Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
SPECIES COMPOSITION AND INFECTION RATES OF MOSQUITOES AND SAND FLIES IN MRIMA HILL, KWALE COUNTY, KENYA ONYIEGO JUDITH ADHIAMBO, B. Ed. (Sc.) REG NO.: I56/CE/14164/2009 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF MASTER OF SCIENCE (APPLIED MEDICAL PARASITOLOGY) IN THE SCHOOL OF PURE AND APPLIED SCIENCES OF KENYATTA UNIVERSITY MAY 2016 2 ii DECLARATION This thesis is my original work and has not been presented for a degree in any other University or for any other award. Onyiego Judith Adhiambo Department of Zoological Sciences Signature: ……………………………….. Date ………………… SUPERVISORS: This thesis has been submitted with our approval as supervisors Professor Jones Mueke Department of Zoological Sciences Kenyatta University Signature: ……………………………….. Date……………………. Dr. Christopher O. Anjili Centre for Biotechnology Research and Development Kenya Medical Research Institute (KEMRI) Signature: ………………….……………… Date………………… iii DEDICATION This thesis is dedicated to my beloved husband Phineas and children Brent, Euan and little Mary Dora. My parents: The late Mr and Mrs Onyiego in their memory for inspiring me. My siblings: Beaty, Izo, Liz, Anuar and Lavenda. iv ACKNOWLEDGMENT My uttermost gratitude goes to the Almighty God for his wholesome support, special and sincere thanks to my supervisors, Professor Jones Mueke, for constantly keeping me on toes when I was lazing and lagging behind schedule and his constant encouragement. I sincerely appreciate Dr Chris Anjili‘s guidance, total devotion and mentoring. Together and individually they inspired me by their commitment towards this work. I appreciate the input of the Department of Zoological Sciences for the intellectual support especially from Dr Joshua Mutiso. I am also indebted to Mr. Kevin Kimenyi for the assistance he offered me during my trips to Mrima hill. My heartfelt gratitude goes to the villagers of Mrima hill, headed by the chairman Mr. Omar Bakari and his nephew Mr. Ahmadi Bakari for being my helper during my work in the village and to my institutional head Mr. Godfrey Njoroge for all the ample time I needed during the whole process. I finally acknowledge the special part played by my family; my husband for all round support, my dear sons Brent Jamba and Euan Tiany for their admiration that inspired me to move on even when things felt hard, my little girl Mary Dora for the sacrifice of time that mum spent away. My sister, Mrs. Elizabeth Obura and her family for financial support and the rest of Konyiego people for their love. Ms Everlyn Samita Khakai, my best friend, for the greatest contribution; constant prodding, objective criticism and positive reinforcements throughout this work. To all, I say thank you. v TABLE OF CONTENTS Declaration .......................................................................................................................... ii Dedication .......................................................................................................................... iii Acknowledgment ............................................................................................................... iv Table of content…………………………………………………………………………...v List of tables ..................................................................................................................... viii List of figure……………………………………………………………………………...ix List of plates ........................................................................................................................ x Abbreviation and Acronyms ............................................................................................. xi Abstract………………………………………………………………………………….xiii CHAPTER ONE: INTRODUCTION ............................................................................. 1 1.1 Background information ............................................................................................... 1 1.2 Statement of the problem .............................................................................................. 4 1.3 Justification of study ..................................................................................................... 5 1.4 Research questions ........................................................................................................ 6 1.5 Hypotheses .................................................................................................................. 6 1.6 Objectives of the study.................................................................................................. 6 1.6.1 General objective ....................................................................................................... 6 1.6.2 Specific objectives………………………………………………………………......7 1.7 Significance of the study............................................................................................... 7 CHAPTER TWO: LITERATURE REVIEW ................................................................ 8 2.1 Mrima Hill .................................................................................................................... 8 2.1.1 Historical background ................................................................................................ 8 2.1.2 The Flora and Fauna of Mrima Hill ......................................................................... 11 2.1.2.1 The Flora ............................................................................................................... 11 2.1.2.2 The Fauna.............................................................................................................. 13 2.2 Natural radiation ........................................................................................................ 15 2.2.1 Thorium.................................................................................................................... 17 2.2.1.1 Physical properties of Thorium ............................................................................. 17 vi 2.2.1.2 Radioactivity ......................................................................................................... 17 2.2.1.3 Effects on humans ................................................................................................ 18 2.2.2 Effects of radiation on living organisms .................................................................. 19 2.2.2.1 Polytene chromosomes and genes ........................................................................ 19 2.2.2.2 The cytogenetic effects of thorium radiation on organisms.................................. 20 2.2.2.3 Insect sterilization by radiation…………………………………………………..22 2.2.2.4 Sterile insect technique ......................................................................................... 23 2.3 Species composition of mosquitoes and sand flies ..................................................... 24 2.3.1 Mosquitoes species and their infections .................................................................. 24 2.3.2 Sand flies species and their infections ..................................................................... 28 2.4 Vector-borne parasitic diseases in Kwale County ...................................................... 29 2.4.1 Prevalence of Malaria .............................................................................................. 29 2.4.2 Prevalence of Filariasis ............................................................................................ 29 2.4.3 Prevalence of Leishmaniasis .................................................................................... 31 2.5 Present study ............................................................................................................... 31 CHAPTER THREE: MATERIALS AND METHODS .............................................. 32 3.1 Study area.................................................................................................................... 32 3.1.1 Location of Mrima Hill ............................................................................................ 32 3.2 Pilot study ................................................................................................................... 36 3.3 Sampling procedure……………………………………………………………….....36 3.4 Laboratory procedures ................................................................................................ 38 3.4.1 Specimen preparation and staining with Giemsa stain ............................................ 38 3.4.2 Preparation of chloral hydrate mounting gum……………………………………..39 3.4.3 Procedure for mounting specimen using chloral hydrate mounting gum ................ 39 3.5 Species identification using anatomical features ........................................................ 40 3.5.1 Mosquitoes ............................................................................................................... 40 3.5.2 Sand flies.................................................................................................................. 40 3.6 Examination for parasites ........................................................................................... 40 3.7 Data Analysis .............................................................................................................. 41 vii CHAPTER FOUR: RESULTS ...................................................................................... 42 4.1 Composition of trapped mosquitoes and sand flies .................................................... 42 4.1.1 Mosquitoes………………………………………………………………………....42 4.1.2 Sand flies.................................................................................................................. 44 4.2 Relationship between number of mosquitoes and sand flies………………………...46 4.3 Relationship between levels of radiation and number of mosquitoes and sand flies...46 4.3.1 Relationship between levels of radiation and number of mosquitoes……………...46 4.3.2 Relationship between levels of radiation and number of sand flies………………..47 4.4 Relationship between Radiation and Elevation .......................................................... 48 4.5 Vector infection rates .................................................................................................. 49 CHAPTER FIVE: DISCUSSION.................................................................................. 50 5.1 Trapped mosquitoes and sand flies ............................................................................. 50 5.1.1 Mosquito species ...................................................................................................... 50 5.1.2 Sand fly species........................................................................................................ 54 5.2 Infection rates.............................................................................................................. 56 5.3 Relationship between radiation and elevation ............................................................ 56 CHAPTER SIX: CONCLUSION AND RECOMMENDATIONS……………….....57 6.1 Conclusions ................................................................................................................. 57 6.2 Recommendations ....................................................................................................... 58 REFERENCES ................................................................................................................ 59 APPENDICES…………………………………………………………………………..69 APPENDIX I…………………………………………………………………………….69 APPENDIX II……………………………………………………………………………70 APPENDIX III…………………………………………………………………………...71 viii LIST OF TABLES Table 4.1: Number of mosquitoes species collected, their percentage, elevation (m (ASL) and radiation level (mSv/yr) around the Mrima study area………………….43 Table 4.2: Number of sand fly species collected, their percentage, elevation (m (ASL) and radiation levels (mSv/yr) around the Mrima study area……………...…..45 ix LIST OF FIGURES Figure 3.1 Map of mrima hill area where the study was carried out in Kwale County ...36 Figure 4.1 Relationship between number of mosquitoes and sand flies………………...46 Figure 4.2 Relationship between levels of Radiation and number of mosquitoes...…….47 Figure 4.3 Relationship between levels of Radiation and number of sand flies......…….48 Figure 4.4 Relationship between levels of Radiation and levels of Elevation...………...49 x LIST OF PLATES Plate 2.1: Agricultural land on the North west of Mrima Hil ………..………………..10 Plate 2.2: A typical homestead on the South East of the Mrima Hill ...………………..11 Plate 2.3: Rhynchocyon petersi ……………………………...………………………...14 Plate 2.4: Galago zanzibaricus ……………………………...………………………....14 Plate 2.5: Colobus angolensis palliates …………...…………………………………...15 Plate 2.6: Myonycteris relicta the fruit bat …..………………………………………...15 Plate 2.7: Features found on the head of Myonycteris relicta fruit bat ……...…………15 Plate 3.1: Images of Mrima Hill …………………………………………..……………35 Plate 3.2: A light trap set in the upper part of the hill to trap mosquitoes and sandflies 37 Plate 3.3: A light trap suspended outside a house to trap mosquitoes and sandflies ...…38 xi ABBREVIATION AND ACRONYMS ARS Acute radiation syndrome ASL Above sea level CEPF Critical Ecosystem Partnership Fund CHIKV Chikungunya CPMs Counters per Minute CRC Computing Research Centres DNA Deoxyribonucleic acid DOD Department of Defence EARPO East Africa Regional Programme Office ELISA Enzyme-linked immunosorbent Assay GPS Geographical Positioning Systems HBRAs High background radiation areas ICPR International Commission for Radiological Protection IUCN International Union for Conservation of Nature IUPAC International Union of Pure and Applied Chemistry KEMRI Kenya Medical Research Institute MASL Metres Above the Sea Level Mrems millirem mSv/yr milliSieverts per year Nb Niobium NEMA National Environmental Management Authority NNDC National Nuclear Data Center xii Pb Lead PBS Phosphate-buffered saline PCR Polymerase Chain Reaction PH Power of Hydrogen PPM Parts per Million Rem Roentgen equivalent man Sr Strontium SAM Solid State Army SI Standard unit SNAP System for Nuclear Auxillary Power SPSS Statistical Package for the Social Sciences SV Sievert SIT Sterile insect technique Th Thorium UEPA United States Environmental Protection Agency UNSCEAR United Nation Science Committee on Environmental Atomic Radiation WHO World Health Organization WNV Western Nile Virus WWF World Wide Forests 2 Chi-square test Yt Yittrium Zn Zinc xiii ABSTRACT Mrima hill in Kwale County, along the Kenyan coast, is known to have high natural background radiation caused mainly by 232 Thorium (Th). Currently, the area also has no proper sanitary disposal system and people use bushes as toilets. There are no fresh water bodies. Despite other parts of the coastal regions of Kenya having been studied for human disease vectors including mosquitoes and sandflies, no studies have been carried out in Mrima hill to establish the prevalence of these important vectors. The main aim of the present study was to determine mosquitoes and sand flies species diversity as well as vector infection rates in order to establish the level of risk of infection with malaria and leishmaniasis for the local inhabitants. The relationship between radiation and population sizes of both mosquitoes and sand flies was determined. This study also established the relationship between levels of radiation and elevation. Thirty eight houses were randomly selected and radiation levels taken using hand held Digilert 100 CPMS reader and recorded. Mosquitoes and sand flies were caught using light traps and counted to establish their population. Species identification was done on mosquitoes and sand flies using standard taxonomic keys. All captured female insects were dissected and examined for presence of parasites to establish the infection rates. Data were analyzed using the Statistical Package for Social sciences (SPSS) Version 20 (2011) utilizing Chi-square and Pearson correlation. A total of 131 mosquitoes and 39 sand flies were captured. Apart from the Aedes aegypti species (1.9%), the only other species of medical importance that were collected in the study area included: Culex pipiens (69.42%), Cx vansomernae (1.5%), Mansonia africana (18.32%) and M. uniformis (9.6%). None of these mosquitoes are known vectors of disease in Kenya. Phlebotomine sand fly species that were collected included Sergentomyia bedfordi (74.36%), S. suberecta (15.35%), S. meilloni (5.12%) S. schwetzi (2.56%) and S. inermis (2.56%). None of these species has been indicated to be of any medical importance in Kenya. The highest radiation level recorded was 17.5 milliSieverts/year whereas the lowest was 5.9 mSv/year; with a mean radiation of 10.52 mSv/year. There was no significant relationship between radiation levels and number of mosquitoes (2 = 103.7; df = 99; P = 0.353) or sandflies (2 = 40.0; df = 55; P = 0.936) collected. There was no significant relationship between the number of mosquitoes and sand flies (2 =36 and P=0.165). In addition, there was no correlation between radiation levels and elevation (r = -0.389; df= 10; P=0.211). From the results of this study it can be concluded that it is unlikely that the insect- borne diseases can occur in Mrima hill, unless the disease causing pathogens are introduced. There is need for further studies to establish the effect of radiation on reproductive capacity and survival of disease vectors in the present study area. 1 CHAPTER ONE: INTRODUCTION 1.1 Background information Species composition of mosquitoes and sand flies have been researched and established in different parts of the world. There are several species of mosquitoes that are vectors of different tropical diseases. Studies have shown that there is low density of vectors across sub-Saharan Africa. A study carried out in Mwea Kenya sampled about twenty one species of mosquitoes where genera Culex, Anopheles, Mansoni, Ficalbia and Aedes constituted the majority of the catches. In the study Culex quiquefasciatus Say was the most abundant species caught (Muturi et al., 2007). There are more than 4000 mosquito species in the world today but only a tenth of these are efficient vectors of diseases that have impact on the welfare and health of humans (Manguin et al., 2009). Malaria and lymphatic filariasis have been ranked as the two most identifiable mosquito-borne parasitic diseases worldwide. These two infections are transmitted by the same vector species of mosquitoes. Anopheles gambiae and A. funestus s.l Giles have been classified as the most important vectors of malaria and lymphatic filariasis. The two species occur all year round in their habitats with peaks of their population abundance coinciding with seasonal rains (Onyango et al., 2013). Culex quinquefasciatus is also known to transmit lymphatic filariasis. Mosquitoes also transmit West Nile Virus through their bites. They acquire the virus from birds which are usually the reservoir (Hayes et al., 2005). West Nile Virus is primarily transmitted by Culex mosquitoes but can also be transmitted by mosquitoes from other genera. In Europe and Africa, the principal vectors are Culex pipiens, Cx. univittatus and Cx. antennatus while 2 in India, it is Cx. vishnui (Hayes et al., 2005). Mosquitoes are known vectors of Chikungunya which was first reported in Tanzania in 1952. Chikungunya is a zoonotic arthropod-borne virus endemic mostly in Africa, India and South East Asia spread and transmitted by mosquito species of Aedes albopictus. In Africa, the virus is maintained in circulation with sylvatic cycle with wild mosquitoes; Aedes furcifer, Ae. luteocephalus, Ae. taylori, Ae. africanus with preferred feeding on primates (Vazeille et al., 2007). Chikingunya virus is mainly transmitted within urban cycles in an inter-human transmission in Asia. The transmission is done by the human biting Ae. aegypti which breeds in man-made sites and the less anthrophilic, Ae. albopictus, also known as the Asian tiger mosquito (the native of South East Asia) (Vazeille et al., 2007). Mosquitoes transmit yellow fever which is the original viral hemorrhagic fever which was feared as the most dangerous disease before the development of an effective vaccine. The disease still affects as many as 200,000 persons annually in tropical regions of Africa and South America. It also poses a significant hazard for unvaccinated travelers. The yellow fever is transmitted by a cycle involving monkeys and mosquitoes but humans can also act as viraemia host for mosquito infection (Monath, 2001). Dengue and Dengue hemorrhagic fever are caused by four serotype viruses of the genus Flavivirus. Dengue fever is majorly an urban disease of the tropics. This virus is caused and maintained in a cycle that involves humans and day-biting mosquito Aedes aegypt, that prefer feeding on human (Gubler and Clark, 1995). Sand flies are also known to be of many species some of which are serious vectors of deadly parasitic diseases. They transmit infections that affect humans and various animal 3 populations throughout much of the tropics and subtropics. The infected female phlebotomine sand flies spread the infection through their bites. Phlebotomine sand flies are dipteran insects of the family psychodidae and approximately 700 species have been described in different places. The female sand flies (Phlebotomine species in the Old world and Lutzomyia species in the new world) acquire Leishmania parasites when they feed on infected mammalian host in search of the bloodmeal (Bates, 2007). The parasites produce a spectrum of diseases in their human hosts including cutaneous and visceral forms which always depend on the species of sand flies that are transmitting these parasites (Sacks, 2001). Sergentomyia sand fly species usually feed on reptiles as well as other vertebrates thereby transmitting Leishmania parasites which are also commonly referred to as Sauro leishmania (Bates, 2007). Although studies on species composition and infection rates have been done elsewhere in the world, no research has been conducted in areas with high natural background radiation. High natural background radiation level in Mrima hill has been reported and is attributed to presence of weathered carbonatite rock with high Thorium concentration (Patel, 1991; Malathi, 2005). High natural background radiation has also been reported in some other areas in Kenya including: Ruri, Kuge, Sokolo and Rangwa on the shores of Lake Victoria. This is associated with carbonate rock containing high concentration of thorium and uranium (Achola et al., 2012). Mrima hill in Kwale County has been reported to have elevated radiation (Patel, 1991). Apart from the total solar radiation (TSI), the area has high radiation believed to have 4 been caused by among other elements; 232 Thorium (Th), which is also the main element releasing radiation into the environment (Patel, 1991). Radioactivity studies have shown that the upper and lower parts of Mrima hill have an annual radiation of 10,670 mrems [106.7 milliSieverts (mSv)] and 1,372 mrems [13.72 milliSieverts (mSv)], respectively. These doses are approximately 53 times higher than the natural background dose of 240 mrems (2.4 mSv per year), what the Commission on International Radiological Protection considers normal (UNSCEAR, 1988). The upper and middle parts of the hill have 106.7 milliSievert yet the annual limit of effective dose is equivalent to 50 milliSieverts (Patel, 1991). The aim of the present study was to establish the composition of species and infection rates of mosquitoes and sand flies in Mrima hill in Kwale County. 1.2 Statement of the problem It has been suggested that Mrima forest conservation zone, particularly Mrima hill has elevated radiation levels (Patel, 1991). There is limited information available on species composition of any vectors of disease in such areas with High Natural Background Radiation. However, there is data on the use of radiation in the laboratory to sterilize insects such as male tsetse flies (the sterile male technique), as a control measure for their population and the disease (Dyck et al., 2005). Data on the endemicity of the human vector-borne parasitic diseases in the Mrima hill and its environs is inadequate even though Kwale County is known to be endemic for diseases such as malaria, filariasis and trypanosomiasis (Wanyiri, 1996; Mwandawiro et al., 1997; Mbogo et al., 2000). As early as 1964, it was reported that phlebotomine sand flies (Diptera: Psychodidae) are present 5 in Kwale (Minter, 1964). Since then, no follow up studies have been carried out to establish the variety of species which are capable of transmitting human leishmaniases in this area. Transmission of vector borne diseases is known to be prevalent in Kwale, but the species of vectors at Mrima hill have not been determined. It is for this reason that studies were undertaken to establish the species composition of mosquitoes and sand fly vectors of human diseases in this area. This study also aimed at investigating the levels of radiation in the area of study and parasite infection levels in the vectors. 1.3 Justification of study Arthropod-borne infections have emerged as a major human health concern worldwide. Those spread by mosquitoes have caused most serious and wide spread parasitic diseases and arbovirus diseases worldwide and are ubiquitous in both feral and urban settings (Ottesen, 2000). The viruses include; Dengue, yellow fever, rift valley fever and chikungunya among others. Malaria and Lymphatic filariasis are among the world‘s most dreaded killer diseases claiming several million lives yearly and disabling and disfiguring more and hence the need to establish their vector status in Mrima Hill. Leishmaniasis, transmitted by infected female phlebotomine sand flies, is also one of the most important vector-borne diseases reported world-wide with estimated half a million new cases and around 50 thousand deaths per year (Bates, 2007). These facts led to the study of the species composition and infection rates of mosquitoes and sand flies in the area of Mrima hill to be able to establish their role in disease transmission in high natural background radiation area owing to the fact that the neighbouring areas have been studied. 6 1.4 Research questions i) What is the species composition of mosquitoes in the study area of Mrima hill, Kwale County? i) What is the species composition of sand flies in the study area of Mrima hill, Kwale County? ii) What is the infection rate of mosquitoes in the study area of Mrima hill, Kwale County? iii) What is the infection rate of sand flies in the study area of Mrima hill, Kwale County? iv) What is the relationship between radiation and elevation in Mrima hill, Kwale County? 1.5 Hypotheses i) There are no mosquito species that are vectors of diseases in Mrima hill. ii) There are no sand fly species that are vectors of diseases in Mrima hill. iii) There are no infections transmitted by mosquitoes in Mrima hill. iv) There are no infections transmitted by sandflies in Mrima hill. v) There is no relationship between radiation and elevation around Mrima hill. 1.6 Objectives of the study 1.6.1 General objective To establish the species composition and infection rates of mosquitoes and sand flies in Mrima hill, Kwale County in Kenya. 7 1.6.2 Specific objectives i) To determine the species composition of mosquitoes in Mrima hill, Kwale County. ii) To determine the species composition of sand flies in Mrima hill, Kwale County. iii) To evaluate the infection rate of mosquitoes in Mrima hill, Kwale County iv) To evaluate the infection rate of sand flies in Mrima hill, Kwale County. v) To determine the relationship between radiation and elevation around a high natural background radiation of Mrima hill, Kwale County. 1.7 Significance of the study Results obtained from the study will provide information on the species composition of mosquitoes and sand flies in the study area and the parasitic infections transmitted by these disease vectors (mosquitoes and sand flies). The information could be used by the Ministry of Public Health and Sanitation to put in place the relevant control measures against vector borne diseases in the area. 8 CHAPTER TWO: LITERATURE REVIEW 2.1 Mrima Hill 2.1.1 Historical background The Mrima hill is a small hill rising within the forest on an alkaline igneous complex that is reported to have significant deposit ores containing manganese and niobium. Considerable mining activities have been undertaken in the area measuring about 390 hectares (Kenya Forest Department, 1994). Mrima hill is biologically important and significant as a kaya. The kayas are relict patches of forest that once sheltered the fortified villages of the Mijikenda people (in Mrima‘s case, the Digo) on the Kenyan coast. The Mrima Forest Reserve was gazetted in 1961 (NEMA, 2001). A survey in 1986 brought the kaya to the attention of the Government as a valuable cultural and natural heritage leading to its gazettement as Mrima Sacred Grove National Monument under the antiquites and Monuments Acts in 1992 (Githitho, 2004). The Kayas have spiritual and ceremonial significance and are customarily protected and managed by a council of elders. National Museums of Kenya manages the National monuments for their historical importance. The site was made a strict nature reserve under the Forests Act in the early 1980s (Githitho, 2004). As a forest reserve, it is managed by the Forest Department but the level of protection is weak, given very insufficient capacity to patrol and ensure protection (Africa News Service, 2010). Mrima hill area was one of the earliest Swahili settlements in Africa. People who lived there depended on the forest for herbs and source of water. The main land use in the area is forestry, nature conservation and research. Survey of Sacred Kaya 9 Forest highlighting the conservation importance for trees led to a comprehensive survey of Kenya Coastal Forest commissioned by World-Wide Forest (Roberson and Luke, 1993). It mainly paid close attention to the plant species and status of the forest. The survey also made recommendation for their conservation (WWF-EARPO, 2002). The forest provides a source of building wood and fuel which include, firewood and charcoal, making the main source of the rural household energy consumption and also the urban energy consumption to growing towns like Mombasa and Lunga lunga (Burgess and Muir, 1994). According to Burgess and Muir (1994), the forest was used for timber production and main local uses such as pole collection and sawing; religious (spiritual) and ceremonial, gathering of medicinal plants; clearing of the forest to grow crops (agriculture); collection of edible plants and honey and mining and building hotels for tourism. Kayas have been seriously encroached by mining for lead and other minerals (Clark and Burgess, 2000). The forest has suffered selective logging and pole cutting. Cutting of trees and destruction of vegetation around the Kaya was prohibited while the surrounding areas slowly turned into farmland, leaving the Kaya site as forested patches of varying size. Restriction by the Government due to overexploitation means much material is harvested illegally from the Forest Reserves and other areas (Kenya Forest Department, 1994; Githitho, 2004). Population density around the hill has increased with encroachment on the lower slopes of the hill to the west. Mining and other activities have also increased due to population increase. There are medium-sized forests occurring in Kwale which have been gazetted as forest reserves for many decades yet have no significant management presence due to the difficulty of looking after the small scattered 10 forests. These sites include, Mrima and Dzombo, a nearby hill (Kenya Forest Department, 1994). Subsistence farming is practised around the forest. Many people in this area are smallholder farmers with most of them owning between 5 and 10 acres of land. However, these farms are becoming smaller and smaller as they are sub-divided and passed onto the male children (NEMA, 2001). Only a few farmers keep large numbers of cattle due to lack of grazing area and lack of water supplies (especially during the dry season). Goats and poultry, however, are found in almost every household. There are no rivers in the area such that all the residents only use water from bore-holes. The bore-holes draw water from rainfall permeating the soil and provide most of the groundwater resources in Kenya (Nyaoro, 1999). A typical agricultural land is shown in Plate 2.1. Plate 2.1: Agricultural land on the North West of Mrima Hill (Adapted from NEMA, 2001). 11 Most of the households have houses that are made of mud and are thatched with coconut plant leaves commonly known in Kiswahili as ‗Makuti‘ (Njenga, 2007). They have no ceilings and usually have many openings (Plate 2.2). Plate 2.2: A typical homestead on the South East of the Mrima hill (Adopted from Njenga, 2007). 2.1.2 The Flora and Fauna of Mrima hill 2.1.2.1 The Flora The vegetation in Mrima hill is diversified. A record from a group of scientists on an expedition in 1989 listed about 270 taxa, with about 25 taxa which are rare globally and within Kenya (BirdLife International, 2012). So many known rare species of trees are 12 found on Mrima hill. These include: Uvariodendron gorgonis and Gigasiphon macrosiphon. It was found that there were only three mature trees. At the 6th East Africa plant Red Listing Authority meeting in April 2012 at the International Union for Conservation of nature Headquarters in Nairobi, there was a report on the existence of the trees in Muhaka, Gongoni and Mrima hill forest. Forests are reducing in size and becoming areas of land used for farming and other uses such as creating villages, constructing tourism facilities and for expanding town and cities. This has caused a reduction in the forest cover, loss of biodiversity in the flora and fauna, reduction of availability of water, soil erosion and loss of land productivity which negatively influence the livelihood of the communities around. The declining forests also affect the biodiversity of conservation and national and global benefits in terms of goods and services (IUCN, 2014). Most of the threatened woody plants species in Kenya occur in coastal forests. Nationally, the threatened species of Kenya‘s coastal forests is made up of plants which make half of the total plant cover and more than half are birds and mammals (Njenga, 2007). The forests are ranked 11th in species endemism by Conservation International and BirdLife International ranks it as one of the most globally important endemic Bird area (Bennun and Njoroge, 1996). According to Clark and Burgess (2000) and CEPF (2003), the area is considered to be a major global conservation priority due to threat of endemism. The Mrima hill forest has farm holdings around it with farms growing maize and there are also rice paddies. Fruits grown in the area include: pawpaw, mangoes, oranges, tangerine, lime, melons, coconut and cashew nut among others. Although many 13 uses of the forest for subsistence or income generation are illegal, they still continue. Assessment of participation of the local community vividly showed that they depend on the forest for building poles and firewood (Waiyaki and Bennun, 2000). 2.1.2.2 The Fauna The main mammalian fauna are the black-and-rufous giant elephant shrew Rhynchocyon petersi Bocage 1880 (Macroscelidea: Macroscelididae) (Plate 2.3), the Zanzibar bushbaby, Galago zanzibaricus Matschie 1893 (Primates: Galagidae) (Plate 2.4) and the Angola black-and white colobus monkey, Colobus angolensis palliates Sclater 1860 (Primates: Cercopithecidae) (Plate 2.5) (Anderson et al., 2005). Bats are found living and hiding in the mineshafts. These include: the rare and localized Myonycteris relicta Bergmans 1980 (Chiroptera: Pteropopididae) (Plate 2.6 and 2.7)(BirdLife International, 2005). There is also the rare butterfly Eresinopsides bichroma. 14 Plate 2.3: Rhynchocyon petersi ( Adopted from planet-mammiferes.org). Plate 2.4: Galago zanzibaricus (Adopted from planet-mammiferes.org). 15 Plate 2.5: Colobus angolensis palliates (Adopted from wikipedia.org). Plate 2.6: Myonycteris relicta the fruit bat, whole body and head. Plate 2.7: Features on the head of the fruit bat Myonycteris relicta ( Adopted from arkive.org). 2.2 Natural radiation Mrima hill is a high natural background radiation area. The radiation is released mainly by radioactive elements. This kind of radiation is known as ionizing radiation. Ionizing 16 radiation is produced from decaying radioactive materials which have sufficient energy to strip away electrons from atoms. This radiation is so harmful that it can destroy any living tissue in the human body. Generally the dose that the body is exposed to determines the severity or type of health effects (UNSCEAR, 2008). The health effects can be stochastic which are associated with long term and low level exposure (considered chronic) to radiation which can cause mutation of the Deoxyribonucleic Acid and cancer (Subhan, 2007). Children are usually more sensitive to radiation than adults because they are growing more rapidly therefore they have more cells that are dividing and poses a greater opportunity for radiation to disrupt the process. Most people receive a fraction of a rem (300 mrem/yr) from background sources which are mostly radon. Roentgen equivalent man measures the biological damage of radiation accounting for both the amount and dose of radiation and also the effects of radiation on the living cells in question. Exposure to a small dose of ionizing radiation to a human being over a life time increases the chances of him/her dying of cancer than would not. The Standard unit of dose equivalent is the sievert (Sv), conversion usually, 1 Sv = 100 rem by definition (UNSCEAR, 2008). The conventional units for dose rate of radiation should be mrem/hr. Regulatory limits and chronic doses are often given in units of mrem/yr or rem/yr. Although radiation may cause cancer at high doses and high dose rates, public health data regarding lower levels of exposure, below about 10 mSv (1,000 mrem), are harder to interpret epidemiologically. Most of the high quality human data available is from high 17 dose exposed individuals, above 0.1 Sv, so any use of the models at low doses is an extrapolation that might be under-conservative or over-conservative (Anand et al., 2008). 2.2.1 Thorium 2.2.1.1 Physical properties of Thorium Thorium is the main radioactive element that is releasing radiation into the environment in the case of Mrima hill. This element is a metal which is air-stable and retains its luster for several months. It is white in color with silvery luster (EPA, 2013). Thorium slowly tarnishes in air when contaminated with the oxide and becomes grey and finally black (Wickleder, 2006). Thorium is dimorphic, changing at 1,360oC from a body-centred tetragonal lattice form that exists at high pressure with impurities driving the exact transition temperature and pressures (Wickleder, 2006). Thorium element is not readily soluble in most common acids, except hydrochloric acid but it can be destroyed by water (Hammond, 2004). However, it dissolves in concentrated nitric acid containing a small amount of catalytic fluoride (Hyde, 1960). 2.2.1.2 Radioactivity Thorium occurs naturally as radioactive chemical element which is found in abundance in the soil throughout the world. Thorium atom (symbol Th) has an atomic number of 90 with 90 protons and 90 electrons, of which 4 are valence electrons. The discovery of the element was done in 1828 and named after Thor, the Norse god of thunder (Hala and Navratil, 2003). Thorium is found in nature as 232 Th (100%). When it decays slowly, it 18 releases an alpha particle (helium-4 nucleus). The element is estimated to be about three to four times more abundant than uranium in the Earth‘s crust (Beck et al., 2007). It is also a by-product of the extraction of rare earths from monazite sands. The most known uses of thorium, for instance as a light emitting material in gas mantles or as an alloying material in several metals, have decreased due to concerns about radioactivity. Thorium is found in small amounts in most rocks and soils and is estimated at average of 12 parts per million (ppm) (Hala and Navratil, 2003). In the event that thorium is inhaled it can lead to increased risk of lung, pancreas and blood cancers due to the fact that lungs and other internal organs can easily be penetrated by alpha particles to induce direct radiation. Internal exposure to thorium can lead to increased risk of the liver and blood cancerous diseases when the exposure occurs at medium and extremely high doses in humans and in experimental animals that have been studied (UEPA, 1990). 2.2.1.3 Effects on humans Contaminated dust, in case of thorium can be inhaled or swallowed with food or water because as earlier enlightened, it is abundant in soil and water. Living near a thorium contaminated site, or working in an industry where thorium is used, will increase one‘s chance of exposure to thorium. If inhaled as dust, some thorium may remain in the lungs for long periods of time, depending on the chemical form (van Kempen et al., 2007). The small amount of thorium left in the body after breathing out will enter the bloodstream and be deposited in the bones where it may remain for many years. There is some evidence that the body may absorb thorium through the skin, but that would not likely be the primary means of entry (UEPA, 1990). 19 The principal concern from low to moderate level exposure to ionizing radiation is increased risk of cancer. Studies have shown that inhaling thorium dust causes an increased risk of developing lung cancer, haemangiosoma of the liver (van Kampen et al., 2007), and cancer of the pancreas (UEPA, 1990). Bone cancer risk is also increased because thorium may be stored in bone. There are special tests that measure the level of thorium in the urine, feaces, and also via exhaled air that can determine if a person has been exposed to thorium. These tests, such as detection by imaging autoradiography (Goto et al., 2002) are useful only if taken within a week after exposure. 2.2.2 Effects of radiation on living organisms Different biological effects have been recorded in studies involving humans who have been exposed to 232 Th or its related compounds such as thorotrast with varying results (van Kampen et al., 2007) and also plants (Evseeva et al., 2003). 2.2.2.1 Polytene chromosomes and genes Polytene chromosomes are many copy chromosomes of giant cells usually in salivary glands of dipteran insects that have been studied widely using the fruit fly, Drosophila melanogaster Meigen 1830 (Diptera: Drosophilidae). There is a distinction between these types of chromosomes and the chromosomes of cells undergoing mitosis which are diploid Drosophila cells have 8 chromosomes. The diploid are 2 copies each of 4 different chromosomes (Strickberger, 1990). In the larvae of dipteran insects, some diploid cells stop dividing and instead grow to giant sizes as larvae grow. These giant 20 cells form much of the digestive system of the larva (Ashburner, 1989). The cells and their nuclei increase in size as the chromosome duplicates repeatedly (polyploidy) without accompanying cell division resulting in many copy chromosomes referred to as polytene. Microscopic examination of several aceto-orcein stained cells showed that chromosomes are in extended interphase of the cell cycle, and as such, are stretched out to their full length because each chromosome actually consists of many strands, and they are referred to as polytene (‗‗many threaded‘‘) chromosomes (Lefevre, 1974). These chromosomes are banded, and each band (chromomere) is associated with a single gene; each cross-band corresponds to one locus (Lefevre, 1974). Because polytene chromosomes are extended and consist of much deoxyribonucleic acid (DNA), they are easily visible under the light microscope. Even though radiation is known to cause gene mutations and deletion of loci, the effects of alpha radiation emitted by 232 Th are not known and therefore it is not known how radiation has affected vectors of disease. 2.2.2.2 The cytogenetic effects of thorium radiation on organisms Considering that 232 Th is a soil radionuclide, most studies on the effects of this element have been done on plant models. A study on effects of thorium by Evseeva et al. (2003) investigated the effects of Th as a soil radionuclide and cadmium (Cd) on the short-term (30 hours) and chronic (30 days) terms using plant models; Tradescantia L. (Commelinales: Commelinaceae) and Allium cepa L. (Liliaceae). The Th ion concentration was equal to 0.18 mg/l and Cd ion-to 60 mg/l in the soil. There was 21 synergic increase of cytogenetic damage frequency in the early response of both somatic and generative plant cells on Th and Cd combined action. During both the chronic and short term actions, the level of genetoxicity and cytotoxic long-term effects turned out to be lower than that of additive one. During the short-term actions, the effects are detected by the intracellular compensating processes and in the chronic action by the mass death of the most damaged buds in the florescence (Evseeva et al., 2003). Another study by Soliman et al. (2011) used three plant species, Cakile maritima Scop. (Capparales: Brassicaceae), Senecio glaucus L. (Asterales: Asteraceae) and Rumex pictus L. (Caryopyllales: Polygonaceae), to investigate the mechanisms by which plants can withstand high concentrations of the absorbed radionuclides. It was shown that exposure causes decrease in the percentage of prophase and also prophase to metaphase ratio. These included the percentage of anaphase and telophase that also increased with soil radioactivity. The results revealed chromosome aberrations. Amino acid profiles of these plants also indicated that radioactive elements stimulated the biosynthesis of some amino acids such as proline, cystine, serine and thereonine while inhibiting some other amino acids such as arginine. Aspartic acid was found to be the most abundant in the three plant species studied (Soliman et al., 2011). All the plants models used in these studies grow on crystalline igneous sands in Egypt with a high concentration of 232Th (Patel, 1991). Both mosquitoes and sand flies are known to use plants as their source of carbohydrates (Schlein and Yuval, 1987; Schlein and Muller, 2008). Not all the plant species that are probed by these insects are known, even though some of the plants have been shown to 22 be toxic to the insects (Schlein and Jacobson, 1994). Flowering plants are usually the favourites because of nector (Junnila et al., 2011). Tamarix jordanis Boiss 1901 (Caryopyllales: Tamaricaceae) is fed on by Culex pipiens L. (Diptera: Culicidae) (Junnila et al., 2011), while the castor bean, Ricinus communis L. (Malpighiales: Euphorbiaceae), the cactus, Opuntia persica var nectarine L. 1753 (Caryophyllales: Cactaceae), and guava, Psidium guajava L. (Myrtales: Myrtaceae) are favoured by sand flies (Schlein and Jacobson, 1994; Junnila et al., 2011). Anopheles gambiae is known to feed on plant sugar. A study has shown that many females feed on plant sugars than males. An. gambiae is the primary vector of Plasmodium falciparum Welch, the parasite that causes human malaria (Fryxell et al., 2012). Mostly, male mosquitoes are known to feed on plant-derived sugars but despite their ravid interest in blood meals, the female mosquitoes also feed on plant derived sugars. However, the role of the plant feeding in the An. gambiae female is poorly understood (Manda et al., 2009). The sugar may play a role in improving their vectoral capacity by extending the female longevity. The primary source of plant derived sugars, however is never from flowers. It is possible that in areas with soil radionuclides, these insects could pick up radioactive elements either from the plant sugars and/or from their breeding sites in the soil or water (Manda, et al., 2009). It is not known how the radioactivity from the plants affects the insect. 2.2.2.3 Insect sterilization by radiation There is literature on the success in control of trypanosomaisis tsetse fly‘s population and the control of the African trypanosomiasis using sterile fly (Kumano et al., 2008). According to research findings by Helsinki et al. (2006), the malaria mosquito Anopheles 23 arabiensis was studied using the Sterile technology radiation- induced sterility (STRIS). The study included exposing male mosquitoes to gamma rays in the pupal or adult stage. The research concluded that release of Sterile insect technology male insects was dependant on their level of sterility and competitiveness. Both stages of the mosquito development can be irradiated but the pupal radiation becomes easier to perform. The sterile insect technology relies on sterilization of insect by chemosterilization, irradiation or modern biotechnological approaches (Helsinki et al., 2006) which has not used mosquito a lot in the researches. Treatment with ionizing radiation damages reproductive cells as well as somatic cell. Sterilization by irradiation is still the most realistic way to sterilize insects at present and it has used Anopheline species. It has also been used on Aedes species, Ae. aegypti. In Ae. aegypti, the main part of spermatogenesis usually takes place in the larval and pupal stages of development. Therefore to effectively sterilize adult mosquito of the species, radiation dose has to be high enough to produce dormant lethal mutation in essentially all spermatozoans (Hamady et al., 2013). This reduces the competitiveness of irradiated mosquito males by damaging somatic cells. However, no studies have been carried out on how high natural background radiation has affected the mosquito populations in their natural habitat. 2.2.2.4 Sterile insect technique Sterile insect technique (SIT) (Dyck et al., 2005; Vreysen et al., 2007) is a method of biological control whereby a big number of sterile male insects are released into the natural environment. Sterile insect technique is an environmentally friendly method of insect extermination and pest management without the use of pesticides. The sterile 24 insects are usually males. The sterile males are released to the environment to compete with wild males for female insects. If a female mates with the sterile male then it will have no offsprings, thus reducing the next generation‘s population. In cases where high or low doses of radiation is applied then the sterilized insects might produce new sterile ones that are weak making them less able to compete with the wild males (Kumano et al., 2008). However, the technique has been successfully used to eradicate the screw worm fly Cochliomyia hominivorax in some areas of North America (Krafsur et al., 1987). There have also been many successes in controlling species of fruit flies, most particularly the medfly Ceratitis capitata and the Mexican fruit fly Anastrepha ludens (Collins et al., 2008). 2.3 Species composition of mosquitoes and sand flies 2.3.1 Mosquitoes species and their infections There are several mosquito species studied in the world some of which are of medical importance. They are vectors of disease in the tropics. Studies have shown however, that there is low density of vectors across sub-Saharan Africa (Onyango et al., 2013). Malaria and Lymphatic filariasis are the most serious mosquito-borne parasitic disease. Studies have shown that almost half of the entire human populations live at the risk of malaria and one million deaths occur yearly particularly among children under five years of age and pregnant women (WHO, 2000). Malaria parasites are transmitted by anopheles females which are definitive host for the parasite. Lymphatic filariasis is regarded the second most common global arthropod-borne infectious disease found in humid tropical areas of Asia and Africa (Ottesen, 2000). 25 Lymphatic filarisis is caused by macroscopic nematode pathogens which are called Wuchereria bancrofti that cause disabilities and deformities. There are three of these parasites with Wuchereria bancrofti being the most prevalent (Manguin et al., 2009). The parasites are responsible for all human lymphatic filarisis infection though a few are due to Brugia parasitism. The parasites are transmitted by Anopheles mosquitoes of approximately 70 species. Bancrotian filariasis is mainly transmitted by Culex and Anopheles mosquito species for the nocturnally periodic form. Anopheles species are usually in rural areas while Culex quinquefasciatus usually attack people in the tropical urban settings. The World Health Organization (WHO) has established two global initiatives, one for reducing malaria and the other for lymphatic filariasis elimination dabbed Roll Back Malaria and Global Programme to eliminate malaria and Filariasis respectively (Manguin et al., 2009). Wuchereria bancrofti can be transmitted by mosquitoes of six different genera including Anopheles, Mansonia, Aedes, Culex among others (Manguin et al., 2009). In East Africa and Middle East (Egypt and Yemen), the infections is transmitted by Cx. quinquefasciatus and Cx. pipiens. Culex pipiens species complex are mosquitoes that are known to have morphological similarities. Culex quinquefasciatus is a member of the complex and is adapted to living in the tropical and subtropical areas while Cx. pipiens prefer the temperate regions. The later is found in most major cities. It transmits Lymphatic filariasis in Pernambuco in Brazil and can also transmit several arboviruses such as West Nile Virus and Saint Louis Encephalitis. Culex quinquefasciatus are able to survive in polluted water where there are no natural predators (Wilke et al., 2014). 26 Mansonia uniformis are mostly found in China and Africa but Mansonia africana is restricted to Africa. They are known vectors of arthropod-borne viruses (arboviruses) such as Chikungunya and Rift valley fever that affect man and livestock (Labeaud et al., 2011). Mosquito species differ in the type of aquatic habitats they prefer for oviposition based on location, the physicochemical conditions of water bodies which include salt, dissolved organic matter, inorganic matter, and degree of eutrophication. The presence of potential predators also affects habitat preference by mosquitoes (Patz et al., 2000). Anopheles culicifacies Giles was positively associated with light and vegetation. Anopheles gambiae in Western Kenya was associated with shallow clean water and sunlit habitats which was the same preference for Anopheles arabiensis in Eritrea. Culex quinquenfasciatus is important in the transmission of Bancrofti filariasis in Kenya though very little is known about its larval ecology (Muturi et al., 2008). Few studies have reported significant association between the genus Culex and environmental factors such as Power of Hydrogen, canopy coverage, debris coverage and decaying organic matter. Culex quinquefasciatus is a predominant nuisance species and potential vector of filariasis and arboviruses in Mwea irrigation scheme in Kenya. In the study, carried out in Mwea, which was a preliminary study in Kenya, it was revealed that the species thrived in a variety of aquatic habitat including rice fields, canals, seepage areas, ditches, marshes, pits and temporary pools (Muturi et al., 2008). Ecological changes affect the vectors in their habitats. Through natural phenomenon or human intervention, there may be changes 27 in ecological balance and context within which vectors and their breed develop and transmit infections. When ecological stability is disturbed, it affects the vector biodiversity, abundance, competence and human biting behavior. These include deforestation, human encroachment, and climate and water bodies‘ location. Deforestation affects parasite vector population and changes in the ecological niches. It also affects conditions for proliferation of newly arriving and adaptive existing vector and their parasites. For instance, an upsurge of malaria has been co-incidental with changes in land use and human settlement subsequent to deforestation in Africa, Asia and Latin America (Patz et al., 2000). Each Anopheles species occupies a unique ecological niche and operates at different level of vector competence. The indigenous Anophelines are not able to successfully adapt to the changes in the ecological environment. Water bodies also affect the kind of vector species, for instance in Africa, An. gambiae prefer sunlit pools with turbid water and no emergent vegetation while An. funestus larvae prefer clear water without organic matter (Patz et al., 2000). Anopheles gambiae shows strong preference for moist soil as a substrate for its oviposition rather than dry soil substrate under the insectary conditions. In the soil, the species remains at dormant stage to resist desiccation. An. gambiae is able to maintain a large effective population size during dry season contrary to the suggestions that they suffer a severe population bottleneck during the dry seasons (Minakwa et al., 2001). Aedes aegypti has increased in population with increased urbanization which has increased sub standard housing, inadequate water, 28 sewer and waste management system in the event increasing Aedes aegypti-borne diseases (Gubler and Clark, 1995). 2.3.2 Sand flies species and their infections There are several species of sand flies with just a few vectors of disease. They transmit the parasitic protozoan of the genus Leishmania responsible for leishmaniasis. The vectors transmit infections in the tropics and subtropics. There are over thirty species of Leishmania named to date with about ten being of medical and vetenery importance (Bates, 2007). Sand flies pick the amastigote forms usually present in the skin of an infected individual. They insert their saw like mouthparts into the skin and agitate them to produce a small wound onto which blood flows from the superficial capilleries. Amastogotes are transformed into motile promastigotes. The major syndromes in human include; cutaneous, mucocutaneous and visceral leishmaniasis (Bates, 2007). Ecological changes also affect leishmaniasis transmission. In some parts of Latin America, deforestation has led to an increase in Leishmania infections (Bates, 2007). Forests are replaced with growth of fox population which is an excellent reservoir host of visceral leishmaniasis -Kala-azar and sylvatic leishmaniasis. Vector sand flies have become peridomestic. Pines and gmelina trees were planted 12 years after large forests in Para states in Brazil had been cleared. The trees were planted for paper industries, the spiny rat which is the reservoir host for cutaneous leishmaniasis parasite had adapted to the changed environment and became infected with Leishmania parasites (Patz et al., 2000). Sudan was an area which was not endemic for Kala-azar but it experienced an 29 outbreak of it due to the introduction of the parasites by immigrants and ecological changes favorable to sand flies. In the Southern Sudan, the sand flies circulate the Leishmania parasites among small animals such as goats, sheep and dogs. The sand flies transmit the parasites to the non-immune population entering the area, for instance, the resettled population and refugees (Patz et al., 2000). They develop large open sores on their faces and arms. 2.4 Vector-borne parasitic diseases in Kwale County 2.4.1 Prevalence of Malaria Epidemiological studies have shown that falciparum malaria caused by Plasmodium falciparum Welch 1897 is endemic along the entire Kenyan Coast. In Kwale County, this disease is transmitted by Anopheles gambiae s.l Giles 1902 (Diptera: Anophelinae) and An. funestus Giles 1902 (Mbogo et al., 2000; Kelly-Hope et al., 2009). In all the endemic areas, An. gambiae has been shown to be the main vector (Mbogo et al., 2000). In this species, the gametocyte frequency is found to be 1.3% and transmission occurs mainly during the rainy season. These results were recorded from Amani, Viriuni, Dumbule, Vuga, Ziwani, Tsuini, Mwaroni, Moyeni, Magaoni and Gazi (Mbogo et al., 2000) at the Kenyan Coast. There are no available reports of malaria incidences and prevalence that are available for Mrima hill. 2.4.2 Prevalence of Filariasis Lymphatic filariasis is a major social and economic burden in tropical and subtropical areas where the disease is well established. Bancroftian filariasis which is caused by 30 Wuchereria bancrofti Wucherer 1873 is also endemic in many parts of Kwale. It is the most common form of human lymphatic filariasis and important cause of morbidity disfiguring deformities of legs arms and genitals. It constitutes 80% of the cases reported in developing world (India and tropical Africa) (WHO, 1992). Studies on vectors of this disease have shown that mosquitoes found infected with W. bancrofti larvae are Anopheles gambiae and Anopheles Funestus. Culex quinquefasciatus Say 1823 is also involved in the transmission of the parasite (Mwandawiro et al., 1997). The prevalence of infection may be on the increase due to inadequate sanitation leading to favourable conditions for the breeding of vector mosquitoes (Ottesen and Ramachandran, 1995). The overall prevalence of microfilaraemia in the population is 16.4% as has been shown in villages that were sampled including Gandini, Dzivani and Lutsangani (Njenga et al., 2000). Several infections of P. falciparum and W. bancrofti have also been detected in mosquitoes in Kwale (Muturi et al., 2006). It has also been shown that An. gambiae is the main vector for both malaria and lymphatic filariasis in both Kilifi and Kwale areas (Kelly-Hope, 2009). Entomological investigations done at the Kenyan coastal region have reported that An. gambiae Sensulato, An. funestus and Culex quinquefasciatus are the vectors involved in transmission of lymphatic filariasis (Mwandawiro et al., 1997; Pederson and Mukoko, 2002). Culex quinquefasciatus is known to breed in a variety of stagnant water habitats where water has been significally polluted like in cess pits and canals near houses with long bathroom sewage water (Njenga, 2011; WHO, 2013). As one moves deep into the 31 rural areas, anopheles mosquitoes become predominant and are the principal vectors in the rural coastal villages in East Africa (Njenga, 2011). It has however, been found that very little or no attention has been paid to the disease in many countries because data on the magnitude of the problem does not exist (WHO, 1992). No data is available on infection and transmission of malaria and lymphatic filariasis around Mrima hill. 2.4.3 Prevalence of Leishmaniasis Even though there are no records to show that there is leishmaniasis in Kwale County, Phlebotomine sandflies were first reported in Mombasa on the Kenya Coast in 1912 (Mantuefeel, 1921). Later in 1930 and 1932, Sinton identified and reported the presence of Sergentomyia schwetzi Adler, (Theodor and Parrot), S. africana Newstead, S. yusafi Sinton and S. bedfordi congolensis Bequaert & Walravens (Sinton, 1930; Sinton, 1932). 2.5 Present study Based on the literature review on the species population and composition of both mosquitoes and sand flies, it is clear that such studies have been conducted in many places in Kenya including the coastal regions and particularly in Kwale County but none has been conducted in Mrima hill. Given that malaria and leishmaniasis are prevalent in other regions neighbouring Mrima hill, it was important to carry out research to establish the population and species composition of mosquitoes and sand flies as well as the infection rates in Mrima hill. 32 CHAPTER THREE: MATERIAL AND METHODS 3.1 Study area Mrima hill is located in Msambweni location in Kwale County, a distance of 70 kilometers south of the City of Mombasa in the Coast of Kenya (390 16.00‘ E, 40 28.00‘ S) (Figure 3.1). People living in the area were mostly peasant farmers practicing both small scale farming and animal keeping. Sampled study sites were areas inhabited by humans around the Mrima hill and homesteads in areas showing radiation levels that were above the normal level of 240 mrems. Sources of water in the area of study included boreholes and swamps with radioactivity which was 50 times higher than the normal natural background radiation as reported by Patel (1991). 3.1.1 Location of Mrima hill Mrima hill is a Forest Reserve which is a mixed coastal forest located in Msambweni Location of Kwale County with an altitude of 58 m above sea level (asl), a distance of 70 kilometers south of the City of Mombasa in the Coast of Kenya. The forest is undifferentiated and has exceptional plant species diversity (BirdLife International, 2005). The coastal plain of Kenya slopes gently seaward and Mrima hill rises from 90 m asl on the northern point to 296 m asl at the highest point near the centre of the hill (BirdLife International, 2005). The hill is ellipsoidal in plan and has a long axis running NW – SE and is approximately 3 Km long and 2.1 Km wide at the widest point. Two small plateaus form the top of hill with elevations of 300 metres and 285 metres, respectively, and are separated by a narrow col. The col broadens out to form a wide shallow valley to the north east. This part of the coastal region is known as the low 33 plateau and lies to the west of the coastal plain at altitudes ranging between about 30 metres to 135 metres asl. Figure 3.1: Map of mrima hill area where the study was carried out in Kwale County – Kenya ( www.Google maps.com). 34 Though most of this region is low lying and very gently rising towards the west, the Mrima hill highest point is averagely 300 metres asl (NEMA, 2001). The total annual rainfall ranges between 965 mm to 1270 mm. The rainfall pattern is characterized by two distinct long and short rainy seasons (bimodal) corresponding to changes in the monsoon winds. The long rains occur from April to July and average 1,100 mm with a peak of 330 mm in May and correspond to the South Easterly monsoons. The short rains are usually experienced in October to December which corresponds to the North Eastern monsoons, which are comparatively dry. January and February are the driest months. Rainfall decreases as one moves inland, away from the influence of onshore winds, with some areas receiving as little as 965 mm per year. Also those areas on the leeward side of the two hills (Mrima and Dzombo) have slightly less rainfall. The area experiences high temperatures with an annual mean of 27.90C (NEMA, 2001). The hottest month is February with a maximum average of 33.10C. The coolest month is July with a minimum of 22.70C. The area is generally hot and humid all the year round due to the high evaporation rate. As Mrima hill is close to the sea the humidity is usually high, except in the three dry months (NEMA, 2001). Mrima hill images are shown in Plate 3.1. 35 Plate 3:1: Images of Mrima hill a) View of Mrima hill from the North East side. b) View of Mrima hill the South West side. c) View of Mrima hill from the South East side (Adapted from NEMA, 2001). 36 3.2 Pilot study Pilot study was carried out in the area around the month of April 2011. The physical observation of the area was done including the area‘s humidity and temperature using weather recording instruments such as barometer and thermometer respectively. The people were generally lean. Oral interview was done on the inhabitants who confirmed that there were cases of malaria in the area. Parasitological information from the Mrima Health Centre for the same period as that of mosquito and sand fly sampling in Mrima hill area revealed Malaria positive diagnosis on people who had not travelled to endemic areas lately. The population mostly slept outside especially men and not many household slept under treated mosquito nets. 3.3 Sampling procedure The study adopted the formulae by Yates et al. (2008) as shown below P =1-N-1/N N-2/N-1…..N-n/N- (N-n) Cancelling =1-N-n/N = n/N =100/1000 =10% The total number of houses in the study area was 384. Taking ten percent of that gave a sample size of 38 houses. A number of households were randomly selected and the radiation levels taken using hand held Digilert 100 CPMs reader and recorded. Selection criterion was based on the total numbers of homesteads in the study area. A total of 12 homesteads with 38 houses were randomly sampled in the village. Human biting night 37 insects (mosquitoes and Phlebotomine sand flies) were trapped for five consecutive days during the rainy season in August 2011 using Solid State Army Miniature (SSAM) light traps (John W. Hock Co., Gainsville, Florida U.S.A). Depending on the numbers of houses in each homestead, a light trap was suspended outside each house to trap peridomestic diptera entering or leaving the houses. The traps were set up one hour before sunset (6.00 pm) because that is usually the time when most dipterans are active and dismantled at 0.5 h after sunrise (6.00 am) as described by Johnson et al. (1993). The trapped insects were kept in papercups and transported every morning to the Kenya Medical Research Institute (KEMRI)‘s Kwale Station Laboratories for processing. They were sorted and identified to species level. Plates 3.1 and 3.2 below show the setting of light traps in the hill and around the households for trapping mosquitoes and sand flies. Plate 3.2: A light trap set in the upper part of the hill for trapping mosquitoes and sand flies. 38 Plate 3.3: A light trap suspended outside a house for trapping mosquitoes and sand flies. 3.4 Laboratory procedures 3.4.1 Specimen preparation and staining with Giemsa stain The mosquitoes and sandflies were dissected into thin films and placed on microscopic slides to air dry before being fixed in absolute methanol for 5 minutes. The fixing was done by first immersing it in absolute methanol to prevent disintegration of the tissues on the slides after which the slide was immersed in a freshly prepared 10% Giemsa stain solution for 30 minutes. The slide was then flushed with tap water and left to air dry before observing it using the light microscopy. 39 3.4.2 Preparation of chloral hydrate mounting gum Chloral hydrate gum, which is usually used to make clear sand fly specimens, was prepared according to the method described by Minter (1964). Eight grams of gum Arabic and 70 grams of chloral hydrate crystals were weighed. They were dissolved in a mixture of distilled water, glycerine and glacial acetic acid. The mixture was stirred using a magnet stirrer and then left to stand for one hour for uniformity. It was then ready to be used to mount the adult sand flies. 3.4.3 Procedure for mounting specimen using chloral hydrate mounting gum A drop of the gum was placed on a slide and mounted on a dissecting microscope before being brought into focus. The sand fly sample was put in the middle of the drop. A pin was used to orient it in the correct position and to make it sink into the gum. Another drop was placed on a cover slip and inverted onto the slide that had the sample so that the two drops came into contact. This was to prevent the formation of bubbles. The gum was left for 24 hours to stand to enable it to spread all over the covered region on the slide. The excess gum was removed by placing a piece of cotton wool on the side of the slide so as to absorb it. The features on the specimen were observed using the dissecting microscope. 40 3.5 Species identification using anatomical features 3.5.1 Mosquitoes Mosquitoes that were captured from the study site were separated by genera using a binocular microscope. They were identified using the standard taxonomic keys as described by Gilles and de Meillon (1968). 3.5.2 Sand flies Any caught female sand flies were washed in 2% liquid soap detergent in normal saline. They were then dissected using pins and a dissecting microscope on ice cold microscope slides before mounting for taxonomy. This was done in order to minimize the chances of motile parasites such as promastigotes in the sand flies dying. All the females caught in all the collection sites were dissected. The heads and genitalia of sand flies were mounted using chloral hydrate gum, covered with cover slips and allowed to dry (these are the parts used for identification of sand flies). Mounted sand flies were left to clear overnight before identification using the taxonomy keys as described by Abonnenc and Minter (1965). 3.6 Examination for parasites All female mosquitoes (131) and sand flies (30) were dissected in a drop of sterile, cold phosphate-buffered saline (PBS) on sterile slides and examined for the presence of parasites in the salivary glands in the case of mosquitoes, and in the guts of sand flies using light microscopy. Slides used for the dissections were further stained with Giemsa for examination of parasites. Giemsa is a differential stain which differentiates the nucleus of merozoites. The merozoite nucleus stains pink. 41 3.7 Data Analysis Comparison of the numbers of mosquitoes and sand flies caught against different levels of radiation was done by Pearson Chi-square test. The relationship of both mosquito and sand fly species and the radiation levels per site and the relationship between radiation and elevation were done using Pearson correlation using the procedure of Statistical Package for the Social Sciences (SPSS) VER 20 (2011). 42 CHAPTER FOUR: RESULTS 4.1 Composition of trapped mosquitoes and sand flies 4.1.1 Mosquitoes The total number of mosquitoes collected was 131. Culex pipiens L was the most abundant at 91 insects, caught from most of the study sites accounting for 69.42% of the total catch. This species was caught in 9 out of the 12 sampling sites at various elevations ranging from an elevation of 33 masl at site 4 up to 82 masl at site 6 and within natural radiation ranging from 5.9 mSv/yr at site 7 to 17.5 mSv/yr at site 4. Mansonia africana Theobald were 24 in number which accounted for 18.32% of the total population of insects caught. The species was collected in only 3 sites, namely 6, 9 and 10 with elevations of 82, 51 and 44 masl and radiation levels of 8.3, 12.2 and 9.9 mSv/yr respectively. Mansonia uniformis Theobald was the next in abundance at 12 insects in total accounting for 9.16% of the total mosquitoes caught and was only collected in site 2 which had an elevation of 45 masl and a radiation level of 7.97 mSv/yr. Aedes aegypti L. and Culex vansomerenae Edwards comprised of 2 insects each accounting for 1.53% each of the total collected mosquitoes. The former was collected in site 3 which had an elevation of 30 masl and a radiation level of 6.48 mSv/yr; whereas Culex vansomerenae was only collected in site 9 which had an elevation of 51 masl and a radiation level of 12.2 mSv/yr. In all these households where the collections of mosquitoes were carried out, despite the fact that the trapping was done during the rainy season, no Anopheles Giles species was caught. This was also despite the differences in 43 radiation levels that ranged from the lowest 5.9 mSv/yr to the highest 17.5 mSv/yr that were recorded. Therefore, no vector of disease was caught (Table 4.1). Table 4.1: Number of mosquito species collected, their percentage, elevation (m (ASL) and radiation levels (mSv/yr) around the Mrima study area Site Elevation Radiation Species m(ASL) (mSv/yr) 75 10.08 Cx. pipiens 45 7.97 Cx. pipiens M. uniformis 30 6.48 Cx. pipiens Aedes aegypti 33 17.5 Cx. pipiens 74 10.9 None 82 8.3 Cx. pipiens M. africana 92 5.9 Cx. pipiens 65 6.5 Cx. pipiens 51 12.2 Cx. vansomerenae M. africana 44 9.9 Cx. pipiens M. africana 38 9.7 Cx. Pipiens 68 7.9 None Frequencies Percentages % 1 31 23.66 2 2 1.53 12 9.16 3 3 2.29 2 1.53 4 9 6.87 5 0 0 6 31 23.66 8 6.1 7 1 0.76 8 10 7.63 9 2 1.53 1 0.76 10 2 1.53 15 11.45 11 2 1.53 12 0 0 Totals 131 100 m(ASL) – metres above sea level, mSv/yr – milliSieverts per year, The numbers in parentheses are percentages of the total catch per site. 44 4.1.2 Sand flies Throughout the entire collection period, in all sites, a total of 39 phlebotomine sand flies were collected. In the study area, only Sergentomyia Franca and Parrot species were collected. Out of these sand fly species, Sergentomyia (Sergentomyia) bedfordi was the most abundant species comprising of 29 insects in number and 74.36% of the total catch. Other species that were collected were Sergentomyia (Parvidens) suberecta at 6 insects accounting for 15.38% of all them caught. A total of 2 Sergentomyia (Grasomyia) meilloni was caught accounting for 5.12% of the total sand fly population caught, Sergentomyia (Parvidens) inermis and Sergentomyia (Sergentomyia) schwetzi were the lowest species caught at a single one each which accounted for 2.56% of the total sand fly species collected. No sand flies harbored parasites and none was blood fed. Only one (1) male Sergentomyia bedfordi was collected in site 5 while all the other sand flies from the other sites were females. There was no Phlebotomus rondani species caught from the study site. Among the sand flies caught none was a known vector of diseases in Kenya (Table 4.2). 45 Table 4.2 Number of Sand fly species collected, their percentage, elevation (m (ASL) and radiation levels (mSv/yr) around the Mrima study area Site Elevation Radiation Species m(ASL) (mSv/yr) 75 10.08 S. meilloni S. suberecta S. bedfordi 45 7.97 None 30 6.48 S. bedfordi 33 17.5 None 74 10.9 S. bedfordi 82 8.3 S. schwetzi S. bedfordi 92 5.9 None 65 6.5 None 51 12.2 None 44 9.9 S. inermis 38 9.7 None 68 7.9 None Frequencies Percentages % 1 2 5.13 6 15.38 22 56.41 2 0 0 3 2 5.13 4 0 0 5 1 2.56 6 1 2.56 4 10.25 7 0 0 8 0 0 9 0 0 10 1 2.56 11 0 0 12 0 0 Totals 39 100 m(ASL) – metres above sea level, mSv/yr – milliSieverts per year, The numbers in parentheses are percentages of the total catch per site. 46 4.2 Relationship between number of mosquitoes and sand flies The radiation levels recorded ranged from the lowest 5.9 mSv/yr to the highest 17.5 mSv/yr whereas elevation levels were recorded from the lowest at 30 m(asl) to the highest at 92 m(asl). Pearson Chi-square statistical test on the number of sand flies and number of mosquitoes collected in Mrima hills indicated that there was no significant association between the two variables ( the number of mosquitoes did not relate to the number of sand flies) (2 =36; df=35; p=0.165; Figure 4.1). Figure 4.1 Relationship between number of mosquitoes and sand flies 47 4.3 Relationship between levels of radiation and number of mosquitoes and sand flies 4.3.1 Relationship between levels of radiation and number of mosquitoes Pearson Correlation analysis on radiation (mSv/yr) and number of mosquitoes collected per site indicated that there was no significant correlation between the two variables (r=0.026; df=10; p=0.937; Figure 4.2, Appendix I). Number of mosquitoes Levels of radiation Figure 4.2: Relationship between levels of radiation and numbers of mosquitoes in Mrima hill. 48 4.3.2 Relationship between levels of radiation and number of sand flies Pearson Correlation analysis on the radiation (mSv/yr) and number of sand flies collected per site indicated that there was no significant correlation between the two variables (r=0.031; df=10; p=0.923; Figure 4.3, Appendix II). Number of sand flies Levels of radiation Figure 4.3 Relationship between levels of radiation and numbers of sandflies in Mrima hill. 4.4 Relationship between levels of radiation and elevation There was variation between elevation and radiation (Table 4.5). There was no significant correlation between elevation and radiation (r = -0.389; df = 10; p = 0.211; Figure 4.4, Appendix III). 49 Levels of elevation Levels of radiation Figure 4.4 Relationship between levels of radiation and levels of elevation in Mrima hill 4.5 Vector infection rates None of the species of both mosquitoes and sand flies trapped and dissected from the study area were infected by parasites of malaria, lymphatic filariasis, mosquito-borne arboviruses or leishmaniasis. 50 CHAPTER FIVE: DISCUSSION 5.1 Trapped mosquitoes and sand flies 5.1.1 Mosquito species The results from the present study indicated that population of both mosquitoes and sand flies in Mrima hill was relatively low and the medically important species of the vectors in Kenya were not found despite the fact that it was during rainy season. The study expected to report availability of the vectors of diseases and a higher number of both mosquitoes and sand flies than reported previously in studies from the neighboring subcounties. The study area had poor sanitary disposal system which could explain the high number of Culex pipiens. According to Syed and Leal (2009), Culex pipiens (the common house mosquito) is usually the most common pest mosquito in urban and suburban settings and an indicator of polluted water in the immediate vicinity. The presence of Cx. pipiens brought the assumption that the level of pollution in the present study area was questionable and therefore, needed to be investigated. Several studies (Wayne, 2013) have shown that the mosquito species lay eggs in batches in polluted or foul water where the larval development takes place in places like septic seepage and other dirty water sources. Lutomia et al. (2013) has shown Culex pipiens to be a competent vector of West Nile Virus in the laboratory and has also been found in nature to be infected by the virus. Culex vansomerenae Edwards was found in very low numbers in Mrima hill. The mosquito species has been shown to be susceptible to infection with Wuchereria 51 bancrofti. However, according to findings by Kinyatta et al. (2011), it does not develop disseminated infection that is necessary for transmission. It is therefore, unlikely that there could be Wuchereria bancrofti transmission taking place within the study area. Culex Vansomerenae has been shown in the laboratory to be susceptible and able to transmit West Nile Virus (WNV) (Lutomia et al., 2011). Even though this mosquito species was found in very low numbers, Mrima hill should be considered an area for West Nile Virus surveillance. Mansonia africana was reported in a higher number than the related species, Mansonia uniformis. It is quite difficult to morphologically distinguish between the two species using the taxonomic key. Interestingly, Mansonia africana was mostly caught in sites with high elevation and moderately high radiation whereas Mansonia uniformis was collected at low levels of elevation and relatively low levels of radiation even though radiation and elevation did not significantly affect the availability of the mosquito species caught from the study area. Mansonia uniformis are mostly found in China and Africa but Mansonia africana is restricted to Africa. They are known vectors of arthropod-borne viruses (arboviruses) such as Chikungunya and Rift Valley Fever that affect man and livestock in Kenya as reported in earlier studies (Labeaud et al., 2011). Even though studies have shown that the two mosquito species are active transmitters of Wuchereria bacrofti in Ghana according to Ughasi et al. (2012), polymerase chain reaction done in Kenya has shown that Mansonia africana and Mansonia uniformis caught in Kwale County are not susceptible to infection with the parasite (Kinyatta et al., 2011). 52 Studies on abundance and bionomics of Aedes aegypti L have been conducted in various parts of the Coastal regions of Kenya (Teesdale, 1955) apart from Mrima hill. Aedes aegypti can be disseminated by ground transportation therefore being able to spread dengue virus beyond the normal flight range of the mosquitoes. They were in low numbers in the present study and it is possible to conclude that the presence of the Aedes aegypti in low numbers was due to transportation in vehicles to the study area, however, the fact needs to be investigated. According to WHO (2006), both the females and male adults of this mosquito feed on nectar of plants; however, female mosquitoes need blood in order to produce eggs and they are active during day time. It is reported by Fontenille et al. (1997) to be an important domestic pest (associated with humans and their dwellings) but it is medically important as a major vector for the yellow fever virus and dengue virus as accounted by Johnson et al. (1982). An outbreak of dengue fever caused by dengue type 2 virus was reported around the coastal towns of Malindi and Kilifi in Kenya (Johnson et al., 1982). It is possible that outbreaks of the two viral diseases can occur in Mrima hill if the population of Aedes aegypti became high. The present study established absence of Anopheline mosquitoes irrespective of the recent report by (Njenga, 2011) suggesting that the deeper one gets into the rural areas, the better the chances of getting Anopheles mosquitoes as they are the predominant and principal vectors in the rural coastal villages. Njenga (2011) concurred with the findings of (Mbogo et al., 2000), that there were Anopheles mosquitoes in Kwale County. Unfortunately, the current study was carried out during rainy season but did not report any Anopheles mosquitoes despite the fact that the mosquito is a perennial fly, abundant 53 mostly during rainy seasons because of availability of their preferred breeding habitats. The absence of anopheline mosquitoes in Mrima hill could have been due to effects of radiation on fertility and fecundity of the insects. It was interesting to note that there was also no mosquito species infected with malaria parasites or filarial worms among the collected species from the present study area. Lack of rivers in Mrima hill left residents with only one option, boreholes as the only sources of water which is normally salty. This may explain why there were no fresh water sources which could be used as breeding sites by Anopheles mosquitoes. Poor sanitary conditions are also normally considered less conducive for Anopheles breeding (Gilles and Coetzee, 1987). Pollutants were observed in the study area which could have contributed to the lack of Anopheles gambiae mosquito species in the study area. However, recent work in other parts of Africa have shown that Anopheles gambiae larvae can develop in polluted temporary pools in peri-urban areas ( Awolala et al., 2007). In another rescent study in other parts of the world (Keating et al., 2004), Anopheles gambiae larvae were collected mainly in temporary habitats containing pollutants such as detergent, human and animal faecal waste and an assortment of domestic debris, an indication that Anopheles gambiae has adapted to tolerating pollutants in breeding environments. It was therefore not clear why Anopheles mosquito species were not found in the present study given that their larvae can survive in polluted habitats. World Health Organization (2010) reported the fact that Anopheles mosquitoes can flourish in any environment as being responsible for rising malaria incidences. Therefore, considering the absence of Anopheles mosquitoes in the study area, it is 54 unlikely that transmission of malaria and lypmphatic filariasis can take place through these species. The results obtained from the present study at Mrima hill have shown that Cx. vansomerenae, Cx. pipiens and Aedes aegypti are the only mosquito species of medical importance caught but did not harbor any parasites of diseases. 5.1.2 Sand fly species As reported by Lewis (1974), Sergentomyia species have been shown to have a feeding preference for reptiles and amphibians and are not known to be susceptible to Leishmania parasites that cause human disease (Lawyer et al., 1990). Sergentomyia bedfordi is known to be a perennial species in other parts of Kenya such as Baringo District and is the most abundant during the dry season and prefers resting mainly in areas with tree canopys (Basimike et al., 1991). Mutinga (1975) reported observations from trapping using Disney traps around Mt Elgon that showed that Sergentomyia bedfordi can be found in all types of buildings, but has never been reported to bite humans. It has also been reported by Kan et al. (2004) to be attracted particularly to goats, Capra hircus L in the Masinga, Mukusi area of Machakos County. This could explain why this species is the most abundant around Mrima hill, where almost every homestead kept goats within their compounds. Field and laboratory studies have shown that Sergentomyia bedfordi feeds on the skink Mabuya striata Peters (Reptilia: Scincidae) and gets infected with Trypanosoma boueti Leger and Leger (Kinetoplastida: Trypanosomatidae). A study report by Ashford et al. (1973) showed the parasites can develop up to the epimastigotepromastigote stage in the foregut but the sand fly cannot transmit it through the bite. 55 Transmission to the skink, which is a reptile, occurs when it feeds on the infected sand fly. Sergentomyia suberecta was first reported within Coast Province by Sinton (1932). The species was collected in Mrima hill but there were no prior records of parasite isolation from it. Similarly, the few that were caught had no infections. Its bionomics is poorly known. In Kenya, Sergentomyia schwetzi is found in two forms, the typical and the atypical, depending on the structures of the seventh and eighth abdominal tergites. The typical form was found in Mrima hill. Laboratory studies have shown that it cannot support the development of Leishmania major Yarkimoff and Schokhor and therefore cannot transmit the parasite (Lawyer et al., 1990). This species has been shown to be susceptible to infection with Rift Valley Fever Virus following oral infection but it cannot transmit the virus to a clean Syrian hamster, Mesocricetus auratus Warehouse (Dohm et al., 2000). Apart from the possibility of this sand fly becoming a biting nuisance, it can be of no major problem to the residents of Mrima hill. Sergentomyia meilloni which occurred in Mrima hill is also mainly found in Coastal regions of Kenya with a few in Eastern regions of Kenya (Anjili et al., 2011). Little is known about the bionomics of this species and also its preferred feeding host. There are no records showing that any parasite has ever been isolated from it. Considering that it was found in very low numbers (5.13%), it is possible that it also possess no major threat to the Mrima hill population. Further studies need to be done to establish whether it can bite man and also to establish whether it is susceptible to viruses of human concern. 56 5.2 Infection rates It was not clear why there were no parasites of diseases present in the species of mosquitoes and sand flies caught and dissected from the study area. It could be probably due to the fact that the medically important species trapped from the study area are not known to transmit infections in Kenya. 5.3 Relationship between radiation and elevation Since there was no significant relationship between radiation and elevation and also between radiation and species of both mosquitoes and sand flies in Mrima hill it remains unknown whether radiation affected the species composition of both mosquito and sand flies in their natural habitat. In the present study, the effect of natural radiation on species of both mosquitoes and sand flies was not established. 57 CHAPTER SIX: CONCLUSIONS AND RECOMMENDATIONS 6.1 Conclusions i. Mosquito species composition in Mrima hill comprised: Cx. pipiens, Cx. vansomerenae, M. uniformis, M. africana and Ae. aegypti. From what is known about the species compositions of mosquitoes around study area, apart from Culex vansomerenae and Culex pipiens, which are of medical importance, the potential of an outbreak of diseases to occur can be considered very remote. ii. The sand fly species which were collected from Mrima hill included: S. meilloni, S. suberecta, S. bedfordi, S. inermis and S. schwetzi. They are not of medical importance in Kenya. Most of the species were Sergentomia which have reptiles and amphibians as feeding preference as discussed. Transmission of leishmaniases is also considered remote because no parasite was found in the dissected insects. iii. Through dissections, microscopic examination of wet and Giemsa stained slides of mosquitoes the study did not detect any malaria or filarial worms. iv. There were no Leishmania parasites detected. It cannot be accurately concluded that this was as a result of the elevated levels of radiation in the study area. 58 v. The radiation readings recorded from the geiger counter indicated the highest radiation level as 17.5 mSv/yr at elevation 33 masl yet the annual limit of effective dose is equivalent to 5.0 milliSieverts. The elevation levels of Mrima hill ranged from as low as 30 masl to the highest at 82 masl. The elevation had no significant relationship with the radiation level as done by the Pearson correlations. 6.2 Recommendations i. From results of this study it is recommended that the trapping of more insects over an extended period of time during different seasons be done. This method will generate period prevalence data that will help understand the epidemiology of disease transmission in the study area. This could provide more information on the possibility of collecting the possible disease vectors in the area. ii. Trapping of insects should be extended to cover a wider area than only in Mrima hill. This is because road construction materials from the quarries on Mrima hill have been used to construct roads in many sections of Kwale County. iii. Studies should be carried out on the effect of radiation on the dipteran arthropod tissues to see if there is any evidence in their polytene chromosomes or aberration of their DNA. 59 REFERENCES Abonnenc, E. and Minter, D.M. (1965). Bilingual keys for the identification of the sandflies of the Ethiopian Region (French and English. Cahier, Office de la Recherche Scintifique et technique d‘ Outre. Mer Entomologie Medicale 5: 1. Achola, S.O.; Patel, J.P.; Mustapha, A.O. and Angeyo, H.K. (2012). Natural radioactivity and external dose in the high background radiation area of lambwe east, south western kenya. Radiation Dosimetry. Africa News Service. (2010). Africa news bulletin. Anand, P.; Kunnumakkara, A.B.; Kunnumakara, A.B.; Sundaram, C.; Harikumar, K.B.; Tharakan, S.T.; Lai, O.S.; Sung, B. and Aggarwal, B.B. (2008). ―Cancer is a preventable disease that requires major lifestyle changes.‖ Pharmacological Research 25 (9): 2097–2116. Anderson, J.; Cowlishaw, G. and Rowcliffe, J.M. (2005). Effects of forest fragmentation on abundance of colobus angolensis palliatus in Kenya‘s Coastal forests. International Journal of Primatology 28: 637-655. Anjili, C.O.; Ngumbi, P.M.; Kaburi, J.C. and Irungu, L.W. (2011). The Phlebotomine Sandfly fauna (Diptera: Psychodidae) of Kenya. Journal of Vector Borne Diseases 48: 183-189. Ashburner, M. (1989). Drosophila melanogaster, Developmental Biology; Laboratory Manuals/Handbooks. University of Cambridge. ISBN 07-87969-322-3; 434. Ashford, R.W.; Bray, M.A. and Forster, W.A. (1973). (Protozoa) parasitic in the skink Mabuya striata (Reptilia) and the sandfly Sergentomyia bedfordi in Ethiopia, Journal of Zoology 171: 285–292. Awolala, T.; Oduola, A.; Obansa, J.; Chukwurar, N. and Unyimadu, J. (2007). Anopheles gambiae ss breeding in polluted water bodies in urban Lagos, South Western Nigeria. Journal of Vector Borne Diseases 44: 241-244. Basimike, M.M.; Mutinga, M.J. and Kumar, R.R. (1991). Distribution of sandflies (Diptera: Psychodidae) in three vegetation habitats in the Marigat area, Baringo District, Kenya. Journal of Medical Entomology 28:330-333. Bates, P.A. (2007). Transmission of Leishmania metacyclic promastigotes by phlebotomine sand flies. International Journal of Parasitology 37(10-3): 1097-1106. Beck, B. R.; Becker, J.A.; Beiersdorfer, P.; Brown, G.V.; Moody, K.J.; Wilhelmy, J.B.; Porter, F.S.; Kilbourne, C.A. and Kelley, R.L. (2007). "Energy Splitting of the Ground-State Doublet in the Nucleus 229Th". Physics Revision Letters 98 (14): 142501. 60 Bennun, L. and Njoroge, P. (1996) Birds to watch in East Africa. A preliminary Red data list. Research Report of the centre for Biodiversity, National Museums of Kenya: Orinthology 23. BirdLife International. (2005). Birdlifes Online WordBird Databasa: the site for bird conservation. www.birdlife. Org accessed on http://www.birdlife.org/datazone/home. BirdLife International. (2012). Nestor notabilis. IUCN Red list of Threatened species. Version 2012.2. International Union for Conservation of Nature. Burgess, N.D. and Clark, G.P. (2000). The coastal forest of Eastern Africa IUCN: Cambridge and Gland. Burgess, N.D. and Muir, C. (1994). The coastal forests of Eastern Africa; biological values and conservation needs. The Society for Environmental Exploration, London. Critical Ecosystem Partnership Fund. (2003). Eastern Arc mountains and Coastal forest of Tanzania and Kenya Biodiversity Hotspot. Ecosystem profile. Oceandocs.org. Clark, G.P. and Robertson, S.A. (2000).Vegetation communities in the Coastal Forest of Eastern Africa.eds.IUCN: Cambridge and Gland; 83-102. Clark, G.P. and Burgess, N.D. (2000).Geology and Geomorphology. In the coastal Forest of East Africa.eds.IUCN: Cambridge and Gland; 29-39. Collins, S. R.; Weldon, C. W.; Banos, C. and Taylor, P. W. (2008). Effects of irradiation dose rate on quality and sterility of Queensland fruit flies, Bactrocera tryoni (Froggatt). Journal of Applied Entomology 132 (5): 398-405. Dohm, D.J.; Rowton, E.D.; Lawyer, P.G.; O’Guinn , M.; Michael J. and Turell, M . (2000). Laboratory Transmission of Rift Valley Fever Virus by Phlebotomus duboscqi, Phlebotomus papatasi, Phlebotomus sergenti, and Sergentomyia schwetzi (Diptera: Psychodidae). Journal of Medical Entomology 37:435-438. Dyck, V.A.; Hendrichs, J. and Robinson, A.S. (2005). Sterile Insect Technique: Principles and Practice in Area-Wide Integrated Pest Management. Dordrecht, The Netherlands: Springer. Evseeva, T.I.; Gres’kin, S.A. and Khramova, E.S. (2003). Comparative evaluation of early and log-term plant cell reactions under the combination of short-term and chronic impact of 232 Th and CD. Tsiitologiia Genetika 37: 61-66. Evironmental Protection Agency. (2013). Thorium radiation protection U.S. Environmental Protection Agency. 61 Fontenille, D.; Diallo, M.;Mondo, M.; Ndiaye, M. and Thonnon, J. (1997). "First evidence of natural vertical transmission of yellow fever virus in Aedes aegypti, its epidemic vector.". Transactions of the Royal Society of Tropical Medicine and Hygiene 91 (5): 533–535. Fryxell, R.T.T.; Nieman, C.C.; Fofana, A,; Lee, Y.; Traore, S.F.; Cornel, A.J.; Luckhart, S. and Lanzaro, G.C. (2012) Differential plasmodium falciparum infection of Anopheles gambiae s.s molecular and chromosomal forms in Mali. Malaria Journal 11(1). Gilles, M.T. and Coetzee, M. (1987). A supplement to the Anphelinae of Africa south of the Sahara (Afrotropical region).The South Africa Institute for Medical Research, Johannesburg No. 55. Gilles, M.F. and de Meillon, B. (1968). The Anophelinae of Africa South of the Sahara. 2nd adition. South African Institute for Medical Research no. 54 Johannesburg, South Africa. Githitho, A. (2004). The coastal terrestrial forests of Kenya. A report on Resources and investments WWF. East African coastal forest programme. Goto, A.; Takebayashi, Y.; Li, L.; Saiga, T.; Mori, T.; Yamadera, A. and Fukumoto, M. (2002). Microdistribution of alpha particles in pathological section tissues from thorotrast patients detected by immging plate autoradiography. Radiation Research 158: 54-60. Gubler, D.J. and Clark, G.G. (1995). Dengue/ Dengue Hemorrhagic fever: The emergence of Global Health problem. Emerging infectious Disease 1(2): 55-57. Hala, J. and Navratil, J. D. (2003). Radioactivity, Ionizing Radiation and Nuclear Energy. Konroj: Brno, Czech Republic. ISBN 80-7302-053.X. Hamady, D.; Ruslam, N.B.; Ahmad, A.; Rawi, C.S.M.; Ahmad, H.; Satho, T.; Miake, F.; Zuharah, W.F.; Fukumitsi, Y.;Saad, A.R.; Rajasaygar, S.; Vargas, R.E.M.; Abmajid, A.H.; Fadzly, N.; Ghani, I.A. and AbuBakar, S. (2013). Colonized Aedes albopictius and its sexual performance in the wild; Implication for STI technology and containment. Parasites and vector borne diseases 6: 206. Hammond, C.R. (2004). The Elements in Handbook of Chemistry and Physics 81st edition. CRC press. Haralambos, M. and Holborn, M. (2000). Sociology: Themes and perspectives. Hammersmith, London: HarperCollins publisher; 998. 62 Hayes, B.E.; Komar, N.; Nasci, S.R.; Montogomery, S.; Daruel, R.; O’leary and Campbell, L.G. (2005). Epidemiology and transmission dynamics of West Nile Virus disease. Emerging infectious diseases CDC 11. Helsinki, E.M.H.; Parker, A.G. and Knols, B.G.J. (2006). Radiation induced sterility for pupal and adult stages of the malaria mosquito Anopheles arabiensis. Malaria Journal 5:41. Hulley, S.B. (2007). Designing Clinical Research. Lippincott Williams and Wilkins; 168169. Hyde, E.K. (1960). The radiochemistry of thorium. Subcommittee on Radiochemistry, National Academy of Sciences—National Research Council. International Union for Conservation of Nature. (2014). IUCN RedList of threatened species version 2010.4. Johnson, R.N.; Ngumbi, P.M.; Mwanyumba, J.P. and Robert, C.R. (1993). Host preference of Phlebotomus guggisbergi, a vector of Leishmania tropica in Kenya. Medical and Veterinary Entomology 7: 216-218. Johnson, B.K.; Ochieng, D.; Gighogo, A.; Musoke, M. and Rees, P.H. (1982). Epidemic Dengue fever caused by dengue type 2 in Kenya: preliminary results of human urological and serological studies. East African Medical Journal 59: 781-784. Junnila, A.; Muller, G.C. and Schlein, Y. (2011). Attraction of Phlebotomus papatasi to common fruit in the field. Journal of Vector Ecology 36(supplement 1): S206-S211. Kan, E.; Anjili, C.O.; Saini, R.K.;Hidaka,T. and Githure, J.I. (2004). Phlebotomine sandflies (Diptera: Psychodidae) collected in Mukusu, Machakos District, Kenya and their nocturnal flight activity. Applied Entomology and Zoology 39: 651-659. Keating, J.; Macintyre, K.; Mbogo, C.M.; Githure, J.F. and Beier, J.C. (2004). Cheracterization of potential larval habitats for Anopheles mosquitoes in relation to urban land use in Malindi, Kenya. International Journal of Health and Geography 39: 9-21. Kelly-Hope, L.A.; Hemmingway, J. and McKenzie, F.E. (2009). Environmental factors associated with the malaria vectors Anophales gambiae and Anopheles funestus in Kenya. Malaria Journal 8: 268- 280. Kenya Forest Department. (1994). The Kenya Forestry plan. FINNIDA/ Forest Department, Karura, Nairobi. Kinyatta, N.M.; Ng’ang’a, Z.W.; Kamau, L.; Kimani, F.T.; Githae, R.W. and Kagai J.M. (2011) Determination of vectoral potential of Mansonia species in the transmission 63 of Wuchereria bancrofti in Tana River Deta District,Coast – Kenya. East Africa Medical journal 88(4) 280-284. Krafsur, E.S.; Whitten, C.J. and Novy, J.E. (1987) Screwworm eradication in North and Central America. Parasitology Today vol. 3(5): 131-137. Kumano, N.; Kawamura, F.; Haraguchi, D. and Kohama, T. (2008). Irradiation does not affect field dispersal ability in the West Indian sweetpotato weevil, Euscepes postfasciatus. Entomologia Experimentalis et Applicata 130 (1): 63-72. LaBeaud, A.D.; Cross, P.C.; Getz, W.M.; Glinka, A. and King, C.H. ( 2011). Rift valley fever virus in African Buffalo syncerus caffer herds in rural South Africa: Evidence of interepidemic transmission. American Journal of Tropical Medicine and Hygiene 84: 541-646. Lawyer, P.G.; Ngumbi, P.M.; Anjili, C.O.; Odongo, S.O.; Mebrahtu, Y.B.; Githure, J.J.; Koech, D.K. and Roberts, C.R. (1990). Development of Leishmania major in Phlebotomus duboscqi and Sergentomyia schwetzi (Diptera: Psychodidae). American Journal of Tropical Medicine and Hygiene 43: 31-43. Lefevre, G. Jr. (1974). The relationship between genes and polytene chromosome bands. Annual Review of Genetics 8: 51-62. Lewis, D.J. (1974). The biology of phlebotomidae in relation to leishmaniasis. Annual Review of Entomology 13: 363-384. Lutomiah, J.L.; Koka, H.; Mutisya, J.; Yalwala, S.; Muthoni, M.; Makio, A.; Limbaso, S.; Musila, L.; Clark, J.W.; Turell, M.J.; Kioko, E.; Schnabel. D. and Sang, R.C. (2011). Ability of selected Kenyan mosquito (Diptera: Culicidae) species to transmit West Nile Virus under laboratory conditions. Journal of Medical Entomology 48: 1197-1201. Lutomiah, J.L.; Bast, J.; Clark, J.; Richardson, J.; Yalwala, S.; Oullo, D.; Mutisya, J.; Mulwa, F.; Musila, L.; Khamadi, S.; Schnabel, D.; Wurupa, E. and Sang, R. (2013). ‗Abundance, diversity, and distribution of Mosquito vectors in selected ecological regions of Kenya: public health implications‘. Journal of Vector Ecology 33(1): 134-142. Malathi, J. (2005). ‗Study of radionuclide distribution around Kudankulam nuclear plant site (Agastheeswaramm taluk of Kanyakumari diatrict India)‘. Radiation Protection Dosimetry 113(4): 415-420. Manda, H.; Gouagna, L.C.; Nyandat, E.; Kabiru, E.W.; Jackson, R.R.; Foster, E.A.; Githure, J.I.; Beier, J.C. and Hassali, A. (2009). Discriminative feeding behavior of Anopheles gambiae s.s on endemic plants in Western Kenya. Medical and Veterinary Entomology 21(1): 103-111. 64 Manguin, S.; Bangs, M.J.; Pothikasikorn, J. and Chareonviriyaphap, T. (2009). Rewiew on global co-transmission of human Plasmodium species and Wuchereria bancrofti by Anopheles mosquitoes. Infection, Genetics and Evolution 10(2010): 159177. Manteufel, P. (1912). Notiz uber ein bisher an der deutschostafrikanschen Kuste nicht bekanntes ‗Sommerfieber‘. Archives Schiffs-U Tropenhygiene 16: 619–622. Mbogo, C.M.; Mwangangi, J.M.; Nzovu, J.; Gu, W.; Yan, G.; Gunter, J.T.; Swalm, C.; Keating, J.; Regens, J.L.; Shililu, J.I.; Githure, J.I. and Beier, J.C. (2000). Spatial and temporal heterogeneity of Anopheles mosquitoes and Plasmodium falciparum transmission along the Kenyan coast. American Journal of Tropical Medicine and Hygiene 68: 734-742. Minakwa, N.; Githure, I.J.; Beier, J.C. and Yan, G. (2001). Anopheles mosquito larval strategies during dry period in Western Kenya. Journal of Medical Entomology 38(3): 388- 992. Minter, D.M. (1963).The distribution of sand flies (Diptera: Psychodidae) in Kenya. Bulletin of Entomological Research 55: 205-217. Monath, P.T. (2001). Yellow fever: an update. The lancet infectious Diseases 1(1): 1120. Mutinga, M.J. (1975). Phlebotomus fauna in the cutaneous leishmaniasis foci of Mt. Elgon, Kenya. East African Medical Journal 56: 153-182. Muturi, E.J.; Mbogo, C.M.; Mwangangi, J.M.; Ng’ang’a, Z.W.; Kabiru, E.W.;Mwandawiro, C. and Beier, J.C. (2006). Concomitant infections of Plasmodium falciparum and Wuchereria bancrofti on the Kenya coast. Filaria Journal 5: 8- 18. Muturi, E.J.; Mwangangi, J.; Shililu, J.; Jacob, B.G.; Mbogo, C.M; Githure, J. and Novak, R. (2007). Evaluation of four sampling Techniques for surveillance of Culex quinquefasciatus (Diptera : Culicidae) and other mosquitoes in African Rice Agrosystems. Journal of Medical Entomology 44(3): 503- 508. Mwandawiro, C.S.; Fujimaki, Y.; Mitsui, Y. and Katsivo, M. (1997). Mosquito vectors of bancroftian filariasis in Kwale District, Kenya. East African Medical Journal 74: 288-392. Nash, D.; Mostashari, F.; Fine, A.; Miller, J.; O’leary, D.; Murray, C.; Huang, A.; Rosenberg, A.; Greenberg, A.; Sherman, M.; Wong, S. and Lyton, M. (2001). Outbreak of West Nile Virus Infection in the New York area in 1999. The New England Journal of Medicine 344(24). 65 NEMA (2001). Environmental Impact Assessment Study Report – Vol II. Mrima Hill Niobium and associated rare earths mining project, in Kwale District, Coast Province. Njenga, S.M.; Muita, M.; Kirigi, G.; Mbugua, J.; Mitsui, Y.; Fujimaki, Y. and Aoki, Y. (2000). Bancroftian filariasis in Kwale District, Kenya. East African Medical Journal 77: 245-249. Njenga, S.M. (2007). ‗Immuno-parasitological assessment of bancrofti filariasis in a highly endemic area along the River Sabaki, in Malindi district, Kenya‘. Annals of Tropical Medicine and Parasitology 101(2): 161-172. Njenga, S. M. (2011). ‗Sustained reduction in prevalence of lymphatic filariasis in spite of missed rounds of mass drug administration in an area under mosquito nets for malaria control‘. Parasite vectors 25(4): 90. Nyaoro, W. (1999). Rainfall permeating the soil provides most of Kenya‘s groundwater resources. ESIA Study Report. Onyango, S.A.; Kitron, U.; Mungai, P.; Muchiri, E.M.; Kokwaro, E.; King, C.H. and Mutuku, F.M. (2013). Monitoring malaria vector control interventions: effectiveness of five different Adult mosquito sampling methods. Journals of Medical Entomology 50(5): 1140-1151. Ottesen, E.A. (2000) The global programme to eliminate lymphatic filariasis. Tropical Medicine Internation Health vol 5: 591-594. Ottesen, E.A. and Ramachandran, C.P. (1995). Lymphatic filariasis in tropical countries. Parasitology Today 11: 129 – 131. Patel, J.P. (1991). Environmental radiation Survey of High Natural Radiation of Mrima Hill of Kenya. Discovery and Innovation 3: 31-35. Patz, A.J.; Graczyk, K.T.; Geller, N. and Vittor, Y.A. (2000). Effects of environmental change on emerging parasitic diseases. International Journal for parasitology 30: 1395-1405. Pederson, E.M. and Mukoko, D.A. (2002). Impact of Insecticide- treated materials on filarial transmission by the various species of vectors in Africa. Annals of Tropical Medical Parasitology 96 (2): 91-95. Robertson, S.A. and Luke, W.R.Q. (1993). Kenya Coastal Forests – report of the NMK/ WWF Coast forest Survey. World Wide Fund for Nature, Nairobi. Unpublished report. Sacks, D.L. (2001). Leishmania –sandfly interactions controlling species specific vector competence. Cellular Microbiology 3(4): 189-196. 66 Schlein, Y. and Yuval, B. (1987). Leishmaniasis in the Jordan Valley. IV. Attraction of Phlebotomus papatasi (Diptera: Psychodidae) to plants in the field. Journal of Medical Entomology 24: 87-91. Schlein, Y. and Jacobson, R.L. (1994). Mortality of Leishmania major in Phlebotomus papatasi caused by plant feeding of sand flies. American Journal of Tropical Medicine and Hygiene 50: 20-27. Schlein, Y. and Muller, G.C. (2008). An approach to mosquito control: Using the dominant attraction of flowering Tamarix jordanis trees against Culex pipiens. Journal of Medical Entomology 45: 384-390. Sinton, J.A. (1930). Some new species and records of Phlebotomus from Africa. Indian Journal of Medical Research 18: 171–193. Sinton, J.A. (1932). Some further records of Phlebotomus from Africa. Indian Journal of Medical Research 20: 565–576. Soliman, M.S.A.; Hegazy, A.K.; Goda, S.K.; Emam, M.H. and Al-Atar, A.A. (2011). Cytotoxicity and mutagenic effects of soil radionuclides on some blac sand plant species. Journal of Mediterranean Ecology 11: 5-20. Strickberger, M.W. (1990). Genetics. McMillan Publishing Company 3: 24-25. Subhan, A. (2007). Ionizing radiation safety,Part II. Journal of Clinical Engineering 32 (3): 103-107. Teijlingen, V.E.; Rennie, A.M.; Hundley, V. and Graham, W. (2001). The importance of conducting and reporting pilot studies: The example of Scottish Birth Survey, Journal of Advanced Nursing 34(3): 289-295. United State Enviromental Protection Agency (1990). Toxicological Profile for Thorium. U.S. Environmental Protection Agency, Agency for Toxic Substances and Disease Registry. Ughasi, J.; Bekerd, H.E.; Coulibaly, M.; Adabie-Gomez, D.; Gyapong, J.; M; Wilson, M.D. and Daniel Adjei Boakye, D.A. (2012). Mansonia africana and Mansonia uniformis are Vectors in the transmission of Wuchereria bancrofti lymphatic filariasis in Ghana, Parasites and Vectors 5: 89-155. United Nation Scientific Committee on the Effects of Atomic Radiation. (1988). Report to the general Assembly with Annexes. United Nations Sales Publication E.88.IX. 7. 67 United Nation Scientific Committee on the Effects of Atomic Radiation. (2008). Effects of ionizing radiation UNSEAR 2006 report to the General Assembly, with scientific annexes. New York: United Nations. 2008. ISBN 978-92-1-142263-4. Van Kampen, R.J.W.; Erdkamp, F.L.G. and Peters, F.J.P. (2007). Thorium-dioxide related haeangiosarcoma. Netherlands Journal of Medicine 65: 279-282. Vazeille, M.; Moutailler, S.; Coudrier, D.; Rlousseauk, C.; Khun, H.; Huerre, M.; Thiria, J.; Dehecq, J.; Fontenille, D.; Schuffenecker, I.; Depres, P. and Failloux, A. (2007). Two Chikungunya isolates from the Outbreak of La Reunion (Indian Ocean) Exhibit Different Patterns of Infections in the Mosquito, Aedes albopictus. Medical Research Centre Detachment / Centres for Disease control, United States of America 2(11): 168. Vreysen , M. J. B. , Robinson , A. S. , and Hendrichs , J. (2007) . ― Area-wide Control of Insect Pests, From Research to Field Implementation . ‖. Springer , Dordrecht, The Netherlands, 789. Waiyaki, E. and Bennun, L.A. (2000). The Avifauna of coastal forests in southern Kenya: status and conservation. Ostrich 71 (1&2): 247-256. Wanyiri, J.W. (1996). A study of the predators of the tsetse fly Glossina austeni Newstead (Diptera: Glossinidae) at Muhaka forest, South Coast, Kenya. MSc Thesis, Kenyatta University, Kenya. Wayne, J.C. (2013). Mosquitoes Research and control, Dept of Entomology. Rutgers, The state University of New Jersey. Wickleder, M.S.; Fourest, B. and Dorhourt, P.K. (2006). "Thorium". In Morss, Lester R.; Edelstein, Norman M.; Fuger, Jean. The Chemistry of the Actinide and Transactinide Elements (3rd ed.). Wilke, A.B.B.; Vidal, P.O.; Suesdek, L. and Marrelli, M.T. (2014). Population genetics of neotropical Culex quinquefasciatus (Diptera: Culicidae). Parasite and Vectors 7(1): 468-476. World Health Organization. (1992). Lymphatic filariasis: The Disease and its control. Fifth Report of the WHO Expert committee on filariasis. World Health Organization, Technical Report Series No. 821. World Health Organization (2000) Expert Committee on Malaria. World Health Organization Technical Report Series 892: i-v, 1-74. World Health Organization. (2006). Multicountry study of Aedes aegypti pupal productivity survey methodology findings and recommendations. TDR/ IRM/ DEN/ 06.1: 1-56. 68 World Health Organization. (2010). World Malaria Report. http://www.who.int/malaria/publication. World Health Organization. (2013). Study of Culex quinquifasciatus findings report. World Wide Forest-East Africa Regional Programme Office. (2002) Eastern Africa Coastal Forest programme; Regional Workshop Report Nairobi; 4-7. Yates, D.S.; Moore, D.S. and Starnes, D.S. (2008). The practice of Statistics, 3RD Edn. Freeman. ISBN 978-0-7167-7301-2. 69 APPINDICES APPENDIX I: Mosquitoes Pearson Correlation between Elevation and Radiation Elevation(m(ASL) Radiation(mSv/yr) Pearson Correlation Sig. (2-tailed) N Pearson Correlation Sig. (2-tailed) N Elevation(m(ASL) 1 17 -.259 .315 17 Radiation(mSv/yr) -.259 .315 17 1 17 70 APPENDIX II: Sand Flies Pearson Correlation between Elevation and Radiation Elevation(m(ASL) Radiation(mSv/yr) Pearson Correlation Sig. (2-tailed) N Pearson Correlation Sig. (2-tailed) N Elevation(m(ASL) 1 15 -.360 .187 15 Radiation(mSv/yr) -.360 .187 15 1 15 71 APPENDIX III: Pearson Correlation between Elevation (m(ASL) and Radiation(mSv/yr) Elevation(m(ASL) Radition(mSv/yr) Pearson Correlation Sig. (2-tailed) N Pearson Correlation Sig. (2-tailed) N Elevation(m(ASL) 1 12 -0.389 .211 12 Radiation(mSv/yr) -.389 .211 12 1 12