ABSTRACT
Long Lasting Insecticidal Nets (LLINs) and indoor residual spraying (IRS) represent powerful tools for controlling malaria vectors in sub-Saharan Africa. The success of these interventions relies on their ability to inhibit indoor feeding and resting of malaria mosquitoes. This study sought to understand the interaction of insecticide resistance with indoor and outdoor resting behavioural responses of malaria vectors from Western Kenya. Mark-release-recapture experiments were used to investigate the plasticity of indoor and outdoor resting behaviour while parity rates were used to estimate the physiological ages of Anopheles mosquitoes collected from Kisumu (Kisian) and Bungoma (Kimaeti) counties in Western Kenya. The status of insecticide resistance among indoor and outdoor resting anopheline mosquitoes was investigated in Anopheles mosquitoes collected from study sites. The level and intensity of resistance were measured using WHO-tube and CDC-bottle bioassays, respectively. The mutations at the voltage gated sodium channel (Vgsc) knock down resistance (kdr) gene and Ace 1 gene were characterized using PCR. Microplate assays were used to measure levels of detoxification enzymes, if present. Sporozoite rates were assessed by ELISAs for Plasmodium falciparum circumsporozoite protein. A total of 1094 samples were discriminated within Anopheles gambiae s.l. and 289 within An. funestus s.l. In Kisian (Kisumu County), the dominant species was Anopheles arabiensis 75.2% (391/520) while in Kimaeti (Bungoma county) collections the dominant sibling species was Anopheles gambiae s.s 96.5% (554/574). The An. funestus s.l samples analyzed were all An. funestus s.s from both sites. Pyrethroid resistance of An. gambiae s.l F1 progeny was observed in all sites. Lower mortality was observed against deltamethrin for the progeny of indoor resting mosquitoes compared to outdoor resting mosquitoes (Mortality rate: 37% vs 51%, P=0.044). The intensity assays showed moderate-intensity resistance to deltamethrin in the progeny of mosquitoes collected from indoors and outdoors in both study sites. In Kisian, the frequency of vgsc-L1014S and vgsc-L1014F mutation were 0.14 and 0.19 respectively in indoor resting An. gambiae s.l mosquitoes while those of the outdoor resting An. gambiae s.l mosquitoes were 0.12 and 0.12 respectively. The ace 1 mutation was present in higher frequency in the An. gambiae s.l F1 of mosquitoes resting indoors (0.23) compared to those of mosquitoes resting outdoors (0.12). In Kimaeti, the frequencies of vgsc-L1014S and vgsc-L1014F were 0.75 and 0.05 respectively for the F1 of An. gambiae s.l collected indoors whereas those of outdoor resting ones were 0.67 and 0.03 respectively. The ace 1 G119S mutation was present in progeny of An. gambiae s.l mosquitoes from Kimaeti resting indoors (0.05) whereas it was absent in those resting outdoors. Monooxygenase activity was elevated by 1.83 folds in Kisian and by 1.33 folds in Kimaeti for An. gambiae s.l mosquitoes resting indoors than those resting outdoors respectively.
The study recorded high resting behavioural plasticity, physiological (phenotypic, metabolic and genotypic) insecticide resistance and sporozoite rate in indoor resting populations of malaria vectors compared to their outdoor resting counterparts. The indication of moderate resistance intensity and for the indoor resting mosquitoes is alarming as it could have an operational impact on the efficacy of the existing indoor pyrethroid based vector control tools.
TABLE OF CONTENTS
DECLARATION ii
DEDICATION iii
ACKNOWLEDGEMENTS iv
LIST OF TABLES viii
LIST OF FIGURES ix
LIST OF APPENDICES x
LIST OF ABBREVIATIONS xi
DEFINITIONS xii
ABSTRACT xiii
CHAPTER ONE: INTRODUCTION
1.1 Problem statement 4
1.2 Conceptual Framework 5
1.3 Justification and significance of the research 6
1.4 Objectives 7
1.4.1 General objective 7
Specific objectives 7
1.5 Hypotheses 8
1.6 Research question 8
1.7 Research assumptions 8
CHAPTER TWO: LITERATURE REVIEW
2.1 Resting behaviour of malaria mosquitoes and vector control 9
2.2 Insecticide resistance and vector control 10
2.3 Mechanisms of insecticide resistance 11
2.5 Monitoring insecticide resistance 13
2.6 Behavioural insecticide resistance and residual malaria transmission by vectors 14
2.7 Insecticide resistance and Sporozoite infection rates of malaria mosquitoes 15
CHAPTER THREE: MATERIALS AND METHODS
3.1 Study sites 16
3.2 Study design 17
3.2.1 Mosquito Sampling 18
3.2.2 Rearing of mosquitoes 19
3.3 Investigating the behavioural plasticity of indoor or outdoor resting of malaria vectors 20
3.3.1 Mark-release-recapture of the F1 progeny of malaria mosquitoes resting indoors and outdoors... 20
3.3.2 Determining the age structure of indoor and outdoor resting wild caught malaria mosquitoes. 21
3.4 Testing phenotypic insecticide resistance in the F1 of indoor and outdoor resting mosquitoes 22
3.4.1 Phenotypic insecticide resistance assays 22
3.4.2 Biochemical tests for resistance associated enzymes by microplate assays 25
3.4.3 Molecular identification and detection of kdr and ace 1 resistance alleles 25
3.5 Investigating Plasmodium sporozoite infection rates in indoor-resting vs outdoor-resting malaria mosquito populations in Western Kenya 26
3.5.1 Preparation of mosquito antigen 27
3.5.2 Preparation of sandwich ELISA plates 27
3.8 Data analysis 27
CHAPTER FOUR: RESULTS
4.1 Plasticity of behaviour in indoor and outdoor resting malaria mosquitoes 30
4.1.1 Mark-release-recapture of the F1 progeny of indoor and outdoor resting malaria mosquitoes 30
4.1.2 Age structure of indoor and outdoor resting wild caught malaria vectors 31
4.2 Species discrimination of An. gambiae s.l. and An. funestus s.l. 34
4.2.2 Phenotypic resistance in the F1 progeny of indoor and outdoor mosquitoes 35
4.2.3 Intensity of insecticide resistance in F1 of An. gambiae s.l. resting indoors and outdoors 39
4.2.4 Target site genotyping for resistance alleles in the indoor and outdoor resting An. gambiae s.l. 41
4.2.5 Biochemical enzyme levels in the F1 of indoor and outdoor resting An. gambiae s.l 43
4.3 Investigating Plasmodium sporozoite infection rates in indoor vs outdoor resting malaria mosquito populations in Western Kenya 45
CHAPTER FIVE: DISCUSSION
5.1 Conclusion 55
5.2 Recommendations 56
REFERENCES 57
APPENDICES 72
Appendix 1: Turnitin Originality report 72
Appendix 2: Originality declaration form 73
LIST OF TABLES
Table 1: Amount of technical grade insecticide required to make 50ml of required concentration of test solutions 23
Table 2: Recapture rates of the progeny of indoor and outdoor resting malaria mosquitoes collected from Western Kenya (Pink colour= F1 of indoor-resting mosquitoes, Green colour= F1 of outdoor-resting mosquitoes) 31
Table 3: Physiological age composition of indoor and outdoor resting malaria mosquitoes (n=number of mosquitoes sampled, % in brackets) 33
Table 4: Species composition of Anopheles gambiae s.l. and Anopheles funestus s.l. from indoor and outdoor resting collections from Western Kenya (% in brackets) 34
Table 5: Frequency of Kdr and Ace 1 resistant alleles in indoor and outdoor-resting An. gambiae s.s and An. arabiensis populations from Western Kenya (n= number of analyzed mosquitoes, p2 + 2pq, Where; p2 = homozygous resistant, 2pq =heterozygous resistant) 42
Table 6: Sporozoite rate between mosquitoes resting indoors and those resting outdoors (n=number of mosquitoes analyzed, % in brackets) 46
LIST OF FIGURES
Figure 1: A conceptual illustration of the interactions between genotypic, phenotypic and behavioral aspects of insecticide resistance highlighting monitoring techniques. (Key focus: Resting behaviour) 5
Figure 2: Map highlighting the study sites, (i) Kisian in Kisumu County and (ii) Kimaeti in Bungoma County, both in Western Kenya 17
Figure 3: Examples of outdoor resting mosquito habitats sampled in Kimaeti, Bungoma County in western Kenya. The habitats in this figure include the pot on the ground and the animal shed that has a roof. These are shown by the red arrows. 19
Figure 4: Interior view of a typical malariasphere at the KEMRI-CGHR centre in Kisumu County, Kenya 21
Figure 5: CDC bottle bioassay to determine the intensity of insecticide resistance in Wheaton bottles coated with 10× concentration of deltamethrin in progress 25
Figure 6: Percentage composition based on the number of egg laying 32
Figure 7: Percentage mortality of An. gambiae s.l from WHO tube bioassays with and without PBO. (Green represent indoors and red represent outdoors). 37
Figure 8: Percentage mortality of An. funestus s.l mortality from WHO tube bioassays with and without PBO 38
Figure 9: Mortality rate of An. gambiae s.l. exposed to ×1, ×5, and ×10 concentration of deltamethrin in CDC intensity bottle bioassays 40
Figure 10Mortality rate of An. funestus s.l. exposed to ×1, ×5, and ×10 concentration of deltamethrin in CDC intensity bottle bioassays 41
Figure 11:Monooxygenase enzyme activity in An. gambiae s.l. (**P<0.05, ***P<0.001) 44
Figure 12: Esterase enzyme activity in An. gambiae s.l. (**P<0.05, NS not significant). 44
Figure 13: Glutathione S-transferase enzyme activity in An. gambiae s.l. (**P<0.05,
***P<0.001) 45
LIST OF APPENDICES
Appendix 1: Turnitin Originality report Appendix 2: Originality declaration form
LIST OF ABBREVIATIONS
1. CI : Confidence Interval
2. CDC : Centers for Disease Control and Prevention
3. CGHR : (KEMRI’s) Centre for Global Health Research
4. CS : Circumsporozoite
5. DC : Diagnostic Concentration
6. DNA : Deoxyribonucleic acid
7. F1 : 1st Filial generation
8. GABA : Gamma-aminobutyric acid
9. GST : Glutathione-S-transferase
10. IRS : Indoor Residual Spraying
11. KDR : Knock Down Resistance
12. KEMRI : Kenya Medical Research Institute
13. LLIN : Long-Lasting Insecticidal Net
14. MRR : Mark-release-recapture
15. PBO : Piperonyl butoxide
16. PBS : Phosphate Buffered Saline
17 PCR : Polymerase Chain Reaction
18. Pf SR : Plasmodium falciparum Sporozoite rate
19. SIT : Sterile Insect Technique
20. TE : Tris-EDTA buffer (Hydroxymethyl-Ethylenediamine tetra-acetic acid)
21. TMBZ : 3, 3’,5 ,5’-tetramethylbenzidine
22. UV : Ultra-Violet
23. VGSC : Voltage-Gated Sodium Channel
24. WHO : World Health Organization
DEFINITIONS
1. Behavioral plasticity : The trait of changing a behavioral preference, usually when conditions become unfavorable.
2. Biparous Mosquito : A female mosquito estimated to have undergone two egg-laying cycles
3.. Knockdown Resistance : A trait that alters the voltage-gated sodium channel properties which reduce pyrethroid effects. either by reducing pyrethroid binding and/or by altering the gating properties
4. Mortality : This the number of dead mosquitoes 24 hours post durational insecticide exposure, penetration, transversion through tissues into the target site causing death.
5. Nulliparous Mosquito : A female mosquito that has not undergone any egg-laying cycle
6. Physiological age: This is the number of gonotrophic (egg laying) cycles a female mosquito has passed through.
7. Resting behaviour : This is the inactive trait of late-stage blood fed, half gravid or fully gravid states of mosquitoes when they are at the period between end of blood feeding and seeking for oviposition sites.
8. Sporozoite rate : Sporozoite rate (Pf SR) is the number of mosquitoes infected with sporozoites divided by the total number of mosquitoes examined
9. Stochastic Phenomenon : This is an occurrence having a random probability distribution or pattern that may be analysed statistically but may not be predicted precisely.
10. Synanthropy : This is the dwelling and benefiting by an organism in close proximity to human beings.
11. Uniparous Mosquito : A female mosquito estimated to have undergone only a single egg- laying cycle.
CHAPTER ONE: INTRODUCTION
Major declines in the incidence as well as the prevalence of malaria within the Sub-Saharan Africa region have been realized owing to the anti-malarial drug administration campaigns and the augmentation of vector management strategies; principally targeting endophagic and endophilic malaria transmitting mosquitoes (WHO, 2019). However, malaria transmission is still persistent in quite a lot of countries of the Sub-Saharan Africa despite the achieved feats in the mitigative wars against malaria (Mwesigwa et al., 2015; Zhou et al., 2011).
The persistence in malaria transmissions has partly been accredited to mosquito deviations with regards to biting and resting patterns; in response to the increase in the usage of insecticides as vector control tools (Reddy et al., 2011; Russell et al., 2011; Sougoufara et al., 2014; Takken & Verhulst, 2013) and the increased insecticide resistance in the malaria mosquitoes (Hughes et al., 2020; Knox et al., 2014; Omondi et al., 2017). Malaria transmission is dependent upon propensity of malaria mosquitoes successfully obtaining blood meals from humans and their particular predilection to living close to human dwellings (Mandal et al., 2011; Takken & Verhulst, 2013).
Insecticide resistance in malaria mosquitoes may develop from one or more mechanisms which include; increase in metabolic detoxification enzyme systems, target site alterations hence insensitivity and behavioural modifications (Liu, 2015). Metabolic enzyme detoxification (Hemingway et al., 2004) and target site insensitivity (Hemingway & Ranson, 2000) are the most responsible mechanisms when it comes to high levels of resistance to insecticides (Brogdon, 1989). Insensitivity to the toxicity of insecticides rely on one or several variations in the hereditary genes within the mosquito genome (Liu, 2015). Detoxification enzymes that are known to confer insecticide resistance are mainly found in three groups of enzymes namely; monooxygenases (cytochrome P450s), beta (β) esterases and glutathione-S-transferases. Approximately 80% of the insecticide resistance genotypes reported in Western Kenya are the knock-down (kdr) mutations the Voltage-Gated Sodium Channel at locus 1014 of Anopheles gambiae s.s., a primary vector of malaria (Bonizzoni et al., 2012; Mathias et al., 2011; Ochomo et al., 2012; Wanjala et al., 2015). The malaria mosquito, An. arabiensis, recently has been reported to developing increments in the levels of knock-down (kdr) mutant genotypes (Hemming-Schroeder et al., 2018). A similarly important vector of malaria in most parts of Africa including Western Kenya, An. funestus mosquitoes, have currently no records of kdr mutants at the locus 1014 in their genome. However, there have been documentation of metabolic resistance in Anopheles funestus mosquitoes (Kawada et al., 2011). The increasing levels of resistance to insecticides by malaria vectors have been linked, by countless accounts, to the incessant exposure to Long Lasting Insecticide Nets ( LLINs) (Lindblade et al., 2015; Moshi et al., 2017) and in agro-chemicals, mainly due to the formation of selection pressures (Diabate et al., 2002; Nkya et al., 2014; Reid & McKenzie, 2016).
Climatic (environmental) fluctuations have as well been drawn in the mosquito behavioural alterations being witnessed. Mosquitoes adapt to prevailing conditions by expressing phenotypes that are better suited for lowering or averting adverse consequences that may be brought about by environmental conditions (Takken & Verhulst, 2013). For example, many East African region studies have documented amplified instances of zoophagy (Stone & Gross, 2018), early feeding in the evening indoors or outdoors-feeding altogether (Monroe et al., 2015; Monroe et al., 2020; Ototo et al., 2015) and changes in resting behavioural preferences, either indoors or outdoors (Bayoh et al., 2014; Killeen et al., 2006; Pates & Curtis, 2005). These shifts in behaviour could have arisen due to selection pressures created from the increased LLIN coverage (Braimah et al., 2005; Killeen et al., 2017; Mayagaya et al., 2015; Perugini et al., 2020). Wide coverage by LLINs in Africa has been shown to alter the vector dominance composition; the highly endophilic An. gambiae s.s. (hereafter An. gambiae) is slowly being replaced by the more exophilic An. arabiensis in Western Kenya (Githeko et al., 2012; Mutuku et al., 2011; Zhou et al., 2011). The arising intervention pressures could selectively eradicate the utmost susceptible mosquitoes from within a population thereby the least susceptible (resistant) mosquitoes that can adapt to the new conditions can survive (Lindblade et al., 2006). Even though these field studies have demonstrated the impact of environmental deviations on the behaviour of mosquitoes, little is known on what relationship there is between resistance to insecticides and the resulting malaria vector behaviours.
Mosquito feeding and resting behaviours are very crucial considerations to the success of malaria transmission reduction and vector control. it is therefore paramount that we understand the relationship between physiological resistance and the resting behaviours seen in mosquitoes and the “how?” of these behavioural observations might affect the already existent frontline measures. The underlying mechanisms of the behavioural modifications observed in mosquitoes are currently a grey zone despite the fact that they might bear epidemiological consequences. In any attempt to sustaining effective insecticide-based control of malaria vectors, the resistance to insecticides should continuously be observed and appropriate mitigative stratagems established (Chanda et al., 2011; Hughes et al., 2020; Ochomo et al., 2014; Ranson & Lissenden, 2016; Russell et al., 2013; Sougoufara et al., 2017; WHO, 2012). The study attempted to answer the how on insecticide use and resistance influencing either the indoor or the outdoor resting behaviours and the implications this may have to malaria transmission. The results of this study are important to provide information on the resting behaviour with regards to levels of insecticide resistance in malaria mosquitoes resting either indoors or outdoors and possibly the infectivity rates of the populations.
1.1 Problem statement
Significant morbidity and mortality resulting from malaria, especially among infants and expectant women, is still recorded in Africa. The transmission of malaria is heavily reliant on the tendency, by malaria vectors, to successfully blood-feed on humans and their preference for living within proximity to human dwellings. Vector control is majorly dependent on the disruption of the cycle of malaria transmission. The use of insecticide-based interventions has been able to avert the transmission cycle by hindering human feeding and deterring resting proximal to human dwellings by either lethal action or repellency. However, recent reports of increasing behavioural shifts and levels of insecticide resistance among malaria vectors threatens the attained successes from these insecticide-centered control interventions. The intensive use of these tools has conceivably led to increased insecticide resistance and shift from indoor-feeding to outdoor-biting by malaria mosquitoes. As we are focusing on insecticide resistance management, we need to as well put effort in determining the effects this has on other behaviours of malaria mosquitoes such as resting, which are key in the interaction circles that facilitate malaria transmission and are determinants of appropriateness of interventions. Unlike other mechanisms of insecticide resistance which have proper workable monitoring tools, behavioural mechanisms lack concrete techniques besides mere vector density surveillance.
1.2 Conceptual Framework
Figure 1: A conceptual illustration of the interactions between genotypic, phenotypic and behavioral aspects of insecticide resistance highlighting monitoring techniques. (Key focus: Resting behaviour)
1.3 Justification and significance of the research
Vector control relies heavily on insecticide-based interventions. These depend on the knowledge of biting patterns and resting behaviour of malaria mosquitoes by dictating how much the exposure of the vectors is to these tools (Trung et al., 2005; WHO, 2012). Insecticide resistance is a hindrance to malaria control efforts (Churcher et al., 2016; Hemingway et al., 2016). Globally, new strategies are required to overcome insecticide resistance in the fight against malaria mosquitoes. The reduced susceptibility and behavioural change responses to common insecticides used in indoor residual spraying (IRS) and in long lasting insecticidal nets (LLINs) have been reported previously (Machani et al., 2020; Ochomo et al., 2012; Ochomo et al., 2014; Ochomo et al., 2015; Omondi et al., 2017). This underscores the importance of inventing new tools for management of malaria mosquitoes. The indoor application of IRS and LLINs in vector control have proved to be effective. However, significantly raised levels of indoor malaria transmissions (Mwesigwa et al., 2015; Zhou et al., 2016) and outdoors are still being observed (Monroe et al., 2019; Moshi et al., 2017; WHO, 2019). Behavioural resistance is a compounding factor that affects fitness of malaria vectors in the presence of indoor insecticide-based interventions. The resting behaviour of these vectors could impact on malaria transmission. Despite having insecticide-based interventions in place, malaria vectors have been seen resting in or within proximity of human dwellings (Russell et al., 2013). In order to tackle the issue of resistance, we must have a clear understanding of the link between insecticide use, insecticide resistance and the resulting behaviour change which in this case is the change in resting behaviour of malaria vectors (Ranson & Lissenden, 2016; Russell et al., 2013). The constant transmission of residual malaria has been attributed to the deviations from known biting phenology and the resting behavioural shifts in mosquitoes owing to increased insecticide vector control (Killeen et al., 2017; Reddy et al., 2011; Russell et al., 2011; Sougoufara et al., 2014; Takken & Verhulst, 2013) and the amplified insecticide resistance (Hughes et al., 2020; Knox et al., 2014; Omondi et al., 2017). Since the resting behaviour is an important consideration when determining appropriate vector control interventions, these findings will be useful in bridging the scientific gap between insecticide-based vector control and the shifts in the resting patterns of mosquitoes. The goal for the study was to find out whether insecticide coverage, through Long Lasting Insecticidal Nets (LLINs), affects the resting behaviour of insecticide resistant mosquitoes and the implications to Plasmodium sporozoite transmission. Understanding behavioural mechanisms of resistance would provide better insights into monitoring and management of insecticide resistance.
1.4 Objectives
1.4.1 General objective
• To determine the effect of indoor insecticide coverage on the resting behaviour of malaria mosquitoes in Western Kenya
Specific objectives
1. To find out whether indoor or outdoor resting behavior of malaria vectors is a plastic phenomenon
2. To compare the status of insecticide resistance in indoor and outdoor-resting malaria vector populations in Western Kenya
3. To investigate the Plasmodium sporozoite infection rates in indoor versus outdoor resting malaria mosquito populations in Western Kenya
1.5 Hypotheses
Ho: There is no difference in the level of insecticide resistance and the sporozoite infection rates between African malaria mosquitoes resting indoors and outdoors.
HA: There is a significant difference in the level of insecticide resistance and sporozoite infection rates between African malaria mosquitoes resting indoor and outdoor.
1.6 Research question
• What is the effect of insecticide coverage through LLINs on the resting behaviour and fitness of female African malaria mosquitoes?
1.7 Research assumptions
• Adult female malaria mosquitoes rest indoors or outdoors regardless of insecticide interventions through LLINs in Western Kenya.
• The mosquito samples collected were homogenous and representative of the mosquito population in study regions of Western Kenya.
• The environments from which mosquitoes were collected had different climatic conditions and geographical indices that are a representation of lowlands and highlands of Western Kenya.
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