i SURVIVAL RATES AND BLOOD MEAL PATTERNS OF Aedes aegypti AND Aedes simpsoni MOSQUITOES IN KERIO VALLEY AND RABAI ARBOVIRUS ECOLOGIES, KENYA WINNIE W. KAMAU (BSc.) A RESEARCH THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF THE DEGREE OF MASTER OF SCIENCE IN INFECTIOUS DISEASES (MEDICAL PARASITOLOGY) IN THE SCHOOL OF HEALTH SCIENCES, KENYATTA UNIVERSITY SEPTEMBER, 2022 ii iii ACKNOWLEDGEMENT It is with gratitude, I appreciate Kenyatta University graduate school for the approval of this study and the opportunity granted to undertake this research project at International Center of Insect Physiology and Ecology (icipe) through Dissertation Research Internship Programme (DRIP), under the capacity building and institutional development, human health theme. I am also grateful for my supervisors; Prof. Rosemary Sang, Dr. David Tchouassi and Dr. Nelson Menza for the support, mentorship and excellent supervision. Special thanks to the staffs and colleagues for the resolute support. iv TABLE OF CONTENTS DECLARATION........................................................................ Error! Bookmark not defined. ACKNOWLEDGEMENT ..................................................................................................... iii LIST OF TABLES ................................................................................................................. vii LIST OF FIGURES ............................................................................................................. viii LIST OF ABBREVIATIONS AND ACRONYMS .............................................................. ix ABSTRACT .............................................................................................................................. x CHAPTER ONE ...................................................................................................................... 1 1.0 INTRODUCTION.............................................................................................................. 1 1.1 Background Information ...................................................................................................... 1 1.2 Statement of the Problem ..................................................................................................... 4 1.3 Justification .......................................................................................................................... 5 1.4 Research Questions .............................................................................................................. 6 1.5 Objectives ............................................................................................................................ 6 1.5.1 General Objective ............................................................................................................. 6 1.5.2 Specific Objectives ........................................................................................................... 6 1.6 Significance of the Study ..................................................................................................... 7 CHAPTER TWO ..................................................................................................................... 8 2.0 LITERATURE REVIEW ................................................................................................. 8 2.1 Global distribution and burden of dengue and yellow fever............................................... 8 2.2 Transmission cycles of dengue and yellow fever viruses .................................................. 10 2.3 Concept of vectorial capacity, blood feeding and survival rates ....................................... 11 v 2.4 Blood meal patterns and significance in pathogen risk assessment ................................... 12 2.6 Survival rates of mosquito ................................................................................................. 14 2.6.1 Methods of estimating mosquito age .............................................................................. 14 2.7 Blood meal analysis of mosquitoes.................................................................................... 16 2.8 Distribution and genetic forms of Ae. aegypti and Ae. simpsoni mosquitoes .................... 17 CHAPTER THREE ............................................................................................................... 19 3.0 MATERIAL AND METHODS ...................................................................................... 19 3.1 Study area........................................................................................................................... 19 3.2 Study design ....................................................................................................................... 19 3.3 Mosquito retrieval and processing ..................................................................................... 19 3.4 Laboratory Analysis ........................................................................................................... 20 3.4.1 Mosquitoes morphological identification ....................................................................... 20 3.4.2 Determination of parity rates of Ae. aegypti and Ae. simpsoni sl mosquitoes ................ 21 3.4.3 Estimate of mosquito daily survival and longevity......................................................... 21 3.4.4 Determination of blood meal sources from Ae. aegypti and Ae. simpsoni ..................... 22 3.4.5 Molecular Identification of Blood-Fed Ae. aegypti and Ae. simpsoni sl ........................ 23 3.5 Genetic and Phylogenetic Analysis ................................................................................... 25 3.6 Data Analysis ..................................................................................................................... 25 CHAPTER FOUR .................................................................................................................. 27 4.0 RESULTS ......................................................................................................................... 27 4.1 Mosquitoes retrieved from Kerio valley and Rabai ........................................................... 27 vi 4.2 Parity rates of Ae. aegypti and Ae. simpsoni sl. ................................................................. 27 4.3 Survival rates and longevity of Ae. aegypti and Ae. simpsoni sl mosquitoes .................... 28 4.4 Blood meal patterns of Ae. aegypti and Ae. simpsoni sl .................................................... 29 4.5 Human blood feeding habits among the genetic forms of Ae. aegypti and subspecies of Ae. simpsoni sl. ........................................................................................................................ 31 CHAPTER FIVE ................................................................................................................... 34 5.0 DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS .............................. 34 5.1 Discussion .......................................................................................................................... 34 5.1.1 Parity rates of Ae. aegypti and Ae. simpsoni ................................................................... 34 5.1.3 Survival rates and longevity of Ae. aegypti and Ae. simpsoni sl mosquitoes ................. 35 5.1.4 Blood meal patterns of Ae. aegypti and Ae. simpsoni sl ................................................. 36 5.1.5 Human blood feeding habits among the genetic forms of Ae. aegypti and subspecies of Ae. simpsoni sl. ........................................................................................................................ 37 5.2 Study Limitations ............................................................................................................... 39 5.3 Conclusions ........................................................................................................................ 40 5.4 Recommendation ............................................................................................................... 40 5.4.1 Recommendations for further research ........................................................................... 41 APPENDICES ........................................................................................................................ 50 Appendix. I: Graduate School Research Approval .................................................................. 50 Appendix. II: Publication ......................................................................................................... 51 vii LIST OF TABLES Table 1: Summary of primers used in this study .................................................................. 246 Table 2: Daily survival rates and life expectancy (longevity) of Ae. aegypti and Ae. simpsoni sl in Rabai and Kerio Valley. ................................................................................................... 30 viii LIST OF FIGURES Figure 1: A map showing global distribution of Dengue fever (Source: WHO 2019) ............. 8 Figure 2: A map showing global distribution of yellow fever (Source: https://www.gs.international/yellow-fever) ............................................................................... 9 Figure 3: A Map showing sampling locations in Rift Valley a history of Yellow fever and coastal Kenya an endemic for Dengue (Source: QGIS 3 software). ........................................ 20 Figure 4: A presentation of tracheolar skeins under microscopy at 40X magnification. ........ 21 Figure 5: Variation in parity rates estimated for Ae. aegypti and Ae. simpsoni s.l. collected at different periods in Kerio Valley (KV) and Rabai. .................................................................. 28 Figure 6: Blood meal patterns of Aedes aegypti in Kerio Valley and Rabai. ......................... 30 Figure 7: Blood meal patterns of Aedes simpsoni sl in Kerio Valley and Rabai. ................... 31 Figure 8: A Maximum likelihood phylogenetic tree of 41 nucleotide mosquitoes COI gene sequences using Tamura-Nei model. ....................................................................................... 32 Figure 9: A Maximum likelihood phylogenetic tree of 46 nucleotide mosquitoes, ITS2 sequences using Tamura-Nei model. ....................................................................................... 33 ix LIST OF ABBREVIATIONS AND ACRONYMS BG Bio Agent CO1 Cytochrome C Oxidase subunit 1 DENV Dengue Virus EIP Extrinsic Incubation Period ELISA Enzyme Linked Immunosorbent Assay GC Gonadotrophic Cycle HBI Human Blood Index IgG Immunoglobulin G ITS Internal Transcribed Spacer 2 NHPs Non- Human Primates PCR Polymerase Chain Reaction PR Parity Rate Ro Reproductive number TAE Tri- Acetate- EDTA VC Vector Capacity WHO World Health Organization YFV Yellow Fever Virus x ABSTRACT Understanding the vectorial capacity for arboviruses transmission can allow for improved prediction and of arboviral disease outbreaks and control. Like other vector-borne diseases, transmission of arboviruses is influenced by vector bionomic traits including age structure and vector feeding habits. The current study investigated the survival rates, blood meal patterns and the human blood feeding habits in field collected populations of Aedes aegypti and Aedes simpsoni mosquitoes, which are vectors of dengue virus (DENV) and yellow fever virus (YFV), respectively, in East Africa. Adult female mosquitoes analysed were trapped during the day using CO2-baited BG Sentinel traps from peri-urban Rabai, Kilifi County (dengue-endemic) and rural Kerio Valley, Baringo County (with a history of yellow fever outbreak) during the period between August 2019 to February 2020. The mean parity rates following dissection and microscopic examination of ovarian tracheoles was high for Ae. simpsoni (85% (n=539) that did not vary between the trapping periods, while in Ae. aegypti was 74.9% (n=735) but varied between the trapping periods. Assuming a 3-day gonotrophic cycle, these translated to a high daily survival rate and longevity ranging between 15.8-19.7 days and 7.7-12.4 days in Ae. simpsoni and Ae. aegypti, respectively. Analysis of blood fed cohorts through DNA sequencing of the12S mitochondrial rRNA fragment showed a diverse host feeding range for Ae. aegypti with estimated human blood index (HBI) of 0.53. HBI did not vary between mitochondrial lineages indicative of domestic and forest genetic forms of Ae. aegypti. The genetic forms of Ae. aegypti were determined by PCR of a cox1 gene fragment and then sequencing followed by phylogenetic reconstruction. Similarly, Ae. simpsoni complex also exhibited a broad host feeding range, with Ae. bromeliae being the most predominant sub-species as determined using Internal Transcribed Spacer 2 (ITS2) PCR sequences, which exhibited a low HBI (0.18 and 0.33 in Rabai and Kerio Valley, respectively). Phylogenetic analysis also suggested the presence of a species which is yet to be described within the Ae. simpsoni complex, demonstrating human blood feeding tendency. The species diversity in the Ae. simpsoni complex may well be greatly higher than earlier thought, which requires more studies. Overall, both species exhibited high survival/longevity that could lead to high vectorial capacity for YFV and DENV transmission. Additionally, the low human blood meals of both Ae. aegypti and Ae. simpsoni cohorts indicated a high capacity for zoonotic transmission of other pathogen and therefore a need for continued efforts to control these vectors. These findings demonstrated the applicability to include other bionomic parameters such as vector competence, which defines vectorial capacity, for an effective understanding of spread and recurrence risk of these arboviruses. In addition to enhance cost effectiveness interventions (e.g. vaccines) and prediction of diseases occurrence, there is urgency to generate surveillance information of vector population founded on genotype analyses. 1 CHAPTER ONE 1.0 INTRODUCTION 1.1 Background Information Dengue (DEN) and Yellow fever (YF) are re-emerging important arboviral diseases worldwide, DEN infecting about 390M people per year (Smith et al., 2004; WHO, 2015; Atieno, 2017). Globally there has been increase of DEN reported cases between year 2013- 2019, from 8 Million to 60 M with 10,000 deaths and about 50-100 Million cases occurs in tropical and sub-tropical countries, every year (Garcia-rejon et al., 2018 ; WHO 2019), incurring a global illness burden of ~$8.9 Billion annually (Gwee et al., 2021). Despite availability of an effective YF human vaccine, it is estimated that 200,000 YF cases occur annually with about 30,000 deaths worldwide, with about >500 Million persons at risk of infections, mostly from South America and sub-Saharan Africa (WHO, 2014; Kraemer et al., 2017). In the last two decades YF outbreaks have been experienced in east Africa including; Kenya in 1992-1995 (Reiter et al., 1998); Uganda in 2011, 2016, 2019, Ethiopia in 2012–2014, 2018, Sudan in 2012 and South Sudan 2020 (WHO, 2015, 2016 ; InterHealth Worldwide, 2016). In addition, Angola and Democratic Republic of Congo (DRC) outbreaks in 2015- 2016, with imports to Kenya (WHO, 2015), revealed the possible spread and risk of YF in the region. These examples indicate that these vector-borne diseases continue to pose public health challenges and afflicting humans (Weetman et al., 2018). The Ae. simpsoni and Ae. aegypti consist of sub-siblings/sub-species that exhibit varying degrees of geographic and reproductive isolation and tend to vary in vectoring abilities (Mukwaya et al., 2000; Hadinegoro et al., 2012). For instance, two genetic forms of Ae. 2 aegypti have been described; the domestic Ae. aegypti aegypti and the forest form Ae. aegypti formosus (Powell & Tabachnick, 2013). Aedes simpsoni consists of about 10 sub-species including the known YF vector Ae. bromeliae (Mukwaya et al., 2000). In spite of the expanding epidemiology of these diseases, important knowledge gaps exist in our understanding of the transmission ecology, dynamics and bionomic role of geographic populations of these known mosquito vectors. Biological traits of these mosquito vectors such as survival rate, genetic diversity, competence, abundance and blood feeding habits are important driving factors of arbovirus transmission and spread (San Martín et al., 2010; Hugo et al., 2014). Surveilling these aspects of mosquito vectors are particularly crucial for risk assessment and guiding vector control or cost-effective vaccinations (e.g. YF). However, geographical transmission and re-emergence of these arboviruses are also modulated by climatic conditions such as rainfall and favourable temperature and human driving activities e.g. travel, land use, urbanisation and global trade (Joyce et al., 2018). The bionomic trait of the vector relating to blood feeding controls opportunities for infection and transmission (Ladeau et al., 2015). It is also a critical parameter used in modelling and control of arboviruses through assessment of vector feeding preference and the way they change over time and space (Kilpatrick et al., 2006). This trait among Ae. aegypti and Ae. simpsoni, and their genetic forms remain poorly characterized for wild populations in East Africa, and Kenya in particular. According to the few studies, an anthropophilic Ae. aegypti habitat near human and typically blood feed on humans ( up to 99%) in Thailand (Harrington, 2005) and in Australia (Stephenson et al., 2019), Nonetheless, it has been found to blood feed on other wild and domestic animals such as cat and dogs in South Texas (Olson et al., 2020) and Senegal (Diallo et al., 2013). 3 In East Africa, West Pokot Kenya, Ae. aegypti exhibited multiple host blood feeding including humans, but majorly feeding on livestock including; rock hyraxes, cattle, goats and sheep, (Chepkorir et al., 2018) with similar zoophilic tendency observed in Kisumu, Kenya (Agha et al., 2019), and about 17% feeding on cattle both in Kisumu and Mombasa (Agha et al., 2019).The feeding behaviour of this species has genetic basis; Ae. aegypti aegypti is reported to be more anthropophilic and Ae. aegypti formosus zoophilic (Huber et al., 2008). Unlike Ae. aegypti, the data on blood feeding behaviour in Ae. simpsoni is limited. Ae. bromeliae a dominant sub-species among Ae. simpsoni complex, breeds near domestic area and is highly an anthropophilic while the sister species Ae. lilii is zoophilic (according to blood feeding preference) and breeds in forest (Mukwaya et al., 2000 ; Walter et al., 2014). The vector survival rate (longevity) is another bionomic trait of importance in pathogen transmission. It is one of the most sensitive determinants of a vector population’s capacity for pathogen transmission (Cook et al., 2013). In the transmission cycle of a pathogen, an infected female mosquito after taking a blood meal must survive the extrinsic incubation period (EIP) of the pathogen before transmission can occur. The increase in the survival/longevity of female adult mosquito linearly increases the vectorial capacity, which explains the incidence of arboviruses and the potential risk of exposure to the infected vector and therefore allows an effective measures in control of vectors (Ndoen et al., 2012) . The survival ability of Ae. aegypti species has been assessed in the recent years globally, such as in Asia (Reinhold & Lazzari, 2018) and central Vietnam (Hugo et al., 2014) displaying a seasonal variation in survivorship, with relatively high survival rates during rainy seasons. Such data is limited or non-existent as for Ae. simpsoni in East Africa, Kenya in particular. Enhanced understanding of these entomological risk factors responsible for the re-emerge of 4 YF and DEN outbreak risks remains a key basis for intervention and preventive strategies against repeated outbreaks of these diseases. 1.2 Statement of the Problem Yellow fever virus (YF) and Dengue virus (DEN) are flaviviruses of medical importance globally. Yellow fever is a public health threat to 34 countries in Africa and 13 countries in central and southern America (Garcia-rejon et al., 2018). Since the first outbreak reported in Kenya (Rift Valley) in 1992-1995 ; Sentinel Surveillance for Yellow Fever in Kenya in 1995 eastern Africa demonstrated an increasing frequency of YF outbreaks including; Uganda, Ethiopia, Sudan, South Sudan and Angola, spreading to Kenya and Congo (WHO, 2015, 2016 ; InterHealth Worldwide, 2016). Over two decades, the number of cases are increasing in east Africa despite the availability of an effective vaccine (WHO, 2016), possibly due to poor health systems, increased urbanization, and low immunity against YF (Neiderud, 2015). In addition, DEN is a global health threat, affecting over 100 countries (Gloria-soria et al., 2016), including; America, Eastern Mediterranean, South-East Asia and Western Pacific and Africa. In eastern Africa, frequent outbreaks of DEN have continued to occur including; Tanzania, Somalia, Djibouti, Eritrea, Sudan, Ethiopia and Kenya (Malik et al., 2011; de Laval et al., 2012; Bosa et al., 2014; Mwanyika et al., 2021; WHO, 2021). Owing to the fact that there is no effective vaccine against DEN or an effective drug (Londono-Renteria et al., 2016), entomological vector control strategies remains the only solution against the increasing outbreaks. The expanding epidemiology of these diseases occurs against the poor understanding of their transmission dynamics, ecology, including bionomic role of geographic populations of 5 known vectors. Survival rate and Blood feeding habits provides opportunities for vector infection and efficient transmission (Kramer & Ebel, 2003; Tchouassi et al., 2020). Little information is in existence regarding these bionomic traits among Ae. aegypti and Ae. simpsoni and their genetic forms in Kenya particularly in Kerio Valley and Rabai. These knowledge gaps limits the capacity to accurately monitor and respond to arboviral infections, outbreaks, and timely predict the spread. There is therefore, urgent need to understand transmission drivers of DENV and YFV, crucial to address on a better and cost effective preventive strategy. 1.3 Justification Owing to recurring outbreaks of YF and DEN in the East African region (LaDeau et al., 2015;WHO, 2015, 2016; Atieno, 2017), high fatality rates (Huhtamo et al., 2013) as well as the established distribution of vectors Ae. simpsoni and Ae. aegypti (Getachew et al., 2015) there is a risk of continued recurrence of these diseases. This is further exacerbated by human activities that potentiate transmission across wider geographical regions (WHO, 2015).The highlighted risks necessitate all possible efforts towards mitigating re-emergence and severity of future outbreaks, owning to, Kerio Valley has a history of YF and Rabai Kenya, a DEN endemic area. The effectiveness of transmission of arboviral diseases is reliant on various bionomic traits intrinsic to the vector (Ladeau et al., 2015). For the YF and DEN vectors of Kerio valley and Rabai Kenya, these traits remain poorly characterized. It is therefore necessary to characterize these traits among the locally established populations of these vectors. In addition, genetic variability has been reported in various mosquito vectors. However, it is not clear whether the existence of such variability contributes to or influence important bionomic traits relevant to 6 pathogen transmission. It is thus of interest to determine the level of genetic diversity among the local populations of DEN and YF vectors and whether such diversity affects traits such as blood feeding and distribution of their subspecies. 1.4 Research Questions i. What are the survival rates of Ae. aegypti and Ae. simpsoni mosquitoes in Kerio Valley and Rabai areas? ii. What are the blood feeding patterns of Ae. aegypti and Ae. simpsoni in Kerio Valley and Rabai areas? iii. What are the human blood feeding habits among the genetic forms of Ae. aegypti and subspecies of Ae. simpsoni in Kerio Valley and Rabai areas? 1.5 Objectives 1.5.1 General Objective To determine the survival rates and blood meal patterns of Aedes aegypti and Aedes simpsoni mosquitoes collected from Kerio Valley and Rabai arbovirus ecologies in Kenya. 1.5.2 Specific Objectives i. To determine the survival rates of Ae. aegypti and Ae. simpsoni mosquitoes in Kerio Valley and Rabai areas. ii. To determine the blood feeding patterns of Ae. aegypti and Ae. simpsoni in Kerio Valley and Rabai areas. iii. To determine the human blood feeding habits among the genetic forms of Ae. aegypti and subspecies of Ae. simpsoni in Kerio Valley and Rabai areas. 7 1.6 Significance of the Study This study unravels the on survival rates and blood meal patterns of Ae. aegypti and Ae. simpsoni mosquitoes in two areas (Kerio valley and Rabai). In addition, the study assesses how these parameters vary among their genetic forms. These two bionomic traits are among the important indicators of the vectorial capacity, as they provide the opportunities for effective vector infections and transmission. A clear understanding of these parameters, may help in modeling transmission essential in diseases risk predictions and an urgent warning. 8 CHAPTER TWO 2.0 LITERATURE REVIEW 2.1 Global distribution and burden of dengue and yellow fever Aedes aegypti and Ae. simpsoni mosquitoes are associated with the transmission of arboviruses including dengue and yellow fever viruses, associated with the diseases (dengue and yellow fever) of global public health threat. Dengue occurs globally both in epidemic and endemic transmission cycles, implicated with principle vector Ae. aegypti majorly in east Africa (Bhatt et al., 2013) and Aedes albopictus as a secondary vector in Asia and European region (WHO 2021). Dengue is estimated to infect 50 million people yearly, globally affecting over 100 countries (Gloria-soria et al., 2016). These include both subtropical and temperate climate areas such as America, Eastern Mediterranean, South-East Asia and Western Pacific and Africa (Ngoi et al., 2016; Garcia-rejon et al., 2018). Figure 1: A map showing global distribution of Dengue fever (Source: WHO 2019) In Africa, since the first outbreak in Zanzibar between the years 1823-1870, outbreaks have been experienced in other countries including Kenya, Tanzania, Somalia, Sudan, Eritrea and Djibouti (Simo et al., 2019). Unlike YF, DEN outbreaks have been rising sporadically in east Africa, Kenya in particular since it was first reported in the coastal region in 1982 (Ngoi et 9 al., 2016) with subsequent outbreaks reported between again 2013-2014 and 2017-2018 along the Kenyan coastline (LaDeau et al., 2015; Atieno, 2017). In addition, a severe outbreak was reported during 2011 in Mandera (north eastern Kenya) that resulted in more than 2100 reported cases (Huhtamo et al., 2013). The increasing outbreaks of dengue in coastal Kenya have implicated the transmission of DEN-1, 2 and 3 serotypes and the increasing distribution of the primary vector Ae. aegypti in the region (Konongoi et al., 2016). Dengue infection is asymptomatic with 50% of infected cases presenting flu-like symptoms. Other symptoms may include, headache, rashes and myalgia (Simo et al., 2019). There is no effective human vaccine or drug against dengue fever; to mitigate the transmission, monitoring and controlling of disease vector remains the major approach. Figure 2: A map showing global distribution of yellow fever (Source: https://www.gs.international/yellow-fever) Yellow fever virus is another Flavivirus in east Africa and it is endemic to tropical regions of Africa, central and southern American countries (Garcia-rejon et al., 2018). YF infections exhibit non-specific symptoms, severely leading to high fever, jaundice, hemorrhaging and nausea, potentially leading to death. In spite of availability of an effective vaccine (live 10 attenuated 17D vaccine), the burden of this disease is estimated at 200,00 cases annually with 30,000 deaths globally, majority occurring in Africa, with annually estimated 84,000 to 170,000 cases and 29,000 deaths (Garske et al., 2014). 2.2 Transmission cycles of dengue and yellow fever viruses Yellow fever (YF) has three transmission cycles in Africa (urban, intermediate or rural, and the sylvatic or jungle), with each cycle involving a specific set of vertebrate hosts and vectors (Garske et al., 2014). The sylvatic cycle (jungle) involves transmission of the virus between non-human primates (NHPs), mediated by tree-hole breeding vectors (Ae. africanus in Africa and Haemagogus spp in America); however, humans can become infected when they encroach into the forests through human activities (Garske et al., 2014). The urban cycle involves human as the reservoir host, and transmission of the virus by domestic Ae. aegypti. Unlike sylvatic, urban transmission cycles are rare, but fatal if occur, which make it difficult to control. Such an outbreak recently occurred in Africa, in Angola and Democratic Republic of Congo (DRC) resulting to > 300 mortalities and > 900 reported cases (with imports to Kenya); (WHO, 2015). These outbreaks generally occur due to both dense urban population and high Ae. aegypti vectors. An intermediate YF cycle also occur (rural transmission cycles) in Africa involving both human and NHPs, vectored by domestic and peri-domestic Aedes mosquito species, including; Ae. metallicus, Ae. simpsoni S.I and Ae. vittatus (Gaythorpe et al., 2021). Dengue virus also originates from sylvatic cycles involving NHPs as reservoir hosts; however, in urban epidemic cycles, human is implicated as the main reservoir. Both endemic and epidemic DEN cycles occur, epidemic cycles arising in urban/peri-urban environments vectored by Aedes aegypti in eastern Africa (Bhatt et al., 2013). 11 2.3 Concept of vectorial capacity, blood feeding and survival rates Vectorial capacity allows an examination of the average rate at which infective mosquitoes initiate infection to a single host. It usually includes the feeding habits, time required for pathogen to fully develop (extrinsic incubation period) and the survival of vector species (Anderson & Rico-Hesse, 2006). Vectorial capacity (VC) is calculated as indicated in the equation, Where; M = vector density a = probability of daily feed p =probability of daily survival of vector n =length of extrinsic incubation period (EIP) The variation of vectorial capacity thus, has an impact on basic reproductive number (Maciel- De-Freitas et al., 2008). Reproductive number (Ro) indicates the expectation of number of hosts that can be infected by only a single host introduced into uninfected population. Therefore, a value of Ro greater than one shows that the number of infected by a pathogen increases and the value of Ro less than one shows a decrease of people infected by the pathogen (Maciel-De-Freitas et al., 2008). The understanding of the Ro and VC in arboviruses endemic areas generate measures to disease control and awareness of disease transmission. In addition, survival and human blood feeding are two of the most sensitive parameters in VC estimation. For pathogen transmission, infected female mosquitoes after taking a blood meal must survive the EIP of pathogen for an effective transmission (David et al., 2009). Female 12 mosquitoes life cycle therefore, indicates the survival rate as an important factor in analysing the stability of female mosquito oviposition, population and pathogen transmission risk (Hugo et al., 2014). This life cycle however, maybe affected by the environmental factors both in their ecosystem and habitats, which includes temperature, humidity and sunlight (Getachew et al., 2015). These factors may thus, affect the survival rates of the female mosquito. 2.4 Blood meal patterns and significance in pathogen risk assessment In pathogen transmission, an adult female mosquito vector requires a blood meal by first biting on an infected hosts and later the recipient host (Stephenson et al., 2019). The host selection of a mosquito is influenced by both extrinsic and intrinsic factors. The host abundance, chemicals released by hosts, the defensive mechanisms of the host and biomass are some of the extrinsic factors that guide host seeking behaviours among mosquito vectors (Stephenson et al., 2019). Other environmental factors include variation of climatic conditions such as relative humidity which influences the availability or unavailability of hosts in their habitants (Joyce et al., 2018). Different mosquito species intrinsically prefer certain hosts as blood meal sources. Mosquito blood feeding patterns play an important role in determination of vectorial capacity (VC), allowing the identification of the host selection and biting rate among mosquitoes. This provides informative insights on vector borne pathogens in epidemiological studies (Cebri, 2020). Knowledge on the human biting preference among mosquitoes allows an estimation of the reproduction rate (Ro). In sub-Saharan Africa, an anthropophilic Ae. aegypti has shown to circulate both in peri- domestic and sylvatic cycles which involves human and wild animals (Sylla et al., 2009). Female Ae. aegypti has been found to have a high human-blood feeding preference (up to 13 99%) both in rural and urban areas (Harrington, 2005; Faraji et al., 2014; Sivan et al., 2015). In Thailand it has been found to prefer feeding on human blood (99%) while less than 1% fed on cats, dogs, rats, and birds (Harrington, 2005). However, a recent study in East Africa in West Pokot County, Kenya, Ae. aegypti exhibited a low human blood feeding preference, with most feeding on rock hyrax (79%), goats (9%), cattle (4%), human (3%) and 2% on hippopotamus and lizard (Chepkorir et al., 2018) with similar zoophilic tendency in Kisumu, Kenya (Agha et al., 2019). The disparity trends of the feeding habits of Ae. aegypti, may be associated with the variety of factors including; difference ecological study site, method of sample collection and the distribution of the genetic forms (Ae. aegypti aegypti and Ae. aegypti formosus). 2.5 Species identification methods Reliable identification of mosquito vectors is a crucial component of disease surveillance and implementation of vector control strategies. Species level identification can be accomplished routinely morphologically or using molecular techniques. Morphological identification involving the use of taxonomic keys and microscopy is based in examination of external features of adult or larval specimens. For instance, the two forms of Ae. aegypti can be discriminated as adults based on scale pattern of the wings being dark in the domestic form and pale in the forest ecotype (McAvin et al., 2005). However, these features are labile depending on the breeding area and difficult to discern. Aedes simpsoni mosquitoes belong to a complex where individual sibling species cannot be reliably identified based on morphology alone. These challenges necessitate the use of molecular techniques to supplement morphological identify to elucidate taxonomic status of vector species including potential genetic variability within species. Molecular 14 characterization or genetic barcoding is most frequently used in identification of mosquito up to species or sub-species level. Common genetic markers include DNA barcoding of the cytochrome c oxidase subunit 1(CO1) gene in mitochondrial genome region and internal transcribed spacer 2 (ITS2) located at in the ribosomal genome region (Bennett et al., 2015b). Although a 860 bp barcode region of mitochondrial CO1 gene has been successfully and effectively utilized in discrimination of closely related species (Paupy et al., 2012), the use of CO1 mitochondrial gene has shown the ability of pseudo gene introgression which limit species discrimination. The region of (ITS2) fast evolve, and was found to be a reliable marker to discriminate some closely related mosquito species including Anopheles complexes and Ae. simpsoni S I (Tchouassi et al., 2014; Beebe, 2018; Ogola et al., 2019). Nested PCR-based method is another method developed to distinguish Ae. simpsoni S I sub- siblings; Ae. bromeliae, Ae. lilii and Ae. simpsoni spp based on the ITS region (Bennett et al., 2015b).Other genetic regions used are cox2 in the mitochondrial region and D3 segment located in nuclear ribosomal region (McAvin et al., 2005). 2.6 Survival rates of mosquito 2.6.1 Methods of estimating mosquito age The chronological and physiological age structure of female mosquitoes have been evaluated using different methods. One of the methods includes the mark-release-recapture method (M. Trpis et al., 1971; Muir & Kay, 1998). The mark-release-recapture is a quick and simple method of determining the age of mosquitoes which involves marking of laboratory reared mosquitoes (within 24 -72 hrs after adult emerge) using florescent pigments, before releasing to the field. Later, within 12-18hrs the marked and unmarked mosquitoes are collected to determine the age structure (Trpis et al., 1971; Muir & Kay, 1998). Using this method, the age of Ae. aegypti has been estimated in Australia, with the mean survival rate of 19 days for 15 female and 14 days for males (Muir & Kay, 1998) and in Shauri Moyo village (Rabai, Kenya) with mean survival rates of 10.7 and 5.8 days in males and female respectively (Trips and Hauserman et al., 1995). Nevertheless, this method is laborious and difficult to conduct in some areas, and since it relies on laboratory reared mosquitoes, it may not accurately represent wild mosquito populations (Joy et al., 2012). The use of reproduction system of a female mosquito has provided a reliable and logistically easier technique to determine the physiological age structure of female mosquito (Ndoen et al., 2012). The technique involves the dissection and examination of adult female mosquito ovaries. The ovarian tracheae are then analysed to determine the female parity status either parous or nulliparous. The parous female mosquitoes, is characterised by uncoiled endings of tracheoles skein, observed under a microscope. This indicates that the female mosquitoes, have taken a blood meal and laid eggs at least once while nulliparous studied under a microscope have folded and coiled tracheoles skeins (Ndoen et al., 2012). To determine the physiological age structure, the approach adopts assumptions including; the mortality rates of the mosquito populations are equal in all ages, the emigration and immigrations are equal and gonadotrophic estimation ranging 3-4 days depending on the mosquito species (Arum et al., 2016). This technique has successfully and broadly been used globally in areas such as West Timor, Central Java (Ndoen et al., 2012) and Eastern African (Arum et al., 2016; Tchouassi et al.,2020). Nonetheless this method may be unsuitable in analysing large mosquito sample size. Molecular based approaches including; cuticle rings counting to represent the mosquito daily growth of layers at the skeletal apodemes, transcriptomic profiling , mosquito protein spectrometric analysis are some of the methods used as an alternative of ovary- based 16 approach. However, the molecular based approaches are expensive and requires a high trained personal making it unsuitable for field collected mosquitoes. Use of mid- infrared spectroscopy approach is another alternative approaches used in estimating mosquito age (Babayan et al., 2019). The infrared spectroscopy analyze the difference in structural and chemical composition in the cuticle, which changes between the species and during mosquito life cycle. These allows prediction of survival and mosquito speciation using machine- learning analysis. Although this approach is not broadly used, according to (Babayan et al., 2019), the approach is suitable to large number of mosquito analysis, both in laboratory and filed collected mosquitoes and cost effective to entomological surveillance compared to molecular based approaches. 2.7 Blood meal analysis of mosquitoes Different techniques have been employed to identify the vertebrate blood meal sources of field collected mosquitoes. These techniques include serological methods such as ELISA (Enzyme Linked Immunosorbent Assay), gel diffusion, precipitin test and molecular techniques (Cebri, 2020). The identification of blood meals via serological methods involves the exposure of immunoglobulin G (IgG) in blood conjugated against the host species (Harrington, 2005 ; Sivan et al., 2015). However, the technique faces a challenge of the availability of serum proteins and antisera against some other targeted species (Cebri, 2020). To increase the specificity of the host identification, molecular techniques are used, which entail the use of specific or universal primers in the amplification of target sequences (Kent, 2009). DNA sequencing is one of the simplest, specific and ultimate molecular techniques for mosquito vectors which feed on a broad range of unknown vertebrate hosts. This include use of a single marker and Sanger sequencing technique to identify single blood feed host. 17 However, for a broad assessment of mosquito vector blood meals, multiple markers and high- throughput sequencing maybe used to identify multiple feeds on different hosts (Muturi et al., 2020 ; Logue et al., 2016). Sequencing approaches are heavily reliant on availability of properly annotated sequence databases and search algorithms/tools for specimen identification usually given as percent identities and/or similarities (Kent, 2009). In addition, sequencing and biological databases enable the inference of evolutionary relatedness through phylogenetic reconstruction analyses. Other molecular methods include real time PCR, PCR- restriction fragment length polymorphism (PCR-RFLM) and high-resolution melting (HRM) analyses on cyt b and 16S ribosomal RNA genes PCR products (Omondi et al., 2015). 2.8 Distribution and genetic forms of Ae. aegypti and Ae. simpsoni mosquitoes Ae. aegypti is found in tropical and subtropical areas worldwide, as well as temperate areas with high latitude (Gloria-Soria et al., 2018). Human activities such as global trade and human movements have facilitated the spread of Ae. aegypti from one continent to another (Joyce et al., 2018), originally from Africa to America and Asia (Gloria-soria et al., 2018). Two subspecies of Ae. aegypti are well defined: the sylvan Ae. aegypti formosus and the domestic Ae. aegypti aegypti (Powell & Tabachnick, 2013). Despite their morphology not reflecting the ecological distinction, the two subspecies have been described according to their geographical distribution (Gloria-soria et al., 2016). The domestic form is more anthropophilic, breeding nearer to human habitats such as in water holding containers (Neira et al., 2014). Aedes aegypti formosus prefers non-human blood feeding and breeds in non- human areas including; natural breeding sites such as tree holes (Huber et al., 2008). Aedes (Stegomyia) simsponi is a species complex (i.e. morphologically identical as adults) consisting of at least ten species, of which three species have originally been described; Ae. 18 simpsoni, Ae. bromeliae and Ae. lilii (Mukwaya et al., 2000). These species are genetically different, and exhibit variation in biting behaviour, oviposition or breeding site preferences and geographic distribution. Among the Ae. simpsoni complex, Ae. bromeliae is known to be anthropophilic and attracted to human bait while Ae. lilii is zoophilic and does not bite humans (Mukwaya et al., 2000). This explains why Ae. bromeliae is the known yellow fever vector within the complex (Mukwaya et al., 2000). Aedes bromeliae breed in domestic areas while Ae. lilii are confined to forest areas (Walter et al., 2014). Although unreliable, morphological identification key has been illustrated to differentiate Ae. simpsoni, by use of the pale coloured scales at the mid and fore tarsomeres and manifestation of tooth of the mid-tarsal claws (Huang et al., 1986). A reliable identification of adult forms of Ae. simpsoni complex using genetic markers, is fundamental due to presence of anthropophilic Ae. bromeliae, a vector of importance in transmission of yellow fever virus to humans and other primate (Bennett et al., 2015a). 19 CHAPTER THREE 3.0 MATERIAL AND METHODS 3.1 Study area This study utilised adult female mosquito samples of the species Aedes aegypti and Aedes simpsoni senso lato (s.l.) that had been collected from Kerio Valley (11.4099° N, 41.2809°E) and Rabai (3.9454° S, 39.5588° E). Kerio Valley is located in Rift Valley while Rabai is located in coastal Kenya, approximately 25 km from Mombasa town (Figure. 3). Kerio Valley has a history of Yellow fever outbreak (Sentinel Surveillance for Yellow Fever in Kenya , 1993 to 1995, 1996) while Rabai in Kilifi County is endemic for dengue fever (Ngoi et al., 2016). 3.2 Study design The study adopted an experimental study design using archived stored mosquitoes collected from Rabai and Kerio valley areas. 3.3 Mosquito retrieval and processing The study used archived freezer stored mosquitoes that had been collected as part of an arboviral surveillance project. The mosquitoes were trapped using CO2-baited BG Sentinel traps set during the day (6:30 am – 18:00), for at least 8 consecutive days at different time points, after the rains. These periods included: Kerio Valley (13th to 28th November, 2019), Rabai (28th August to 4th September2019) and Rabai (6th to 17th February 2020). This trap is suitable for trapping these Stegomyia mosquito species which are active during the day (Agha et al., 2017). After trapping the mosquito samples were transported in liquid Nitrogen to International Centre of Insect Physiology and Ecology (Emerging Infectious Diseases laboratory) Nairobi. 20 Figure 3: A Map showing sampling locations in Rift Valley a history of Yellow fever and coastal Kenya an endemic for Dengue (Source: QGIS 3 software). 3.4 Laboratory Analysis 3.4.1 Mosquitoes morphological identification All the mosquito samples collected from the field were retrieved from liquid nitrogen and morphologically identified to species level, according to (Edwards, 1941) and were stored at - 80 °C freezers. 21 3.4.2 Determination of parity rates of Ae. aegypti and Ae. simpsoni sl mosquitoes A total of 1,008 adult female Ae. aegypti and 570 Ae. simpsoni sl mosquitoes were retrieved from -80 °C freezers, and each allowed to thaw for 10 minutes, by placing on petri dish on ice. The ovaries from each mosquito was extracted and dissected under a stereomicroscope on 2/3 drop of saline on a sterile microscope slide. After drying out, parity was scored via microscopy at 40X magnification by observing for the presence or absence of coiled tracheolar skeins or dilation of the avarioles (Detinova, 1962; Tchouassi et al., 2020). Specimens with dilated tracheolar skeins were classified as parous, an indication of an earlier blood feeding and egg production or nulliparous with presence of coiled tracheolar skeins indicating mosquitoes without any previous blood feed and egg production. The thorax and legs from each dissected sample were preserved individually and stored at -80°C for further molecular analysis. Figure 4: A presentation of tracheolar skeins under microscopy at 40X magnification. (A) Indicates Nulliparous and (B) Parous. 3.4.3 Estimate of mosquito daily survival and longevity Generated data on parous rates from the different sampling periods for each area were used to estimate the daily survival rate for both mosquito species as described previously (Davidson, Coiled tracheolar skeins Dilated tracheolar skeins A B 22 1954). Using formula, Pn = M, where p is the daily survival rate, n the gonotrophic cycle, which was assumed as 3 days according to (Tchouassi et al., 2020) and M is the parity rate. Lastly, using 1/-lnp formula, the longevity in days was estimated, where P represented estimated survival rate (Davidson, 1954). 3.4.4 Determination of blood meal sources from Ae. aegypti and Ae. simpsoni Preserved blood-fed specimens (individually at -80°C freezer) encountered during trapping in both areas were processed to identify the host meal sources. The blood-fed specimens were retrieved at -80°C freezers and abdomen was separated from the thorax or head using a sterile scalpel. To avoid contamination, the scalpel was disinfected between each sample using 70% ethanol applied on cotton wool. The head/thorax was preserved frozen for further molecular analysis while the abdomen (with blood) was processed for DNA extraction using DNeasy Blood and Tissue Kit (Bioline, Germany) according to the manufacturer’s instructions. To identify the blood meal sources of the engorged mosquitoes, the extracted DNA was used as the template in Polymerase chain reaction (PCR), to amplify a 500 bp of the 12S mitochondrial rRNA gene using established 12S3F and 12S5FR primers (Table 1) as described previously (Roca et al., 2004); Tchouassi et al., 2020). The PCR was done using MyTaq HS Mix kit (Bioline, Germany), in amplification reaction volume of 10 μl, which consisted of 2 μl of 2xMytaq HS mix polymerase, 10 M of each primer, 0.2 μl of Mytaq polymerase and 1 μl the template DNA (~20ng). Thermal cycling conditions were 95°C for 3 min followed by 40 cycles at 95°C for 20 s, 59°C for 30 s and 72°C for 30 s and 72°C for 7 min. PCR products were then run on 1.2% agarose gel electrophoresis in Tri- Acetate- EDTA (TAE) stained with Ethidium bromide against a 100 bp DNA ladder (Fisher Scientific, UK) and gel visualised in UV trans illuminator. The 23 amplicons were purified using the Sure Clean Plus kit (Meridian Bioscience) and sequencing were outsourced to Microgen (Tchouassi et al., 2020). 3.4.5 Molecular Identification of Blood-Fed Ae. aegypti and Ae. simpsoni sl The remaining portion (head/thorax) of each mosquito used for host blood meal determination was processed to identify the sibling species (Ae. simpsoni sl) or genetic forms (Ae. aegypti). First, DNA was extracted using an in-house sodium dodecyl sulcate method adapted from (Adams et al., 2008) with slight modifications. Briefly, each specimen was homogenized for 20 s using 4 to 5, 2-mm zirconium beads in 50 μl PBS in a Mini- beadbeater-16 (BioSpecs inc USA). A 300 μl of Cell Lysate Buffer (CLB) consisting of 5mM Ethylene Diamine Tetrameric Acid (EDTA), 10 mM Tris-HCl, PH 8.0, 0.05%SDS, was added into the homogenate and incubated at 65°C for 1.5 h. A 100 μl of Protein Precipitation Solution (PPS) consisting of 1M EDTA and 8M ammonium acetate was then added, the mixture vortexed for 1 min and incubated on ice for 30 min. Afterwards, the mixture was centrifuged at 16400rpm x g at 4°C for 10 min. All the supernatant was transferred into 300 μl isopropanol, in a new sterile 1.5ml micro centrifuge tube. The mixture was then mixed thoroughly by inverting 100 times, centrifuged for 30 min at 16400 rpm x g at 4°C. The supernatant was discarded and pellets suspended using 300 μl of 70% ethanol (ice-cold). The mixture was inverted 50 times to mix thoroughly, centrifuged for 30 min at 4°C, 16400 rpm x g, and thereafter the supernatant was pipetted off and pellets were inverted and left to air-dry overnight. The DNA pellets were eluted using 50 μl PCR grade water and using NanoDrop ™ 2000 Spectrophotometer, the extracted DNA were quantified (Adams et al., 2008) For Ae. aegypti specimens, PCR targeting a 860 bp barcode region of mitochondrial Cytochrome C Oxidase subunit 1 gene (CO1 gene) was amplified using the CO1-FOR and 24 CO1 –REV primer (Table 1); (Paupy et al., 2012).The PCR was performed using Mytaq HS Mix kit (Bioline, Germany), the reaction volume comprised of 5μl of 2xMytaq HS mix polymerase, 10 M of each primer and a template DNA of 2 μl using the thermal cycling conditions of 95°C for 2 min followed by 40 cycles at 95°C for 30 sec, 60°C for 30 sec and 72°C for 35 sec and 72°C for 7 min. However, for Ae. simpsoni sl specimens, PCR was conducted targeting a portion of the ITS2 region using the primers ITS2A-FOR and ITS2B-REV (Beebe & Saul, 1995) as shown in Table 1. The PCR was performed comprising of 20 μl reaction volume including 3μl 5x Hot Firepol Blend master mix kit (Estonia) 0.5 μl of 10 M of each forward and reverse primer, 5μl of 2xMytaq HS mix polymerase and 1 μl DNA template (~20ng) . The thermal cycling conditions were 95°C for 15 minutes followed by 40 cycles at 95°C for 30 seconds, 60°C for 30 seconds and 72°C for 45 seconds and 72°C for 7 minutes. The PCR products for both targets were run in 2% gel electrophoresis as described earlier. Finally, the PCR products were purified using Sure Clean Plus kit and outsourced to Microgen for sequencing however, in both the forward and reverse direction, as shown in Table 1. Table 1: Summary of primers used in this study Primer Oligonucleotide sequence Reference CO1-22-F CO1-22-R 5’-TGTAATTGTAACAGCTCATGCA-3’ 5’-AATGATCATAGAAGGGCTGGAC-3’ Paupy et al., 2012 ITS2A-F ITS2B- R 5’-TGTGAACTGCAGGACACAT-3’ 5’-TATGCTTAAATTCAGGGGGT-3’ Beebe & Saul, 1995 25 3.5 Genetic and Phylogenetic Analysis The nucleotide sequences were cleaned and aligned using Molecular Evolutionary Genetics Analysis Version 6.0 (Tamura et al., 2013). The nucleotide sequences were searched against the GenBank database using the Basic Local Alignment Search Tool, BLAST (www.ncbi.nlm.nih.gov/blast). Evaluation of sequence matches closest on or near matches of threshold >98% was used, to conclude the species individuality of the blood meal source. The CO1 nucleotide sequences were viewed, cleaned and aligned using MEGA v.6 preceding to the phylogenetic analysis. In the GenBank database through BLASTn searches, corresponding CO1 gene sequences of Ae. aegypti formosus (GenBank accession no: AY056597 and domestic Ae. aegypti (GenBank accession no: AF390098 and MF 194022) were identified and included in the alignments to infer a Maximum Likelihood (ML) phylogenetic tree. For Ae. simpsoni sl homologous ITS2 sequence of Ae. simpsoni clone GenBank accession no: AF 439601, Aedes bromeliae GenBank accession no: KF135509 and Aedes lilii (MH277635, MH277623 and MH277625) were included in a Maximum Likelihood phylogenetic tree (Tamura et al., 2013). 3.6 Data Analysis The percentage of total number of parous mosquitoes scored of the total number mosquitoes dissected was calculated as the parity rate (PR). This was determined both for each species and for each trapping period. For each species, the PR (proxy for survival) was compared between the trapping periods, using Pearson Chi-square test (χ2) at P=0.05. The human blood 12S3F 12S5R 5′-GGGATTAGATACCCCACTATGC-3′ 5′-TGCTTACACATGTTACGACTT-3′ Roca et al., 2004 26 index was calculated as the proportion of human blood meals of the total number of blood- fed mosquitoes examined. The proportions of human blood meal between the mitochondrial CO1 lineages of Ae. aegypti and established genetic forms of Ae. simpsoni were compare and tested significant differences using Chi-square test (χ2) at P= 0.05, using R programing software (R Core Team, 2013). 27 CHAPTER FOUR 4.0 RESULTS 4.1 Mosquitoes retrieved from Kerio valley and Rabai Out of 1,578 adult female mosquitoes (morphologically identified Ae. aegypti and Ae. simpsoni sl) were retrieved from the -80 freezers at EID lab in ICIPE, a total of 1,520 mosquitoes were successfully dissected and scored for parity. However, 58 mosquitoes were not successfully identified for parity owing to physical damage. Out of 1,008 of Ae. aegypti, 981 (97.3%) and for Ae. simpsoni sl out of 570, 539 (94.2%) were successfully dissected and scored for parity both in Kerio valley and Rabai. For the blood fed cohorts, a total of 80 engorged mosquitoes (morphologically identified Ae. aegypti and Ae. simpsoni sl), were retrieved from Kerio Valley and Rabai, these comprised of 53 Ae. aegypti and 27 Ae. simpsoni sl. The blood-meal sources from Ae. aegypti 48 of 53 (90.6%) and Ae. simpsoni sl 24 of 27(88.9%) were successfully identified. 4.2 Parity rates of Ae. aegypti and Ae. simpsoni sl. A total of 1,520 mosquitoes were successfully dissected and scored for parity from Kerio Valley and Rabai across the three sampling periods. This comprised 981 Ae. aegypti and 539 Ae. simpsoni sl. The parous/ parity rate (PR) were calculated as the percentage of total number of parous mosquitoes scored of the total number of mosquitoes dissected. Regardless of site or sampling period, parous rate for Ae. aegypti was 74.9% (735/981) and 85% (458/539) for Ae. simpsoni sl. For Ae. simpsoni sl parous rates were 82.5% (71/86) in August- September, 2019 in Rabai, 85.1% (189/222) in February 2020 in Rabai and 85.7% (198/231) in Kerio Valley in November 2019. There was no significant difference in the parous rates across the sampling periods p>0.05 (χ2 =0.50, df = 2, P=0.78). Aedes aegypti parous rates were 67.7 % (212/313) in August-September, 2019 in Rabai, 77.8% % (277/356) in February 28 2020 in Rabai and 78.8% (246/312) in Kerio Valley in November 2019. Analysis showed that there was a significant difference in the parous rates across the sampling periods p<0.05 (χ2 = 12.75, df = 2, P=0.002) as shown in figure 5 below. Figure 5: Variation in parity rates estimated for Ae. aegypti and Ae. simpsoni s.l. collected at different periods in Kerio Valley (KV) and Rabai. (n) Represents, total number of mosquitoes dissected and Aug-Sept: 2019, Feb: 2020 and Nov: 2019, represents August-September: 2019, February: 2020 and November: 2020 respectively. 4.3 Survival rates and longevity of Ae. aegypti and Ae. simpsoni sl mosquitoes From the parous data estimated daily survival rates for Ae. simpsoni sl was 0.947 in February 2020 in Rabai, 0.94 in August –September 2019 in Rabai and 0.95 in November -2019 in Kerio Valley, translating to longevity of 18.6, 16.2 and 19.5 days, respectively. In Rabai, Aedes aegypti had estimated daily survival rates of 0.92 in February -2020 and 0.87 in August – September 2019 while value for Kerio Valley in November-2019 was 0.923 translating to longevity of 12,7.7 and 12.4 days, respectively (Table 2). 67.7 77.9 78.8 82.5 85.1 85.7 0 10 20 30 40 50 60 70 80 90 100 Aedes aegypti Aedes simpsoni P ar it y R at es ( Su rv iv al ) % p = 0.002 p= 0.78 m (n =3 1 2 ) (n =3 5 6 ) (n =8 6 ) (n =2 2 2 ) (n =2 3 1 ) (n =3 1 3 ) 29 Table 2: Daily survival rates and life expectancy (longevity) of Ae. aegypti and Ae. simpsoni sl in Rabai and Kerio Valley. 4.4 Blood meal patterns of Ae. aegypti and Ae. simpsoni sl A total of 80 blood fed mosquitoes were analysed from Kerio Valley and Rabai, these consisted 53 Ae. aegypti and 27 Ae. simpsoni sl. The blood meal sources were successfully identified from 48 of 53 (90.6%) and 24 of 27(88.9%) Ae. aegypti and Ae. simpsoni sl respectively, revealing a total of 13 different host species. Of the 48 blood fed Ae. aegypti successfully identified, three were from Kerio Valley and forty-five from Rabai. The 3 Ae. Species Trapping period Parity Rate (PR) Daily Survival Rates (P) Longevity (Days) Aedes aegypti Rabai August-September :2019 0.677 0.878 7.7 Rabai February :2020 0.779 0.92 12 Kerio Valley November :2019 0.788 0.923 12.4 Aedes simpsoni Rabai August- September:2019 0.825 0.94 16.2 Rabai February :2020 0.851 0.947 18.6 Kerio Valley November :2019 0.857 0.95 19.5 30 aegypti from Kerio Valley had 3 different host species; rodent, goat and squirrel each at 33.3% (1/3). In Rabai majority were human blood fed at 53.3% (24/45), followed by domestic dog 17.7 % (8/45) rodent 8.9% (4/45), lizard 6.7% (3/45), domestic cat at 4.4% (2/45) and finally bat, tortoise, goat and hedgehog had each at 2.2% (1/45) as shown in Figure.6 below. Figure 6: Blood meal patterns of Aedes aegypti in Kerio Valley and Rabai. Out of 24 Ae. simpsoni sl successfully identified for blood meal sources, 15 were from Rabai and 9 in Kerio Valley, showed narrow specific blood meal host species. In Kerio Valley those found to feed on human was highest at 44.4% (4/9), then rodents at 33.3% (3/9), goat and domestic cat each at 11.1% (1/9). In Rabai, those feed on human were 26.6% (4/15), squirrel 20% (3/15), mongoose and rodent each at 13.3 %( 2/15), wild cat, lizard, cow and goat each were at 6.6 %( 1/15) as shown in figure 7 below. 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Kerio Valley Rabai blood meal sources % Human Domestic Dog Rodent lizard Domestic cat Bat Tortoise Goat 31 Figure 7: Blood meal patterns of Aedes simpsoni sl in Kerio Valley and Rabai. 4.5 Human blood feeding habits among the genetic forms of Ae. aegypti and subspecies of Ae. simpsoni sl. Phylogenetic analysis of 38 blood-fed Ae. aegypti from the two study areas, showed three distinct lineages (Figure 8). One of these, clustered with the domestic form (Ae. aegypti aegypti GenBank No: Af390098 and MF 194022), and Kerio Valley (n=1 and majority of the samples from Rabai (n=25) and Lineage 2 had samples from Rabai (n=5) while lineage 3 contained samples from Rabai (n=7), and clustered with the forest form (Ae. aegypti formosus GenBank No: AY056597). Data for human blood index for each of the mitochondrial lineages are presented in Figure 8. In Rabai, lineage 1 which clustered with the domestic form had a HBI of 0.59(13/22). HBI were 0.75(3/4) and 0.43(3/7) for lineage 2 and 3, respectively. The proportions of human feeds did not vary among the mitochondrial lineages p>0.05 (χ2= 1.13, df = 2, p= 0.565,) In Kerio Valley had one sample in lineage 3, which fed on a non-human host (rodent). 0 20 40 60 80 100 120 Kerio Valley Rabai blood meal sources % Human Rodent Goat Domestic cat Squirrel Mongoose Wild cat Lizard Cow 32 Figure 8: A Maximum likelihood phylogenetic tree of 41 nucleotide mosquitoes COI gene sequences using Tamura-Nei model. GenBank accessions and species are highlighted in blue and red. The sequences acquired in the study specified in black. Ae. albopictus was used as out group. Bootstrap values = 1000 replicates. Adjacent are the presentation of human blood feeding proportions, among the Ae. aegypti mitochondrial lineages in Rabai. A total of n=27 Ae. simpsoni blood-fed samples were profiled for phylogenetic analysis Kerio Valley (n=9) and majority of samples from Rabai (n=15). The Ae. simpsoni samples resolved into 3 clades with well supported bootstrap values (Figure 9). One of these, clustered with Aedes bromeliae (Aedes bromeliae GenBank No: KF135509, Ae. simpsoni clone GenBank accession No: AF 43960) and majority of the samples from Rabai (n=11) and Kerio Valley 33 (n=6). Clade 2 contained samples entirely from Rabai (n=5) and designated Ae. simpsoni spp 1, while clade 3 had samples solely from Kerio Valley (n=3) (Ae. simpsoni spp 2). Data for human blood index for each of the ITS clades are presented in Figure 9 and In Rabai, clade1 which clustered with Ae. bromeliae had a HBI of 0.18(2/11) and in Kerio Valley 0.33 (2/6). Similar data for the other clades were 0.67(2/3) in Rabai and 0.67(2/3) in Kerio Valley for clade 2 and 3, respectively. Figure 9: A Maximum likelihood phylogenetic tree of 46 nucleotide mosquitoes, ITS2 sequences using Tamura-Nei model. GenBank accessions and species are highlighted in blue and red.The sequences obtained in the study specified in black. Ae. aegypti and Ae. metallicus were used as out group. An asterisks indicated an additional sequence. Bootstrap values = 1000 replicates. Adjacent are the presentation of human blood feeding proportions, among the clades. 34 CHAPTER FIVE 5.0 DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS 5.1 Discussion 5.1.1 Parity rates of Ae. aegypti and Ae. simpsoni Pathogen transmission patterns are greatly influenced by ecological, biological and behavioral characteristics of vectors. Here, the estimates of survival and blood feeding of Ae. aegypti and Ae. simpsoni collected from the two areas that contrast in level of urbanization. High parity and the survival rates/longevity was found in both species although with a notable variation among populations of Ae. aegypti based on trapping periods. The mosquitoes which where scored parous, would have been involved in the circulation of pathogens in the selected study area (Rift Valley and Coastal regions). This is the reason that the parous mosquito would have obtained blood from infected hosts during their first fresh blood meal and perhaps transmit the pathogen to another host during the second blood meal, which would have kept the diseases to the circulation. The infectious mosquito mirrors the age structure of adult female mosquito (Smith et al., 2004), and regardless of the modest human feeding rates observed in this study, the findings suggests an increased pathogen/disease transmission (vectorial capacity) by these two vectors. Estimates of parity rates (PR) for natural populations of these species are few or non-existent (e.g Ae. simpsoni) especially in endemic countries of Africa. The values estimated for Ae. aegypti generally mirror those in recent published literature although the rates are dependent on the habitat and prevailing climatic conditions (e.g., temperature, rainfall and humidity) (Hugo et al., 2014). For instance (Garcia-rejon et al. 2018) observed mean parity rates of 0.58 (range: 0-0.88) and 0.61 (range: 0.11-1.00) in the dry and rainy seasons, respectively, in a Mexico cemetery. Relatively lower Ae. aegypti parity rate (27%) was reported in the city of 35 São Paulo, in Brazil (Andrade et al., 2018) . It is also worth noting that PR may be affected by mosquito sampling method (Maciel-de-Freitas et al., 2006; David et al., 2009). The analyzed samples were representative of captures over a number of days for each trapping period. Overall, the study provides new estimates for PR for Ae. simpsoni and the high values for both species indicate high contact rates with vertebrate hosts, creating opportunities for infection and pathogen transmission. 5.1.3 Survival rates and longevity of Ae. aegypti and Ae. simpsoni sl mosquitoes Parity rate was used to estimate the vectors’ daily survival rates assuming a gonotrophic cycle of 3 days. Estimates of the gonotrophic cycle likely vary between these mosquito species with published estimates for Ae. aegypti varying between 3-4 days (Garcia-rejon et al., 2018). No such data exist for Ae. simpsoni. The derived survival rates for Ae. aegypti are consistent with data for this species in Rabai in the mid 1980’s based on mark-release recapture method, with female survival of 0.8 (Trpis and Hausermann, 1986). A mean adult female age was 10.7 days (max. value 42 days) generally in agreement with our present data for this species. The model used to estimate survival from parity assumes that mortality is the same for all ages i.e., age-independent survival (Brie, 2002) which may not necessarily be accurate (Charlwood et al., 1985). The variation in survival rates observed in this study could be modulated by other environmental factors e.g., quality of plant diet sources (Nyasembe et al., 2021; Wanjiku et al., 2021). The variation of these parameters could be a function of trapping period, indicating seasonal transmission risk as previously noted, (Agha et al., 2017; Agha et al 2019) especially for the coastal endemic area which has been prone to several DEN outbreaks with annual rises experienced after the short and long rains between February and June (Konongoi et al,. 2016; WHO, 2021). 36 5.1.4 Blood meal patterns of Ae. aegypti and Ae. simpsoni sl The feeding behaviour is another bionomic trait and a significant principle of vector species explored in this study, which allows to understand the interaction between potential hosts and vector species in the risk posed in transmission of pathogen (Kilpatrick et al., 2006). For instance Ae. aegypti has been involved in transmission of dengue virus especially in costal Kenya (Kyungah et al., 2020) and reported to prefer feeding on human ( Scott et al., 1993; Thomas W Scott & Takken, 2012; Musa et al., 2020). However, according to this study on blood feeding patterns of Ae. aegypti in Rabai, it shows that, an anthropophilic mosquito, equally feeding on human and diversely feeding on other hosts such us domestic dog, rodents, domestic cat, lizard, goat, bat, hedgehog, squirrel and tortoise, and it’s not exclusive as observed in Mombasa and contrary observed on other studies done in endemic areas such as Thailand (Harrington, 2005) and Australia (Stephenson et al., 2019) where Ae. aegypti was found to exclusively blood feed on human at proportions rate of 99%. Although the actual data on the densities of domestic animal is currently unavailable in the study areas, the moderate human feeding behaviour observed in this study may be associated by higher availability of non- human hosts or low human density in the study area, and this may indicate a high potential risk in transmission of enzootic or zoonotic diseases. Similar studies associated with high non-human feeding among Ae. aegypti had been reported where majority 50 % were found to blood feed on domestic dog (Olson et al., 2020). This diverse blood meals sources of Ae.aegypti demonstrates the feeding preference is commonly based on their genetic forms occupying different habitats, Ae. aegypti aegypti reportedly more anthropophilic and Ae. aegypti formosus zoophilic (Joyce et al., 2018). 37 In addition, the blood meal pattern displayed Ae. simpsoni feeding on various hosts. Although the overall engorged Ae. simpsoni mosquitoes analysed was fairly small (n=24), which was related with difficulty in trapping blood meal mosquito samples, the blood meal pattern showed a moderate human blood index. The human blood feeding and non-human blood meal sources including ; rodents, squirrel, mongoose, goat, cow, domestic cat, lizard and wild cat, could increase the risk to humans of diverse vector- borne pathogens such as dengue, zika and chikungunya viruses, which could include zoonotic ones circulating in livestock or rodents hosts. 5.1.5 Human blood feeding habits among the genetic forms of Ae. aegypti and subspecies of Ae. simpsoni sl. Blood meal data showed feeding on diverse hosts for the two vectors with correspondingly low human blood index (Ae. aegypti 0.53 in Rabai, Ae. simpsoni 0.44 in Kerio Valley and 0.26 in Rabai). Similar values for Ae. aegypti was recorded in a recent study in Rabai in coastal Kenya (Agha et al., 2019). However, the estimated HBI contrasts findings, elsewhere this species is known to be primarily anthropophilic with rates (>0.87-1) in Australia (Stephenson et al., 2019) and Thailand (Harrington, 2005). This is consistent with most previous data on the urban populations of this species classified as anthropophilic (Diallo et al., 2020). The difference could relate to where the samples were captured (indoor vs outdoor) and habitat (urban vs rural/sylvatic) (Joyce et al., 2018). Surprisingly, recent data suggest outdoor rather than indoor resting habitats for populations of Ae. aegypti in urban and rural areas of Kenya (Ngugi et al., 2020), in support of our trapping that focused on the outdoors. This may point to fundamental differences in the behavior of Ae. aegypti from those in west Africa and outside Africa requiring further elucidation. Nonetheless, few studies on the trophic behavior of Ae. aegypti in Africa found that this species fed mainly on 38 animals such as dog, wild and domestic animals in Nigeria (Davis and Philip 1931) and Senegal (Diallo et al., 2013). The degree of anthropophagy in Ae. aegypti has been posited to have a genetic basis with the domestic form generally considered to prefer human over the forest form with a more zoophilic habit (Joyce et al., 2018). Phylogenetic analysis of blood-fed specimens resolved mainly as mitochondrial lineages mirroring these genetic forms, with no differences in the estimated HBI between the lineages. While the sample size was small to allow definitive conclusion on the trophic behavior between the species, the co-occurrence in domestic habitat (Agha et al., 2019) poses enhance risk of pathogens to humans. Feeding on humans by both forms escalates the risk of transmission to humans of various vector-borne pathogens such as DENV, CHIKV, and ZIKV that are known to have animal reservoirs. Blood-fed specimens of Ae. simpsoni were mostly Ae. bromeliae the principal YFV vector in eastern Africa and seems to be most abundant sub-species in Ae. simpsoni complex. This species has been described as solely biting sub-species in this complex, although low HBI was attributed to the species. Remarkably, based on the phylogenetic tree with well supported clades, there seems to have a presence of a yet-to-be described species exhibiting human feeding tendencies. Trapping of blood-fed cohorts are very difficult. Generally, the blood-fed mosquitoes samples analyzed was fairly small (n= 80) reflected by the trapping method that is biased towards host-seeking females. Further studies should include resting collections to increase freshly blood- fed mosquitoes (Diallo et al., 2021). Despite the low number of engorged females tested, a better understanding of host selection by Aedes species was possible implicating these vectors including Ae. aegypti in these foci as principled blood- 39 feeding vector, blood feeding on a broad variety of host including mammals, rodents and reptiles. Our feeding profiles were exclusively single feeds on the hosts described with no record of mixed blood meals. This may have been attributed to the molecular method of analysis using a single marker only and Sanger sequencing technique. Multiple feeds on different hosts have been unraveled using ELISA or multiple markers including next generation sequencing (Logue et al., 2016). A high-throughput sequencing has been used for comprehensively evaluating the composition of insect blood meals (Muturi et al., 2020). Molecular assays only test for the presence of a few pre-selected species (Logue et al., 2016). A minor proportion of blood meals were unsuccessfully analyzed and could be related to the advanced digestion of some blood meals, or due to unknown causes. 5.2 Study Limitations i. The overall engorged Ae. simpsoni samples analyzed were relatively small(n=24), reflecting the hitches encountered in trapping blood-fed mosquitoes. This could be associated with the trapping methods used probably biased towards host- seeking females. However the study provided valuable baseline data indicating a diverse blood feeding habit. ii. Nested PCR-based method has also been developed to distinguish Ae. simpsoni S I sub- siblings; Ae. bromeliae, Ae. lilii and Ae. simpsoni spp based on the ITS region (Bennett et al., 2015b). The application of this protocol in this study resulted to mixed and inconclusive because some of the samples showed amplification, both in the specific primers used in targeting their sub-siblings. 40 5.3 Conclusions i. Overall, it was evident that in both species the parity rates which translated to survival rates /longevity, were generally high with variation based on the trapping period among Ae. aegypti populations. ii. High values of survival/parity rates indicated the possibility of high contact rates with vertebrate hosts, allowing tremendous chances for the transmission of pathogen and infections. These designate a high vectorial capacity for YFV and DENV transmissions by these vectors. iii. The analysis of blood fed cohorts showed a diverse host feeding range for Ae. aegypti with estimated low HBI, which did not vary between mitochondrial lineages indicative of domestic and forest genetic forms. iv. The contemporary results of both genetic forms of Ae. aegypti species feeding on human, indicated an enhanced risk of a diverse vector borne disease transmission in human. Ae. simpsoni complex similarly, had a broad host feeding range, with Ae. bromeliae being the utmost predominant sub-species although unveiling low HBI. v. The phylogenetic analysis suggested the presence of new sub-species which is not yet described within the Ae. simpsoni complex, affiliated with human blood-feeding. 5.4 Recommendation i. These findings demonstrate the applicability to incorporate other bionomic parameters such as vector competence, which describes vectorial capacity, for an effective understanding of spread and recurrence risk of these arboviruses. ii. To enhance disease prediction and interventions which are cost effectiveness, this study demonstrates the urgency to actuate surveillance information of vector population based on genotype analyses. 41 iii. To fully understand the effect of high survival ability and human feeding found in Ae aegypti at different periods of the year, other environmental factors should be incorporated such us temperature, relative humidity, to intensively report on the intense dengue virus transmission in coastal regions. iv. 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