This document presents a thesis proposal on assessing entomological indicators of malaria transmission in relation to human activities at sites of a sugar development project in South Omo, Ethiopia. The proposal includes an introduction outlining the background and problem statement, objectives to investigate mosquito species composition and behaviors, a literature review on malaria burden and vectors, and proposed methods for mosquito collection, processing, and data analysis. If approved, the research aims to provide insights on local vector bionomics and behaviors to inform malaria control strategies for the project area.

1. i
ARBA MINCH UNIVERSITY
SCHOOL OF GRADUATE STUDIES
DEPARTMENT OF BIOLOGY
ENTOMOLOGICAL INDICATORS OF MALARIA TRANSMISSION IN
SOUTH OMO KURAZ SUGAR DEVELOPMENT PROJECT SITES IN
RELATION TO HUMAN ACTIVITIES
By
MULUALEM TOLOLE
FEBRUARY, 2021
ARBA MINCH, ETHIOPIA

2. ii
Entomological indicators of malaria transmission in South Omo Kuraz sugar
development project sites in relation to human activities
By
MulualemTolole
This proposalis submitted to the College of Natural Sciences Department of
Biology Schoolof PostGraduate Studies, Arba Minch University to fulfillthe
requirements for the Degree of Master of Science in Medical Entomology and
Vector control
Advisor
Fekadu Massebo (PhD)
February, 2021
Arba Minch, Ethiopia

3. iii
ARBA MINCH UNIVERSITY
SCHOOL OF GRADUATE STUDIES
ADVISORS’ PROPOSAL APPROVAL SHEET
This is to certify that the thesis proposal entitled, “Entomological indicators of malaria
transmission in South Omo Kuraz sugar development project sites in relation to human
activities” has been developed by MulualemTolole, PRNS/054/12 under my supervision.
Therefore, I recommend that the student’s proposal can be presented for review and open oral
presentation.
__________________________________________________________
Name of Principal advisor Signature Date
__________________________________________________________________
Name of co-advisor Signature Date

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ARBA MINCH UNIVERSITY
SCHOOL OF GRADUATE STUDIES
APPROVAL SHEET OF REVIEWED THESIS
Name of the Candidate: MulualemTololeTolcha
Department: Biology
Thesis Title: Entomological indicators of malaria transmission in South Omo Kuraz sugar
development project sites in relation to human activities
Date of Review _______________________
1. Comment on proposal and open presentation
________________________________________
________________________________________
2. Suggestions made by the reviewer/s
_______________________________________
_______________________________________
3. Modification(s) to be made
_______________________________________
_______________________________________
4. Final decision by the Reviewer/s
_______________________________________
_______________________________________
Name of the Reviewer/s Signature Date
_______________________________________
Department Head’s Name Signature Date
________________________________________

5. 1
TABLE OF CONTENTS
1. INTRODUCTION .......................................................................................................................3
1.1. Background .......................................................................................................................... 3
1.2. Statement of the problem......................................................................................................5
1.3. Objectives..............................................................................................................................6
General objective......................................................................................................................... 6
Specific objectives....................................................................................................................... 6
1.4. Research questions................................................................................................................6
1.5. Significance of the study.......................................................................................................7
2. Literature review...................................................................................................................8
2.2. Burden of Malaria ............................................................................................................ 8
2.3. Malaria Mosquitoes...............................................................................................................9
2.3.1. Dominant Malaria Mosquitoes ..................................................................................... 9
2.3.2 Secondary malaria vectors………………………………………………………………10
2.3.3. Entomological variables of Malaria Mosquitoes........................................................ 10
2.3.4. Feeding Behaviors ...................................................................................................... 11
2.3.5. Resting behavior ......................................................................................................... 11
2.3.6. Longevity.................................................................................................................... 12
2.3.7. Entomological Inoculation Rate ................................................................................. 12
2.3.8. Malaria Mosquito and Human activities…………..………………………………...…12
2.3.9. Malaria vector control................................................................................................. 13
3. MATERIALS AND METHODS........................................................................................14
3.1. Study area description.................................................................................................... 14
3.2. Study Design.......................................................................................................................14
3.3. Adult mosquito collection...................................................................................................14
3.3.1. Mosquito collection by Human Landing Catches...............................................................15
3.3.2. Mosquito collection by CDC light trap...............................................................................15
3.3.3. Mosquito collection by Prokopack .....................................................................................15
3.4. Larval and pupal sampling..................................................................................................16
3.5. Mosquito processing ...........................................................................................................16
3.5.1. Morphological identifications.............................................................................................16

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3.5.2. Molecular speciation...........................................................................................................17
3.5.3. CSPs detections...................................................................................................................17
3.5.4. Blood meal origin identification .........................................................................................18
3.6. Data analysis .......................................................................................................................19
3.7. Ethical considerations .........................................................................................................19
4. Work Plan ...........................................................................................................................20
5. Financial requirements........................................................................................................21
6. References.............................................................................................................................2

7. 3
1. INTRODUCTION
1.1. Background
Malaria is a public health problem caused by Plasmodium parasites and transmitted from an
infected person to normal by bites of female Anopheles mosquitoes. There are
five Plasmodium parasites responsible for the occurrence of human malaria, but two of them
namely P. falciparum and P. vivax are the most common and accounting for greater threat in
malaria-endemic countries in sub-Saharan Africa (WHO, 2020). The high burden of malaria in
sub-Saharan Africa countries is because of the favorable environmental, climatic, seasonal, and
ecological conditions for the occurrence and intensity of malaria transmission. Moreover, the
environmental modifications, such as the construction of dams and irrigation schemes, climatic
factors like precipitation and temperature are suitable for mosquito larval development and
parasite growth within the vectors (Kibret et al., 2019). Similarly, malaria remains a long-
standing problem in Ethiopia, where P. falciparum is the dominant parasite species, followed
by P. vivax. The two other parasite species such as P. malariae and P. ovale are rare (WHO,
2020).
Improving the understanding of the local malaria vectors behavior, and their ecology would
permit a better understanding of malaria transmission and optimize control strategies to halt
human-vector contact. Modifying the environment for sugarcane production using irrigation
might enhance malaria transmission by increasing the number and diversity of mosquito
breeding habitats and increasing vector composition, density, and longevity (Fornace et al., 2021;
Kibret et al., 2021). For example, villages practicing irrigation-based agriculture had more
malaria vector density, and risk of malaria infection, compared with non-irrigated villages
(Demissew et al., 2020). Moreover, human population movements from higher malaria
transmission settings or malaria-free highlands into the endemic areas make malaria control
programs more complex. The development project areasare where population movement is
occurring. People are moving from different parts of the country into developmental project sites
to get jobs. Those people moving from low malaria transmission areas to high malaria
transmission settings is more susceptible than residents, while those moving from high malaria
transmission settings increase the burden of malaria (Ward et al., 2013). Furthermore, changing

8. 4
the environmental conditions may also affect the vectors' behavior, which might contribute to
residual malaria transmission.The population movement into new sites may establish new human
behaviors and activities. People might stay outside at night due to the warming climatic
condition and night irrigation activities, which might increase outdoor exposure to malaria
mosquitoes. This might change the vectors biting and resting behaviors. The vectors avoid
contact with the insecticide sprayed indoors and bed nets as the vectors tend to feed and rest
outdoorsand tend to bite early hours of the night (Sokhna et al., 2013). Moreover, asthis study
will be conducted in a big development project corridor, the South Omo sugary factory, there
might be a direct connection with the port in Djibouti where the An. stephensi is already
established (Faulde et al., 2014). This species is also widely distributed in the Eastern part of the
country Ethiopia (Balkew et al., 2020), which is thought to be introduced recentlywhich believed
to be genetically more similar with southern Asia species (Carter et al., 2021). Therefore, the
present study aimed to assess the feeding and resting behaviors of malaria mosquitoes in the
development project site in South Omo concerning human activities. It also investigates the
artificial and natural breeding habitats to see the species composition of Anopheles mosquitoes
with special emphasis on An. stephensi.
1.2. Statement of the problem
Malaria is the most widespread vector borne human disease throughout tropical and sub-tropical
regions of the world with huge medical, economic and social impacts. As malaria is the disease
mostly occurs in poor countries, it is a big problem on the economic development of the
countries. A number of people become physically morbid due to malaria and unhealthy to
perform daily activities to contributefor country economy. Malaria patients spent more time
athome, clinics and health centers to get treatment.The evolving behavior of malaria vectors is
another big challenge in the malaria control and preventionprogram.Anophelesarabiensis is the
principal main vector with evolving behaviors.ThisAnopheles species has diverse feeding and
resting behaviors. The diversified Anopheles species and ecosystem makes the transmission of
malaria variable and challenging for the control programs.The environmental modifications for
the development projects including clearing forests, agricultural intensification, and irrigation
schemes make the area suitable for the distribution of malaria vectors. Although, migration of

9. 5
humans to the development project site to get job from different parts of the countries make the
transmission dynamics more complex and challenging for the control program.
The modification of mosquito behavior which favors outdoor feeding and resting might
compromise indoor based interventions. Using non-human hosts as alternative blood meal
sources allowed mosquitoes to avoidLLINs that mainly target indoor feeding and resting
mosquitoes. The current studywill assess the bionomics, biting and resting behaviors of malaria
mosquitoesin the development project site in South Omo in relation to human activities.
1.3. Objectives
General objective
To assess the entomological indicators of malaria transmission in the development project site in
South Omo in relation to human activities.
Specific objectives
 To investigate the bionimics and species compositionof malaria mosquitoes in the
development project sitein South Omo
 To examine the feeding behavior and biting rhythm of malaria mosquitoes in the
development project site in South Omo
 To estimatethe blood meal sources,infection and entomological inoculation rates of
malaria mosquitoes in the development project site in South Omo
 To assess the night human activities in the development project site in South Omo
 To assess the artificial mosquitoes breeding habitats to identify the mosquitoes species
with special emphasis on An. stephensi.
1.4. Researchquestions
 Which Anophelesspecies are there in the study area?
 Where and when mosquitoes do feed on humans predominantly?
 Do the Anopheles mosquitoes are infectious?
 What are the blood meal sources of Anophelesmosquito in the study area?

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 What risky human night behaviors/activities are therein the study area?
 Does Anopheles stephensispecies exists in the study area?
1.5. Significance of the study
This study will have several benefitsfor the malaria control program. First, the output of this
researchenables to identify the dominant and principal malaria vectors in the project
areas.Second, it also provides information onbiting behavior of malaria vectors concerning
thehuman activities, so that help design appropriate intervention to reduce the malaria
infection.Third, this research will inform the control program about the peak biting hours of the
mosquitoes in relation with the human outdoor activities. Human population around the project
site will be benefitedbecause understanding of the local vectors behavior and bionomics could
help the people to improve their outdoor activities, life style and to use personal protective tools
to reduce the human-vectorcontact.Ultimately, project is relevant to designing participatory
interventions.In addition, the research will provide relevant information about the breeding
habitats of malaria mosquitoes to people in the project sites, so that they can plan for safe
irrigation development project. This study also provides evidence on the presence or absence of
An.stephensi, which provide information on the distribution map of the species.

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2. LITERATURE REVIEW
2.1. Burden of malaria
Malaria is a life threatening disease caused by Plasmodium parasite which spread to people
through the bite of malaria vectors. In 2019, there were an estimated 229 million cases and about
409 000 deaths globally (WHO, 2020). It is a deadly disease mostly affecting children under 5
years. Africa shares high number of global malaria burden and considered as home for 94% of
malaria cases and deaths in 2019.Thisindicatesthat sub-Saharan Africa suffers most due to
malaria related cases and deaths. Malaria may affect any human who has contact with the vector,
but there are populations considerably at higher risk in contracting malaria and developing sever
disease than others. These groups are infants, children under 5 years age, pregnant women and
patients with HIV/AIDS as well as the non-immunemigrants and mobile population (WHO,
2020).
In Ethiopia, malaria is considered as one of the main public health problem and also affects the
economy of the country.The distribution pattern and intensityof malaria is depending on climate,
rainfall patterns and altitude.There are two principal human malaria parasites, P. falciparum and
P. vivax, co-exist in the country. The burden of these species varies due to the geographical
location and their distribution is extremely heterogeneous (Taffeseet al., 2018; Ketemaet al.,
2021).
2.2. Malaria mosquitoes
The primary malaria vectors which play a major role in transmission of malaria in Africa are
An.gambiaes.s, An.coluzzi, An.arabiensis, An.funestus, An.moucheti and An.nili(Sinka et al.,
2010)). They are anthrophilic, anthropophagic, endophilic and endophagic.In sub-Saharan
Africa, An.gambiae, An.arabiensis and An.funetus are documented as primary malaria vectors. In
Ethiopia, An.arabiensis, a member of An.gambiaecomplex,is the primary malaria
vector(Abraham et al., 2017; Massebo et al., 2013). Since dominant malaria vectors prefer
human habitation, biting humans, indoor resting and indoor biting,ITNs and IRS are the primary
strategy used to limit transmission of malaria (Bhatt et al., 2015).

12. 8
There are vectors playing a minor role in malaria transmission across Africa( Sinka et al.,, 2010).
Secondary malaria vectors preferred to bite and resting outdoor mostly associated with zoophily
and zoophagy which may sustain malaria transmission outside the protection of indoor
interventions (Massebo et al., 2015). Some example of secondary vectors is An. coustani, An.
ziemanni, An. pharoensis, An. rivulorumand An. squamosus(Mustapha et al., 2021).
2.3. Entomological indicators ofmalaria transmission
Malaria is one of the major public health problemstransmitted by malaria vectors.There are two
main malaria vector control interventions, such as IRS and ITNs. The effectiveness of these
interventions can be measured using the entomological variables. These variables are the indoor
density of host seeking and resting malaria vectors, longevity, entomological inoculation rate,
and infection rates (Garietal., 2016).
2.3.1. Feeding behaviors
In sub-Saharan Africa, there are primary and secondary malaria vectors which are exceptionally
high and efficient in malaria transmission. The reason beyond the efficient transmission and their
role is that they are anthropophagic and anthropophilic in their behavior (Sink et al., 2010). Due
to these behaviors they prefer explore nearby human habitats.These malaria vectors mainly feed
on human blood and rest in the indoor predicts the intensity of malaria transmission. But, the use
of anti-vector interventions is very important for sustainable vector control. The use of IRS in the
house reduces the malaria vectors those feed on humans and rest inside the house. These results
in reduction of host availability for malaria vectors and also provide the vectors with
modification of host selection (phenotypic plasticity)(Lefèvre et al., 2009). Then some of those
malaria vectors do not enter house and survive outdoor and some of them become opportunistic,
and resistant to insecticides (Monroe et al., 2019).As the malaria vectors feed on different hosts,
blood meal in mosquitoesmay determine the adult survival, fecundity, and hatching rates
(Javedet al., 2021).

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2.3.2. Resting behavior
The malaria transmission is highly affected by environmental temperature, since malaria vectors
are poikilothermic in their nature. As the environmental temperature changes, it results in
changes in mosquitoes physiology and parasite development(Beck-Johnson et al., 2013). If the
environmental temperature increases, it influences the gonotrophic cycle of mosquito(Diouf et
al., 2021).The influence of environmental temperature differs, however, variaes based on their
resting position. Malaria vectors those rest inside in human dwelling after feeding blood meal
until the start of ovipositionare endophilic and others spend this time out of human dwelling and
called exophilic. Of the members of malaria vectors Anopheles gambiaes.s are thought to be
more endophilic and spend long time indoor after feeding (Paaijmans and Thomas, 2011).
2.3.3. Longevity
A lot of environmental factors can influence the vectorial capacity of malaria vectors. Mainly
temperature affects the vectorial capacity traits such as longevity, fertility, vector competence
and biting behavior(Beck-Johnson et al., 2013). Longevity is the major determinants of the
evolutionary fitness of the vector and along with biting rate has the strongest influence on
vectorial capacity(Ohm et al., 2018). It is expected that longevity is positively correlated with
mosquito’s body size and as mosquitoes becomes larger they become more efficient in
accumulating reserves.
2.3.4. Entomological inoculation rate
Entomological inoculation rate is a very important parameter to understand the intensity of
malaria transmission(Doumbe-Belisse et al., 2021). It is used to measure the risk of malaria
infection.The entomological inoculation rate of a vector depends on the human biting frequency
and susceptibility of the vector to Plasmodiumgametocytes and calculated as a product of human
biting rate (HBR) and sporozoite rate (SR)(Doumbe-Belisse et al., 2021). Entomological
inoculation rate is influenced by the vector control interventions such as ITNsand IRS, since EIR
is one of the entomological transmission indicators of malaria. Estimating entomological
inoculation rate is significant to quantify the potential level of human exposure to infected
mosquito and measures the intensity of malaria transmission in particular area.

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2.4. Malaria mosquito and human activities
Mosquitoes are highly sensitive to environmental changes. Human activities such as
deforestation, wetland cultivation, water resource development, and cropcover and land use
changes for agricultural purpose causes environment suitable for malaria mosquito development
(Himeidan & Kweka, 2012). Deforestation is the most potent factor that includes logging,
transmigration programs, road construction, agricultural development, mining andhydropower
development. The human activities changes local ecosystems such as microclimate, soil and
aquatic conditions for the growth of malaria vectors. Mainly, deforestation and land
transformation have influences on Anopheles vectors larva and adult survivorship (Paul et al.,
2018).
2.5. Malaria vector control
There are a number of ways to reduce human-vector contacts. The two main vector control
interventions are ITNsand IRS to target mosquitoes biting and resting indoor(WHO,
2021).Though, remarkable advanced in the fight against malaria have been achieved by using
ITNs and IRS in many countries (Bhatt et al., 2015), these interventions cannot prevent outdoor
malaria transmission (Gari and Lindtjørn2018). These vectorcontrol interventions take
advantages on the susceptible mosquitoes behavior based on theobservations that malaria vector
prefers to bite humans indoor late at night and often rest insidehouses after blood feeding in
which the vectors will be exposed to sufficient levels of insecticides that will either kill and
reduce their longevity. Continuous use of indoor interventions may add stress on the indoor
feeding andresting malaria vectors leading them to either behavioral defense or physiological
defense(Rodriguez, 2021).The vectors have shown to adapt to changing environment due to both
behavioral avoidance and selection of mutation or recombination that favor their survival in the
presence of insecticidesmight threatening the efficacy of the current indoor based vector control
tools and the resulting increasein residual transmission(Aloutet al., 2017).
With increased use up of indoor based vector control tools mosquitoes have changed behaviors,
some are biting and resting indoors whilst others have changed to preferbiting and behavioral

15. 11
modifications including changes in biting time and location, changes hostchoice and shifted from
endophilic to exophilic behavior have been associated with long term use of insecticide based
interventions(Rodriguez, 2021).The behavioral resistance of mosquitoesis expected when
environmental conditionsare variable resulting in constitutive behavioral resistance traits and
inducible resistance traits. The most behavioral resistance phenotypes are grouped into
qualitative behavioral resistance such as shifts in biting time, site and shifts in bitten
hosts(Carrasco et al., 2019). In general, those behavioral shifts can be grouped into three key
categories. These are changes in biting rhythms and degree of exophagy and endophagy (maybe
confounded by outdoorhumans activities), changes in host preference, and the sensory detection
of control tools implemented(Carrasco et al., 2019).

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3. MATERIALS AND METHODS
3.1. Study area description
This study will be conducted in Southern, Nations, Nationalities and Peoples Regional state
(SNNPRs) in South Omo Zone.South Omo zone is borderingwith neighboring countries like
Kenya, and South Sudan, and Bench Majizone and Oromoia region of Ethiopia. The name of the
zone is derived from the Omo River which flows to Lake Turkana on the western side.Jinka is
the capitalof South Omo zone, which is 768kmsSouth of Addis Ababa,the capital of Ethiopia,
and about 275 kms from Arba Minch, the capital of Gamo zone. There is Kuraz Sugar
Development project around Salamago, the largest agricultural development scheme launched as
five year Growth and Transformational Plan (GTP) plans by Ethiopian government. It comprises
six sugar factories, sugar cane plantations, housing units, villages, canals and roads.The project
also contains the community development components such as irrigation lands to cultivate
agricultural products for the nearby societies like Mursi, Bodi,Menti as a well as Nangatom
communities.This study will be conducted in Kuraz Sugar Development project site in south
Omo zone.
There is a huge movement of populations from different parts of the country to get job. Some of
the people moves from malaria endemic areas to the project site, which may fuel the malaria
transmission.The development of huge irrigation canals and improper management them provide
ideal environment for malaria vectors breeding and which may in turn leading to the spread of
malaria infection. Moreover, the susceptible population from malaria free highlands may get
infection on their arrival, which also affect the productivity of the development project and may
increase extra cost for treatment.
3.2. Study design
A longitudinal entomological sampling will be conducted to assess the indoor and outdoor biting
behaviors, the density of Anopheles species, the feeding patterns and the species composition of
the Anopheles mosquitoes. Moreover, the potential artificial containers will be sampled to see
whether the newly introduced species, An. stephensi, colonized the project sites. The assumption
for assessing the exotic species in this site is due to the connection with the port in Djibouti,

17. 13
where An. stephensiis established as a vector of malaria. There is huge machinery transportation
to the project site fromDjibouti.
3.3. Adult mosquito collection
Different entomological methods are available to collectmosquito to provide information for
malaria control and prevention programs.These methods are human landing catches (HLCs),
CDC light traps, prokopack and pyrethrum space spray and others. For this study, HLC, CDC
light traps and prokopack will be used for adult collections.
3.3.1. Mosquito collection by HLCs
The data collected by human landing catches (HLCs)provides information on indoor and outdoor
biting patterns of mosquitoes.Two households will be selected in the project site, one from the
center and another from the edge of the project site. Human volunteers identified from the
project sites and trained on how to collect mosquitoes by HLCs. Then, they allowed sitting on
chair by exposing the lower part legs and collect mosquitoes that come to feed on them during
the night.They will be informed to wear long-sleeve shirts to avoid mosquito bites on exposed
part of their arms.
The collection will be done both indoor and outdoor twice per month in each house for six
months. One person will sit indoor and the other sit outdoor and exchange their position each
hour to minimize the bias due to the collection skill and individual variation in attractiveness to
mosquitoes. The collection will start at 18:00 and end 6:00 in the next morning. In each house,
two collectors will do the collection until mid-night and the other two take rest and replaced at
mid-night and continueuntil 6:00. This is to minimize the problem related with sleeping. There
will be intensive supervision to minimize the bias due to collectors’ behavior and avoid infection
of collectors. Collectors will be instructed to transfer mosquitoes to a new paper cup each hour.
Collectors will be given malaria prophylaxis. Collected mosquitoes will be identified
morphologically and individually preserved in a vial with Silica gel.

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3.3.2. Mosquito collection by CDC light traps
Center for Disease Control and prevention light traps (CDC-LT) is one of the widely used types
of trapsto collect indoor and outdoor host seeking mosquitoes.Five households will be selected
randomly in the project site to collect mosquitoes twice a month. Two CDC light traps (one
inside and one outside) will be hanged both indoor and outdoor in each house. Inside a house, a
CDC light trap will be hanged beside a personprotected by untreated bed net. Outdoor CDC light
trap will be hanged in either animal shelter or under shade at about ten meters from the house.
CDC light traps will be hanged at 18:00 and collected the next morning at 6:00.Collection bags
will be collected at the early morning.Collected mosquitoes will be identified morphologically
and individually preserved in a vial with Silica gel.
3.3.3. Mosquito collection by prokopack
Prokopack aspirator isdeveloped to collect resting mosquitoes. Five households will be selected
randomly. Two field technicians will be trained on how to collect mosquitoes by prokopack. The
trained technicians will aspiratemosquitoes resting indoors. The selected households will be
aspirated twice a month. The aspiration will be done starting from 6:00 to 8:00in the morning.
Mosquitoes will be identified and preserved in a vial with Silica gel for further analysis.
3.3.4. Larval and pupal sampling
Collection of immature stages will be done from natural and artificial breeding habitats. All
potential natural small water bodies, irrigation canals, ponds and other available breeding
habitats will be sampled. Artificial containers like discarded tyres, barrels, small containers
containing water will be sampled. The sampling will be done by dipping. As the objective of this
study is not to identify the productive breeding habitats, the collected sample will be pooled
together and wait for adult emergence for morphological identification. The newly emerging
adults will be provided with 10% sugar solution.

19. 15
3.4. Mosquito processing
3.4.1. Morphological identification
Morphological identification of adult mosquito is play a great role in determining the species
composition of mosquito fauna at a given place and time.The morphological identification of
mosquito will focus on the series of couplets each one giving two choices of characters(Coetzee,
2020).Light microscope will be used to observe the morphological structures relevant to
taxonomy.
3.4.2. Molecular speciation
The wing and thorax then will be homogenized with a motorized polypropylene pestle for 30 s.
the homogenate will be briefly centrifuged before it is lysed at 100 °c for 10 min. Then,after the
sample will be kept on ice for 2 min. The debris or wastes will be extracted by centrifugation.
The universal primers of DNA will be amplified. The PCR volume 15 µl consisting of 1 x PCR
buffer, 2.5 mM Mgcl2, 200 µM each dNTP, 0.5 µM of each primer will be adjusted. The thermal
protocol of PCR will be 94°c for 2 min and 15 s followed by 30 cycles at 94°c for 30 s, 57°c for
25 s, 72°c for 45°c and will be elongated to 75 °c for 5 min. The PCR product will be visualized
on 1.5% agarose gel.An. gambiaeand An. funestuscomplexeswill be a subject for the molecular
speciation.
3.4.3. CSPs detection
The infection of malaria and its risk partly depends on the proportion of mosquito vectors that
contain the infectious sporozoite stages of Plasmodium parasites.CSPs analysis procedure
described briefly as follows. ELISA plates will be coated with 100µl of 5µg/mL in PBS and will
be incubated for 3 h at room temperature (RT). The plates will be washed 3 times with PBS, and
subsequently 150µl of blocking buffer will be added. Then the plates will be incubated for 1 h at
RT and will be washed three times with PBS. 50 µl of mosquito homogenate will be transferred
to the ELISA plate.Multiple blank wells (homogenate) and pooled negative control wells
(homogenate from uninfected) mosquitoes will be used on each plate together with an eight step
standard curve of recombinant CSP. The plates will be incubated overnight at 4°c and will be

20. 16
washed four times with PBS in the following day.A 100 µl conjugate monoclonal antibody will
be added to the wells and will be left for 3 h at RT. Plates will be subsequently washed four
times with PBS. A 100 µL of substrate will be added to the wells and will be left for 20 min at
RT. Then finally, 50µl of 0.2 M H2SO4will be added to stop the reaction.
3.4.4. Blood meal origin identification
Blood meal source identification in malaria vector is very important to create better
understandings in host-vector interactions and malaria epidemiology and control.It will happen
by using direct enzyme linked immunosorbent assay (ELISA). First, the abdomen of freshly fed
each mosquito will be crushed in 50µl of Phosphate buffer saline (PBS) solution. The solution
will be diluted further to 950µl PBS. Fifty microliters of sample will be added to each well in a
96 well microtiterplate to be incubated overnight at room temperature.Each well will be washed
twice by PBS and will be incubated for 1 hr. After incubation for one hour, each well will be
washed three times with PBS. Finally, 100µl of peroxidase substrate will be added to each well.
After 30 min, the absorbance of 405nm will be recorded with an ELISA plate reader.
If the absorbance value exceeded the mean plus three times the standard deviation of the four
negative controls, the blood meal sample will be considered as positive for human and bovine
blood.
3.5. Data analysis
The data will be checked for missed information’s, outliers, recording errors and normality. The
data will be entered and analyzed using IBM SPSS statistics version 20. The sporozoite rate and
entomological inoculation rate will be calculated.Quantitative data analysis techniques will be
manipulated to analyze the data.The entomological inoculation rate of Plasmodiumparasite will
be calculated as product of the human biting rate and the plasmodium species sporozoite
infection rate. Then human biting rate of mosquitoes will be calculated as the total number of
anophelines captured per person per hour divided by the number of collectors.

21. 17
3.6. Ethical considerations
The Ethical approval will be obtained from Research and Ethical review committee of Arba
Minch University.All of the study participants will be informed about the aim of the study and its
procedure. Confidentiality will be kept if any and participation will be voluntarily. Mosquito
collectors will be given anti-malarial prophylaxis voluntarily.

22. 18
4. Work Plan
Activity Time duration Remark
Dec Jan Feb Mar Apr May Jun
Proposal development 
Proposal presentation 
Data collection   
Data analysis    
Thesis defense 

23. 19
4. Financial requirements
Cost items Unit Quantity Cost per unit Total cost Remark
Per Diem
researcher
Days 10 days/month 4500 27,000
Transportation Km 29.38/Km 1000 6000
Ento_field
assistants
(HLCs)
Night 2nights/month (8
people/night)
9600 9600
Ento_field
assistants (CDC
light traps and
prokopack)
Days 2days/month (8
people/day)
9600 9600
Total cost 52200

24. 20
5. References
Abraham, M., Massebo, F. and Lindtjorn, B. (2017). High entomological inoculation rate of
malaria vectors in area of high coverage of interventions in southwest Ethiopia: Implication for
residual malaria transmission. Parasite Epidemiology and Control2(2): 61-69.
Adugna, T., Yewhelew, D. and Getu E. (2021). Blood meal sources and feeding behavior of
anopheline mosquitoes in Bure district, northwestern Ethiopia. Parasite & Vectors13: 166.
Barreaux, A. M. G., Stone, C. M., Barreaux, P. and Koella, J. C. (2018). The relationship
between size and longevity of the malaria vector Anopheles gambiae( s.s) depends on the larval
environment. Parasites & Vectors11: 485.
Bartilol, B., Omedo, I., Mbogo, C., Mwangangi, J. and Rono, MK. (2021).Bionomics and
ecology of Anophlesmerus along East and Southern Africa coast.Parasites and vectors14: 84.
Carrasco, D., Lefevre, T., Moiroux, N., Pennetier, C., Chandre, F. and Cohuet, A. (2019).
Behavioral adaptations of mosquito vectors to insecticide control. Current Opinion in Science34:
48-54.
Composition, blood meal hosts and Plasmodium infection rates of Anopheles mosquitoes in
Ghibe River Basin Southwestern Ethiopia. Parasite and Vectors 12: 259.
Demissew, A., Hawaria, D. and Kibret, S. et al. ( 2020). Impact of sugarcane irrigation on
malaria vector Anopheles mosquito fauna, abundance and seasonality in Arjo-Didessa,
Ethiopia.Malaria Journal19: 344.
Fasil, A K., Tamirat, H W/, Endalkachew, N S and Melaku, W F. (2021). Prevalence of Malaria
among Adults in Ethiopia: A Systematic Review and Meta- Analysis. Journal of Tropical
Medicine.
Gari, T. and Lindtjorn, B. (2018).Reshaping the vector control strategy for malaria elimination in
Ethiopia in the context of current evidence and new tools: opportunities and challenges.Malaria
Journal17:454.
Gari, T., Kenea, O., Loha, E. et al. (2016). Malaria incidence and entomological findings in an
area targeted for a cluster – randomized controlled trial to prevent malaria in Ethiopia: results
from a pilot study. Malaria journal15: 145.
Getachew, D., Gebre-Michael, T., Balkew, M. and Tekie, H. (2019). Species
Kibret, S., Wilson, G.G., Ryder, D., Tekie, H. and Petros, B. (2019). Environmental and
metrological factors linked to malaria transmission around large dams at three ecological settings
in Ethiopia. Malaria Journal18: 54.
Machaniet al., (2020). Resting behavior of malaria vectors in highland and lowland sites western
Kenya: Implication on malaria vector control measures. PLOS ONE10: 1371.

25. 21
Massebo, F., Balkew, M., Gebre-Michael, T. and Lindtjorn, B. (2013).The Entomological
Innoculation Rates of Anopheles arabiensis in Southwestern Ethiopia.The American Journal of
Tropical Medicine and Hygiene89(3): 466- 473.
Mustapha, A M., Nyamachel, A K., Machani, M G., Kosgei, J., Wamuyu, L., Ochomo, E. and
Lobo, N F. (2021). Secondary malaria vectors in western Kenya include novel species with
unexpected high densities and parasite infection rates. Parasite & Vectors14: 252.
Paaijmans, K P. and Thomas, M B. (2011).The influence of mosquito resting behavior and
associated microclimate for malaria risk.Malaria journal10: 183.
Sokhna, C., Ndiath, MO.And Rogier, C. (2013). The changes in mosquito vector behavior and
the emerging resistance to insecticides will challenge the decline of malaria. Clinical
Microbiology and Infection19 (10): 902 – 907.
World Health Organization.(2019). World malaria report.
World Health Organization.(2021). Malaria reports.
Paul, P., Kangalawe, R.Y.M. and Mboera, L.E.G. (2018). Land use patterns and their implication
on malaria transmission in Kilosa District, Tanzania. Trop Dis Travel Med Vaccine4: 6
Beck-Johnson LM, Nelson WA, Paaijmans KP, Read AF, Thomas MB, Bjørnstad ON ( 2013).
The effect of temperature on Anopheles mosquito population dynamics and the potential for
malaria transmission.PLoS One.14; 8(11).