluzzii. It was assumed that a higher infection rate would result in shorter survival of An. coluzzii. Both
of the experiments had shorter survival of An. coluzzii in all treatments than negative control. However,
there was a noticeable mortality in the negative control between two experiments. The possible
explanation could be stress (sugar water deprivation) of the female mosquitoes during experiments. In
the flight room female mosquitoes were released for 24hrs and there was no sugar water provided
during the experiments. Previous studied (Okech et al., 2003) reported that availability of sugar water
reduced the mortality of female An. gambiae. On the other hand there was a variation observed for
temperature in flight room. Apart from sugar water deprivation, temperature variation (Max. 270
C and
Min. 210
C) could also cause mortality in negative control and other treatments as well.
Moreover there was no significant difference between either addition of “heat” or “heat and odour”
treatments with “spore only” treatments for the survival of An. coluzzii. The possible explanation could
be spores might have an individual effect on An. coluzzii attraction. George et al., (2013), reported that
female An. stephensi was highly attracted to spores of B. bassiana. They concluded that spraying with
oil-formulated B. bassiana spores on black cotton cloth, resulted 95% attraction which becoming
infected after-one minute visit. But they could not reveal the exact mechanism of attraction towards
spores.
31. 31
In conclusion, it seems that addition of “heat” or “heat and odour” cues could be increased the landing
response of An. coluzzii. As they were attracted towards black cotton cloth, then chances of getting
spores were higher. It ultimately caused infection and shorter survival time in above two experiments.
Spitzen et al., 2013, concluded that using the combination of “heat and human odour” helped to trap
An. gambiae due to enhancement of the landing response. They concluded that a landing response of
An. gambiae could be evoked at 340
C. In our study system black cotton cloth was also heated at 340
C.
4.3. Correlation between infection rate (%) and median survival time (days)
From the infection rates of both two experiments, it was observed that survival of the group of
mosquitoes was related to the infection rate of that group of mosquitoes. But in the second experiment
the survival was significantly correlated with infection rate. However, in both experiments the infections
of An. coluzzii never exceeded 30%. We already mentioned in paragraph 4.2 that An. coluzzii might be
died for sugar water deprivation and temperature variation. The flight room was not optimal in terms of
temperature (max. 270
C and min. 210
C) and humidity (variable but not all data recorded) and this could
also result for mortality in “negative control” treatments that eventually caused quick mortality of rest
of the treatments. Previous studied showed that temperature variation reduced the survival of
female An. gambiae (Okech et al., 2003).
Unlike bug-dorm cage it was not possible to provide sugar water in free flight room. The lack of sugar
water for 1 day could also be the reason for less infection and shorter survival of An. coluzzii. As
mosquitoes became stressed for sugar water deprivation and temperature before contacting with
spores then it could be died without infection. To compare the results of previous two experiments, an
additional experiment was done to see the effect of sugar water deprivation experienced before fungal
exposure in the PVC tube bio-assay. It showed that An. coluzzii that deprived from sugar water had
shortest survival time than with sugar water. This conclusion of forced exposure experiments could also
explain that a significant number of An. coluzzii died due to sugar water deprivation in free flight
exposure experiment.Previous studied elucidated that the cumulative survival of female An. gambiae
was 51.5% with sugar and 25.6% without sugar after 10 days for sugar water deprivation (Stone et al.,
2009).
4.4. Optimization of B. bassiana production
An additional activity to make studies on entomopathogenic fungi possible, production of spores
needed to be set-up and optimized at the Laboratory of Entomology. Therefore, I conducted three trials
for the production of fungal isolates. In almost all trials there was noticeable infection recorded for
unknown reasons. Only from the second trial we obtained few spores (0.28 gram) but still infection was
found in the tube. Mass production of fungal isolates might be contaminated by other fungus or bacteria
during preparation. A number of steps should be followed for mass production and all steps had
possibilities for contamination. The possible contamination could be transferred to the impregnated
hemp into the 0.2L glass tube. During impregnation of hemp with spores the stock solution should be
kept free from contamination. Among the three trials spores was added in prepared media under flame.
However, there could also chance for infection. It would not a critical problem but better to add spores
32. 32
in a flow cabinet. A roller bank was needed to properly mix the spores with hemp where we used hand
due to lack of roller bank in entomology lab.
4.5. Concluding remarks
The first objective of my study was to increase infection by inclusion of “heat” or “heat and odour”. I
concluded that infection was indeed increased when “heat and odour” was combined. But the result
was different for first experiments where “spore only” had highest infection when “heat and odour”
used separately. The next objective of my study was how infection can influence the survival of An.
coluzzii. I concluded that addition of either “heat” or “heat and odour” during B. bassiana spores
delivery significantly reduced the survival of An. coluzzii compared with uninfected mosquitoes. It means
that “heat and odour” might be increased the suitable landing site of An. coluzzii when black cotton
cloth was heated at 340
C.
An additional activity of my study was to optimize mass production of B. bassiana spores in the
Entomology lab. I concluded that adding of spores in hemp and transferring impregnated hemp into 0.2L
glass tube should be done in sterile condition to avoid contamination. There should also focus on the
stock solution from where spores are added in the media.
4.6. Recommendation
Inclusion of “heat and odour” during delivery of B. bassiana spores might be increased the landing
response of An. coluzzii. As a result infection was increased that reduced the survival of An. coluzzii.
However, this bioassay results was only for two B. bassiana isolates. More investigation with different
types of B. bassiana isolates may be give clear effect of “heat and odour” during delivery of B. bassiana
spores. The future research with attraction and mechanism between An. coluzzii and B. bassiana spores
may be also addressed. The optimization of climatic factors; temperature and humidity and availability
of sugar water should be considered before start experiment in free flight room. For mass production of
spores there should need to more focus on stock solution and also need to work always under flow
cabinet.
33. 33
Acknowledgements
I would like to thank my supervisor Sander Koenraadt and Claudio Valero Jimenez in the first place for
supervising me. Their constructive suggestion during experiments helps me to successful completion of
my experiments. Sander helped me to reviewing my proposal and important comments on final report.
During experiments he always helped me to solve my problem. He also helped me to data analysis.
Claudio helped me to spores production. A lot of problem during mass production of spore was solved
by him.
I would also like to specially thank to Jeroen Spitzen for his technical help for setting up experiments and
answering a lot of question during experiments. Aside from that I would also like to give very special
thanks to Django Kaasschieter for his time to explain me about the procedure of preparation spores
suspensions, spraying black cotton cloth and infecting mosquitoes.
At last I want to thank vector group for arranging weekly meetings, and the Laboratory of Entomology
for a nice and exciting working place.
34. 34
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Appendix 1.
Protocol spore mass production in 0.2l system (Floor, Gilian and Claudio, 2014)
Materials for 12 setups
12 bottletrays
36 0.5 l bottles
12 0.2 l glass tubes
60 Silicone tubes with connectors
Cotton wool
Aluminum foil
Demi water
Preparing setup
Fill the bottles with demi water and place them in bottle trays
Place a small glass tube + sieve in the glass 0.2l tube
Place two rubber at both ends of the glass 0.2l tube(ad a little white connector to the inside of
the bottom one)
Connect the bottles and 0.2l tube with the right silicone tubesaccording to figure 1
Connect setup to air flow tubes to test setup
Loosen the tube connectors(especially the one between de second bottle and the 0.2l tube,
stick a piece of autoclave tape at this point)
Put cotton wool in the end of the last silicone tube and cover with aluminum foil
Autoclave for 30 minutes at 121°C
Tighten the tube connecters
Pre-sterilization of hemp:
Sieve the hemp and fill12 1l plastic bottles with 50 grams of this dry, sieved hemp (on yellow
balance)
Add 196 ml distilled water to each bottle
Autoclave for 30 minutes at 121°C
Add to each 1l bottle pre-sterilized hemp:
10 gram yeast extract(on yellow balance)
10 gram bacterial peptone (on yellow balance)
Optional: 357 µl gentamycin
Autoclave for 30 minutes at 121°C(Together with glucose stock)
Prepare glucose stock in 2l plastic pots
o For each setup: 80g glucose + 139g water (Use 1% extra to compensate for losses)
Autoclave for 30 minutes at 121°C (Together with hemp mixture)
Add 219.23g of glucose stock to each 1l bottle with hemp(Place balance in flow cabinet and use
50ml pipettes with pipette boy)
Add 0.9ml of spore solution (Vortex first!) (10^8 - 10^9 spores/ml)
Mark bottles with strain code
38. 38
Optional: add 1 ml/l antibiotics strepto/peniciline
Mix overnight on rollerbank at room temperature
Fill the fermenters (Work sterile and in flow cabinet!)
Drain the 1l bottles filled with hemp, using a 50ml plastic pipette and pipette boy
Fill the 0.2l glass tubes in the setup with the hemp, using30cm tweezers. Sterilize the 30cm
tweezers by flaming with 90% ethanol before use.
Remove excess fluid from 0.2l glass tubes using sterile 5ml syringes
Mark all setups with strain code
Clean everything between different strains!
Place all setups in climate chambers (25 °C)
Remove aluminum foil
Attach air flow tubes and run for 3 weeks
Spore harvesting
39. 39
Figure 19: The detailed step for the production of selected fungal isolates. The mass production of B.
bassiana isolates starts from the stock solution which was stored in −80°C in the freeze. (Valero
Jimenez et al., 2014).
40. 40
Appendix 2A.
Figure 20: Kaplan Meier survival curve of all three replicates of each treatment (experiment-1). A: Negative
control (p>0.05), B: Only spore (p<0.05), C: Spore and odour (p<0.05) and D: Spore, heat and odour (p<0.05). P-
value for log-rank (Mantel-Cox) test.
A B
C D
41. 41
Appendix 2B.
Figure 21: Kaplan Meier survival curve of all three replicates of each treatment (experiment-2). A: Negative
control (p>0.05), B: Only spore (p>0.05), C: Spore and heat (p>0.05) D: Spore and odour (p>0.05) and E: Spore,
heat and odour (p>0.05). P-value for log-rank (Mantel-Cox) test.
A
B
C D
E
