Bioprospecting for Anti-Mosquito Phytochemicals Associated with Olfaction and Larvicidal Activities from Selected Kenyan Plants

BIOPROSPECTING FOR ANTI-MOSQUITO PHYTOCHEMICALS ASSOCIATED WITH OLFACTION AND LARVICIDAL ACTIVITIES FROM SELECTED KENYAN PLANTS

JOHN BWIRE OCHOLA (MSc. B.Sc.)

I84/27752/2013

A THESIS SUBMITTED IN FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF THE DEGREE OF DOCTOR OF PHILOSOPHY (CHEMISTRY) IN THE SCHOOL OF PURE AND APPLIED SCIENCE OF KENYATTA UNIVERSITY

JANUARY 2020

DECLARATION

I certify that this thesis is my original work and has not been presented for award

of a degree in any other university. Besides where due acknowledgement has been

made, the work is the result of my labour which has been accomplished from the

commencement date of the permitted studies application.

Signature ……………………………… Date ………………………………… John Bwire Ochola Reg I84/27752/2013

Department of Chemistry

Declaration by supervisors

We confirm that the work reported in this thesis was carried out by the student under our supervision and has been submitted for examination with our approval as supervisors

Signature ……………………………………. Date …………………… Prof. Ahmed Hassanali Department of Chemistry, Kenyatta University, Nairobi, Kenya

Signature ……………………………………. Date …………………… Dr. Wilber Lwande Applied Bioprospecting Programme International Centre of Insect Physiology and Ecology, Nairobi, Kenya.

Signature ……………………………………. Date …………………… Dr. Barbara Frei Haller Institute of Pharmaceutical Sciences, ETH Zurich Switzerland iii

DEDICATION

“To my wife Jackline and sons, Nathan, Seth, Lawrence and Ronnie, you kept my spirit alive.”

ACKNOWLEGDEMENTS

I express my sincere gratitude to Dr Wilber Lwande of ICIPE, Prof. Ahmed.

Hassanali of the Department of Chemistry, Kenyatta University and Dr. Barbara

Frei Haller of the Institute of Pharmaceutical Sciences, ETH Zurich, Switzerland for their support and mentorship throughout the research period and in the writing of this thesis. I would also like to thank Dr Margret Ng’ang’a for her guidance and support.

My sincere thanks go to Dr Patrick Guerin and Thomas Krober for hosting me at

Neuchâtel University, Switzerland and for providing practical knowledge on GC-

EAD technique. Dr. Pie Muller for hosting me at the Swiss Tropical and Public

Health Institute, Switzerland, and sharing enthusiastically his experiences in Vector

Control Research, mainly on repellent testing.

My most sincere appreciation goes to Dr Barbara Frei Haller for her dedication to fund my tuition fee without whose support I would not have come this far. Special thanks also go to UBS Optimus Foundation for funding part of my research work within the African Repellents Project. ICIPE is highly appreciated for provision of the research environment. I am equally indebted to my colleagues; Mr. Wanyama for carrying out GC-MS analyses of my samples, Rose Marubu, Nixon Onyimbo,

Fred Nduguli and Lamberts Moreka at the Bioprospecting Programme at ICIPE. I owe special gratitude to my family particularly my wife, Jackline Majuma Bwire for her love, support and patience mixed with a dose of “go for it!” Lots of thanks also go to my dear parents (posthumous), siblings and friends. Finally, I give all glory and honor to God Almighty who continues to guide, sustain and carry me through life’s challenges. v

TABLE OF CONTENT

DECLARATION ...... ii

DEDICATION ...... iii

ACKNOWLEGDEMENTS ...... iv

TABLE OF CONTENT ...... v

LIST OF FIGURES ...... xi

LIST OF PLATES ...... xiii

LIST OF SCHEMES ...... xiv

LIST OF APPENDICES ...... xv

CHAPTER ONE ...... 1

INTRODUCTION ...... 1

1.1 Background information ...... 1 1.2 Statement of the Problem ...... 5 1.3 Justification of the Study ...... 5 1.4 Significance of the study...... 6 1.5 Hypotheses ...... 6 1.6 General objective ...... 7 1.6.1 Specific objectives ...... 7 1.7 Scope and limitation ...... 8

CHAPTER TWO ...... 9

LITERATURE REVIEW ...... 9

2.1 Mosquitoes as vectors of diseases ...... 9 2.2 Biology and life cycle of a mosquito ...... 11 2.3 Economic importance of mosquitoes ...... 13 2.4 Host-seeking behaviour of mosquito ...... 13 2.5 Mosquito vector borne disease burden ...... 16 2.5.1 Malaria ...... 17 2.5.2 Dengue ...... 18 2.5.3 Chikungunya ...... 18 vi

2.5.4 Yellow fever ...... 19 2.5.5 Zika virus disease...... 19 2.5.6 West Nile virus ...... 20 2.6 Distribution of mosquito borne diseases ...... 20 2.7 Control of mosquito vectors ...... 22 2.7.1 Chemical control of mosquitoes ...... 22 2.7.2 Environmental control ...... 25 2.7.3 Biological/Genetic Control ...... 25 2.7.4 Vector Resistance ...... 26 2.8 Use of Natural products in Mosquito Control ...... 27 2.8.1 Mosquito Repellents ...... 28 2.8.2 Larvicides...... 31 2.9 Plants used in the study ...... 32 2.9.1 The lamiaceae family ...... 32 2.9.1.1 The genus Satureja ...... 32 2.9.1.1.1 Biological studies and clinical properties of Satureja Species ...... 33 2.9.1.1.2 Previous phytochemical studies of Satureja species ...... 33 2.9.1.2 The genus Plectranthus ...... 34 2.9.1.3 The genus Ocimum ...... 35 2.9.1.3.1 Ocimum kilimandscharicum ...... 35 2.9.1.3.2 Ethnobotany of O. kilimandscharicum ...... 35 2.9.2 The family Asteraceae ...... 38 2.9.2.1 The genus Conyza ...... 38 2.9. Terpenoid biosynthesis ...... 39 2.11 Intraspecific variability of plant essential oil composition ...... 40

CHAPTER THREE ...... 42

MATERIALS AND METHODS ...... 42

3.1 General experimental procedures ...... 42 3.2 Chemicals and solvents ...... 42 3.3 Insects ...... 42 3.4 Plant materials...... 43 3.5 Extraction and identification of volatile phytochemicals ...... 48 3.5.1 Hydro-distillation ...... 48 3.5.2 Analyses of essential oils using Gas Chromatography – Mass Spectrometry ...... 49 vii

3.5.3 Electrophysiological recordings using GC-EAG ...... 50 3.5.4 Variability in the composition of the essential oils...... 52 3.6 Repellent tests ...... 52 3.6.1 Estimation of effective dose using vitro ‘K and D module’ ...... 52 3.6.2 Warm body in vitro assay for evaluating mosquito repellents effective dose ...... 55 3.6.3 Estimation of repellent efficacy and protection time ...... 56 3.6.4 Subtractive-combination repellence bioassay ...... 58 3.7 Larvicidal and insect growth regulation assays ...... 58 3.7.1 Formulation of essential oil plant and vegetable oil ...... 58 3.7.2 Laboratory Larvicidal and insect growth regulation assays ...... 59 3.7.3 Semi-field mosquito larvicidal evaluation of water miscible formulations ...... 60 3.8 Statistical analysis ...... 62

CHAPTER FOUR ...... 63

RESULTS AND DISCUSSION ...... 63

4.1 Plant essential oil yields ...... 63 4.2 Chemical compositions of essential oils of selected plants ...... 64 4.2.1 Chemical composition of essential oil of Satureja biflora ...... 64 4.2.2 Chemical composition of essential oil of Conyza newii ...... 68 4.2.3 Chemical composition of essential oil of Satureja abyssinica ...... 70 4.2.4 Chemical composition of essential oil of Plectranthus marrubioides ...... 72 4.2.5 Chemical composition of essential oil of Ocimum kilimandscharicum ...... 74 4.3 Analysis of essential oil chemical composition variability ...... 76 4.4 Larvicidal efficacies of essential oils against mosquito larvae ...... 78 4.4.1 Larvicidal efficacies of essential oils against mosquito larvae under laboratory conditions ...... 78 4.4.2 Dose response larvicidal activity ...... 79 4.4.2.1 Effects of lower doses ...... 81 4.4.3 Semi-field evaluation of O. kilimandscharicum mosquito larvicide formulation ... 82 4.5 Repellent activities of selected plant essential oils against mosquitoes ...... 84 4.5.1 Estimation of effective dose using Klun and Debboun Module ...... 84 4.5.2 Testing sensitivity of mosquito to insect repellent employing a warm body ...... 86 4.6 Estimation of protection time in WHO arm-in-cage tests ...... 87 4.7 Further improved formulation ...... 91 viii

4.8 Subtractive bioassay on the constituents of Satureja biflora and Conyza newii with significant contribution to mosquito repellence ...... 92 4.8.1 Summary on EAG-active compounds ...... 92 4.8.2 Electrophysiological responses of the constituents of essential oils to An. gambiae ...... 92 4.8.3 Repellence of S. biflora and C. newii essential oils and their EAG active blends .. 97 4.9 Discussion ...... 99

CHAPTER FIVE ...... 106

CONCLUSIONS AND RECOMMENDATION ...... 106

5.1 Conclusions ...... 106 5.2 Recommendations from this study ...... 108 5.3. Recommendations for future Research ...... 109

REFERENCES ...... 110

APPENDICES ...... 129 ix

LIST OF TABLES

Table 2.1: Traditional uses of O. kilimandscharicum ...... 35

Table 3.1: Sample locations and GPS information ...... 45

Table 3.2: Plant essential oils concentrations tested for repellence against Ae. aegypti,

An. gambiae s.s and Cx quinquefasciatus ...... 51

Table 4.1. Plant essential oils yields ...... 62

Table 4.2: Chemical composition of essential oil of S. biflora ...... 63

Table 4.3: Chemical composition of essential oil of C. newii ...... 68

Table 4.4: Chemical composition of essential oil of S. abyssinica ...... 70

Table 4.5: Chemical composition of essential oil of P. marrubioides ...... 72

Table 4.6. Chemical composition of the essential oil of O. kilimandscharicum ...... 74

Table 4.7: The LC50 and LC95 values of the essential oil extract of

O. kilimandscharicum and water miscible formulation against 3rd instar

larvae of An. gambiae under laboratory conditions ...... 78

Table 4.8: The LC50 values of O. kilimandscharicum essential oil against 3rd instar

larvae of An. gambiae, An. arabiensis, An. aegyptei and C. quinquifasciatus

mosquitoes after 24 and 48 hrs exposure under laboratory conditions ...... 79

Table 4.9: Larvidal activity of O. kilimandscharicum formulation Bti granules and

untreated control against An. gambiae mosquito larvae under semi-field

conditions in Kilifi Kenya ...... 85

Table 4.10: Effective dose (ED) results against Ae aegypti and An. gambiae s.s.

using the K and D module...... 87

Table 4.11: Protection time of formulated plant extracts measured as the complete

protection time (CPT) and the time during which protection was at least 90%.92

Table 4.12: Compounds present in S. biflora essential oil identified by GC-MS that

elicited electrophysiological responses in antennae of female An. gambiae. ... 95 x

Table 4.13: Compounds present in C. newii essential oil that elicited electrophysiological

responses in antennae of female An. gambiae...... 96

Table 4.14: RD50 and RD75 (95% CI) of the crude essential oils and synthetic repellent

DEET ...... 96

Table 4.15: RD50 and RD75 (95% CI) of C. newii oil, its selected electrophysiological

active synthetic blends...... 96

Table 4.16: RD50 and RD75 (95% CI) of S. biflora oil, its selected electrophysiological

active synthetic blends ...... 97

LIST OF FIGURES

Figure 2.1: Life cycle of a mosquito ...... 11

Figure 2.2: Distribution of the Culex Mosquito -Carrierr of West Nile Virus ...... 20

Figure 2.3: Distribution of Dengue fever -caused by the Aedes mosquito ...... 21

Figure 2.4: Distribution of the Anopheles - Carrier of Malaria ...... 21

Figure 3.1: Map showing sample collections location in Aberdare range region ...... 43

Figure 3.2: Map sample collection location in Kakamega forest ...... 43

Figure 4.1: GC profile of S. biflora essential oil ...... 64

Figure 4.2: The GC profile of C. newii oil ...... 67

Figure 4.3: Scatter plot of the study samples for the two principal components

extracted in the PCA to which cluster belongs ...... 75

Figure 4.4: Dendrogram representing chemical composition similarity relationships

among S. biflora (SB), S. abbysinica (SA), C. newii (CN) and

P. marrubioides (PM) leaf oil samples ...... 76

Figure 4.5: Larval developmental disruption by low dose of O. kilimandscharicum

water miscible formulation on An. gambiae s.s ...... 80

Figure 4.6: Larvicidal activity against An. gambiae mosquito larvae under semi-field

conditions in Kilifi, Kenya for a period of 8 days ...... 82

Figure 4.7: Essential oils Dose-response from K and D assay for Ae. aegypti ...... 84

Figure 4.8: Essential oils Dose-response from K and D assay for An. gambiae s.s...... 84

Figure 4.9: In vitro test for mosquito repellents using a warm body ...... 85

Figure 4.10: Complete protection time (CPT). Dots represent measurements of CPT for

each volunteer and formulation against Ae aegypti and Cx quinquefasciatus . 88

Figure 4.11: Complete protection time (CPT). Dots represent measurements of

CPT for each volunteer and formulation ...... 89

Figure 4.12: Relative repellency (median protection time) of volatile oils with and xii

DEET formulations with addition of Vanillin against 3 mosquito species

under laboratory conditions ...... 90

Figure 4.13: EAG track (upper one) and its GC Chromatogram (lowest one) recorded

from antennae of female An. gambiae in response to essential oil of S. biflora .. 92

Figure 4.14. EAG active compounds of essential oil components of S. biflora ...... 93

Figure 4.15: EAG track (lower one) and its GC chromatogram (upper one) recorded from

antennae of female A. gambiae in response to essential oil of C. newii ...... 93

Figure 4.16: EAG active compounds of essential oil components of C. newii ...... 94

Figure 4.17: Compounds of other minor EAG-active constituents of C. newii and S. biflora

plant essential oils ...... 95

xiii

LIST OF PLATES

Plate 2.1: An. gambiae s.s...... 9

Plate 2.2: An. arabiensis ...... 9

Plate 2.3: Anopheles (celliaI funestus Giles) ...... 9

Plate 2.4: Ae aegypti ...... 10

Plate 2.5: Cx. quinquefasciatus ...... 10

Plate 3.1: Plant collection and identification by a taxonomist at Kinale ...... 44

Plate 3.2: Harvesting of C. newii at Nyahururu ...... 44

Plate 3.3: P. marrubioides ...... 45

Plate 3.4: C. newii ...... 45

Plate 3.5: O. kilimandscharicam ...... 45

Plate 3.6: S. biflora ...... 45

Plate 3.7: S. abyssinica ...... 46

Plate 3.8: Hydro-distillation using Clevenger apparatus ...... 46

Plate 3.9: Drying oil using a rotor evaporator ...... 47

Plate 3.10: Schematic diagram of a GC-MS Equipment showing the sample analysis

components ...... 47

Plate 3.11(a): Gas Chromatography linked to Electroantennogram ...... 49

Plate 3.11(b): Schematic diagram of GC linked to Electroantennogram (GC-EAG) ...... 50

Plate 3.12 (a-b): Marking for K and D assay ...... 52

Plate 3.13: K and D bioassay set up ...... 52

Plate 3. 14: The warm body bioassay setup ...... 56

Plate 3.15: WHO ‘arm in the cage’ Mosquito cage...... 56

Plate 3.16 WHO arm in the cage exposure of the treated arm ...... 56

Plate 3.17: Semi-field bioassay set-up for evaluation of mosquito larvicides in Kilifi

District, Kenya ...... 60 xiv

LIST OF SCHEMES

Scheme 2.1: Terpenoid biosynthesis and Catalytic reactions of PTSs ...... 39

LIST OF APPENDICES

APPENDIX A: ETHICAL APPROVAL ...... 135

APPENDIX B: DISSEMINATION OF RESEARCH FINDINGS ...... 136

Appendix B1: Conferences -1 ...... 136

Appendix B2: Conference - 2 ...... 137

APPENDIX C: ABSTRACTS AND SUBMITTED MANUSCRIPTS ...... 138

Appendix C1: Abstract Manuscript 1: ...... 138

Appendix C2: Abstract Manuscript 2: ...... 139

Appendix C3: Abstract Manuscript 3: ...... 140

APPENDIX D: PATENT FILED ...... 141

APPENDIX E: STANDARD OPERATING PROCEDURES (SOPS) ...... 141

Appendix E1: The Klun and Debboun Module (K and D Module) for evaluation of

repellent efficacy using human subjects ...... 141

Appendix E2: SOP - Laboratory Testing of Formulated Mosquito Larvicides ...... 145

Appendix E3: SOP - Aliquots of various strength solutions added to 100 ml water

to yield final concentration ...... 150

Appendix E4: SOP - Laboratory evaluation of the efficacy of larvicides against mosquito

larvae ...... 151

Appendix E5: SOP - Laboratory evaluation of the efficacy of insect growth regulators

against mosquito larvae ...... 146

xvi

ABBREVAIATIONS AND ACRONYMS

ACTs Artemisinin-based Combination Therapies

AMCA American Mosquito Control Association

ANOVA Analysis of Variance

CN/NHR Conyza newii/ Nyahururu

CPT Complete Protection Time

DEET N, N-diethyl-meta-toluamide

EAD Electro Antennogram Detector

ED50 / ED99.9) Effective Dose

EO Essential Oils

FB Full Blend

FID Flame Ionization Detector

GC Gas Chromatograph

GC-EAD Gas Chromatography Electro-Antennogram Detector

GC-MS Gas Chromatography - Mass Spectrometer

HCA Hierarchical Cluster Analysis

ICIPE International Centre of Insect Physiology and Ecology

IRS Indoor Residual Spraying

ITNs Insecticide Treated Mosquito Nets

IVM Integrated Vector Management

LLINs Long-Lasting Insecticidal Nets

MEP Methylerythritol phosphate

MEV Mevalonate

MSDS Material Safety Data Sheet

NMK National Museum of Kenya xvii

PCA Principal Component Analysis

PCM Percent Control Mean

PE Protective Efficacy

PM/NVS Plectrunthus marrubioides/ Naivasha

PPM Parts Per Million

PTM Percent Test Mean

STPHI Swiss Tropical and Public Health Institute

SA/NHR Satureja abbysinica/ Nyahururu

SB/KG S. biflora/ Kieni Gakoe

SB/KNL Satureja biflora/ Kinale

WHO World Health Organization xviii

ABSTRACT Mosquitoes are of high public health concern since they are significant disease vectors of different tropical and subtropical life-threatening diseases like malaria, dengue, yellow fever, chikungunya, zika virus and encephalitis. Use of synthetic insecticides for control of mosquitoes causes development of resistance in vector species and have negative effects to the environment and human. This study aimed to find alternative, effective tools against these vectors from plant essential oils. Plant-based products are promising alternatives of low- toxicity, eco-friendly and low-cost. Oils from air-dried aerial parts of Satureja biflora, Satureja abbysinica, Conyza newii and Plectranthus marrubioides from Aberdare and Ocimum kilimandscharicum from Kakamega in Kenya were extracted using Clevenger apparatus. Oil yields were determined and analyzed by Gas Chromatography/Mass Spectrometry. Repellency of the oils was assessed on adult mosquitoes in ‘WHO arm in the cage’ method, while 3rd instar larvae were used to assess larvicidal activities based on WHO protocol. Oil with better larvicidal activity was formulated into a water-miscible solution for laboratory and semi-field testing. GC linked to EAG detector was used to determine the constituents that elicited chemosensory responses from the antenna of An. gambiae. Individual constituent contribution to mosquito repellence was established through a subtractive-combination bioassay. From the results, oils from different habitats and seasons showed qualitative and quantitative differences. Thirty three (33) compounds were identified in S. biflora oil with 3 chemotypes based on location: geranial(31%)/neral (24%)/linalool (12%) of Kinale, linalool (28%)/neridol (21%)/β- pinene (7.4%) of Nyahururu, and geranial (27%) neridol (Z) (21%)/linalool (16%) of Kieni- Gakoe. S. abyssinica had also 33 compounds comprising majorly menthone (44.1%) and pulegone (33.3%). C. newii had 19 components forming 2 chemotypes p-mentha-1,8-dien-7-yl acetate (24%)/limonene (23%)/5-methyl-2-phenyl-2-hexenal (21%) associated with Nyahururu, and p-mentha-1,8-dien-7-yl acetate (27%)/ limonene (38%). P. marruboides and O. kilimandscharicum had 35 and 41 components respectively. The major compounds in P. marrubioides were carene-2-δ (18.7%) and camphor (17.9%), whereas for O. kilimandscharicum was camphor (36.6%) and limonene (18.6%). C. newii (Nayahururu) S. biflora (Kieni) and S. biflora (Nyahururu) gave a high repellence of ED50 at < 1.95 ppm, whereas S. biflora (Kinale) had ED50 at < 2.35 ppm against Ae. aegyptei and An. gambiae. Besides, C. newii had strong repellence with ED50= 0.5 ppm against An. gambiae. Comparatively DEET gave the highest protection time of 389 min against An. gambiae, followed by C. newii (cream) against the 3 mosquito species; 241, 206 and 60 minutes for An. gambiae, Cx. quinquefasciatus and Ae. aegyptei respectively. S. biflora cream gave 208, 180 and 56 minutes against An. gambiae, Cx. quinquefasciatus and Ae. aegypti respectively. Addition of vanillin to the cream formulation resulted in a longer protection time of 382 min against An. gambiae. Nine EAG-active constituents were identified. Perillyl alcohol and α- pinene increased repellency, while neral, geraniol, perill aldehyde and cinnamaldehyde-α- pentyl reduced the repellency of the respective blends. Limonene and linalool interchangeably increased or decreased the repellence based on the resulting blend. O. kilimandscharicum oil showed the highest larvicidal effect against 4 mosquito species, with LD50 of 0.292 and 0.41ppm at 24 and 48 hrs respectively against An. gambiae larvae. The O. kilimandscharicum water miscible formulation recorded a LC50 of 0.13, 0.14, 0.16 and 0.13ppm against larvae of An. gambiae, Ae. aegypti, Cx. quinquefasciatus and An. arabiensis respectively. In a semi-field setup, the larvicidal formulation at 0.5ppm on day 8 attained 100% larval reduction when B.ti had 96.5%. The larvicidal and repellent results of the plant formulations generated in this study have demonstrated high potential for practical application in control of mosquito vector borne diseases and need to be deployed for large scale field trials and registered with the relevant bodies for adoption for control of mosquitoes.

CHAPTER ONE

INTRODUCTION

1.1 Background information

Mosquitoes are of key concern in public health since they are important disease vectors

(Riehle & Jacobs-Lorena, 2005).. They are vectors of many tropical and subtropical life- threatening diseases like malaria, dengue, yellow fever, chikungunya, Zika virus and encephalitis that lead to millions of worldwide infections annually subsequently causing morbidity, mortality, social disruption and economic loss among the population (Becker et al., 2003; Tuetun, et al., 2005, Singh, 2015). Mosquito bites also lead to allergic responses like local skin reactions and urticarial systemic reactions. (Sritabutra and Soonwera, 2013).

An. gambiae senso stricto has been singled out as the main vector associated with transmission of malaria in most of sub-Saharan Africa. In 2015, over 438 000 deaths were attributed to malaria (WHO, 2015). An. gambiae is a member of a complex species comprising of at least seven morphologically indistinguishable species. An. gambiae s.s is anthrophonic and one of the most effective malaria vectors known. Malaria, a vector borne disease caused by Anopheles mosquitoes is currently endemic in 96 countries. The malaria disease burden is high, with more than 500 million cases each year. It is estimated that almost half of the world population of approximately 3.2 billion people are at danger of contracting malaria (WHO, 2015). Besides lowering productivity in socio-economic activities, it causes masses of deaths, particularly to children under five years of age and expectant women (WHO, 2010). On the other hand, Ae. aegypti and Cx. quinquefasciatus are key urban vectors of dengue fever, dengue hemorrhagic fever, chikungunya and

Japanese encephalitis (Sritabutra, and Soonwera, 2013). Incidences of dengue have risen

30-fold worldwide in the past 30 years. Dengue virus haemorrhagic fever had been

eliminated in the mid-20th century in some regions but the same regions are now reporting occurrence of epidemics. Moreover, throughout the Americas, West Nile virus has become endemic (Reisen, 2013). Chikungunya virus affects millions in Indian Ocean basin and

Japanese encephalitis is widespread in the Indian subcontinent and Australasia. Filariasis was a focus of a global elimination campaign (Tolle, 2009). Currently Rift Valley Fever is predominant in Africa and the Middle East where it’s a source of sicknesses in human and domestic animals (LaBeaud et al., 2011).

Breeding environment for mosquitoes comprise water, moist surfaces, tree holes and containers (Jahn et al., 2010). Human activities intensify breeding areas by increase in debris and other material that hold water (Tolle, 2009). Mosquitoes life cycle include eggs, larvae, pupae and adults. Female mosquitoes lay eggs in their whole adult life although they only mate once. They get a blood-meal from vertebrates which is used for nourishment and egg development (Foster and Walker, 2002). Mosquitoes spread diseases when some pathogens complete their life cycles inside the mosquito and are passed onto humans during the time of biting (Tolle, 2009). Males do not suck blood but feed on plant juices (Foster and Walker, 2002).

Mosquito control can be realized by numerous ways: biological, physical and chemical methods (Jahn et al., 2010). Biological control comprises use of predators like fish that feed on larvae, parasites and/or pathogens. However, some predators can also feed on useful organisms and cannot be used in contaminated water and temporary aquatic areas such as puddles and vehicle grooves that form only in rainy periods (Maniafu et al., 2009). Vector resistance results where there is frequent use of chemicals for mosquito control (Maniafu et al., 2009). The persistence of chemicals in soil, plant and animal tissues causes death to fish

and other aquatic life (Matasyoh et al., 2008). Even though bed nets treated with insecticide are effective in regulating mosquitoes, they affect ventilation causing breathing difficulties

(Ogoma et al., 2010).

The fast spread of parasite resistance to drugs and vector resistance to synthetic chemical insecticides has also significantly increased the cost, and challenges of mosquito vector borne diseases management (Carter and Mendis, 2002; Korenromp et al., 2003; Ter Kuile et al., 2004; Burfield et al., 2005). The chemical methods for controlling mosquito disease vectors rely mainly on disruption of the disease transmission cycle by either aiming at the mosquito larvae through spraying breeding sites or by use of synthetic repellents such as

N,N,-Diethyl-toluamide (DEET) (1) for personal defense to lessen disease transmission

(Josepha et al., 2004). However, presently, the use of synthetic chemicals to control insects and arthropods raises several concerns related to environmental and human health (Nerio et al., 2010).

N 1

Plants have co-evolved with insects over long periods, thus attaining a variety of chemical protection mechanisms which can be considered for control of insect vectors and pests

(Arnason et al., 1989). Phytochemicals resulting from various botanical sources have provided several beneficial uses ranging from pharmaceuticals to insecticides. In this respect, over 2000 plant species have been described to have potential to yield chemical factors and metabolites of value in pest control programs (Roark, 1947; Arnason et al.,

1989; Sukumar et al., 1991, Hedlin. et al., 1997; Hikal et al., 2017). Members of the plant families including Solanaceae, Asteraceae, Cladophoraceae, Labiatae, Miliaceae,

Oocystaceae and Rutaceae have been reported to show various kinds of larval, adulticidal or repellent activities to counter different mosquito species.

Powdered chrysanthemum has been used by Chinese as an insecticide (Seyoum et al.,

2002). Moreover, fresh twigs of Ocimum species and Azadirachta indica A. Juss, have been reported to be positioned in corners of rooms or burnt to block mosquitoes from entering the houses in Tanzania (Moore and Lenglet, 2004). Furthermore, plant parts or mosquito coils made from dried plants are burned to repel mosquitoes (Seyoum et al., 2002). In 2005 it was reported that O. forskolei and O. fischeri essential oils exhibited greater repellent activity than DEET (Odalo et al., 2005), whereas, p-menthane-3,8-diol (PMD) (2), also known as Quwenling isolated from Eucalyptus essential oil, was repellent to mosquitoes and as effective as DEET (1) (Coetzee, 2000). In addition, citronella oil extracted from

Cymbopogon nardus (L.) Rendle is commercially used as a natural repellent for children since it is safer than DEET (Maia and Moore, 2011).

OH 2

Despite the knowledge concerning plants, there has been negligible endeavours to assess and encourage use of plant-derived agents with activity against mosquitoes. The present study was intended to generally assess repellent and larvicidal activity of the selected plant extracts against adult mosquitoes and larvae respectively. The effective extracts were formulated into safe, effective, and cheap mosquito repellent and a mosquito larvicide for use to control mosquito disease vectors.

1.2 Statement of the Problem

Mosquito–borne diseases have been shown to be re-emerging owing to numerous factors including resistance development to applied synthetic insecticides and change of mosquito behavior. The high dependence on synthetic insecticides also continues to raise environmental and public health. In some areas, the resistance is widespread, and has been reported for all classes of synthetic insecticides that WHO has permitted for public use

(WHO, 2015). Furthermore, the contrasting behavior of different mosquito vector species leads to disparity in the effectiveness of the diverse means in controlling local mosquito populations (Dekker et al., 2011).

1.3 Justification of the Study

Over half of the world’s population live in areas infested with numerous mosquito species that can transmit mosquito borne diseases. Control of mosquito transmitted diseases can effectively be realized through controlling the vector itself. Moreover synthetic-based products are costly with potential for being toxic and causing irritation, and other adverse side effects (WHO, 1996; Qiu et al., 1998). Therefore, there is an urgent need for discovery of other mosquito deterrents, repellents and/or spatial inhibitor and larvicides, especially those that are naturally occurring, more selective, available, of low cost and ecologically sound. This study is part of a bioprospecting effort to evaluate four plant species, Satureja biflora (Micromeria biflora) Buch.-Ham (Lamiaceae), Conyza newii Oliv. & Hier

(Asteraceae), Ocimum kilimandscharicum Gürke(Lamiaceae family) and Plectranthus marrubioides Hochst (Labiatae family) as sources of contact or spatial repellency and possible larvicidal activity. The study was designed to identify repellent essential oils and their constituents, develop effective larvicide formulation, establish the possible mode of actions of the components and describe the most viable test plant chemotype based on

location and season. It also explored and analyzed the repellent and larvicidal potential of

the volatile compounds and blends from the plants considering their chemical components

and their respective variation percentages based on abiotic and biotic factors. It aimed at

providing locally available easy-to-use effective repellent and/or larvicidal products with

good toxicological tolerance.

1.4 Significance of the study

Results generated from this study provide information on essential oils intraspecific

composition variations and their potential impact in downstream product development. The

experimental results bridge the information gaps on contact and space repellence of

essential oils of the plants studied and their larvicidal activities, which facilitated

identification of the optimum blend for repelling the target mosquito’s species and

effectively controlling the larval stages. The study lays down significant groundwork for

developing products that can be deployed in the control of the mosquito disease vectors

studied.

1.5 Hypotheses

(i) Epigenetic variations of plant essential oils are dependent on ecological growing

conditions.

(ii) Some plants may provide alternatives products to synthetic insecticides that are less

toxic, and environmentally safe for control of pests and disease vectors.

(iii) The bioactivity of some plant is through interference with normal physiological

processes of various development stages of a mosquito and constitute an effective

agent for control of the vector.

(iv) Mosquito repellence is attributed to individual and/or blend of compounds from the

plant essential oils.

(v) Appropriate formulations of plan-based products can enhance the bioactivity against

pests and disease vectors.

1.6 General objective

The main objective was to bio-prospect for phytochemicals from selected Kenyan plants for repellent and larvicidal activities with potential for mosquito control and determine their chemotypic variation and underlying mode of action.

1.6.1 Specific objectives

(i) To determine chemical constituents of essential oil of S. biflora, S. abysinnica, C.

newii, O. kilimandscharicum and P. marrubioides using GC-MS.

(ii) To assess the epigenetic variations of essential oil of S. biflora, S. abysinnica, C.

newii, O. kilimandscharicum and P. marrubioides.

(iii) To assess larvicidal activities of essential oils and formulated water-soluble products

of S. biflora, C. newii, O. kilimandscharicum and P. marrubioides on larval stage

of mosquitoes.

(iv) To evaluate the repellent activity of essential oils of S. biflora, C. newii, and P.

marrubioides collected from different habitats against An. gambiae s. s., Ae.

aegyptei, and Cx quinquefasciatus adult mosquitoes.

(v) To identify the most potent plant essential oils constituents of the selected plants that

make significant contribution to mosquito repellency through subtractive

bioassays.

(vi) To explore the modes of action of repellent constituents and blends and formulated

larvicidal essential oils on adult and larval stage of mosquitoes respectively.

1.7 Scope and limitation

The research study was confined on the mosquito repellent and larvicidal properties of the essential oils from C. newii, S. biflora, S. abysinica and P. marrubioides leaves. Clever apparatus was the favoured method in obtaining the essential oil. For repellence the oils and their resulting formulations were used in comparison with the standard commercial repellent 20% DEET against 3 mosquitos’ species; An. gambiae, Ae aegyptei and Cx. quinquefasciatus. Using Klun and Debbourn dose response and WHO -arm in the cage method the percentage repellence and Complete Protection Time was recorded. As for the larvicidal activity, the oils were tested for their bioactivity against 3rd instar larvae of An. gambiae, An. arabiensis, Ae. aegyptei and Cx. quinquefasciatus using WHO laboratory and semi-field larvicidal evaluation methods. GC-MS, GC-EAG analysis were utilized to characterize the constituents of the essential oils. This research did not deal with the other activities of the plant material but rather limited to its mosquito repellent and larvicidal properties. The bioactivity for S. abyssinica was not evaluated since the oil obtained was only enough for analysis and not for bioassay.

CHAPTER TWO

LITERATURE REVIEW

2.1 Mosquitoes as vectors of diseases

Mosquitoes belong to the order Diptera, suborder ematocera. This suborder also includes other vectors like midges, sand-flies, and blackflies. Mosquitoes are in the family Culicidae which is divided into three subfamilies: Anophelinae, Culicinae, and Toxorhynchitinae. The family Culicinae includes the genera Culex, Aedes, and Mansonia (Gwadz and Collins,

1996). The three most significant malaria vectors in Africa are An. gambiae s.s (Plate 2.1),

An. arabiensis (Plate 2.2) and An. funestus (Plate 2.3).

Plate 2.1: An. gambiae s.s. Plate 2.2: An. arabiensis

Plate 2.3: Anopheles funestus Giles

These mosquito species are extensively dispersed in tropical Africa and other parts of the world (Manson-Bhar and Bell, 1987). Some 60 species of Anopheles are important vectors of human malaria. Ae. aegyptei (Plate 2.4), the yellow fever mosquito can spread dengue fever, chikungunya, Zika fever, Mayaro and yellow fever viruses, and other disease agents.

Whereas Culex is a genus of mosquitoes, several species of which include Culex quinquefetaus (Plate 2.5), which serve as vectors of one or more important diseases of birds, humans, and other animals.

Plate 2.4: Ae. aegyptei Plate 2.5: Cx. quinquefasciatus Credit: James Gathany, CDC, Public Health Image Library (https://phil.cdc.gov/)

Both sexes of mosquitoes feed on nectar and other plant fluids, but the female mosquito sucks blood from mammals including man. Female mosquitoes require at least one blood meal before oviposition. Most Anopheles species are nocturnal. Endophilic mosquitoes rest indoors while exophilic mosquitoes prefer outdoor feeding (Garret-Jones et al., 1980).

Some species are both anthropophagic and zoophagic (Garret-Jones et al., 1980). Recent research has revealed the presence of Plasmodium parasites in apes and monkeys. These are thought to act as reservoirs for the Plasmodium parasites thus further complicating the malaria control equation. Diseases that are spread by mosquitoes are passed from person to person through the bite of an infected mosquito. The parasite is passed into the blood stream, where it travels to the liver and multiplies. From the liver, the parasite passes again

through the blood stream and attacks the red blood cells. It ruptures the cells causing malaria symptoms (Manson-Bhar Bell, 1987). Much of the long-standing morbidity from malaria is attributed to the anemia it causes.

2.2 Biology and life cycle of a mosquito

The larval habitats for mosquitoes are shallow bodies of water with little or no water movement. They reside in shallow pools, swamplands and water filled tree holes. Most species are adapted to fresh water, but a few species live in salt marshes or inland saline pools. When a larva hatches from the egg (Figure 2.1), it adapts to live in the water.

Figure 2.1: Life cycle of a mosquito (Illustration by: Scott Charlesworth, Purdue

University

The larvae use atmospheric oxygen to breathe and feed on aquatic microorganisms such as bacteria, diatoms, algae and particles of decayed plant tissues. Anopheline larvae lie horizontally at the surface of the water where the dorsal surface and the spiracles of the abdomen are in contact with the air (Clements, 1992). Anopheline larvae tend to hover horizontally at the surface of the water when not in motion. However, culicinae larvae float with head low-slung and only the siphon at the tail is held at the surface. The larva molts four times. During the pupal stage, adult organs are constructed (Nasci and Miller, 1996).

When the adult is completely developed inside the pupal cuticle, the insect starts to

'swallow air', which raise the internal pressure triggering a split along the cuticle. The adult emerges out of the pupal cuticle onto the water surface (Clements, 1992).

Adult mosquitoes are fluid feeders. Their mouthparts consist of a proboscis suitable for feeding on nectar. Both males and females use sugars from plant juices, nectar and honeydew as an energy source. Similarly, male and female mosquitoes become sexually active approximately 2-3 days after emergence. Mating usually occurs when a female enters a swarm of flying males. Female mosquitoes need protein to develop egg batches, and they feed on vertebrate blood.

Blood meal provides the protein needed for egg production. Digestion of blood meal results in amino acids that are kept in the mosquito's fat body as proteinaceous yolk. The yolk is then transported to the ovaries and oocytes. When a female has mature eggs, she searches for a suitable oviposition site (Clements, 1992; Roitberg and Friend, 1992).

2.3 Economic importance of mosquitoes

Blood sucking insects transmit many important arthropod-borne pathogens. A variety of pathogens and parasites, including viruses, protozoa and nematodes have been isolated from mosquitoes (Niangi et al., 2018). Some of these organisms are entirely parasitic, and others have alternate life cycles between parasitic and free-living phases. Mosquito-borne diseases are caused by bacteria, viruses or parasites spread by the insects. They comprise: malaria, dengue, West Nile virus, chikungunya, yellow fever, (AMCA, 2018), filariasis, tularemia, dirofilariasis, Japanese encephalitis, Saint Louis encephalitis, Western equine encephalitis, Eastern equine encephalitis, Venezuelan equine encephalitis, Ross River fever,

Barmah Forest fever, La Crosse encephalitis, and Zika fever ( AMCA, 2018).

2.4 Host-seeking behaviour of mosquito

Mosquitoes trace their hosts by means of heat, moisture, visual and olfactory cues (Eiras and Jepson, 1994; Service, 2000). For the later, the volatile chemicals emanating from hosts are detected by the mosquito’s antennae and maxillary palps (McIver, 1982). The chemicals, that alter the behaviour of the insect when perceived, are referred to as semio- chemicals (Takken, 1991; Takken and Knols, 1999), and the chemicals used by mosquitoes to discover their host are kairomones, for example compounds which profit the receiver (the mosquito) to the disadvantage of the organism discharging the compound (the human host).

The capability of a mosquito to detect kairomones from hosts has a direct consequence on its ability to spread the pathogens that cause disease (Zwiebel and Takken, 2004; Bohbot et al., 2007).

Several semio-chemicals that are released by vertebrates and fascinate mosquitoes have been identified and one of the most significant, which is associated with mosquito flying action, is carbon dioxide (CO2) (3) (Takken and Kline, 1989; Eiras and Jepson, 1991).

O C O 3

Nevertheless, CO2 from breath accounts for only 50% of the attraction to hosts in exceedingly anthropophilic species (Costantini et al., 1996), demonstrating that other chemical cues from the host must play an important role in olfactory host-seeking behaviour

(Costantini et al., 1998). An. gambiae are attracted to volatile chemicals given off in odors from human skin (Maibach et al., 1966; Mayer and James, 1969; Schreck et al., 1990).

The human body produces about 350 volatile compounds (Bernier et al., 2000), and many of which have been attractive to mosquitoes (Geier et al., 1996; Bernier et al., 2002; Logan et al., 2008). Mosquitoes demonstrate partialities for certain distinct human hosts, which might be owed to difference in the proportions of these attractive chemicals (Qiu et al.,

2006; Williams et al., 2006; Logan et al., 2008). For some of the chemicals found in human sweat, for example geranyl acetone (4), 6-methyl-5-hepten-2-one (5), octanal (6), nonanal

(7) and decanal (8), people with greater levels than normal are repellent to mosquitoes, and these compounds resulting from human can be used as mosquito repellents (Logan et al.,

2008). One of the significant ways to manage insects is to manipulate their host-seeking capabilities, either by disturbing their recognition of attractants, or by instigating direct repellency. However, mechanism by which mosquitoes perceive, analyze and act upon chemical signals is still not well understood, and this is a vital subject to explore further.

O 4 O 5

O 6 O 7 O 8

It has been recommended that chemo-stimuli such as body odors and carbon dioxide stimulate mosquitoes, and spatial temperature variances caused by the hosts guides movements at short distances (Bos and Laarman, 1975; Bar-Zeev et al., 1977). Host odors are may be long-range factors. Studies have revealed that CO2 can entice mosquitoes to traps in the field.

Body odor and CO2 (3) passed in the wind arouse the receptors on the antennae of the female mosquitoes. When they are near the host, visual cues and convection streams of the host help find the host (Allan et al., 1987; Clements, 1992). The main olfactory signals involved in host-seeking are CO2 (3), octanol (9), lactic acid (10), butanone (11), acetone

(12), and phenolic components of urine (Bowen, 1991; Klowden, 1996; Lehane, 1991).

Studies have been undertaken on human blood, sweat, urine and CO2 (3) and many other compounds (Parker, 1948; Gouck and Bowman, 1959; Skinner et al., 1965). Skin lipids were found to be repellent to mosquitoes (Skinner et al., 1965). Urine fractions, especially steroids, were found to be attractive, even at low concentrations. L-lactic acid (10) is known to be an attractant to mosquitoes. Acree et al., (1968) showed that 1g of L-lactic acid (10) attracted 29-75% of the mosquitoes in 3 min. It was also found that L-lactic acid was 5 times more attractant than D-lactic acid.

O O HO OH 9 OH 11 12 10

Mosquitoes have been observed to respond to floral odors in flight and landed on the flowers in the absence of visual stimuli. This discovery suggests that mosquitoes can sense floral odors in long-range location of floral nectar sources (Healy and Jepson, 1988).

Besides CO2 (3), additional kairomones influence host selection. It has been observed that, in still air, biting occurs preferentially on ankles and feet of a bare immobile human host.

This preference was linked to combinations of 13 skin temperature and eccrine sweat gland densities. The response of female An. gambiae to Limburger cheese, which has a strong

'foot' odor, was tested in a wind tunnel olfactometer. Mosquitoes were attracted to the odor of non-human source, and this strongly indicated that bacteria are accountable to produce some odors involved in host seeking of this species (Knols and Jong, 1996).

2.5 Mosquito vector borne disease burden

Over the centuries, the mosquito vector-borne diseases are imposing a grave public health danger to humankind in terms of illness and deaths worldwide. In addition to their undesirable public health impact, these diseases are also posing serious impediment to socio-economic development in countries wherever they are rampant in nature

(Karunamoorthi and Sabesan, 2010). Malaria remains a major global public health threat with 3.2 billion people at risk in 96 endemic countries (WHO, 2015). It leads not only to severe public health impact but also negative socio-economic development in the resource- poor backgrounds of Africa, Asia, Latin America and beyond (Karunamoorthi and Bekele,

2009). It remains as one of the significant causes of maternal and childhood morbidity and

mortality in sub-Saharan Africa. It also contributes to stillbirths, low birth weight, and early infant mortality (Menendez, 1995).

The WHO Malaria Report (2015) approximates that malaria associated illness and mortality have been intensely reduced by the joint efforts of supply of long-lasting insecticide treated mosquito nets (LLINs), effective case management with effective anti-malarial, source reduction and selective intradomicile spraying. However, the heavy dependence on synthetic pyrethroids has led to the emergence of resistance in a wide variety of malaria- endemic settings of Africa and the rest of the world. This is well-thought-out, to be a major threat to the global public (Karunamoorthi and Sabesan, 2013).

2.5.1 Malaria

Anopheles mosquitoes are the principal vector of malaria. These mosquitoes bite mostly at night, from dusk to dawn. Children of less than five years and expectant women are at a higher risk of disease in countries with higher rates of malaria transmission. In 2015, more than 3.2 billion people were at risk, and current malaria transmission was found in 95 countries and regions (WHO, 2015). Sub-Saharan Africa carries an unreasonably higher portion of the global malaria burden with 92% of cases and 93% of global malaria deaths

WHO, 2018).

Four different species of Plasmodium produce infections in humans: P. vivax (Tertian), P. ovale (Mid Tertian), P. falciparum (Malignant Tertian) and P. malariae (Quartan). P. falciparum is the greatest prevalent parasite in Africa and is accountable for most malaria deaths globally. The female Anopheles mosquitoes is an effective vector that likes tropical, rural, and urban situations. Malaria infections are common amongst disadvantaged groups

of people that have recurrent contact with high anopheline populations (Wagner, 1992).

Anopheles that carry P. vivax are found generally outside Africa.

2.5.2 Dengue

The Ae. aegypti mosquito remains the main vector of dengue. The virus is conveyed to humans through the bite of an infected female mosquito. Ae. aegypti bites generally during the day. Female Ae. aegypti, and Ae. albopictus are found in Latin America, the United

States of the America, Europe, Africa and Asia. Dengue is prevalent throughout the tropics, in countryside and city areas. After a drop in the number of cases in 2017-18, sharp increase in cases is being observed in 2019. In the Western Pacific region, increase in cases have been observed in Australia, Cambodia, China, Lao PDR, Malaysia, Philippines, Singapore,

Vietnam while Den-2 was reported in New Caledonia and Den-1 in French Polynesia.

Dengue outbreaks have also been reported in Congo, Côte d’Ivoire and Tanzania in the

African region. Several countries of the American region have also observed an increase in the number of cases. An estimated 500,000 people with severe dengue requires hospitalization each year, and with an estimated 2.5% case fatality, annually. However, many countries have reduced the case fatality rate to less than 1% and globally, 28% decline in case fatality have been recorded between 2010 and 2016 with significant improvement in case management through capacity building at country level

(WHO, 2019).

2.5.3 Chikungunya

The female Ae. aegypti and Ae. albopictus causes Chikungunya which is found in over 60 countries in Asia, Africa, Europe and the Americas (WHO, 2013). Mostly they are found biting outdoors, but Ae. aegypti likewise feeds indoors. Mosquito breeding habitats that are

adjacent to homes are a substantial danger factor for chikungunya. Numerous Chikungunya epidemics have been reported in several countries in Southern and South East Asia. Distinct strains of Chikungunya virus within varying transmission cycles have been reported from different locations. The African variant has managed to persist over the years with frequent outbreaks due to a sylvatic cycle maintained between monkeys and wild mosquitoes.

Conversely, the Asian variant causes epidemics that are maintained by an urban cycle, characterized by long inter-epidemic quiescence for more than 10 years or so (CDC, 2019).

2.5.4 Yellow fever

Yellow fever is primarily spread through the bite of the Ae. aegypti mosquito. Yellow fever is found in Africa, Latin America, in urban, jungle, forest, semi-humid conditions and around houses (WHO, 2013). Mosquitoes infect monkeys and then go on to transmit the disease from monkeys to humans, and from human to human. Haemagogus species occurs in Central and South America, Trinidad, Brazil, and Argentina, and transmit the sylvan or

"jungle" yellow fever, carried by monkeys (Goenaga et al., 2012).

2.5.5 Zika virus disease

Epidemics of Zika virus disease have been documented in Africa, America, Asia and the

Pacific. Female Ae. aegypti and Ae. albopictus that transmit Zika are found in over 130 countries. Aedes bites habitually during the day. There has been a sharp increase in local

Zika transmission in the America; since 2015, 62 countries and territories reported mosquito transmitted Zika virus (Mishra and Behera, 2016). Mosquitoes infect persons and in turn can infect each other through sexual contact. Zika has been spotted in blood, saliva, semen, spinal and other body fluids. Mother to child transmission in early pregnancy has also been reported (Epolboin et al., 2017).

2.5.6 West Nile virus

West Nile virus is transmitted by female Culex. The Culex also transmits Japanese encephalitis. It is 1 of 3 most common mosquitoes to be found globally, excluding for the extreme northern parts of the temperature zone. West Nile virus is present in Africa,

Europe, the Middle East, North America and West Asia (WHO, 2013). Yellow fever virus is estimated to cause 200,000 cases of disease and 30,000 deaths each year, with 90% occurring in Africa. 20% to 50% of infected persons who develop severe disease die. Yellow fever virus is transmitted to people primarily through the bite of infected Aedes or Haemagogus mosquitoes (https://www.cdc.gov/globalhealth/what/default.htm).

2.6 Distribution of mosquito borne diseases

Distribution of vector-borne diseases is determined by complex demographic, environmental and social factors and neighbourhood disease intensity variables that influence emergence dynamics Figure: 2.2, 2.3 and 2.4.

Figure 2.2. Global distribution of the Culex Mosquito -Carrierr of West Nile Virus : (WHO, 2013)

Figure 2.3: Distribution of Dengue fever -caused by the Aedes mosquito (WHO, 2013)

Figure 2.4: Distribution of the Anopheles - Carrier of Malaria ( WHO, 2013)

2.7 Control of mosquito vectors

Vector control is one of the most commonly used effective measure to prevent mosquito transmitted diseases, when coverage is sufficiently high. Such control aims at eliminating mosquitoes capable of spreading pathogens and parasites. Vector-control tactics are developed over an integrated vector management (IVM) strategy which pursues to better the efficacy, cost-effectiveness, ecological soundness and sustainability of regulating disease vectors. Integrated Vector Management (IVM) includes environmental management, chemical and biological control and personal safety.

2.7.1 Chemical control of mosquitoes

Mosquitoes are controlled using chemicals mainly by use of insecticides and larvicides to destroy the adults and larvae respectively. Larvicides are the most generally used method of mosquito management (Service, 2000), eradicating the insects before they develop into

adults and be able to spread diseases or reproduce. The regularly used larvicides are synthetic organophosphates, for instance temephos (13), and carbamates like propoxur (14).

Individuals are hesitant to pollute drinking water with chemicals whereas environmentally safe substitutes like growth regulators are relatively expensive. Furthermore, larvicides have not so far been completely established and employed entirely for all mosquito species

(Morrison et al., 2008).

N O MeO OMe O O H p p O MeO OMe S S S

13 14

There are a variety of insecticides existing to target adult mosquitoes, these comprise organochlorine compounds (such as dichlorodiphenyltrichloroethane, DDT), organophosphates, carbamates, pyrethroids, neonicotinoids and biological pesticides.

However, most of the non-biological pesticides are highly toxic to wildlife and persist in the environment. Thus, currently the only pesticides endorsed for use by the WHO are pyrethroids, which have small mammalian toxicity and minimum persistence (WHO,

2005). The concentrations of pyrethroids for domestic use also display irritancy and repellent properties, even in pyrethroid-resistant strains, but little is known of the mechanisms responsible for these behaviours (Chareonviriyaphap et al., 2004; Grieco et al., 2007; Mongkalangoon et al., 2009). The use of pyrethroid-treated bednets is extensive, since they guarantee complete protection from mosquitoes of the person inside. Damaged nets may however provide a shield since they kill mosquitoes on contact. Even though the use of Insecticides and Insecticide Treated bed Nets (ITNs) has remained very successful

in some areas in regulation of mosquitoes, extensive resistance against synthetic pyrethroid has been developed rendering them ineffective (Hemingway et al., 2004).

The use of ITNs for the last decade has been revealed to be an effective instrument to control childhood mortality and morbidity due to malaria. Their distribution and use for the successful control of An. gambiae has been reported in five large scale studies, carried out in four African countries, Gambia, Ghana, Burkina Faso and Kenya (Lengeler, 1996 a,b), providing renewed hope for the development of alternative malaria control tools. In

Gambia an overall mortality reduction of 63% was observed (Alonso et al., 1991). In the coastal Kenya where transmission pressure was in the order of 10-30 infectious bites per person per year, and exposure of the population was high, reduction of overall mortality was in the order of 3% (Nevill et al., 1996). In Ghana, with the highest transmission pressure of 100-300 infectious bites per person per year and coverage, overall mortality reduction was in the order of 17% (Binka et al., 2007).

The most extensively marketed synthetic insect repellent is DEET (1) which has been used worldwide since 1957. It is presently effective and obtainable in commercial formulations for example solutions, lotions, gels, creams, aerosol sprays, sticks or permeated towelettes.

However, there are problems of cost, logistics and toxicity (Qiu et al., 1998; Nentwig,

2003). Cases of resistance against DEET (1) have also been reported (Klun et al., 2004;

Stanczyk et al., 2010).

Resistance by mosquitoes is in most classes of insecticides. Meanwhile, by 2010 mosquito resistance to insecticides used in public health has been recognized in 60 nations around the globe. Nevertheless, many of the countries affected do not regularly carry out satisfactory

testing, which means that the comprehension of the level of insecticide resistance is inadequate.

2.7.2 Environmental control

Environmental management of mosquitoes comprises of the elimination or monitoring of vessels of abandoned tyres which would provide oviposition spots. This can also be achieved through modification of appropriate mosquito habitats such as marshlands or pools by emptying them, presenting predatory fish, or allowing water flow so that still pools cannot form (Service, 2000). Difficulties with environmental control comprise complications in upkeep, necessitating teaching on health and proper communication with local people (Erlanger et al., 2008). This is done even though modification of local environment might lead to unwanted increase in plenty of diverse mosquito species

(Service, 2000).

2.7.3 Biological/Genetic Control

There are several studies on the genetic manipulation of mosquitoes, for instance by varying their lifetime. With the manipulation they are expected to live less longer to spread disease

(Corby-Harris et al., 2010). The most common technique of genetic control is the use of sterilized males into the wild, which will mate with females but not fertilise their eggs

(Lofgren et al., 1974). This method has only functioned successfully alone in extremely remote areas such as islands, perhaps because of high genetic variability (Walter et al.,

1978) and the movement of insects (Bailey et al., 1980). Therefore, the high cost and the struggle in producing enough males to contend with wild mosquitoes (particularly as they can have an inferior suitability) makes it impractical as an impartial control technique, and

it is therefore frequently suggested as part of a combined control plan together with other control measures (Townson, 2009).

2.7.4 Vector Resistance

Although some remarkable success of malaria control by use of vector control methods has been registered in Africa (Bradley, 1991) and in India (Sharma, 1991), these methods and efforts have not provided a lasting solution to malaria prevalence in the tropics. Malaria has proved to be a disease with a complex parasite life cycle, whose control has been found to be extremely difficult. This owes to the fact that mosquitoes have vast breeding sites in the wide and favorable African environment in which the malaria parasite vector thrives.

Matters have become worse with the resurgence of vector resistance to insecticides, which is reported to be spreading at an alarming rate in Asia (Reacher et al., 1981; Kuile, 1994) and Africa (Kihami and Gill, 1981; Irare et al., 1988; Bloland et al., 2001. At present, synthetic pyrethroids (SPs) are the most commonly used anti-mosquito agents on ITNs.

However, even though bed nets are now available in many rural areas, the insecticide is not.

Under-dosing and untimely re-impregnation may increase the risk of susceptibility to malaria parasites.

Development of resistant strains of mosquitoes and the public fear of environmental hazards of the insecticides themselves is a greater barrier to the success of malaria elimination.

Steadily rising costs of materials and labour have also made current control efforts increasingly difficult.

Mosquitoes need and obtain important resources from the surrounding location to attain their full lifecycle. The acquired resources, sequentially, enable spread of P. falciparum

parasites to humans. These resources comprise water breeding spots, carbohydrate sugar sources, blood hosts, and resting habitats which impact the capacity of mosquitoes to spread malaria parasites. Entomological observation studies direct that advent of behaviourally resistant and antagonistic vectors that escape beset killing of IRS and Long-Lasting

Impregnated Nets (LLINs) has led to high rates of out-of-doors P. falciparum spread in diverse epidemiological areas (Gatton et al., 2013; Durnez et al., 2013). As such, interruption of malaria transmission would involve integrative intercessions that deter mosquitoes from obtaining these resources. Consequently, in addition to the first line intercessions (IRS and LLINs), use of larvicides and the mosquito olfactory system appears to be the targetable weak point (Killeen et al., 2002) that could be discovered to significantly advance control of vector populations and malaria vector annual inoculation rates.

Thus, the evaluation of the potential of using local plants as repellents, larvicides, insecticides or natural insect growth regulators (IGRs) will contribute novel approaches in providing cheap and reliable control strategies for mosquitoes in the rural communities.

2.8 Use of Natural products in Mosquito Control

Naturally occurring bioactive compounds which may be less likely to cause ecological damage, provide a multifactorial selective pressure that slows the development of resistance in pests (Arnason et al., 1989). Mostly, plant-based insecticides are target-specific and comparatively non-toxic. (Duke et al., 2010).

The plant world offers a rich unexploited pool of phytochemicals that could offer uses in place of synthetic insecticides in the management of disease vectors and plant pests

(Kishore et al., 2011). Constituents of phytochemical blends affect insect physiology in diverse ways and at numerous receptor sites, the major of which is irregularity in the nervous system. Thus, it takes extended time for insects to build resistance to such a blend of bioactive compounds, because they have broad modes of action and work synergistically

(Singh, 2014). Although, numerous studies have documented the effectiveness of plant extracts as the reservoir pool of bioactive toxic mediators against mosquito larvae and adults, a limited number have been steered downstream, commercially produced and used in vector control programs.

Therefore, there is a renewed attention observed by numerous researchers to develop green pesticides with minimum risks. Thus, thousands of plant species have been assessed as possible larvicides, antifeedants, repellents, oviposition deterrents, growth regulators and for anti-vector activities (Karunamoorthi, 2012). It is important to note that in contrast to synthetic repellents, alternative repellents from native plants of good efficacy and environmentally friendly, have the advantages of minimal cost, local availability and no documented cases of resistance (Dekker et al., 2011).

2.8.1 Mosquito Repellents

Repellents are chemical materials that act nearby (contact) or at a distance (spatial), preventing an arthropod from flying to, landing on or piercing human or animal skin (or a surface in general) (Blackwell et al., 2003; Choochote et al., 2007). Typically, insect repellents work by providing a gaseous barricade preventing the arthropod from meeting the surface (Brown and Hebert, 1997).

Integrated vector control, mixing most of the above methods, is measured as the most successful way of controlling mosquito populations (Service, 2000). A vital element of this is personal defence, such as the use of screens, bed nets and repellents, to guard against mosquito bites. The use of ITNs and repellents developed from plants during the hours when mosquitoes are very active has been acknowledged to have led to decline in spread of malaria by up to 80% (Hill et al., 2007). This demonstrates that these approaches can be effective in the control and spread of malaria.

The use of plants as insect repellents has existed more than 2000 years ago (Nentwig,

2003). In countryside communities of Africa, plants have been used for individual protection in form of lotions of entire plant oil or formulations containing the repellent oil

(Božović and Ragno, 2017). In addition, they have been used as space adulticides by through burning of plant material, warm air expulsion using burning surfaces and hanging repellents plants in houses to repel mosquitoes (Seyoum et al., 2003, Pino et al., 2001).

Equally, in many cases, the peels, oils or scents of citrus fruits are applied by the animals to their integument or skin for the purpose of deterring ecto-parasites. The major or chief component of these citrus fruits is limonene (4-isopropenyl-l-methyl-cyclohexane) (15) generally more than 55% and usually about 95%-99% in the peel of these fruits.

From this it was assumed or inferred that limonene (15) might be an effective repellent or spatial inhibitor for mosquitoes. However, when limonene (15) was tested against mosquitoes, it was not found to be either an effective repellent or spatial inhibitor (Feinsod and Spielman, 1979). As such, repellent action of essential oils may be credited to specific compounds, while a reinforcing phenomenon amongst these metabolites may give a higher bioactivity in comparison to the individual components (Omolo et al., 2004; Odalo et al.,

2005; Pohlit et al., 2011). For the last 15 years there has been considerable advancement in developing alternate insect repellents as well as other chemicals that belong in the amide, piperdine, diol, and terpene grouping. The innovative repellent compounds endorsed by the

Center for Disease Control and Prevention that belong to these classifications comprise

IR3535, picaradin, and p-Menthane-3,8-diol (2). There has also been adva ncement in the study of repellent chemistry mostly in the less-studied chemical classes like piperdines and terpenes.

Subsequent to the development of DEET (1), there has been curiosity in having insight in repellent mode of action and how repellents work (Davis, 1985). In order to elucidate the mode of action of a chemical repellent, it is essential to initially identify which component of the of insects’ behavior is susceptible to an exogenous stimulus, or candidate repellent, and then deduce this behavior with information of how these kind of compounds can influence insect olfactory mechanisms (Davis, 1985). Most olfactometers used in the bioassays for assessing repellents generally do not quantity mosquito repellency, but rather quantify non-attraction. Hence, it’s imperative to engage olfactometer that can split host- seeking behavior of mosquitoes into attraction and repellency at adjacent range to be able to measure true repellency. There is also an innovative term to the jargon of host-seeking behavior, that is 'inhibition', which happens when at least two chemicals combine leading to

failure of mosquitoes to respond to an earlier attractant inducement which needs to be investigated (DeGennaro, 2015).

Enhanced knowledge of the volatile compounds produced by plants could improve their use in the field situations. It may also lead to the identification of novel compounds with potential for development as synthetic repellents, as well as deciphering the olfactory mechanisms underlying repulsion. In this work, the repellent activity, synergistic phenomenon of botanical repellents, sesquiterpenes, is investigated, including a spectrum of activity and toxicity. Further, efforts to explain how sesquiterpenes impact insect orientation behavior is addressed in this study.

2.8.2 Larvicides

Considerable investigation has been undertaken on the use of botanical by-products against mosquitoes, and more than 344 plant species have been itemized that show mosquitocidal activity (Mittal and Subbarao, 2003; Sukumar et al., 1991; Das et al., 2007). Studies have also been reported on natural plant products as larvicides against mosquito vectors

(Sukumar et al., 1991; Das et al., 2007; Anupam et al., 2012; Gutierrez et al., 2014).

Numerous studies have been reported on the larvicidal properties of plant essential oils against Anopheles mosquitoes. Larvicidal activity of essential oils extracted from leaves of

Azadirachta indica and rhizomes of Curcuma longa were confirmed against An. gambiae

(Ajaiyeoba et al., 2008; Okumu et al., 2007). Whereas essential oils of Eucalyptus camaldulensis, Plectranthus amboinicus, Zanthoxylum armatum, Eucalyptus tereticornis and Tagetes patula were found to possess larvicidal activity against An. stephensi (Cheng et al., 2009). Further studies were designed to shed more light on the mechanism of essential

oil’s action/physiological disturbances with mosquito larvae (Pugazhvendan and Elumali,

2013).

2.9 Plants used in the study

2.9.1 The lamiaceae family

Lamiaceae, formerly called Labiatae, the mint family of flowering plants, has 236 genera and more than 7,000 species, and is the largest family of the order Lamiales. Lamiaceae is spread almost worldwide, and several species are grown for their scented leaves and beautiful flowers. The family is mostly significant to humans as source of herbal plants, valuable for flavour, fragrance, or medicinal characteristics (Raja, 2012). The majority of members of the family are perennial or yearly herbs with square stems, however some species are woody shrubs or subshrubs. The leaves are naturally simple and oppositely organized; most are fragrant and comprise volatile oils. The flowers are typically arranged in bunches and feature two-lipped, open-mouthed, tubular corollas (united petals) with five- lobed bell-like calyxes (united sepals). The fruit is usually a dry nutlet (Raja, 2012).

2.9.1.1 The genus Satureja

The genus Satureja L. (savory) belongs to the family Lamiaceae (Labiatae), subfamily

Nepetoidae and tribe Mentheae. The Satureja spp. are annual or perennial aromatic herbaceous plants with dark green or grey greenish leaves which grow in the arid, sunny, stony and rocky environments. The species grow in temperate regions up to 45 - 60 cm high. They grow in well-drained neutral alkaline dry soil (Momtaz and Abdollahi, 2008).

This genus comprises about 200 species of aromatic herbs and shrubs, mainly spread in an area extending from the Mediterranean region to Europe, West Asia, tropical Africa, Africa, the Canary Islands, and South America. More than 30 species of this genus are spread in

eastern parts of Mediterranean area (Momtaz and Abdollahi, 2008). Exclusively dried parts and characteristically, above ground parts comprise medicinal values and are used as medicines (Momtaz and Abdollahi, 2008). They are primarily aromatic herbs and shrubs, some of which are used as flavoring agents and some for medicinal use. Among the

Satureja species which occur in Kenya (Matasyoh et al., 2007) has been investigated under screen house conditions. The composition of many Satureja oils has been reported

(Matasyoh et al., 2007).

2.9.1.1.1 Biological studies and clinical properties of Satureja Species

Many plant parts of the majority of Satureja species are traditionally used to treat patients with various diseases and difficulties (Momtaz and Abdollahi, 2008). Several Satureja species have been pharmacologically assessed for the existence of diverse classes of metabolites and their ethnomedical and ethnopharmacological uses. A total of 13 Satureja species have been assessed pharmacologically and that S. khuzestanica, S. bachtiarica, S. montana and S. hortensis seemed to be the most active, both clinically and phytopharmacologically (Momtaz and Abdollahi, 2008). Most assessment of the plants have concentrated on the antimicrobial properties. Nevertheless, the antioxidant, antidiabetic and anticholesterolemic properties of the investigated species of the above species were established to be good (Jafari et al., 2016).

2.9.1.1.2 Previous phytochemical studies of Satureja species

Hydro-distilled essential oil from S. biflora (Lamiaceae) growing in Kenya was assessed and found to be composed mainly of monoterpenes, which accounted for 62.02 % of the oils. This monoterpene portion was characterized by a high percentage of linalool (50.60 %) such that it was categorized as a linalool chemotype. The other major monoterpenes were α-

terpineol (2.80 %), β-ocimene (2.25 %), β-pinene (1.96 %) and cis-linalool oxide (1.91 %).

Sesquiterpenes were found in good amounts of 1.5 to 5 percent were germacrene D, α- cadinol (β-bourbonene, δ-cadinene, τ-cadinol, endo-1-bourbonanol and β-caryophyllene.

Aliphatic alcohols and acids accounted for 7.23 % of the oil, of which the main one was linoleic acid at 4.48 percent (Matasyoh et al, 2007). However, the variation of the chemical composition with different environmental changes has not been assessed.

2.9.1.2 The genus Plectranthus

The genus Plectranthus belongs to the Lamiaceae family which grows globally and has species that are adapted to nearly all habitats and height above sea level. The genus

Plectranthus L' He'r. belongs to subfamily Nepetoideae of tribe Ocimeae (Cantino et al.,

1992). It encompasses about eighty species worldwide. Many Plectranthus species are of commercial value and medicinal plants. Quite a lot of species might be cultivated as ornamentals (Smith et al., 1996; Liu et al., 1996).

The essential oil from P. marrubioides (Plate 3.3) was analysed by GC, GC-MS and GC co- injection with the known standards. A total of 70 compounds amounting to 87.17% of the oil were identified. Camphor (48.80%), 1,8-cineole (9.0%), p-cymene (3.08%), α-terpinene

(2.58%), limonene dioxide (2.5%), fenchone (1.75%), isocaryophyllene (1.67%), viridiflorol (1.61%), camphene (1.58%), p-selinene (1.50%), trans-sabinene hydrate

(1.19%), caryophyllene oxide (1.13%) and terpen-1-ol (1.08%) were the main components

(Omolo et al., 2005).

2.9.1.3 The genus Ocimum

Ocimum is a genus of aromatic annual and perennial herbs and shrubs in the family

Lamiaceae, native to the tropical and warm temperate regions of all 6 inhabited continents,

with the greatest number of species being found in Africa. The genus comprises of up to

about 150 species with most native to central and south America, Africa and Asia (Makri

and Kintzios, 2008).

Herbs of the Ocimum family (Labiatae) have many traditional medicinal usages in Africa

and Asia. In East and West Africa, they are also used as mosquito repellents (Dalziel, 1937;

Kokwaro, 1993). In Northern Tanzania, basil-like herbs (Ocimum spp. and Hyptis

soaveolens) and neem (A. indica) leaves were used for repelling mosquitoes. When smeared

on the legs, the juice of Ocimum spp reduced biting by caged An. gambiae. Later, the

repellence was attributed to eugenol (Chogo and Crank, 1981; Hassanali, 1990), which are

main components of clove and other essential oils with repellent properties.

2.9.1.3.1 Ocimum kilimandscharicum

O. kilimandscharicum (Plate 3.5), is a commercially significant medicinal perennial herb

that is extensively dispersed in East Africa, India and Thailand. It is widely grown in the

tropics (Sumit et al., 2011). Ocimum spp. has been recorded as traditional repellent plant,

and is effective against mosquito, black flies, and ticks (Pugazhvendan and Elumali,

2013).Numerous names have been given to O. kilimandscharicum conferring to the

neighbourhood wherever it is grown. In Kenya Kikuyu called it ‘Gacuki; Luhya ‘Mwonyi’

and Meru ‘Makori’ (Kashyap et al., 2011).

2.9.1.3.2 Ethnobotany of O. kilimandscharicum

2.9.1.3.3. Traditional uses

Traditionally, this species is taken in various formulae as herbal tea, dried powder, fresh leaf or mixed with honey or essential oils. Table 2.1 summarises the traditional usages of O. kilimandscharicum plant.

Table 2.1. Traditional uses of O. kilimandscharicum Country Part of Traditional use Preparation References plant Kenya Leaves Congested chest, cough, Inhaling Kokwaro, et al measles, to repel insects, colds, vapour, cold 2009, Jembee abdominal pains and diarrhoea, infusion dry. et al, 1995. protecting grains against pest, used I apiculture to attract bees to beehives.

Tanzania Leaves As repellent against nuisance Essential Kweka et al., biting insects and treat malaria oils 2008

India Leaves Ear pain, insecticidal, mosquito - Kashyap et al., repellent, and as aromatic 2011 2.9.1.3.4 Other uses of O. kilimandscharicum

O. kilimandscharicum is an economical origin of natural camphor for industries; it is also used as flavoring for rice, vegetables and brewed as tea (Lawal et al., 2014). By means of modern science and technology, a new brand of medicines called Naturub® was derived from purified extracts of O. kilimandscharicum founded on the traditional information and practices. Naturub® is registered as a medicine. Naturub® is certified and registered as the first natural product in Kenya by the Pharmacy and Poisons Board of Kenya - it is sold extensively in corporate retail chains in Kenya. Its balm is used for relieving flu, cold, chest congestion, aches and pain, insect bites and muscular pain. An ointment derived from O. kilimandscharicum is used for the fast relief of muscular strain, rheumatism, arthritic joint, fibrositis, bruises, lumbargo, neuralgia and sciatica (Agarwal, 2017). Due to the varied

therapeutic qualities of these species numerous researchers have been studying the

biological activities and the chemical composition of their essential oils (Singh et al., 2014).

2.9.1.3.5. Biological studies of O. kilimandscharicum essential oils

The biological activities of essential oils of O. kilimandscharicum oil harvested in Kenya

exhibited insecticidal activity against Sitophilus zeamais and Rhyzopertha dominica (Bekele

and Hassanali, 2001). The essential oil of the plant gathered in different regions of the

world confirmed antimicrobial, wound healing, mosquito repellent, arcaricidal,

radioprotective and antimelanoma activities (Seyoum et al., 2003; Vatsya et al., 2006;

Paschapur et al., 2009; Morrison et al., 2008). Work on larvicidal activity has been done but

more exploration needed to harness this useful shrub for larval control.

2.9.1.3.6 Previous phytochemical studies of the genus Ocimum essential oils

The composition of O. kilimandscharicum essential oil have previously been determined by

other researchers, who came up with variations based on localities where collected. The

wide range in the constituents of the O. kilimandscharicum oils reported several chemo-

types which could be due to diverse environmental and hereditary factors, diverse

chemotypes and the nutritional status of the plants as well as other factors that can influence

the oil composition (Moghaddam and Mehdizadeh, 2017).

2.9.2 The family Asteraceae

The family Asteraceae is also called compositae, the aster, daisy, or family of the flowering- plant order Asterales. The family is composed of 1,620 genera and 23,600 species of herbs, shrubs, and trees spread all over the world. Asteraceaeis is one of the biggest plant families.

2.9.2.1 The genus Conyza

The genera Conyza belongs to the family Compositae comprising of about 15 species which are found in tropical Himalaya. It is a cosmopolitan genus of annual, biennial or perennial herbs, particularly inhabiting temperate and mountainous regions. The plant C. newii has been described to possess mosquito repellent and fumigant toxicity properties (Omolo et al.,

2005, Mayeku et al., 2013).

In Kenya, the essential oil of C. newii (Asterale: Asteracea, Oliv. & Hiern) (Plate 3.4) growing in the northern part of West Pokot was shown to be extremely repellent to An. gambiae s.s. Fumigant toxicity of the oil towards the mosquito was also established (Omolo et al., 2005). The main components of the oil were found to be monoterpenoids, comprising

(S)-(-)-perillyl alcohol, (S)-(-)-perillaldehyde, geraniol, (R)-(+)-limonene, trans-β-ocimene and 1,8-cineol (Omolo et al., 2005). The results suggested substantial epigenetic

(chemotypic) discrepancies in the repellency and composition of C. newii essential oils growing in diverse areas of Kenya (Omolo et al., 2005; Mayeku et al., 2013).

Following the above discovery, studies on essential oils and their isolates have laid bare their great potential as insect control agents (Tiwari et al., 2012). Considering the chemical predictions, in addition to the principal compounds, the genera Satureja, Conyza,

Plectranthus and Ocimum spp comprises numerous secondary metabolites such as

perillaldehyde, limonene, 2-methyl-5-(methylethyl)-2-cyclohexene-1-ol, 1,8-cineole,

perillyl alcohol, germacrene B, trans-bocimene, geraniol, β-myrecene and α-amorphene

(Omolo et al., 2005; Mwangi et al., 1986). Some of these compounds have been connected

to repellency but no complete report on repellency has been reported on S biflora. The

secondary metabolites found in Conyza and Plectranthus plants have been found to have

repellency effects on adults of An. gambiae (Omolo et al., 2005). As such, there is a need to

have a comprehensive knowledge of the potential useful essential oils for mosquito

repellent development, their effect when used, and their seasonal and environmental

variability in composition.

2.9. Terpenoid biosynthesis

The schematic diagram (Scheme 2.1) shows the catalytic reactions of PTSs and outlines the

terpenoid biosynthesis that leads to diverse products. The basic five-carbon building blocks

of C5-IPP and its isomer, C5-DMAPP, are synthesized in vivo via the cytosolic mevalonate

(MEV) pathway or the methylerythritol phosphate (MEP) pathway.

The several chain lengths of linear prenyl pyrophosphates acting as the critical precursor for

terpenoid biosynthesis are catalysed by their respective PTSs (Fu-Lien et al., 2011). For

C30- to C50-terpenes, the hydrophobic tail of quinine is derived from the long-chain linear

prenyl pyrophosphates. The number of carbons in the prenyl tail differs from plastoquinone

to ubiquinone and varies among the plant species. The abbreviation OPP indicates the

pyrophosphate group (Fu-Lien et al., 2011.

Scheme 2.1: Catalytic reactions of PTSs and the terpenoid biosynthesis, Source: Uploaded by Tao-Hsin Chang 2.11 Intraspecific variability of plant essential oil composition

The composition of medicinal plants in relation to active principles displays high intraspecific variations which are controlled not only genetically but as well by environmental settings, collection site and season. The endogenous influences are associated to anatomical and physiological features of the plants and to the biosynthetic pathways of the volatiles, which may change in whichever the different tissues of the plants or in changed seasons, but also might be influenced by DNA adaptation. The exogenous

influences, given a long period, might affect some of the genes that are responsible for volatiles formation. Those factors cause ecotypes or chemotypes in the same plant species

(Barra, 2009). Nevertheless, while the chemotypes are vastly influenced by environmental circumstances, they also hinge on genetic polymorphism (Lee and Mitchell-Olds, 2012).

Certainly, different chemotypes can breed in the same habitation (Salgueiro et al., 1995;

Salgueiro et al., 1997) and quite several authors have confirmed the genotypical determination of the chemical diversity of the populations in cold climates (Boira and

Blanquer, 1998). In this respect, for the last few years, chemotaxonomy has been used extensively to categorize plants with essential oils based on intra-specific chemical polymorphism traits (Barra, 2009).

It is vital to put into consideration the chemical variations in essential oils caused by genetic, physiological or environmental factors when domesticating and cultivating species of medicinal interest. Consequently, it is required to describe and find the existence of chemotypes, particularly when referring to plant material used in chemical, pharmacological and agronomic investigations that purpose to produce herbal medicines, because pharmacological activities of the same species can vary due to alterations in essential oil composition (Lima et al., 2003; Vieira and Costa, 2006).

CHAPTER THREE

MATERIALS AND METHODS

3.1 General experimental procedures

All recyclable glassware was washed in warm soapy water, then rinsed with distilled water and acetone. The glassware for collection and storage of the sample extracts was chemically cleaned by soaking in freshly prepared chromic acid overnight and cleaned with distilled water, then rinsed with the appropriate solvent and dried in the oven at 100C for one hour before use.

3.2 Chemicals and solvents

The solvents used in this work and chemical constituent standards of the essential oils that were used for the Gas Chromatography (GC) co-injection and bioassay was of High-

Performance Liquid Chromatography (HPLC) analytical grade purchased from Sigma-

Aldrich Chemical Company (USA).

3.3 Insects

Five to ten days starved female An. gambiae s.s. [ex - Kisumu (Kenya) strain], Cx. quinquefasciatus and Ae. Aegypti (SWISS TPH insectary) mosquitoes were reared under standard conditions at the Swiss Tropical and Public Health institute (STPHI) and used for repellency assays. Additional repellency assay in Kenya also conducted using 5-10 days starved female An. gambiae mosquitoes [ex-ifakara (Tanzania) strain] and An. arabiensis

(from icipe insectary) that were reared following standard settings at the Animal Rearing and Containment Unit of the International Centre of Insect Physiology and Ecology (icipe)

Mosquito insectary. Larvae for An. gambiae, Cx. quinquifasciatus, Ae. aegypti and An. arabiensis mosquitoes were obtained from the above colonies maintained at the Insect Mass

Rearing Unit of ICIPE. Larvae that hatched were shifted to larger pans (37 × 31 × 6 cm) at masses of 200-300 at 2nd instar stage. Tetramin® fish food (Terta GmbH, Germany) was used to feed them whereas the rearing water temperature was upheld at 28±2°C during larval development.

3.4 Plant materials

The following four plant species namely Satureja biflora, Satureja abyssinica, P. marrubioides, Ocimum kilimandscharicum and Conyza newii were selected for investigation based on previous mosquito repellent and larvicidal studies (Omolo et al.,

2005) and traditional knowledge and usage. Satureja biflora, Satureja abyssinica, P. marrubioides Conyza newii were collected from different locations of Nyahururu, Kieni-

Gakoe and Naivasha in the Aberdare ranges region in Central Kenya (Figure 3.1) while

Ocimum kilimandscharicum was collected Isecheno in Kakamega forest by Muliru Farmers

Conservation Group in western Kenya (Figure 3.2). The aerial parts of the plant samples were gathered randomly from five diverse locations in the area at their different phases of development (Plate 3.2). The ecological zone, geographical positioning system (GPS) coordinates, sample part and taxa of the collected plants were recorded (Table 3.1). The plants were identified by a taxonomist, the late Mr. Simon Mathenge (Plate 3.1), formerly of Botany Department, University of Nairobi, and voucher specimens deposited at the

Herbarium of the National Museums of Kenya (NMK). The collected plant materials were air-dried under shade at room temperature (25±2oC) for 7 days before extraction.

Figure 3.1: Map showing sample collection location in Aberdare ranges region (Wangari, 2018)

Figure 3.2: Map showing sample collection locations in Kakamega forest (Uploaded by Kawawa et al., 2016)

Plate 3.1: Plant collection and identification by a taxonomist at Kinale

Plate 3.2: Harvesting of C. newii at Nyahururu

Table 3.1. Sample location and GPS information Voucher GPS

Family Plant Species Location Specimen no Longitude Latitude Alt(m)

Lamiaceae Satureja biflora Nyahururu SB/NHR/02/10 N00°02.651’ E036°17.216’ 2443

Satureja biflora Kinale SB/KNL/01/10 S00°54.992’ E036°36.612‘ 2625

Satureja biflora Kieni-Gakoe SB/KNG/03/11 S00°51.226’ E036°40.112 2704

Satureja abyssinica Nyahururu SA/NHR/02/10 N00°02.651’ E036°17.216’ 2443

Plectranthus Naivasha PM/NVS/01/10 S00°43.000’ E036°26.000’ 2086 marrubioides

Ocimum Kakamega OK/KAK/01/11 N00°16.000’ E034°55.000' 1605 kilimandscharicum

Asteraceae Conyza newi Nyahururu CN/NHR/02/10 N00°02.651’ E036°17.216’ 2443

Conyza newii Kinale CN/KNL/01/10 S00°54.992‘ E036°36.612‘ 2625

Plate 3.5: Ocimum kilimandscharicam Plate 3.6: Satureja biflora

Plate 3.3: Plectranthus marrubioides Plate 3.4: Conyza newii

Plate 3.7: Satureja abyssinica

3.5 Extraction and identification of volatile phytochemicals

3.5.1 Hydro-distillation

The dried aerial parts were extracted by hydro distillation using Clevenger apparatus (Plate

3.8).

Plate 3.8: Hydro distillation using Clevenger apparatus

Various quantities (700 – 1500 g) of each of the plant material were put into a 10-liter round-bottom flask and 4000 ml of water added. The flask was then fitted with the

Clevenger apparatus and a double pocket condenser (Plate 3.8). The plant materials were hydro distilled for 4 hours. The condensing oil was collected over hexane (Aldrich, HPLC grade). Anhydrous sodium sulphate (about 0.5-2 g) was added to the oil-hexane mixture and filtered through a filter paper to remove traces of water. Hexane was removed under reduced pressure with a rotor evaporator (Plate 3.9) at temperature of 40 °C, and the residual oil collected, weighed and stored in amber-colored vials at a temperature of 2 to 4

°C until time for use.

Plate 3.8: Solvent removal from oil using a rotor evaporator

3.5.2 Analyses of essential oils using Gas Chromatography – Mass Spectrometry

Characterization, identification and determination of the constituents of the essential oils was done using a gas chromatography linked to Mass Spectrometer (MS) (GC) (HP-7890A,

Agilent technologies, Wilmington, USA) (Plate 3.10). The GC was equipped with a non- polar HP-5MS capillary column, 30 m x 0.25 mm i.d., 0.25 µm film thickness (J and W

Scientific, Folsom, USA) with 5%-phenyl methyl silicone as stationary phase.

Plate 3.10: Schematic diagram of a GC-MS Equipment

Nitrogen was used as carrier gas for the GC, and helium (1.2 ml min-1) for the GC-mass spectrometer (MS). The oven temperature programme involved initial temperature of 35 ◦C

(for 5 mins), which was risen to 280 ◦ C at 10 ◦C/min, and held isothermally at 280 ◦C for

10.5 min. An aliquot (1 µl) of individual oil was injected in the splitless mode into the column. The ion source temperature was 230 ◦C; electron ionization mass spectra were equipped at 70 eV with a mass range of 38 350 Daltons (Da) during a scan time of 0.73 scans s-1. Identity of the oil constituents was based on the retaining indexes (Adams, 1995) and by comparison of mass spectra with databases (NIST, 2005, NIST 05a, and Adams MS

HP, USA) and by comparison with those of authentic samples analyzed in a similar way.

The percentage composition of each constituent in the oil was determined from quantification of its peak area comparative to the total composition.

3.5.3 Electrophysiological recordings using GC-EAG

Gas chromatography connected to electroantennogram (GC-EAG) was used to identify which volatile compounds within a sample caused an electrophysiological response from antenna of a female An. gambiae s.s. Using attached Gas Chromatography-

Electroantennogram (GC-EAG) system (Plate 3.10 a-b). Unfed, 1-3-day old, mated female

Anopheles mosquitoes were used. A mosquito was mounted under an Olympus light microscope at 400 x magnification. The insects were anaesthetized by chilling and the head was removed and put in the tip of indifferent electrode made using Ag-AgCl glass electrodes filled with ringer solution (Maddrell, 1969). GC effluent was split into two. One portion was sent to a flame ionization detector (FID), while another portion was directed into the air stream over the mosquito head as defined by Wadhams (1990). GC-traces and antennal responses were visualized on a computer monitor.

For Electroantennogram (EAG) recordings, the signals were passed through a high impendence amplifier (UN-06, Syntech, and The Netherlands) and analyzed by using a customized software package (Syntech, The Netherlands). Co-occurring FID peaks and antennal responses were identified using a HP 5970 GC-MS fitted with a solgelwax column

(Agilent). The column used was a 50 m x 0.32 mm i.d. HP-1 column (non-polar). Oven temperature that was maintained at 40 ◦C for 2 min and then programmed at 5 ◦C/min to 100

◦C and then at 10 ◦C/min to 250 ◦C. The carrier gas was hydrogen (15 psi). A standard stimulation was done at the beginning and at the end of each recording to correct for loss of sensitivity of the preparation. The above was repeated six times. Identification of components of essential oils which provided peaks related to EAG-activities was established by peak enrichment upon GC co-injection of the crude essential oils with standard samples (Pickett, 1990). The signals were visualized and saved on a PC with

Syntech software (Syntech, The Netherlands).

GC EAG station Syntec machine

Plate 3.11a: Gas Chromatography linked to Electroantennogram (GC-EAG)

Plate 3.11 b: Schematic diagram of GC linked to Electroantennogram (GC-EAG)

3.5.4 Variability in the composition of the essential oils

The quantitative and qualitative chemical composition of the volatile oil from plant samples gathered from Aberdare ranges region, Kenya, were processed as detailed in section 3.5 and

3.6. They were exposed to Principal Component Analysis and Cluster Analysis to find out any variation therein (Lele and Rechsteiner, 1995). This is executed out on the data matrix to find the various groups (chemotypes) to which the diverse samples belong. Principal component analysis (PCA) was carried out to identify probable relations amongst the constituents. The number of factors extracted for the analysis are chosen bearing in mind only those factors which offered a growing eigen value higher than 1 (Ait-Sahalia and Xiu

2015). The Cluster analysis and PCA was performed with R version 3.4.0 (2017-04-21).

3.6 Repellent tests

3.6.1 Estimation of effective dose using vitro ‘K and D module’

To estimate the effective dose of the crude plant extract, K and D module bioassays were used (Jerome and Mustapha, 2000). All the essential oils were obtained through hydro- distillation. All were tested in five concentrations for their repellence activity. Firstly,

varying concentrations of the samples were prepared.: 10%, 5%, 2.5% and 1.25% and

0.63% as shown in Table 3.2. The test samples were ethanolic dilutions and the percentages given as weight by weight (w/w). Ten percent concentrations were prepared by adding 1ml of essential in 9 ml ethanol. Likewise, 5% concentration had 0.5 ml of essential oil in 9.5 ml of ethanol, 2.5% concentration had 0.25 ml of essential oil in 9.75 ml of ethanol, 1.25% concentration had 0.125 ml of essential oil in 9.875 ml, while 0.63% concentration had 0.63 ml of essential oil 9.94 ethanol.

Table 3.2: Plant essential oil concentrations tested for repellence against Ae. aegypti, An. gambiae s.s Cx quinquefasciatus Plant species Location Concentrations C. newii Nyahururu 0.63%, 1.25%, 2.5%, 5% and 10%

P. marrubioides Naivasha 0.63%, 1.25%, 2.5%, 5% and 10%

S. biflora Kieni - 1.25%, 2.5%, 5% and 10%

Kinale 0.63%, 1.25%, 2.5%, 5% and 10%

Nyahururu 0.63%, 1.25%, 2.5%, 5% and 10%

DEET 20%

A Klun and Debboun (K&D) test module, previously developed and used for quantitative measurement of the efficacy of mosquito repellents on human volunteers, was adapted to approximate the effective dose of the repellent oils. In the assay, a prototype was used to demarcate 3 cm x 4 cm areas on the skin that match to each of the six cell openings on the bottom of the module Plate 3.12(a). Five areas were treated with different concentrations of the repellent whereas one area acted as a control succeeding a complete randomized block design with negligible treatment interaction and 6 repeats (for example 5 mosquitoes per concentration) as indicated in Plate 3.12(b). A complete K and D assay set up is shown

Plate 3.13.

Plate 3.12(a) Plate 3.12(b) Marking for K and D assay

Plate 3.13: K and D assay set up The module, with five host-seeking, 5 to 10 days old mosquitoes to each cell, was then placed over the treated skin zone. Each cell door was opened and the number of mosquitoes biting within a 2-minute experience was documented, after which the doors were shut (Plate

3.11). This procedure was repeated until six replicate exposures and observations were made for each of three human volunteers. The effective dose (for example. ED50 and ED99.9) was then estimated using a modified Gauss-Newton probability investigation with a

Gompertz cumulative probability distribution in the statistical software R (http://www.r- project.org).

3.6.2 Warm body in vitro assay for evaluating mosquito repellents effective dose

An in vitro behavioral assay for evaluating mosquito repellents applied in a dose-based style to a warm body (34 oC) (Plate 3. 16) in test cages was used to evaluate the sensitivity of 4–

6-day old An. gambiae to the insect repellent N, N-diethyl-meta-toluamide (DEET) and essential oils. These tests were made in the absence and presence of additional carbon dioxide (CO2) applied as a pulse to stimulate mosquitoes in the cages (Krober et al., 2010).

Plate 3. 16: Warm body bioassay Setup

Reduction in the number of landings by the African malaria mosquito, An. gambiae, females on a warm body shown in Plate 3.13 treated with essential oils (EO’s) from Kenyan wild plants (CN/NHR, PM/NVS, SB/KIN, SB/KG and SB/NHR) compared to treatments with DEET and to the solvent control (ethanol p.a.) was recorded.

3.6.3 Estimation of repellent efficacy and protection time

The protection time of the two most promising formulated repellent products from the K and D assay (excipial lotion) was estimated using the WHO protocol (WHO, 2009). The

WHO arm-in-cage test was carried out with three different genera of anthropophilic mosquitoes (Ae. aegypti, Cx. quinquefasciatus and An. gambiae) and 10 human volunteers.

Mosquito cages (size: 40 x 40 x 40 cm) with a solid bottom (mirror for ease of observation) and top, screen on the posterior, a clear acrylic sheet (used for seeing) on the right and left flanks and a cloth wrapper for admittance on the front as shown in Plate 3.12 (a). 200 non- blood-fed females, at the age of 5 to 10 days, were used. The repellent was smeared to the forearm amid the wrist and elbow at a concentration of 1.5 g lotion per 600 cm2 while the hand was protected by a latex glove through which the mosquitoes cannot bite as shown in

Plate 3.15.

Thirty minutes after treating with the repellent volunteers inserted their untreated and treated forearm for 3 minutes into the cage to determine biting activity. The numbers of landings/probing were recorded for each interval. Preceding every exposure of the treated arm, the eagerness of mosquitoes to bite was assessed via introducing an untreated arm into the cage for 30 seconds or till 10 landing/probing were added up. This process was repeated every 30 minutes up to 8 hours. The end of this behavioral assay was an estimate of the repellent’s complete protection time and repellent efficacy over time. The complete guard

time was demarcated as the duration of the repellent protection until the initial (confirmed) bite.

Plate 3.14: WHO ‘arm in the cage’ Plate 3.15: WHO arm in the cage Mosquito cage exposure of the treated arm

Relative protection of the tested formulation was defined as the reduction in mosquito landings/probing as compared to the untreated forearm. The percentage (%) protective efficacy (P.E) (equation 1) was calculated as follows;

PE = (PCM - PTM)/ PCM. ……………………………………….... Equation 1.

Percent control mean (PCM). and Percent Test Mean (PTM) of mosquitoes landing on the control and treated arms respectively.

3.6.4 Subtractive-combination repellence bioassay

A subtractive-combination repellence bioassay was used to determine spatial repellents and attraction inhibitors of commercially available constituents (Aldrich) of the essential oils and blends of these at their natural proportions (GC) in the oils and in amounts present in the minimum 100% lethal dose of the oils, with each component absent (subtracted) from the blend in turn to determine its relative contribution to the overall toxicity of the natural oil (Owaga et al., 1988). In this experiment, the role of the individual EAG active constituents of oils in conferring repellence to the Full Repellent Blend (RB) was assessed in a Latin square design experiment with the following treatments:

For S. biflora: Full Blend (FB); (b) FB minus linalool (15), (c) FB minus neral (16), (d) FB

minus geraniol (17) and (e) FB minus neridol (18).

For C. newii: 5-Components, FB, (b) FB minus pinene (20), (c) FB minus perilla alcohol

(21), (d) FB minus limonene (21), (e) FB minus perillaldehyde (19) and (f)

FB minus cinnamaldehyde-α-pentyl (22)

The blends were evaluated in the concentration range of 10-5 – 10-1 g/ml.

3.7 Larvicidal and insect growth regulation assays

3.7.1 Formulation of essential oil plant and vegetable oil

The essential oil extract was liquified in acetone and their water-miscible formulation were assessed for mosquito larvicidal activity in the laboratory. The standard World Health

Organization (WHO) method was used (WHO, 2005).

The essential oil of O. kilimandscharicum was formulated using Polysorbate 80, a nonionic surfactant and emulsifier, into a water-miscible concentrate consisting of 33% v/v

O. kilimandscharicum essential oil, with aqueous polysorbate 80. Vegetable oil (Elianto,

Bidco, East Africa), used as the experimental control, was also similarly formulated with aqueous polysorbate 80.

3.7.2 Laboratory Larvicidal and insect growth regulation assays

Initially mosquito larvae were subjected to a broad range of concentrations of the product to find its activity profile. A narrower range of 4 - 5 concentrations, giving between 10% and

95% mortality in 24 h or 48 h were then used to determine LC50 and LC90 values of the product.

Batches of 25 third instar larvae were moved by means of droppers to 200 ml beakers holding 100 ml of water. The level of the water in the beakers was maintained between 5 cm3 and 10 cm3, as deeper levels might lead to unwarranted mortality. The suitable volume of dilution was made-up to 100 ml in the beakers to attain the anticipated target dosage, preliminary with the lower most concentration. Four duplicates were set up for each concentration and an equivalent number of controls arranged concurrently with tap water, to which 1 ml acetone was added. Each test was repeated three times on separate days. Larval food was added to each test beaker, mostly if high mortality was recorded in the control

(Puggioli et al., 2013).

The test containers were kept at 25 - 28 0C and a photoperiod of 12 hours well-lit followed by 12 hours darkness (12L:12D). Larval mortality was recorded after 24 hours and 48 hours experience, the number of moribund larvae were added to dead larvae for working out percentage mortality. Dead larvae were those that could not be made to move when probed with a spike in the siphon or the cervical area. Moribund larvae could not rise to the surface

or show the typical diving response when the water was agitated. The results were recorded from which the LC50, and LC95 values, and gradient and heterogeneity investigation were also recorded. Larvae that had transformed into pupae in the test period nullified the test. If more than 10% of the control larvae turned into pupae when the experiment was running, the test was cancelled and redone. If the control death was in the range of 5-20% the mortalities of treated groups were adjusted as per Abbott’s formula (Abbot, 1925).

Mortality (%) = (X – Y)/X * 100 ………………………………………Equation 2.

where X = percentage survival in the untreated control. Y = percentage survival in the treated sample. The test was tripled from separately reared batches of larvae. Mortality delay was studied by recording cumulatively the number of dead larvae, abnormalities and developed adults after each 24 h for 14 days. Throughout the experiment, the room temperature was upheld at 33 ± 2 0C, and water at 28± 2 0C. The larvae were fed on the fish food, Tetramin® at 1mg/ beaker per day.

3.7.3 Semi-field mosquito larvicidal evaluation of water miscible formulations

Semi-field evaluation of the emulsifier concentrates formulation of the essential oils was undertaken based on the WHO mosquito larvicidal semi-field trials method (WHO, 2005).

The evaluation was performed on a small scale against An. gambiae s.s. mosquitoes in representative natural breeding sites at Jaribuni, Kilifi county in Kenya. In these trials, 5 litres multiple artificial clay pots of water were placed in the field and the samples were tested against laboratory reared larvae. The water-filled pots were left at least 24 hours for acclimatizing or ageing. A set of 50 laboratory-reared 3rd instar larvae of An. gambiae s.s. mosquitoes were freed into each of the pots and larval food added. Treatment

concentrations were calculated based on the quantity of active constituent per capacity of water in the pot, that is for everyone litre,1 ppm of the concentrate was introduced or surface area in the case of Bti granules as the positive control. After 2-3 hours of larval acclimatization, the pots were treated with selected dosages of the oil water miscible formulation and Bti granules in a randomized manner using perforated jars and by broadcasting of the selected dosage of solid Bti granules on the water surfaces respectively.

The pots were shielded with nylon mesh screen to avoid other mosquitoes or other insects from depositing eggs. The water was also guarded from dropping debris. The water level in the pots was maintained by making additions when needed. Five duplicates of each dosage and five controls were used as indicated in Plate 3.17 below.

Plate 3.17: Semi-field bioassay set-up for evaluation of mosquito larvicides in Kilifi District, Kenya

All the pots were examined after 24 hrs and 48 hrs where living larvae were tallied to score post-treatment larval mortality. For slow-acting trial sample ingredients or quantities, the survival of the larvae, pupae and pupal skins was evaluated for seven days or more posttreatment, whereby all larvae had turned into pupae and emerged as adults. The pH and water temperature readings were documented during the assessment.

3.8 Statistical analysis

The repellence for the treated and the control was compared using the two-way analysis of variance (ANOVA) (Swallow I., 1984). The means was ranked using Student–Newman-

Keuls (SNK) test (SAS Institute, 2000). The RD50 values for the essential oils were calculated by Probit analysis (Busvine, 1971; Finney, 1971). The effective dose (i.e. ED50 and ED99.9) was then estimated using a modified Gauss-Newton probability analysis with a

Gompertz cumulative probability distribution in the statistical software R (http://www.r- project.org). Data for larvae mortality and deformities was subjected to ANOVA and the mean percentage mortality compared using Student–Newman–Keuls test in R. The Cluster analysis and Principal Component Analysis was performed with the open source package R version 3.4.0 R (2017-04-21).

CHAPTER FOUR

RESULTS AND DISCUSSION

4.1 Plant essential oil yields

The hydro-distilled plant aerial parts afforded essential oils, whose yields of which are

summarised in Table 4.1.

Table 4.1: Plant essential oil yields

Plant species Location Sample MO Sea Qty(Kg) % code No. NTH son Oil yield Satureja biflora Nyahururu SB/NHR/02/10 OCT Dry 0.61 1.50±0.00b

SB/NHR/02/05 MAY Wet 0.49 1.20±0.14b

Kinale SB/KNL/01/10 OCT Dry 0.70 1.25±0.58b

SB/KNL/01/05 MAY Wet 0.68 1.21±0.09b

Kieni-Gakoe SB/KNG/03/05 MAY Wet 1.17 0.88±0.22b

SB/KNG/03/03 MAR Dry 1.52 1.14±0.01a

Conyza newii Nyahururu CN/NHR/02/10 OCT Dry 0.83 1.61±0.22b

CN/NHR/02/05 OCT Wet 0.70 1.35±0.14b

Kinale CN/KNL/01/10 OCT Dry 0.65 1.50±0.00b

CN/KNL/01/05 MAY Wet 0.61 1.40±0.04b

Satureja Nyahururu SA/NHR/02/10 OCT Dry 0.31 0.29±0.00a abyssinica

Plectthrus. Naivasha PM/NVS/01/10 OCT Dry 2.28 0.43±0.08a marrubioides PM/NVS/02/05 MAY Wet 3.76 0.71±0.18b

Ocimum Kakamega OK/KAK/01/05 MAY Wet 0.30 3.51±0.56c kilimandscharicum OK/KAK/02/10 OCT Dry 0.31 3.68±0.33c

Each value for the subgenera represents the mean oil yield of the samples collected from mature trees. Species sharing the same letter aa, bb or cc are not significantly different at P < 0.05.

The color of all the extracted essential oils was brown yellow. The mean percentage essential oil yield of the studied species ranged from 0.29 - 3.68 %. The maximum yield was obtained from O. kilimandscharicum (3.68%), followed by C. newii (1.61. %) and S. biflora (1.50%). The yield from S. abbysinica collected from Nyahururu during dry season was the lowest (0.29%). Results from analysis of variance showed that the overall differences in oil yields were significant (P<0.5). There are four seasons in Kenya namely two dry seasons (December-March and July-October) and two rainy seasons (April-June and November). Across the seasons, C. newii gave comparatively higher average oil yield than S. biflora and P. maruboides. In the Aberdare region, the oil yield from C. newii was highest during the dry season in October and showed lower yields in the wet season of May.

The same applied to S. biflora, whereby in dry season of October in Nyahururu Kinale and of March in Kieni-Gakoye provided slightly higher oil yields compared to lower yields in the wet seasons of May for the same location. The oil yield of O. kilimandscharicum that was harvested in the wet season was higher than that collected in the dry season.

4.2 Chemical compositions of essential oils of selected plants

4.2.1 Chemical composition of essential oil of Satureja biflora

The essential oils of S. biflora was obtained from samples collected from three different habitats in the Aberdare ranges (Kinale, Kieni-Kagoe and Nyahururu). Thirty-three (33) constituents were identified in the essential oil of aerial parts of S. biflora by GC-MS and

GC co-injections with available authentic standards. The GC-MS profile for S. biflora oil is shown in Fig. 4.1 and the identified compounds are listed in Table 4.2.

Figure 4.1. GC profile of Satureja biflora essential oil.

Table 4.2: Chemical composition of essential oil of Satureja biflora % composition COMPOUND SB/KNL/10 SB/KNG/10 SB/NHR/10 cis -Linalool oxide t t 1.36 trans- Linalool oxide 0.60 1.40 Linalool 12.81 16.30 28.60 Trans- Pinocarveol t t 1.34 Terpinen-4-ol t t 3.72 Dec-1-en-3-ol t t 1.28 Isocitral 4.55 3.94 t -Terpineol 1.46 1.34 0.85 Myrtenal t t 1.18 Neral 23.82 20.64 3.63 Geraniol 4.60 1.15 t Geranial 31.19 27.42 4.78 -Bourbonene 0.69 Caryophyllene(E-) 1.71 1.87 2.02 Aromadendrene 0.61 1.05 Geranyl propanoate t 3.21 t Germacrene D 0.69 0.86 1.36 Bicyclogermacrene 0.61 1.33 Bulnesene t t 1.53 Nerolidol 5.56 21.78 Nerolidol t 15.11 t Caryophyllene oxide 0.94 1.00 2.18 Guaiol t t 2.71 Geranyl valerate 3.37 t t 3-Octanone 1.97 1.39 t Sabinene 1.15 1.40 0.75 -Pinene t t 7.43 -Phellandrene t 1.10 t -Ocimene<(E) 0.58 t 2.26 Terpinene<γ, -> 0.65 t 1.04 Bicyclo [2.2.1] heptan-2-ol, 1,7,7-trimethyl-, (1S-endo)- t t 1.07 Bicyclo [4.1.0] heptane, 3- methyl- t 1.072 t Cyclooctane, ethenyl- 1.41 t t Total 98.28 97.802 95.34 GC co-injection identification done by GC-MS only; t = trace amounts (< 0.1%). NHR, KNL and GNK refer to Nyahururu, Kinale and Kieni-gakoe respectfully.

The major components of the oil collected from the three habitats were geranial (27-31%), linalool (12-28%), neral (20-23%), nerolidol (Z) (5-21%), isocitral (3-5%), β-pinene (0-

7%), geraniol (1-5%), caryophyllene (1-2%), caryophyllene oxide (0.9-2%), terpinen-4-ol

(0-4%), α-terpineol (0.8-1.5%), geranyl valerate (0-3%), 3-octanone (1-2%), sabinene (1-

1.2%), and geranyl propanoate (0-3%). Several other monoterpenoid and sesqui-terpenoid compounds were identified (Table 4.2) but these were present in amounts < 1%. Twenty- three compounds were present in trace (t) amounts (<0.1 %), six compounds in 0.1 - 0.2%, four compounds at 0.2 - 0.4%, two compounds at 0.4 - 0.6%, three compounds at 0.6 - 0.8% and two at 0.8 - 1% (Table 4.2). Out of the identified compounds, 56% were oxygenated terpenes, which constituted most of the major components.

There were significant variations in oil composition of plant samples collected from different locations in the same month (October). The major constituents of S. biflora collected from Nyahururu were linalool (28.6%) and neridol (21.7%); however, samples collected from Kinale and Kieni-Gakoe, gave relatively lower amounts of linalool (12.9% and 16.3%, respectively) and nerolidol (5.6% and 15.1%, respectively). This could be attributed to environmental differences and altitude. Kinale and Kieni-Gakoe at higher altitudes and comparatively cold and wet as compared to Nyahururu, which has a mild climate and generally warm and temperate (The Köppen Climate Classification). The composition differs with a S. biflora collected at Egerton University, Njoro at an altitude of

2217m where the oil was dominated by monoterpenes which accounted for 62.02 % of the oil with a high percentage of linalool (50.60 %). For other species of the same genius, the main component of the oil of Satureja icarica, Satureja boissieri and Satureja pilosa from

Turkey was carvacrol (59.2 %, 44.8 %, 42.1 %), respectively (Azaz et al., 2002).

4.2.2 Chemical composition of essential oil of Conyza newii

Nineteen (19) compounds were identified in the essential oil of C. newii by GC, GC-MS and GC co-injections (GC-CO) with available authentic standards. The GC-MS chromatogram of the oil is shown in Fig. 4.2 and the identified compounds listed in Table

4.3.

Figure 4.2. The GC profile of C. newii oil

Table 4.3:Chemical composition of essential oil of Conyza newii

% composition COMPOUND CN/NHR/10 CN/KNL/10 Menthatriene<1,3,8-para-> 0.89 1.04 Limonen-10-ol 1.38 t Perilla alcohol 14.67 5.25 1H-Indene-4-carboxaldehyde, 2,3-dihydro- 2.45 t Curcumene 0.22 t Germacrene D 1.42 1.84 2-Methylindene t 2.00 5-Methyl-2-phenyl-2-hexenal 21.21 t Myrcene 1.39 1.29 Limonene 23.15 37.76 Ocimene<(E)-beta-> 1.31 2.19 9-Methyltricyclo [4.2.1.1(2,5)] deca-3,7-diene- t 6.36 9,10-diol Benzene, 1-(1-methyl-2-propenyl)-4-(2- t 3.80 methylpropyl)- Benzeneacetonitrile-α-acetyl- 1.41 t Bicyclo[3.2.1]oct-2-ene, 3-methyl-4-methylene- 1.62 1.40 Bicyclo[4.2.1]nona-2,4,7-triene, 7-ethyl- 1.04 t Ethanone, 1-(2-methoxy-1H-inden-3-yl)- t 2.44 Naphthalene, 2,3-dimethoxy- t 6.97 p-Mentha-1,8-dien-7-yl acetate 24.57 26.81 Total 96.73 99.15 GC co-injection identification done by GC-MS only; t = trace amounts (< 0.1%). NHR and KNL refer to Nyahururu and Kinale respectfully.

The major components in the oil were limonene (23-38%), p-mentha-1,8-dien-7-yl acetate

(24-27%), 5-methyl-2-phenyl-2-hexenal (0-21%), perillyl alcohol (6-14%), naphthalene,

2,3-dimethoxy- (0-7%), 9-methyltricyclo[4.2.1.1(2,5)]deca-3,7-diene-9,10-diol(0-6.5%), benzene, 1-(1-methyl-2-propenyl)-4-(2-methylpropyl) (0-4%), β-ocimene (1-2%),

germacrene-D (1-1.9%), bicyclo[3.2.1]oct-2-ene, 3-methyl-4-methylene- (1-1.5%), myrcene (1-1.3%), 1H-indene-4-carboxaldehyde, 2,3-dihydro- (0-2.5%), ethanone (0-

2.5%), 1-(2-methoxy-1H-inden-3-yl)-2-methylindene (0-2%), menthatriene 1,3,8-para, benzeneacetonitrile, α-acetyl- (1-1.4%) and limonen-10-ol (0.1.38%), Several other monoterpenoid and sesquiterpenoid compounds were identified (Table 4.3) but these were present in amounts less than 1%. Thirteen compounds were present in trace (t) amounts

(<0.1 %), while six compounds were at 0.1 - 0.3%. Out of the identified compounds, 56% were oxygenated terpenes, which constituted most of the major components. The largest variation was found in the oil which was associated with variable amount of 5-methyl-2- phenyl-2-hexenal, with 21% in Nyahururu but none at Kinale. In addition, the composition of perillyl alcohol was high (14%) at Nyahururu as compared to 5% at Kinale. Relatively,

Kinale and Kieni-Gakoe are at a higher altitude as compared to Nyahururu. The main components of the oil were found to be monoterpenoids, comprising (S)-(-)-perillyl alcohol,

(S)-(-)-perillaldehyde, geraniol, (R)-(+)-limonene, trans-β-ocimene and 1,8-cineol (Omolo et al., 2005). The results suggested substantial epigenetic (chemotypic) discrepancies in composition of C. newii essential oils growing in diverse areas of Kenya Omolo et al.,

2005; Mayeku et al., 2013).

4.2.3 Chemical composition of essential oil of Satureja abyssinica

Thirty-three (33) compounds were identified in the essential oil of S. abyssinica by GC,

GC-MS and GC co-injections (GC-CO) with available authentic standards as shown in

Table 4.4. The major components of the oil were menthone (44.1%), pulegone (33.3%), germacrene D (5.2%), piperitone (2.8 %), β -bourbonene (1.9%), amorpha-4,7(11)-diene

(1.4%) and β-ocimene (1.3%). Several other monoterpenoid and sesquiterpenoid compounds were identified (Table 4.4), but these were present in amounts < 1%.

Table 4.4: Chemical composition of essential oil of Satureja abyssinica

% Peak area COMPOUND SA/NHR/10 1,6-Octadien-3-ol, 3,7-dimethyl- 0.44 Menthone 44.08 Terpineol 0.20 Pulegone 33.26 Piperidone 2.85 Perilla alcohol 0.15 2,6-Octadienal, 3,7-dimethyl-, (Z)- 0.24 Myrtenyl acetate 0.15 Piperitenone 0.29 Eugenol 0.14 Piperitenone oxide 0.88 Bourbonene 1.89 Copaene 0.78 Amorpha-4,7(11)-diene 1.37 Germacrene D 5.15 Muurola-4(14),5-diene 0.29 Cadinene<γ, -> 0.43 δ -Cadinene 0.48 Cadinol (=tau-cadinol) 0.26 Nuciferol 0.54 Muurolene<14-hydroxy-alpha-> 0.26 Hexadecanoic acid 0.12 Pinene 0.39 5-Hydroxy-3-methyl-1-indanone 0.15 5-Isopropyl-3,3-dimethyl-2-methylene-2,3-dihydrofuran 0.47 Pinene 0.71 Myrcene 0.28 Limonene 0.59 Ocimene<(Z)-beta-> 0.13 Ocimene<(E)-beta-> 1.30 Bicyclo[3.3.1]non-6-ene-3,9-dione 0.29 Bicyclo[6.3.0] undec-1(8)-en-3-one, 2,2,5,5-tetramethyl- 0.11 Linalyl acetate 0.95 Total 99.62 GC co-injection identification done by GC-MS only; t = trace amounts (< 0.1%). NHR refer to Nyahururu.

Thirty-two compounds were present in trace (t) amounts (< 0.1 %). Similarly, analysis of S. abyssinica collected from Gorfo mountain near Guder in Ethiopia at an altitude of 3500 m above sea level had high amounts of pulegone (43.5%) and isomenthone (40.7%), (Tolossa et al., 2007). The altitude of the two locations differ by almost 1000m hence the contrasting composition of S. abyssinica at Nyahururu at an altitude of 2443m with more of menthone

(44.1%) and less pulegone (33.3%).

4.2.4 Chemical composition of essential oil of Plectranthus marrubioides

Thirty-five (35) compounds were identified in the essential oil of P. marubioides by GC,

GC-MS and GC co-injections (GC-CO) using available authentic standards as shown in

Table 4.5. The major components of the oil were Carene-2-δ (18.7%), camphor (17.9%), o-cymene (8%), cadinol (7.9%), α-cardinal (6.9%), caryophyllene (E) (6.9%),

3,4-pentadien-1-ol, 2,2-dimethyl- (4.7%), β-selinene (3.9%), δ-cadinene (2.62%), cuparene

(2.2%), caryophyllene oxide (2.1%), 1-azabicyclo [2.2.2] octan-3-one (2.0%), 1,8-cineole

(1.9%), γ-terpinene (1.6%), camphene (1.3%), γ-cadinene (1.3%), globulol (1%) and terpinolene (1%). Several other monoterpenoid and sesquiterpenoid compounds were identified (Table 4.5) but these were present in amounts < 1%. Twenty-two compounds were present in trace (t) amounts (< 0.1 %).

Overall, the chemical composition of P. marrubioides oil samples collected in the wet

(May) and dry (October) seasons did not vary significantly. However, there was notable presence of (E)-caryophyllene in samples collected in the wet seasons, but it was totally absent in those collected in the dry season.

Table 4.5: Chemical composition of essential oil of Plectranthus marrubioides % Peak area COMPOUND PM/NVS/10 PM/NVS/05 Terpinolene 1.02 1.30 Camphor 17.86 19.88 Terpinen-4-ol 0.68 0.72 1-Azabicyclo [2.2.2] octan-3-one 2.02 t Caryophyllene(E-) 6.89 t Muurolene<γ, -> t 1.35 Selinene 3.97 4.27 Bicyclogermacrene t 0.79 Muurolene 0.80 t Cuparene 2.22 t Cadinene<γ, -> 1.31 t δ -Cadinene 2.26 2.29 3-(But-3-enyl)-cyclohexanone t 2.05 3,4-Pentadien-1-ol, 2,2-dimethyl- 4.66 4.68 Palustrol 0.53 0.40 Caryophyllene oxide 2.09 2.03 Globulol 1.07 t Viridiflorol t 1.11 Cadinol (=tau-cadinol) 7.92 7.86 Cadinol 6.97 6.70 Calamenen-10-one<10-nor-> t 1.72 Muurolene<14-hydroxy-alpha-> 0.71 t Pinene 0.49 0.41 Camphene 1.32 1.16 Myrcene 0.61 0.57 Carene-2- δ 18.68 17.21 7-Oxabicyclo [4.1.0] heptan-2-one, 3-methyl- 0.78 0.84 6-(1-methylethyl)- Cymene 7.96 8.30 Cineole<1, 8-> 1.97 1.89 Terpinene<γ, -> 1.56 1.63 Androstane-3,17-dione, (5.α.)- 0.58 t β- Selinene t 7.29 Bicyclo [7.2.0] undec-4-ene, 4,11,11- 0.76 t trimethyl-8-methylene- Estriol t 0.62 Naphthalene t 0.40 Total 97.69 97.47

In a previous study on the essential oil from P. marrubioides had a total of 70 compounds amounting to 87.17% of the oil constituents identified. It comprised; Camphor (48.80%,

1,8-cineole (9.0%), p-cymene (3.08%), α-terpinene (2.58%), limonene dioxide (2.5%), fenchone (1.75%), isocaryophyllene (1.67%), viridiflorol (1.61%), camphene (1.58%), p- selinene (1.50%), trans-sabinene hydrate (1.19%), caryophyllene oxide (1.13%) and terpen-

1-ol (1.08%) were the main components (Omolo et al., 2005).

4.2.5 Chemical composition of essential oil of Ocimum kilimandscharicum

Forty-one (41) compounds were identified in the leaf oil of O. kilimandscharicum by GC,

GC-MS and GC co-injections (GC-CO) with available authentic standards as shown in

Table 4.6. The major components of the oil were camphor (36.584%), limonene (18.6%), camphene (7.1%), linalool (5.72%), terpinen-4-ol (3.9%), α-terpineol (3.6%), terpinolene

(2.2%), sesquisabinene (2.1%), α-pinene (1.9%), (E)-caryophyllene (1.943%), myrcene

(1.87%), 2,6-octadien-1-ol (1.28%), germacrene D (1.18%) and γ-terpinene (1.05%).

Several other monoterpenoid and sesquiterpenoid compounds were identified (Table 4.6) but these were present in amounts < 1%. Eighteen compounds were present in trace (t) amounts (<1 %).

The composition of O. kilimandscharicum essential oil has previously been determined by other researchers, who came up with variations based on localities where the plant was collected. The wide range in the constituents of the O. kilimandscharicum oils and several chemo-types could be due to the diverse environmental and hereditary factors, chemotypes and the nutritional status of the plants as well as other factors that can influence the oil composition (Moghaddam and Mehdizadeh, 2017).

Table 4.6. Chemical composition of the essential oil of Ocimum kilimandscharicum

Compound % Composition Camphor 36.58 Limonene 18.61 Camphene 7.14 Linalool 4.32 Terpinen-4-ol 3.90 α-Terpineol 3.60 Terpinolene 2.23 Sesquisabinene 2.12 α-Pinene 1.94 Caryophyllene(E-) 1.94 Myrcene 1.88 Linalool 1.40 2,6-Octadien-1-ol, 3,7-dimethyl- 1.28 Germacrene D 1.17 γ-Terpinene 1.05 Borneol 0.97 (E)-β-Ocimene 0.92 Trans-Sabinene hydrate 0.86 β-Copaene 0.70 Tricyclene 0.69 δ-Carene 0.68 6-Camphenol 0.60 α-Phellandrene 0.55 α-Copaene 0.50 Bicyclogermacrene 0.47 Viridiflorene 0.46 α-Humulene 0.38 β-Copaene 0.37 α-Copaene 0.34 Tricyclene 0.30 Trans-Muurola-4(14),5-diene 0.26 para-Cymen-8-ol 0.23 α-Phellandrene 0.21 Geranial 0.21 (E)-2-Butenoic acid, 2-methyl-, 2-methylpropyl ester, 0.17 Isoborneol 0.14 Eugenol 0.14 γ-Muurolene 0.12 Dauca-5,8-diene 0.12 α-Bisabolene 0.12 Dauca-5,8-diene 0.11 Total 99.93

4.3 Analysis of essential oil chemical composition variability

The percentage contents of the 125 identified components were significantly different (p <

0.05) among species as shown in Table 4.2 to 4.6. The 125 components were used for the

PCA (Principal Component Analysis) and the HCA (Hierarchical Cluster Analysis) analyses. The PCA horizontal axis accounted for 23.9% of the total variance while the vertical axis accounted for a further 20.7%. Figure 4.3 portrays a visual impression of how well the canonical functions are discriminated in different locations and months using the species as a priori grouping. In Figure 4.3. Vertical axes refer to scores from samples.

Horizonal axes refer to scores from discriminant oil components which are represented as vectors from their origin.

Figure 4.3: Scatter plot of the study samples for the two principal components extracted in the PCA to which cluster belongs.

The dendrogram below (Figure 4.4) represent chemical composition similarity relationships among the identified chemical compounds. The PCA results substantiated the HCA analysis and differentiated the populations as per the chemical composition of their essential oil into

5 clusters (I, II, III, IV and V) with a well-defined geographical dispersal and a variation of

11.0 (Figure 4.2) associated with their essential oil chemotypes.

Figure 4.4: Dendrogram representing chemical composition similarity relationships, among S. biflora (SB), S. abbysinica (SA), C. newii (CN) and P. marrubioides (PM) leaf oil. Samples from Nyahururu (NHR), Kinale (KNL) and Kieni Gakoe (KNG) and Naivasha (NVS) Wet season: Apri-(04), May (05), Jun (06), Nov (11) Dry season: Jan (01), Feb (02), Mar (03), July (07), Aug (08), Sept (09), Oct (10) and Dec (12). The clusters are denoted as I-V.

Cluster I was composed of one species, S. biflora, collected in Kinale both in the wet and dry season, which was characterized by s high mean percentage of geranial (27.4 -31%), neral (20-23.8%), linalool oxide (12.8-16%), isocitral 1,8-cineole (3-4.5%). Cluster II wasmade up of S. biflora collected at Nyahururu during the wet and dry seasons, with essential oils characterized by a high percentage of neral (31.29%), geranial (31.6%), neridol (21.7%), and nerolidal (19.5%). Cluster III was made up of C. newii collected at

Nyahururu and Kinale during the wet and dry seasons, with essential oils characterized majorly by limonene (23-37%), p-mentha-1,8-dien-7-yl acetate (24-26%) and perilla alcohol (5-15%). Cluster IV is made up of only S. abyssinica collected at Nyahururu during the dry season, with essential oils characterized by a high percentage of menthone (44%%) and pulgone (33.2%). Cluster V is made up of P. marrubioides collected at Naivasha during the wet and dry season, with essential oils characterized mainly by camphor (17.8%) and δ-

2-careen (18%).

4.4 Larvicidal efficacies of essential oils against mosquito larvae

4.4.1 Larvicidal efficacies of essential oils against mosquito larvae under laboratory conditions

The mosquito larvicidal activity of the essential oils of S. biflora, C. newii, O. kilimandscharicum and P. marubioides was evaluated under laboratory conditions. After 24 and 48 hours of exposure to third instar of An. gambiae mosquito larvae, the essential oils exhibited significant activity with an LC50 of 10.333, 15.128, 0.292 and 0.814 respectively.

The oil of O. kilimandscharicum gave activity of LC50 of 0.292 and 0.414 at 24 and 48 hrs respectively.

4.4.2 Dose response larvicidal activity

The dose response of the acetone solution of O. kilimandscharicum, water miscible and vegetable oil formulations against An. gambiae larvae are summarized in Table 4.7.

Table 4.7: The LC50 and LC95 values of the essential oil extract of O. kilimandscharicum and water miscible formulation against 3rd instar larvae of An. gambiae under laboratory conditions

LC50 (ppm) LC95 (ppm) Test sample (UCL- LCL) (UCL- LCL) 24 hrs 48 hrs 24 hrs 48 hrs Essential oil 0.29 - 0.41 - (1% in (0.000 - 0.704) (0.00 - 0.85) acetone)

Essential oil 0.13 0.07 0.36 0.14 formulation (0.116 - 0.146), (0.07 - 0.09) (0.280 - 0.530) (0.12 - 0.23)

Vegetable oil 1204.01 1001.27 1592.00 900.97 formulation (100.32-111.37) (92.44-102.34) (132.44 -142.34) (801.44 -107.34)

LC50- lethal concentration that killed 50% of mosquito larvae population, UCI-LCL – upper and lower Confidence Interval. Mortality means are significantly different at p ≤ 0.05 (Student-Newman-Keuls test)

The essential oil formulation exhibited significantly higher mosquito larvicidal activity compared with the vegetable oil formulation against 3rd instar of An. gambiae larvae as summarized in Table 4.7 (example. LC50 of water miscible essential formulation was 0.129 ppm and that of the vegetable oil was 1204 ppm after 24 hours exposures).

The essential oil water-miscible formulation was also found to show comparable levels of activity against Ae. aegypti, Cx. quinquefasciatus and An. arabiensis as summarized in

Table 4.8. A 100% larval survival was noted in the negative control (vegetable oil) group for the entire experiment period. Relative to controls, the O. kilimandscharicum essential oil water miscible formulation significantly reduced survival rates of An. gambiae s.s

(ANOVA, F (4,70) = 77.034, P < 0.001) and An. arabiensis larvae (ANOVA, F (4,70) =

16.422, P < 0.001). There was significant susceptibility difference among the mosquito species to the formulation (ANOVA, F (8,111) = 12.6398, P < 0.001). A time- and dose- dependent reduction in survival rates of water miscible formulation treated mosquito larvae was established (Table 4.8).

Table 4.8: The LC50 values of the O. kilimandscharicum water-miscible product against 3rd instar larvae of An. gambiae, An. arabiensis, Ae. aegypti and Cx. quinquefasciatus mosquitoes after 48 hrs exposure under laboratory conditions

Mosquito species LC50 (ppm) LC95 (ppm) (UCL-LCL) (UCL-LCL) An. gambiae 0.129 0.086 (0.116-1.462) (0.0675 - 0.926)

Ae. aegypti 0.137 0.0716 (0.092 - 0.203) (0.07155 - 0.214)

Cx. quinquefasciatus 0.156 0.064 (0.176 -1.463) (0.166 - 1.334)

An. arabiensis 0.126 0.031 (0.974 - 1.762) (0.274 - 2.463)

Vegetable oil 1204.01 1001.27 formulation (100.32-111.37) (92.44-102.34)

4.4.2.1 Effects of lower doses

In addition to toxicity of high doses, it was observed that lower doses of O. kilimandschericam formulation which did not lead to mortality still showed growth disruption of the larvae as illustrated in Figure 4.5. There was unusually increased development rate of larvae to pupae resulting into inadequate myelinization as shown in

Fig. 4.5 (i), as compared to a normal An. gambiae s.s larvae in control condition shown in

Fig 4.59(ii). Whereas Figure 4.5 (iii) shows a normal An. gambiae s.s larval-pupal intermediate, Figure 4.5(iv) Abnormal An. gambiae s.s larval-pupal intermediate shows uncharacteristic dead larval pupal intermediates as represented in Fig. 4.5 (i) in comparison to a normal An. gambiae s.s larvae in control condition as in Fig. 4.5 (ii).

Fig. 4.5. Larval developmental disruption by low dose of O. kilimandscharicum oil and its water miscible formulation on An. gambiae (s.s.).

In the Figure (i) Demelanized An. gambiae s.s larvae (ii) A normal An. gambiae larvae (iii)

Normal An. gambiae s.s larval-pupal intermediatecontrol larvae (iv) Abnormal An. gambiae s.s larval-pupal intermediate (v) Arrested adult emergence in An. gambiae and (vi) Failed adult emergence in An. gambiae.

Despite moulting having been sustained normally, the development of the juvenile stages was significantly affected. The microscopic inspection of the dead undeveloped stages at

30x intensification which revealed morphological imperfections manifesting as abnormal dead larval-pupal intermediates in leading to arrested adult emergence in An. gambiae with mouthparts and wings crumpled inside the pupal exuvium Fig. 4.5 (v). he adults that fortunately developed from the treated water were unable to escape from the pupal caste and died on the surface of test solution Fig 4.5. (vi). Overall, the formulation at low doses induced extended larval stage duration by 4 days preceding to pupation as compared to the negative controls.

4.4.3 Semi-field evaluation of O. kilimandscharicum mosquito larvicide formulation

The results of the semi-field evaluation are tabulated in Table 4.9 and illustrated in Figure

4.6. They indicated that the formulated O. kilimandscharicum product had impressive efficacy compared with B.ti granules used as positive control. After 1 day of treatments, the formulation at 0.5 ppm led to 98%% larval mortality, while Bti at 28 ppm showed larval mortality of 54%. However, after 8 days Bti attained 96.5% mortality while the formulation had attained 100% mortality on day three.

Table 4.9: Larvidal activity of Ocimum kilimandscharicum water miscible formulation and Bti granules against An. gambiae mosquito larvae under semi-field conditions in Kilifi, Kenya

Percentage larval density (mean ± SD) 24hrs Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Treatment

O. 2 0.5 kilimandscharicum formulation 100 (±5.0) (±1.99) 0

23.5 20 15 .5 Bti granules 46 29 17.5 11.5 3.5 (28ppm) 100 (±2.22) (±1.99) (± 3.56) (±4.50) (±4.56) (±3.19) (±5.02) (±4.44)

93.5 85 80.5 79.5 78.5 68.5 65 45 Untreated control 100 (±4.75) (±5.01) (±5.89) (±2.99) (±4.11) (±3.98) (±3.24) (±3.12)

Data expressed as % mean mortality ± standard deviation (S.D)

Figure 4.6: Larvicidal activity against An. gambiae mosquito larvae under semi-field conditions in Kilifi, Kenya for a period of 8 days. Each point on the plots represents a percentage mean (±SD) larval mortality of mosquito treated with larvicide formulation, Bti granules and untreated control

4.5 Repellent activities of selected plant essential oils against mosquitoes

4.5.1 Estimation of effective dose using Klun and Debboun Module

For each of the four plant essential oils, concentrations and volunteers, 30 female mosquitoes were tested against Ae. aegypti and An. gambiae, that is. 90 mosquitoes per plant (Table 4.10). All four plant essential oils showed repellent properties when assessed in the K and D tests. Four extracts showed very similar dose-response patterns. However, P. marrubioides was several folds less active, both against Ae. aegypti and An. gambiae as shown in Table 4.10 and illustrated in Figure 4.4 and 4.5.

Table 4.10: Effective dose (ED) results against Ae aegypti and An. gambiae s.s. using the K and D module.

Mosquito species Plant Species Location ED50 (95% CI) ED95 (95%CI) Ae. aegypti C. newii Nyahururu 1.28 (0.93 - 1.77) 23.74 (10.69 – 52.72)

P. marrubioides Naivasha 6.18 (2.84 - 13.46) 443.34 (36.38 –5402.71)

S. biflora Kieni 1.95 (1.48 - 2.58) 14.75 (8.16 - 26.67)

Kinale 2.35 (1.99 - 2.78) 15.33 (10.15 - 23.15)

Nyahururu 1.71 (1.25 - 2.32) 8.90 (5.80 -13.65)

An. gambiae C. newii Nyahururu 0.50 (0.20 - 1.23) 11.03 (4.22 - 28.85)

P. marrubioides Naivasha 5.44 (1.94 - 15.24) 595.43 (20.04 - 17691.7)

S. biflora Kieni 1.34 (0.87 - 2.05) 9.01 (5.12 – 15.85)

Kinale 1.84 (1.43 - 0.38) 9.95 (6.22 – 15.92)

Nyahururu 1.74 (1.23 - 2.45) 16.30 (9.32 – 2

Effective dose (ED) are given as percentage crude plant oil in EtOH (w/w).

The four plant extracts, S. biflora (Nyahururu, Kinale and Kieni) as well as C. newii

(Kinale) collected during dry season showed comparable repellent effects at a concentration

of 20% oil in ethanol and comparable to 20% ethanolic DEET solution, showing that the

essential oils of plants are equally effective as DEET. In contrast, the data for P.

marruboides shows that even the crude, undiluted plant essential oil (that is 100%) would

not provide a 95% bite avoidance effect (Table 4.11). Remarkably, the dose-response

curves are very similar between mosquito species indicating that the repellent effect works

across species, which is desirable if the repellent formulation is to be used as a broad band

protection against biting mosquitoes. A caveat underlying the data from the K and D assay

is that the variability is relatively high which is particularly pronounced in the confidence

intervals for the ED95 estimates. Taking this into account, it was decided to prepare 20%

formulations for testing in the WHO cage-in-arm method. While S. biflora and C. newii

showed similar protection in both mosquito species, P. marrubioides provided several folds

less protection Figure 4.7 and 4.8). Ethanol was used as the negative control

Figure 4.7: Essential oils Dose-response from Figure 4.8: Essential oils Dose-response K and D assay for Ae. aegypti, from K and D assay for An. gambiae

4.5.2 Testing sensitivity of mosquito to insect repellent employing a warm body

An. gambiae, 4–6-day-old insect was tested against repellent N, N-Diethyl-meta-toluamide

(DEET) and essential oils for sensitivity. The EO’s were all tested at 3.8 μg/cm², equivalent to 20 nmol DEET. In Figure 4.6, the limits of the boxes specify the twenty-fifth and seventy-fifth percentiles, the solid line in the box is the median, the capped bars show the tenth and the ninetieth percentiles, and data points outside these limits are plotted as circles

(Figure 4.6).

Landing response 2 min by A.gambiae females on a warm body treated with DEET and diff EOS

300

250

200

150

landings

100

Control DEET 20 nmol CN/NHR @ 3.8 ug PM/NVS @ 3.8 ug SB/KIN @ 3.8 ug SB/KML @ 3.8 ug SB/NHR @ 3.8 ug

mean 118.22 2.52 19.2 127.5 99.33 105.25 112.33 median 121 0 16 121.5 110 105 106 min 0 0 12 117 77 98 105 max 330 21 30 150 111 113 126 sum 50718 116 96 510 298 421 337 10% perc. 19.8 0 13.2 117.3 83.6 98.3 105.2 90% perc. 182 9.5 27.2 142.5 110.8 112.4 122 stdev.spl 63.38 5.02 7.26 15.42 19.35 7.85 11.85 valid n 429 46 5 4 3 4 3 total n 435 49 5 4 3 4 3

Figure 4.9: In vitro test for mosquito repellents using a warm body

4.6 Estimation of protection time in WHO arm-in-cage tests

With regard, to complete protection time, the oil formulations of all four plant, S. biflora

(Nyahururu), S. biflora (Kinale), C. newii, P. marrubioides oil formulations gave only complete protection time (CPT) of 30 minutes against the highly aggressive Ae. aegypti.

Since measurements were taken only every 30 min and no test was performed at 0 minutes, absence of complete protection may not be excluded (Table 4.11 and Figure 4.7). In contrast, there is clear evidence for complete protection against Cx. quinquefasciatus with mean CPTs ranging between 90 and 120 minutes. The 20% DEET ethanolic solution showed a higher CPT than all the plant oil formulations against both mosquito species.

DEET is a known standard repellent product and was used in this experiment as the positive control (Zhu et al., 2018).

The values obtained for a given repellent (for example a 20% DEET in ethanol solution) may vary between studies depending on various biotic and abiotic factors such as mosquito species and strain, larval rearing and nutrition, mosquito density and cage size, innate differences of human volunteers, bioassay procedures, etc. Therefore, a 20% ethanolic

DEET solution was included as a positive control in this study to facilitate comparison with other reports and published data. For example, Cilek et al., (2004) found an average complete protection time of 183±14.2 minutes which is slightly more than the 135±46 minutes that was observed in this study.

A caveat underlying CPT measurement is that it may not represent very well the average behaviour of the mosquito batch used, rather it may pick up the extremes. Taking this and variations in attractiveness between volunteers into account, another measurement of longevity was used: the 90% protective time (PT90) which is the time after application of

the formulation until protection relative to the negative control drops below 90%. When estimating PT90, it becomes clear that the plant formulations provide protection against Cx. quinquefasciatus, An. gambiae as well as Ae. aegypti (Table 4.11 and Figure 4.10).

Generally, the repellent formulations work better against An. gambiae and Cx. quinquefasciatus as compared to Ae. aegypti which is in line with other reported studies

(Sokny et al., 2014).

Among the plant formulations, there is clear difference between ethanolic solutions and cream formulations. This is because of reduced release rate where the cream formulation may be long lasting. Figure 4.10 shows the individual CPT for every volunteer and formulations when they were tested against Ae. aegyptei and Cx. quinquefasciatus.

Table 4.11: Protection time (in minutes) of formulated plant oils measured as the complete protection time (CPT) and the time when protection was at least 90%. Mosquito species Plant Species Formulation Complete protection ≥90% protection time (95% CI) Minutes) time (95% CI) ______Ae. aegypti C. newii 20% cream 30 (30 -30) 60 (26 -94) 20% EtOH solution 30 (30 - 30) 38 (24 - 51) DEET 20% EtOH solution 135 (45 - 225) 229 (184 - 274) S. biflora 20% Cream 34 (27 - 41) 56 (30 - 83) 20% EtOH solution 30 (30 - 30) 38 (24 - 51) Cx. quinquefasciatus C. newii 20% cream 120 (86 - 154) 206 (135 -276) 20% EtOH solution 113 (56 - 169) 165 (88 - 242) DEET 20%EtOH solution 218 (152 -283) 323 (260 -385) S. biflora 20% cream 128 (103 - 152) 180 (138 - 222) 20% EtOH solution 90 (69 -111) 128 (84 - 171) An. gambiae C. newii 20% cream 160 (73 - 141) 241 (111 -2009) 20% EtOH solution 135 (76- 1171) 119 (69 - 220) DEET 20% EtOH solution 240 (142 -261) 380 (233 -366) S. biflora 20% cream 150 (90 - 131) 208 (122 - 201) 20% EtOH solution 107 (74 -117) 159 (88 - 161)

Figure 4.10: Complete protection time (CPT). Dots represent measurements of CPT for each volunteer and formulation against Ae aegypti and Cx quinquefasciatus

Protection time of formulated plant oils was measured as complete protection time

(CPT) and was at least 90%. Figure 4.11 shows the duration of 90% plus protection time (hours) for each volunteer and formulation.

Figure 4.11: Complete protection time (CPT). Dots represent measurements of

CPT for each volunteer and formulation

4.7 Further improved formulation

Improvement in formulation of the repellent plant products may lead to controlled release of volatiles with resulting increase in time of protection by the repellent. This was demonstrated when 5% vanillin was added to cream formulation of S. biflora which led to increased repellence time (Figure 4.12). The results are comparable to those found when vanillin was added to DEET formulated products (Tawatsin et. al.,

2001).

Figure 4.12: Relative repellency (median protection time) of volatile oils with/without vanillin compared with DEET formulations against 3 mosquito species under laboratory conditions.

4.8 Subtractive bioassay on the constituents of Satureja biflora and Conyza newii with significant contribution to mosquito repellence

4.8.1 Summary on EAG-active compounds

Selected electro-physiologically (EAG) active compounds and blends of synthetic standards, in the ratio found in the GC-MS analyses, were assessed for their repellency action on the forearms of human volunteers against female An. gambiae s.s. mosquitoes. Absence of some constituents in the blends occasioned either a surge or a decrease in repellency of subsequent blends, while absence of some components was inconsequential. Synthetic blends of electrophysiological active constituents of C. newii and S. biflora showed significant activities more than their matching oils.

4.8.2 Electrophysiological responses of the constituents of essential oils to An.

gambiae

Gas chromatography electroantennography (GC-EAG) analyses exhibited that An. gambiae mosquito responded to some of the compounds present in the essentials of S. biflora and C. newii. Some of the minor compounds that are yet to be identified similarly elicited strong responses from the mosquitoes.

In the S. biflora essential oil, EAG responses were detected recurrently with constituents at 4 different retention times (Figure 4.13).

Figure 4.13: EAG track (lower one) and its GC chromatogram (upper one) recorded from antennae of female An. gambiae in response to essential oil of S. biflora.

The EAG active compounds identified included linalool (16), neral (17), geraniol (18) and neridol (19) which were constituted into a four-component synthetic blend as summarized in Table 4.12.

Table 4.12. Compounds present in S. biflora essential oil identified by GC-EAG that elicited electrophysiological responses in antennae of female An. gambiae.

Retention Time (GC-EAD) Compound Identified No of antennae responding N= 6 13.00 Linalool (16) 3

15.19 Neral (17) 2

15.42 Geraniol (18) 3

19.45 Neridol (19) 5

The structures of the EAG active compounds in S. biflora oil are listed in Figure 4.14

HO O

16 17

HO 19 HO

Figure 4.14. EAG active compounds of essential oil constituents of S. biflora

For C. newii essential oil, the EAG active compounds were detected simultaneously in

GC-EAG setup with constituents appearing at 5 different retention times (Figure

4.15).

Figure 4.15: EAG track (lower one) and its GC chromatogram (upper one) recorded from antennae of female A. gambiae in response to essential oil of C. newii

The EAG active compounds identified included limonene (15) perilla aldehyde (20),

α-pinene (21, perilla alcohol (22), and cinnamaldehyde-α-pentyl (23) that formed the five-component synthetic blend of C. newii essential oil. They are listed in Table 4.13.

The identified compound structures are illustrated in Figure 4.12.

15 20

22 21

Figure 4.16. EAG active compounds of essential oil components of C. newii

Table 4.13. Compounds present in C. newii essential oil that elicited electrophysiological responses in antennae of female An. gambiae.

Retention Time Compound Identified No of antennae (GC-EAG) responding N= 6 10.69 α-Pinene (21) 1 11.75 Limonene (15) 2 15.71 Perillaldehyde (22) 4 16.12 Perilla alcohol (20) 3 20.44 Cinnamaldehyde-α-pentyl (23) 3

Other minor compounds that were identified that had influence on olfaction were caryophyllene (24), hexyl acetate (25), Methyl chavicol (26), Ethyl isovalerate (27), α-Humulene (28), 1,8- Cineole (29) and (E,E)-Decadienal (30) are shown in Figure 4.17

O 25 24

27 H3CO 26

28 29

Figure 4.17: Compounds of other minor EAG-active constituents of C. newii and S. biflora plant essential oils

4.8.3 Repellence of S. biflora and C. newii essential oils and their EAG active blends

The comparative results of the repellence of the two better performing crude essential oils and that of DEET are shown in Table 4.14

Table 4.14. RD50 and RD75 of the S. biflora and C. newii oils and DEET

-5 -2 -5 -2 Essential oil /DEET RD50 (×10 mg cm ) RD75 (×10 mg cm ) S. biflora 5.22 (0.0672, 31) 751.0 (138, 2000) C. newii 2.37 (6.27, 0.612) 9.49 (9.5, 57.0) DEET 1.19 (0.289, 2.85) 7.3 (3.34, 15.2) Values in parentheses represent lower and upper limits at 95% confidence

The repellency data of C. newii essential oil and blends and some of its EAG active constituents in subtractive combination bioassays are provided in Table 4.15.

Table 4.15. RD50 and RD75 (95% CI) of C. newii oil, and blends of its selected electrophysiologic ally active compounds with each EAG-active constituent missing.

-5 -2 -5 -2 Essential oil and blends RD50 (×10 mg cm ) RD75 (×10 mg cm ) C. newii 2.37 (0.677, 6.72) 10.5 (10.5, 63) 5-Components [5CB] 20.3 (4.02, 60.3) 571 (172, 4080) FB minus pinene (21) 682.0 (5.33, 1250) 36.8 (4.39, 246) FB minus perilla alcohol (22) 168.0 (76.1, 362) 318 (678, 5500) FB minus limonene (15) 71.9 (34.9, 147) 5160 (243, 146000) FB minus perillaldehyde (20) 13.3 (5.89, 26.6) 122 (60.1, 2700) FB minus cinnamaldehyde-α- 16.4 (6.17, 35.8) 24.4 (142.0, 937) pentyl (23) Values in parentheses represent lower and upper limits at 95% confidence, FB refers to Full Blend The EAG active five-component synthetic blend [5CB] exhibited lower repellency

-5 -2 -5 activity (RD50 20.3×10 mg cm , 95% CI) than the parent oil (RD50 2.37 ×10 mg

98 cm-2, 95% CI). The highest increase in the repellency of the blend was observed with

-5 -2 subtraction of perillaldehyde (19) (RD50 13.3 ×10 mg cm ), followed by that of

-5 -2 cinnamaldehyde-α-pentyl, (22) (RD50 16.4 ×10 mg cm ) and then limonene (15)

-5 -2 -5 -2 (RD50 71.9 ×10 mg cm ), perilla alcohol, (21) (RD50 168.0 ×10 mg cm ) and lastly

-5 -2 pinene (20) (RD50 682.0 ×10 mg cm ). Table 4.16 shows the repellency results of the crude oil, four-component synthetic blend [full blend (FB)] and the subtractive effect of the EAG active compounds of S. biflora.

Table 4.16. RD50 and RD75 (95% CI) of S. biflora oil and its selected electrophysiologic ally active synthetic blends.

RD50 RD75 Essential oil/blends (×10-5 mg cm-2) (×10-5 mg cm-2) S. biflora 5.68 (0.0782, 34) 851.0 (158, 26100)

[Full Blend (FB)] 66.6 (0.02, 235000) 65000 (1400, 722000)

FB minus linalool (16) 25.1 (0.188, 1000) 15000 (1000,

FB minus neral (17) 0.838 (0.437, 7.2) 450 (70.4, 440000)

FB minus geraniol (18) 0.412 (0.122, 940) 112000

FB minus neridol (19) 14.3 (4.89, 26.6) 123 (61.1, 2500)

Values in parentheses represent lower and upper confidence limits at 95%

The results showed that S. biflora essential oil exhibited lower repellency (RD50 66.6

-5 -2 -5 -2 ×10 mg cm , 95% CI) than the parent oil (RD50 5.68 ×10 mg cm , 95% CI).

The highest increase in the repellency of the blend was observed with exclusion of

-5 -2 geraniol, (18) (RD50 0.412 ×10 mg cm , 95% CI), followed by that of neral, (17)

-5 -2 -5 -2 (RD50 0.838 ×10 mg cm , 95% CI), then neridol (18) (RD50 14.3×10 mg cm , 95%

-5 -2 CI and finally linalool, (16) (RD50 25.1 ×10 mg cm , 95% CI).

4.9 Discussion

Several previous studies have shown large differences in seasonal yields of essential oils of different plants (McGimpsey et al., 1994; Perry et al., 1999; Sangwan et al.,

2001; Verma et al., 2011). However, in the present study the four plant species: S. biflora, C, newii, P. marrubioides and S. abyssinica growing in the Aberdare ranges and Kakamega region in Kenya showed insignificant differences in oil yields in both the wet and dry season.

The chemical compositions of all the essential oils of S. biflora, C, newii, P. marrubioides and S. abyssinica were analysed using both GC/FID and GC/MS techniques. All the essential oil samples were characterized by very high percentage of monoterpenes (49.91-90.33%) especially the oxygenated ones (32.01-62.18%), which constituted the predominant class as was found previously for S. biflora (Ali et al., 2014). The sesquiterpenes were also represented mainly by oxygenated sesquiterpenes (2.92-21.84%) in contrast to what was previously observed where the amount of oxygenated sesquiterpenes did not exceed 4.6% of the total essential oil of

S. biflora (Ali et al., 2014). However, the structural profiles of the oxygenated monoterpenes showed significant variations. The essential oil chemotypes detected in

S. biflora, C. newii and P. maruboides populations based on the composition of their major compounds, as well as the clusters based on all the constituents. Regarding the morphological polymorphism, the sampling sites did not relate to any morphological variation within each population. As such in this study, the presence of chemotypic variation cannot be resolved, because the plants grow in different environments at each sampling site.

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However, the results direct that the Nyahururu site and Kinale together with Kieni-

Gakoe represent two separate ecotypes in relation to S. biflora. Nyahururu is on a lower altitude as compared to the other two thus, geographically separating the population. At least in part, this spatial barrier could contribute to an ecological segregation, a precondition for speciation. Indeed, chemo-variation and ultimate speciation was observed. Considering cluster, I and II, there is a clear demonstration of two different chemotypes of S. biflora, one based on geranial (27.4 -31%)/neral

(20-23.8%) chemotype and a second characterized by neral (31.29%)/geranial/

(31.6%)/neridol (z) (21.7%).

Thus, these disproportions in our results may be associated with factors other than the genetic changes in terpenoid biosynthetic pathway (Scheme 2.1) which might modify during plant growth, or under herbivory pressure (Sturgeon, 1979; Lawal et al., 2014), or because of changes in environmental circumstances (Figuei-redo et al., 1997;

Robles and Garzino, 2000). Although it has been suggested that terpene chemotypes are powerfully controlled by genetic factors (Hannover, 1992). Hannover also described occurrences of environmental disparity in terpene expression under risky habitation situations (Hannover, 1992). Influence of environmental factors in the chemical composition of essential oils have also been described in the Asteraceae family of plants (Haider et al., 2004) and are well recognized for Lamiaceae (Loziene and Venskutonis, 2005; Karousou et al., 2005).

Moreover, essential oil composition variability is well recognised within plant populations (Perry et al., 1999; Muñoz-Bertomeu et al., 2007), so in breeding and assortment programmes, the chemical description of the population and their related

101 bioactivities is the initial stage in the direction of the choice of the target genotypic/epigenetic type. According to our results, populations from the Abedares

Range region are preferred source of S. biflora essential oils, that have high neral, neridal, geranial and linalool oxides levels, whereas C. newii oils from Nyahururu populations are more suitable to produce oils rich in limonene (15), camphor and borneol. HCA and PCA outcomes demonstrated in Figure 4.3 and Figure 4.4 show a direct association among geographical detachments of the populations and their grouping patterns. Neighbouring populations have added similar chemical profiles owing to either an advanced similarity amongst genotypes of the populations or to similar environmental settings.

The use of larvicides for the larval control of vectors of malaria is a well-proven disease preventative method that has received little attention, but which needs re- consideration for malaria control programmes in the current times (Walker and

Lynch, 2007). Such control has mainly been using synthetic chemicals including organophosphates (mainly temephos), and a biopesticide, Bacillus thuringiensis israelensis (Bti) (WHO, 2013). However, the potential of resistance developing against temephos and Bti in their continued operational use is a matter of concern

(Marcombe et al., 2011).

The larvicidal activity of the water-miscible formulation of O. kilimandscharicum is comparatively better than that previously reported in a previously highly rated emulsified concentrate formulation of neem (A. indica) oil developed by the BMR and Company of Pune, India (Virendra et al., 2009). The median lethal concentrations

(LC50) of the neem oil formulation against An. stephensi, Cx. quinquefasciatus and

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Ae. aegypti were reported to be 1.6, 1.8 and 1.7 ppm, respectively. These values were much higher than those found in this study for the O. kilimandschericum water- miscible formulation on An. gambiae, Cx. quinquefasciatus and Ae. aegypti as summarized in Table 4.8. The median lethal concentrations (LC50) of the O. kilimandschericum formulation against An. gambiae, Ae. Aegypti, Cx.

Quinquefasciatus and An. arabiensis was 0.129, 0.137, 0.203, 0.156 and 0.126 respectively.

Previously, the use of oils even at low concentrations was found to form droplets on the surface, which would coalesce at higher doses to form thin layers. The resulting drop in the surface tension made it very difficult for the mosquito larvae to effectively float on the surface and breathe. So, probably, the observed effects on larvae were triggered primarily by inability of the larvae to breathe on the surface rather than any insecticidal effects during the period of exposure (Reddy et al., 2011). Thus, water miscible formulations constitute the way forward in the effective evaluation of essential oils and other water insoluble larvicidal candidates. Thus, O. kilimandscharicum formulation shows potential for practical field application in the control of mosquito larvae. Since the yield of the essential oil from O. kilimandscharicum is relatively high, there is a potential for large-scale extraction of the essential oil from the plant to produce the water-miscible formulation for the control of mosquito larvae. The formulation showed potential as a larvicide against other important vectors of malaria and yellow fever.

Moreover, the O. kilimandschericum formulation being of plant origin and with a range of constituents in the blends, can provide resistance-mitigating effects over long periods (Bekele and Hassanali, 2001). In addition, it has the advantage of being eco-

103 friendly and easily biodegradable and may be cheaper than other larvicidal agents, such as temephos and Bti (Ghosh et al., 2012). Furthermore, different constituents of phytochemical blends affect insect physiology in different ways and at different receptor sites, the principal of which is abnormality in the nervous system. Thus, it takes extensive time for insects to build resistance to such a blend of bioactive compounds, because they have broad modes of action and work synergistically

(Singh, 2014). Although numerous studies have recorded the effectiveness of plant extracts as the reservoir pool of bioactive toxic mediators against mosquito larvae, only a limited number have been steered downstream, commercially produced and used in vector control programmes.

In the present study, there is clear evidence that the essential oils from C. newii and S. biflora are strongly repellent against at least three medically important mosqutoes, An. gambiae s.s., Ae. aegypti and Cx. quinquefasciatus. However, while the effective dose is comparable to that of DEET, the protection time is significantly shorter, suggesting faster evaporation of the active compounds, which is a general property of constituents of essential oils. Addition of 5% vanillin to a cream formulation of S. biflora led to increased repellency time . Similar results were found when vanillin was added to DEET formulated products (Khan et al., 1975; Tawatsin et al., 2001).

However, there is need to evaluate other formulations of the oils, such as microencapsulation with different polymeric products, for better controlled release of the active constituents that would extend the duration of the repellent blends.

Subtractive assays provided information on the relative contribution of some EAG- active constituents of the essential oils of S. biflora, and C. newii to the overall

104 repellency of the parent oils. The lower repellent activities of the synthetic blends of available EAG-active compounds of S. bioflora and C. newii relative to their corresponding parent oils, could be attributed to additional EAG-active constituents that were not included in the blends. To characterize the full active blend, it would be important to source these constituents either commercially or by synthesis and evaluate their effects on repellence of EAG-active blends. Moreover, EAG- constituents of the oils have variable effects on overall repellence.

The fact that absence of α-pinene (21), perilla alcohol (22) and limonene (15) substantially lowered the activities of the resultant blends of C. newiii essential oil indicated that these components had significant contribution to the repellency of the parent oil. However, the significant upsurge in the repellency on subtraction of geraniol (18) from the blend of S. biflora could be due to enhanced synergy between the other compounds resulting from reduced antagonistic effect of geraniol. Similar observations have been reported in the case of mosquito larvicidal activity of dichloromethane extract of the root bark of Lantana viburnoides against An. gambiae where removal of some compounds lead to improved activity (Innocent et al., 2008).

In addition, (E)-caryophyllene (24) has been described to contribute towards the overall repellency of Nepeta cataria oil against An. gambiae (Birkett et al., 2011).

Further, studies elsewhere showed that (E)-caryophyllene (24) independently does not display at all repellent activity against An. gambiae (Omolo et al., 2004). This indicates synergistic effects of different constituents, like an earlier report by Bekele and Hassanali presented that the lethal activity of quaternary combination of methyl chavicol (26), ethyl isovalerate (27), α-humulene (28), and 1,8-cineole (29) counter the postharvest pest Rhyzopertha dominica, whereas none of the compounds were

105 independently lethal to the pest (Bekele and Hassanali, 2001). Nevertheless, the lower activity of S. biflora essential oil relative to the synthetic blend of available EAG-

-5 active compounds minus neral at RD50 0.838 × 10 mg cm-2) suggested that other constituents of the essential oil might hinder the repellence of active compounds and combinations. Overall, the results from the subtractive-combination bioassays showed that presence of perilla alcohol (22) and α-pinene (21) contribute to major increase in repellency of their respective blends against An. gambiae, while neral (17), geraniol

(18), perillaldehyde (20), cinnamaldehyde-α-pentyl (23) were found to significantly reduce the repellency of the respective blend.. Likewise, presence of limonene (15) and linalool (16) indicated moderate increase in repellency activity of their respective blends.

This study lays down useful groundwork for characterizing the compositions of the essential oils of C. newii and S. biflora associated with considerable repellence and for their downstream quality-controlled development and application in effective repulsion of mosquitoes. Equally, the water miscible formulated larvicide from O. kilimandscharicum proved to be an effective and eco-friendly larvicide, which could be used as an alternative for malaria control.

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CHAPTER FIVE

CONCLUSIONS AND RECOMMENDATION

5.1 Conclusions

The phytochemical investigation reported in the current study comprised of extraction, characterization of essential oils using analytical techniques like GC and

GC-MS; screening for bioactivity (repellence and larvicidal) of essential oils and constituents of 4 plant species from the Meliacea family, namely S. biflora. O. kilimandascharucam, P. maruboides and Astracea Family, namely C. newii, on An. gambiae, Ae. Aegypti and Cx quinquefassciatus mosquitoes. The behavioural response to EAG-active constituents and blends in subtractive bioassays following exposure to allelochemicals was determined.

From the results obtained and discussion thereto, the following was concluded.

i) The yields of each of the plant essential oils from different habitats and

seasons were determined to be similar qualitatively but with significant

quantitative differences.

ii) There was a clear demonstration of chemotypic variation based on location

among the two-plant species namely C. newii and S. biflora. For S. biflora,

there were 3 chemotypes based on location are defined as: S. biflora, the

chemotypes based on location are defined as geranial (31%)/neral

(24%)/linalool (12%) of Kinale. linalool (28%)/neridol (21%)/β-pinene of

Nyahururu, and geranial (27%) neridol (Z) (21%)/linalool (16%) chemotype

of Kieni Gakoe. For C. newii, two chemotypes were identified as p-mentha-

1,8-dien-7-yl acetate (24%)/limonene (23%)/5-methyl-2-phenyl-2-hexenal

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(21%) associated with Nyahururu, and p-mentha-1,8-dien-7-yl acetate (27%)/

limonene (38%) associated with Kinale.

iii) The water-miscible formulation of O. kilimandscharicum essential oil has

potential for practical application for the control of larvae of mosquito species.

The yield of the essential oil was relatively high (3.5%) indicating that there

was a potential for large scale extraction of the oil for production of a bio-

larvicide

iv) The study suggests that part of the mode of action of larvicidal activity is

through disruption of larval development.

v) While the repellence activity of C. newii and S. biflora formulations are

comparable to DEET the duration of protection is, however, shorter than a

typical N, N-diethly-3-methylbenzamide (DEET) formulation. It was

demonstrated that use of cream and incorporation of vanilla significantly

improved and prolonged duration of repellence.

vi) The cream with added vanillin formulations of C. newii and S. biflora essential

oils showed high activity as repellents in personal and/or space protection

against at least three medically important taxa, An. gambiae s.s., Ae. aegypti

and Cx. quinquefasciatus mosquitoes.

vii) The synergistic and antagonistic effects of the essential oil components was

demonstrated when some of the EAG-active constituents, perilla alcohol (22)

108

and α-pinene (21), contributed to major increase in repellency of their

respective blends against An. gambiae, while neral (17), geraniol (18),

perillaldehyde (20) and cinnamaldehyde-α-pentyl (23) were found to

significantly reduce the repellence of the respective blend.

viii) The study lays down useful groundwork for characterizing the compositions of

the essential oils of plants associated with enhanced repellence and quality-

controlled downstream application in effective repulsion of mosquitoes.

5.2 Recommendations from this study

Based on the conclusions above, we recommend that:

i) The highly effective and potential larvicidal product from O. kilindscharicum

oil that was formulated need to be deployed for large scale field trials and

registered with the Pest Control Products Board of Kenya for adoption as a

mosquito larval manager. The most effective dose being 1 ppm of the

concentrate to everyone 1 litre of water hosting the larvae.

ii) The improved repellent cream with added vanillin formulation of essential oils

from S. biflora and C. newii had prolonged protection duration efficacy

comparable to DEET. There is need to prepare a detailed dossier for

registration application with the relevant authorities for use by communities

with high prevalence of mosquito borne diseases.

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5.3 Recommendations for future Research

i) There is need to undertake detailed experiment to establish the mode of action

of the bio-larvicide and any effects on non-target organisms.

ii) Undertake detailed study on biochemical mechanism of action of the

developed larvicide and repellents to understand how they function.

iii) The work on S. biflora and C. newii was developed on essential oils isolated

from a single year in only two seasons. Further investigations need to be

undertaken in successive seasons, including soil and weather data of the

trial points, to clarify the impact of genetic inheritance and abiotic

influences on the compositions of the essential oils.

iv) Further study is required in the formulation of essential oils and their blends

using nanoencapsulation techniques for their slow controlled release for

improved performance in terms of prolonged time of use in the field.

v) Exploiting the use of attractant constituents in a “push-pull” set up to divert

the mosquitoes from human dwellings to baited traps.

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APPENDICES

APPENDIX A: ETHICAL APPROVAL

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APPENDIX B: DISSEMINATION OF RESEARCH FINDINGS

Appendix B1: Conferences-1

5-8TH October 2014, 1ST Pan-African Mosquito Control Association Conference - “Challenges of Vector control in the malaria eradication era”. Panari Hotel, Nairobi Kenya.

Title: Development of plant-derived products for control of mosquito larvae

Abstract:

Background: The use of synthetic insecticides to control mosquito larvae has resulted in serious environmental problems such as air, soil and ground water pollution as well as toxicity to aquatic ecosystems, animals and humans. Their repeated use has also led to development of insecticide resistance in mosquito vectors. Potential risks due to the use of toxic synthetic pesticides have necessitated the development of eco-friendly and efficient alternatives. Although lot of research work has been undertaken in Africa and elsewhere to discover new products from plants for control of mosquito larvae, very few plants derived products have been developed and applied for this purpose. In this study, we report on the development of several plan-derived products that have shown highly effective activity against mosquito larvae in the laboratory and under semi-field and field conditions.

Method: Plant products were extracted using standard methods and formulated into water-based products using proprietary methods. The larvicidal assay tests were carried out using Anopheline and culicine mosquito larvae following standard WHO larval bioassay test method. Semi-field and field evaluation were undertaken in Kilifi and Malindi, Kenya and Tolay, Ethiopia using standard WHO methods.

Results: In the laboratory, the product exhibited activities with LD50 of as low as 0.086 ppm (0.000086%) while under semi-field, they showed 100% mortality after 48hours at concertation as low as 1 ppm (0.0001%). under field conditions, 78 to 100% mortality is achieved after 48 to 72hours with concertation of 1pppm of the product. The product had no observable adverse effects on non-target organisms such

131 as bees, tad poles and fish. Registration of some of the products with relevant bodies has been initiated.

Conclusion: Results from this study demonstrate the potential of plan derived products as biodegradable Larvicidal natural products that can be used in local mosquito management.

Key words: mosquitoes, plant-derived, larvicide, vector control

Appendix B2: Conference 2

1-7TH August 2016, 15th Congress of the international Society of Ethnobiology Conference “Ethnobiology knowledge for improved human well-being and development”. Department of Plant Science, microbiology and Biotechnology, Makerere University, Uganda.

Title: Promotion of ethno-botanical plant-derived community enterprises in Kenya and Tanzania for livelihood improvement and biodiversity improvement.

Abstract:

ICIPE and partners have been facilitating community members in Kenya and Tanzania to domesticate and cultivate several useful ethnobotanicals to reduce their overexploitation from the wild to support demand, and to provide alternative income generating opportunities

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APPENDIX C: ABSTRACTS AND SUBMITTED MANUSCRIPTS

Appendix C1- Abstract Manuscript 1:

Title: Mosquito larvicidal activity of Ocimum kilimandscharicum essential oil and its water-miscible formulation under laboratory and semi-field conditions (Journal: ChemEcology)

Background: Mosquitoes act as vectors of many life-threatening diseases globally, including malaria, yellow fever, dengue fever and lymphatic filariasis. Mosquito control strategies include the use of synthetic chemical repellents and insecticides, which have been associated with resistance development and undesirable ecological effects. There has been increasing interest in the use of phytochemicals as alternatives. However, there have been limited R&D studies to facilitate downstream development of phytochemicals for practical use. In this study toxicity and growth disruption activities of essential oil of O. kilimandscharicum water-miscible formulation against Anopheles gambiae sensu secto, Culex quinquefaciatus, Aedes eagytei and Anopheles. arabiensis larvae were evaluated. Methods: The study first compared the larvicidal efficacies of essential oil of O. kilimandscharicum dissolved in acetone under laboratory conditions on An. gambiae s.s. The water-miscible product was formulated as an emulsifiable concentrate using Polysorbate 80, a nonionic surfactant and emulsifier. The formulated product was tested in the laboratory against An. gambiae s.s., Ae. aegypti, An. arabinoses, Cx. quinquefasciatus larvae and against An. gambiae s. s. under semi-field conditions. The larval mortality was scored after 24 h and 48 h. The data was corrected for control mortality using Abbott’s formula. The percentages of mortality were transformed to arcsine square- foot values for analysis of variance (ANOVA). Concentration–mortality data were subjected to probit analysis. The essential oil composition was analyzed using gas chromatography–mass spectrometry (GC-MS). Results: The water miscible formulated product showed higher mortality against the third instar An. gambiae s.s larvae than unformulated essential oil. The activity was dose dependent with highest mortality of 100%. It was also effective against Ae. aegypti, Cx quinquefasciatus and An. arabiensis larvae. The activity of the formulated oil on An. gambiae s.s. larvae were also confirmed in a semi-field setup alongside Bacillus thurigeniss granules. Analysis of the essential oil of O. kilimandscharicum by GC-MS revealed 44

133 constituents with the major compounds being D-camphor (36.6%), limonene (18.6%), camphene (7.1%), linalool (4.3%), terpinen-4-ol (3.9%), α-terpineol (3.6%), terpinolene (2.2%) and sesquisabinene (2.1%). The LC50 values of the formulation ranged between (0.11574 -1.462) for An. gambiae s.s, 1.2928 - 0.15634 (0.176 - 1.463) for Cx. quinquefasciatus. 0.13674 (0.092 - 0.203) for Ae. aegypti and 0.12634 (0.974 - 1.762) for An. arabiensis There was development disruption of the juvenile stage of larval -pupal intermediate with adult emergence inhibition. Conclusions: The results indicated that the water-miscible formulations of essential oils had significant larvicidal activity through disrupted growth and adult emergence inhibition. showing potential for practical application in the field for the control of larvae of mosquito species.

Key words: Larvicidal essential oils, water-miscible formulations, mosquito larval control

Appendix C2- Abstract Manuscript 2:

Title: Laboratory efficacy evaluation of plant essential oils as mosquito Repellents (Journal: Vector and Parasites)

Introduction: Mosquitoes are the most important single group of insects well-known for their public health importance. More than 100 out of the 3000 species of mosquitoes worldwide are vectors of different tropical and subtropical life-threatening diseases like malaria, yellow fever, dengue, chikungunya, encephalitis, and Zika virus. Synthetic insecticides used for control of mosquitoes have negative effects to the environment. They also lead to resistance development of mosquitoes. There is therefore a need to find alternative, effective tools against these disease vectors. Plants have been a subject of research for eco-friendly alternatives to the conventional synthetic insecticides. In the present study we determined the mosquito repellent activity of essential oils from three plants against three medically important mosquito species female An. gambiae s.s, Aedes aegypti and Culex quinquefasciatus. Methods: In a first series of tests the effective doses of five crude plant extracts from three different plant species, S. biflora, C. newii and P. marrubioides (S. biflora was collected from three different locations), was assessed in a K and D module assay with human volunteers against two mosquito species, An. gambiae and Ae. aegypti.

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The practical potential of the selected volatiles and formulations in personal and in space protection (spatial repellence) was assessed using WHO arm-in-cage and fumigant toxicity assays. On a volunteer’s forearm, 0.1 mL of each essential oil was applied to 3 cm0 cm of exposed skin. The protection time was recorded for 3 min after every 30 min. The better performing oils were evaluated for dermal toxicity. Results: Essential oils from C. newii and S. biflora were strongly repellent to the three-mosquito species, and comparable to DEET (1). However, the durations of protection were shorter than a typical DEET (1) formulation. Addition of vanilla oil to the essential oils resulted in longer protection time. Essential oil from C. newii and S. biflora gave the longest protection of 76.50 min and 96.00 min respectively against Aedes aegypti. PM, C, N exhibited protection against Culex quinquefasciatus at 165.00, 105.00, and 112.50 min respectively. Conclusions: The results clearly indicated that C. newii and S. biflora were the most promising repellence against mosquito species. These oils could be used to develop a new formulation to control mosquitoes. Key words: Essential oils, Repellent, Aedes aegypti, Culex quinquefasciatus, Protection time

Appendix C3- Manuscript 3:

Title: Laboratory efficacy evaluation of plant extracts as mosquito Repellents

The present laboratory study evaluated the repellent efficacy of crude and formulated essential oils extracted from plants sampled in Aberdare range in Kenya. In a first series of tests the effective doses of five crude plant extracts from three different plant species, Satureja biflora, Conyza newii and Plectranthus marrubioides (S. biflora was collected from three different locations), was assessed in a K&D module assay with human volunteers against two mosquito species, Anopheles gambiae and Aedes aegypti. In second series of tests the two most promising extracts from the K&D assay, S. biflora and C. newii, were tested for their protection duration on humans against Ae. aegypti and Cx. Quinquefasciatus in a WHO arm-in-cage test. (Journal: Malaria journal)

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APPENDIX D: PATENT FILED

Composition and Methods for Controlling Larvae

Inventors: Ochola J. B, Lwande W., Marubu R. M., Moreka L., Nduguli F.W. and Ligare J., Composition and Method for Controlling Larvae. Patent No: KE/UM/2016/00569

Abstract

The invention pertains to a composition containing an extract from Ocimum kilimandscharicum, where the composition is suitable for use as a larvicide or insecticide for controlling or eliminating mosquito larvae in locations such as bodies of water, and the composition is suitable for administration to such locations. Compositions, methods of using the compositions, and methods of making the compositions are provided.

Claims a. A composition for controlling mosquito larvae in water, comprising: an effective amount of an extract from a basil plant. b. The composition of claim 1, wherein the basil plant is Ocimum kilimandscharicum, and wherein the extract is an essential oil. c. The composition of claim 1, wherein the composition is toxic to mosquito larvae. d. The composition of claim 1, wherein the composition further comprises one or more other additional essential oils or active agents that is toxic to mosquito larvae. e. The composition of claim 1, wherein the composition is suitable for use as a larvicide and insecticide for controlling or eliminating mosquito larvae, and wherein the composition is suitable for application to a body of water, and wherein the composition is in the form of an emulsion, solution, soluble powder, microencapsulated material, oil dispersion, water dispersible granules, dry flowable solid, or water-soluble packet. f. The composition of claim 1, wherein the composition is an essential oil and comprises at least 0.01% each of the following compounds: camphor, limonene, camphene, linalool, terpinen-4-ol, α-terpineol, terpinolene,

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sesquisabinene, α-pinene, caryophyllene(E-), myrcene, linalool, 2,6-octadien-1- ol, 3,7-dimethyl-, germacrene D, γ-terpinene borneol, (E)-β-ocimene, trans- sabinene hydrate, β-copaene, tricyclene, δ-carene, 6-camphenol, α- phellandrene, α-copaene, bicyclogermacrene, viridiflorene, α-humulene, β- copaene, α-copaene, tricyclene, trans-muurola-4(14),5-diene, para-cymen-8-ol, α-phellandrene, geranial, (E)-2-butenoic acid, 2-methyl-, 2-methylpropyl ester, isoborneol, eugenol, γ-muurolene, dauca-5,8-diene, α-bisabolene, dauca-5,8- diene, humulene epoxide II, 1,5,5-trimethyl-6-methylene-cyclohexene and α- cubebene. g. A method for controlling mosquito larvae, the method comprising applying an effective amount of the composition of claim 1 to a location containing mosquito larvae. h. The method of claim 7, wherein the location is a body of water, and wherein the method controls or eliminates mosquito larvae. i. The method of claim 7, wherein the basil plant is Ocimum kilimandscharicum, and wherein the extract is an essential oil. j. A kit comprising the composition of claim 1 in packaging suitable for application to a body of water.

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APPENDIX E: STANDARD OPERATING PROCEDURES SOPS

Appendix E1 - The Klun and Debboun Module (K and D Module) for evaluation of repellent efficacy using human subjects

Issued: 22.09.16 First Edition

Introduction:

The K and D Module is used for quantitative evaluation of arthropod repellents in human subjects and used to determine effective repellent concentrations on human skin or repellent-treated cloth over human skin. Biting responses to varied doses of the repellent on human skin is quantified, and the effectiveness of the repellent is compared with the standard.

AUTHOR: John Bwire Ochola

Signature: …………………. Date: ……………………….

AUTHORISED BY: Dr Pie Müller

Signature: …………………. Date: ……………………….

DISTRIBUTION:

NSTRUCTIONS 1. Safety 1.1 Repellent formulations should be handled with care.

1.2 Use a fresh finger cot for to apply each repellent formulation.

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2. Principle The K and D method consist of a unit with 6 test chambers and permits the simultaneous comparison of up to 5 repellent doses or chemical types and a control using a complete randomized block design with minimal treatment interaction and with 6 replicates per human subject. The numbers of insects biting in each cell within a 2-min exposure are recorded, after which the slides are closed.

3. Procedure 3.1 Test animals Observations are made using 5 to 10-day-old (post-eclosion) host-seeking females which have been starved from sugar water for the preceding 12 hours and provided with water only. For testing, mosquitoes are collected from a stock population cage in which both sexes have been maintained to allow mating to occur but not blood fed.

3.2 Materials and reagents a) K and D test modules b) Template for marking areas of application c) Sleeved cage to release mosquitoes from the Kand D module after test d) Mouth aspirator e) Data record sheet f) Timer g) Cotton wool h) 70% ethanol solution/isopropanol wipes i) 200 µl pipettor and yellow tips j) Finger cots k) Thermo-/hygrometer 3.3 Method a) Hungry females are first attracted by holding a hand close to the cage and then collected with a mouth aspirator;

b) Each cell of a K and D unit is provided with 5 female mosquitoes and the hole plugged with cotton wool;

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c) Prior the test the volunteer’s body temperature is measured in the right ear using the Braun ThermoScan thermometer. If the measured body temperature is above 37.6 °C the volunteer is excluded from the test; d) A human volunteer wearing shorts seats with legs horizontally extended; e) Using the template and a water-soluble marker, skin areas (4 cm x 5 cm) representing floor openings of the Kand D test module are outlined on the outer, top and inner thigh of each leg; f) The test solutions are uniformly spotted within the marked areas of skin in volumes of 33.3 µl using a P200 pipettor. A thin layer is spread evenly using finger cots. g) Replace tips and finger cots after each application. This process should not take longer than 1 min. per area. Start with the outer, left thigh; h) After areas for two replicate K and D test modules are treated, the first is exposed to mosquitoes by placing the K and D test module over it; i) The human test subject holds the K and D module in place on the thigh and assists with opening the slide doors of the cells. All slide doors are opened at the same time. The volunteer assists with opening the slides. The timer which has previously been set at 2 minutes is started when all slides are open; j) The number of insects biting in each cell within the 2-min exposure is recorded, after which the slide doors are closed. Care must be taken not to squash the mosquitoes; k) Repeat steps (f) to (k) once until two K and D test modules are completed; l) Continue with steps (f) to (k) until a maximum of 6 K and D test

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modules are completed from the left, outer to the right, outer thigh;

m) At the end of the test series on one volunteer, mosquitoes are released by opening the cells of the K and D test module in an empty sleeved screened cage;

n) Before reusing the K and D test units they are cleaned according to the protocol below.

4. Cleaning the K and D test units 4.1. At the end of each assay, the bottom of each module that had contacted treated skin is cleaned with an ethanol-soaked tissue, dried with a fresh tissue and blow-dried with a warm-air hair drier. 4.2. At the end of the assay session, the K and D test units that have been used by 1-3 human subjects, are washed with warm aqueous detergent, rinsed and dried. 5. Precautions 4.1 Avoid taking items and protective clothing used in the test to the main insectary. 4.2 Test insects should be used only once and safely disposed of immediately after the trial. 4.3 When testing in subdued light, care should be taken not to compromise the investigators’ ability to observe insect activity. 5. Reporting Only data sets in which 3 or more females bite in each control cell are analyzed. If there are less than 3 bites in the negative control, then that run is discarded and repeated.

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APPENDIX E2-SOP Laboratory Testing of Formulated Mosquito Larvicides

Issued: 01.06.17 Second Edition TITLE: LABORATORY TESTING OF FORMULATED MOSQUITO LARVICIDES

Introduction:

The objective of laboratory testing is to determine the inherent biopotency of the technical material or, in the case of formulated larvicides, their activity. The larvicidal activity of formulated product plant-based biopesticide will be evaluated and compared to a positive control like Bti used against Anopheline and other mosquitoes.

AUTHOR: John Bwire

Signature: ………………….. Date: ……………………….

AUTHORISED BY:

Signature: ………………….. Date: ……………………….

DISTRIBUTION:

INSTRUCTIONS 1. Safety 1.3 Larvicide formulations should be handled with care. 1.4 Avoid skin and mouth contact with the formulation.

2. The aims of the tests are:  To establish dose–response line(s) against susceptible vector species;  To determine the lethal concentration (LC) of the larvicide for 50% and 90% mortality (LC50 and LC90) or for 50% and 90% inhibition of adult emergence (IE50 and IE90);

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 To establish a diagnostic concentration for monitoring susceptibility to the mosquito larvicide in the field; and  To assess cross-resistance with commonly used insecticides.

3. Principle  To evaluate the biological activity of a mosquito larvicide, laboratory-reared mosquito larvae of known age or instar are exposed for 24 h to 48 h or longer in water treated with the larvicide at various concentrations within its activity range, and mortality is recorded.  For IGRs and other materials with delayed activity, mortality should be assessed until the emergence of adults. It is important to use homogenous populations of mosquito larvae or a given instar. These are obtained using standardized rearing methods.

4. Materials and reagents  One pipette delivering 100–1000 μl.  Disposable tips (100 μl, 500 μl) for measuring aliquots of dilute solutions.  Five 1 ml pipettes for insecticides and one for the control.  Three droppers with rubber suction bulbs.  The following materials to make a strainer: two wire loops, one piece of nylon netting (30 cm2) and one tube of cement. It is suggested that two pieces of netting be cut and cemented to opposite sides of the larger end of the wire loops. More cement should then be applied around the edges of the loops  to join the two pieces of netting. When dry, the netting may be trimmed with scissors.  If a strainer is not available, a loop of plastic screen may be used to transfer test larvae into test cups or vessels.  Data recording forms (see Annex).  Disposable cups (preferred as they avoid contamination) or, if not available, glass bowls or beakers of two capacities: 120 ml (holding 100 ml) and 250 ml (holding 200 ml).  Graduated measuring cylinder.  Log–probit software or paper.

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5.2 METHOD Preparation of stock solutions or suspensions and test concentrations The stock solution is then serially diluted (ten-fold) in ethanol or other solvents (2 ml solution to 18 ml solvent). Test concentrations are then obtained by adding 0.1– 1.0 ml (100–1000 μl) of the appropriate dilution to 100 ml or 200 ml chlorine-free or distilled water (see Table A2.1). For other volumes of test water, aliquots of dilutions added should be adjusted per Table A2.1. When making a series of concentrations, the lowest concentration should be prepared first. Small volumes of dilutions should be transferred to test cups by means of pipettes with disposable tips. The addition of small volumes of solution to 100 ml, 200 ml or greater volumes of water will not cause noticeable variability in the final concentration. Six concentrations of test sample, 0.625, 0.125, 0.25, 0.5, 1 and 2 ppm were prepared in 250 ml water At least two control replicates were run simultaneously which included water controls for test sample

5.3. EXPERIMENTAL DESIGN A). Larvicide bioassays  Initially, the mosquito larvae are exposed to a wide range of test concentrations and a control to find out the activity range of the materials under test. After determining the mortality of larvae in this wide range of concentrations, a narrower range (of 4–5 concentrations, yielding between 10% and 95% mortality in 24 h or 48 h) is used to determine LC50 and LC90 values.  Batches of 25 third or fourth instar larvae are transferred by means of strainers, screen loops or droppers to small disposable test cups or vessels, each containing 100–200 ml of water. Small, unhealthy or damaged larvae should be removed and replaced.  The depth of the water in the cups or vessels should remain between 5 cm and 10 cm; deeper levels may cause undue mortality.  The appropriate volume of dilution is added (see Table A2.1) to 100 ml or 200 ml water in the cups to obtain the desired target dosage, starting with the lowest concentration.

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 Four or more replicates are set up for each concentration and an equal number of controls are set up simultaneously with tap water, to which 1 ml alcohol (or the organic solvent used) is added.  Each test should be run three times on different days. For long exposures, larval food should be added to each test cup, particularly if high mortality is noted in control.  The test containers are held at 25–28 oC and preferably a photoperiod of 12 h light followed by 12 h dark (12L:12D).  After 24 h exposure, larval mortality is recorded. For slow-acting insecticides, 48 h reading may be required.  Moribund larvae are counted and added to dead larvae for calculating percentage mortality (Dead larvae are those that cannot be induced to move when they are probed with a needle in the siphon or the cervical region. Moribund larvae are those incapables of rising to the surface or not showing the characteristic diving reaction when the water is disturbed).

 The results are recorded on the data sheet (Fig. A4.1), where the LC50, LC90

and LC99 values, and slope and heterogeneity analysis are also noted.  The form will accommodate three separate tests of six concentrations, each of four replicates.  Larvae that have pupated during the test period will negate the test. If more than 10% of the control larvae pupate in the course of the experiment, the test should be discarded and repeated. If the control mortality is between 5% and 20%, the mortalities of treated groups should be corrected per Abbott’s formula (3): X – Y Mortality (%) = ------x 100, X where X = percentage survival in the untreated control and, Y = percentage survival in the treated sample. Data analysis  Data from all replicates should be pooled for analysis.  LC50 and LC90 values are calculated from a log dosage–probit mortality regression line using computer software programs or estimated using log– probit paper.

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 Bioassays should be repeated at least three times, using new solutions or suspensions and different batches of larvae each time.  Standard deviation or confidence intervals of the means of LC50 values are calculated and recorded on a form (Fig.A4.1).  A test series is valid if the relative standard deviation (or coefficient of variation) is less than 25% or if confidence limits of 12 LC50 overlap (significant level at P < 0.05).  The potency of the chemical against the larvae of a vector and strain can then be compared with the LC50 or LC90 values of other insecticides.

B) Insect growth regulators Note: Testing methods for the juvenile hormone (JH) analogues (juvenoids) and the chitin synthesis inhibitors differ.  JH analogues interfere with the transformation of late instar larvae to pupae and then to adult,  whereas chitin synthesis inhibitors inhibit cuticle formation and affect all instars and immature stages of the mosquito.

The delayed action of IGRs on treated larvae means that mortality is assessed every other day or every three days until the completion of adult emergence. The effect of both types of IGR on mosquito larvae is expressed in terms of the percentage of larvae that do not develop into successfully emerging adults, or adult emergence inhibition (IE%).

C– T IE (%) = — x 100, C where C = percentage emerging or living in control habitats and T = percentage emerging or living in treated habitats.

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Appendix E3: SOP Aliquots of various strength solutions added to 100 ml water to yield final concentration

Initial solution Aliquot (ml) Final concentration % PPM (PPM) in 100 ml 1.0 10 000.0 1.0 100.0 0.5 50.0 0.1 10.0

0.1 1 000.0 1.0 10.0 0.5 5.0 0.1 1.0

0.01 100.0 1.0 1.0 0.5 0.5 0.1 0.1

0.001 10.0 1.0 0.1 0.5 0.05 0.1 0.01

0.0001 1.0 1.0 0.01 0.5 0.005 0.1 0.001

0.00001 0.1 1.0 0.001 0.5 0.0005 0.1 0.0001

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Appendix E4: SOP - Laboratory evaluation of the efficacy of larvicides against mosquito larvae

DATA RECORDING FORMS

Laboratory evaluation of the efficacy of larvicides against mosquito larvae

Experiment No: ______Investigator: ______Location: ______Treatment date: ______Material: ______Formulation: ______Temp: ______Lighting: ______Species: ______Larval instar: ______Larvae/cup or vessel: ______Water: Tap/Distilled Volume of water: ______ml Food: ______Date stock solution made: ______

No of dead larvae at various conc. (mg/L) post exposure (hr.) 24 hr 48 hr Date Replicate 1 2 3 4 5 6 7 8 9 10 11 12 Total Ave % Mortality LC50 (CL 95%): LC50 (CL LC90 (CL 95%): 95%): LC99: LC90 (CL Slope: Heterogeneity: 95%): Heterogeneity: LC99: Slope:

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Appendix E5: SOP – Laboratory evaluation of the efficacy of insect growth regulators against mosquito larvae

Data recording forms

Experiment No: ______Investigator: ______Location: ______Treatment date: ______Material:____ Formulation:______Sampling technique: ______Species: ______Larval instar: ______No. of larvae released/exposed: ______Setting date: ______

Date Grand Total Conc Alive Dead Alive Dead Alive Dead Alive Dead Alive Dead Alive Dead (mg/L) Rep L P A L P A L P A L P A L P A L P A L P A L P A L P A L P A L P A L P A 00 1 2 3 4 Total Mean 01 1 2 3 4 Total Mean 12 1 2 3 4 Total Mean

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13 1 2 3 4 Total Mean 14 1 2 3 4 Total Mean 15 1 2 3 4 Total Mean

Cumulative number of dead / alive mosquitoes after treatment (date or days pre or posttreatment or setting) L=larvae, P=pupae, A=adult