University of Ghana http://ugspace.ug.edu.gh Optimisation of Sugar and Blood Feeding Regimen in Anopheles gambiae Mass Production System BY SEDOFIA BRANSFORD KWASHIE (10363170) BSc. Agriculture (University of Cape Coast), 2002 This thesis is submitted to the University of Ghana, Legon, in partial fulfilment of the requirement for the award of MPhil Radiation Processing Degree July, 2013 i University of Ghana http://ugspace.ug.edu.gh DECLARATION This is to certify that this thesis is the result of research work undertaken by BRANSFORD KWASHIE SEDOFIA in the Department of Nuclear Agriculture and Radiation Processing, School of Nuclear and Allied Sciences, University of Ghana, under the supervision of Dr. Delphina A. Adabie-Gomez and Dr. D. D. Wilson, towards the award of Master of Philosophy Degree. This work has not been submitted either in part or full, to this University or elsewhere for the award of any other degree. Literature cited from other people‟s work has been duly acknowledged. ………………………......... Date………………………... BRANSFORD K. SEDOFIA (STUDENT) ………………………....... Date.………………………. DR. DELPHINA A. ADABIE-GOMEZ (SUPERVISOR) …………………………..... Date……………………….. DR. DAVID. D WILSON (SUPERVISOR) ii University of Ghana http://ugspace.ug.edu.gh DEDICATION I first and foremost gratefully dedicate this thesis to the Almighty God for bringing me this far. I equally dedicate it to my two daughters Eyram and Makafui, my mother Mrs. Mawunyo Sedofia and my wife Mrs. Josephine Sedofia for their unflinching support and encouragement throughout my study. iii University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENTS I am most grateful to my supervisors; Dr. Delphina A. Adabie-Gomez, Senior Lecturer and Former Deputy Director of BNARI, GAEC and Dr. D. D. Wilson, Senior Lecturer at the Department of Zoology, University of Ghana for their, patience, suggestions, constructive criticisms and dedicated support in compiling this thesis. I would like to also express my profound gratitude to Mr. Michael Osae, Lecturer, School of Nuclear and Allied Sciences – University of Ghana, Alessi A. Kwawukume, Mr. E. A. Ewusie and Mr. Godfrey K. Damnyag, all of the Radiation Entomology and Pest Management Centre (REPMC) of GAEC for their immense and unflinching support in the laboratory work. My profound appreciation also goes to Mr. Justice Okona Frimpong of Nuclear Agriculture Centre, BNARI-GAEC for his immeasurable contributions to this work. I am very grateful to Dr. Alexander Egyir- Yawson (Centre manager, REPMC) for giving me access to the facilities at the Centre for my work. A note of special thanks is further extended to all REPMC staff, family members and those who endeavoured to assist me in one way or the other, May the good Lord bless you all. Finally, my utmost appreciation goes to the Almighty God for bringing me this far, having shown me such abundant grace, protection and divine guidance to go through this programme of study successfully. iv University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENTS TITLE PAGE i DECLARATION ii DEDICATION iii ACKNOWLEDGEMENTS iv TABLE OF CONTENTS v LIST OF TABLES ix LIST OF PLATES x LIST OF FIGURES xii LIST OF APPENDICES xiv ABSTRACT xv CHAPTER ONE 1 1.0 INTRODUCTION 1 1.1 Background 1 1.2 Statement of the problem 6 1.3 Relevance and justifications 7 1.4 Objectives 10 1.4.1 General Objective 10 1.4.2 Specific Objectives 10 CHAPTER TWO 11 2.0 LITERATURE REVIEW 11 2.1 Brief biology of Anopheline mosquitoes 11 v University of Ghana http://ugspace.ug.edu.gh 2.2 Role of Anopheline mosquitoes as vectors in malaria transmission 13 2.3 Need for novel control strategies 14 2.4 Sugar feeding in adult Anopheles mosquitoes 16 2.5 Sugars as nutritive food resource for adult mosquitoes 18 2.6 Blood feeding in female Anopheles mosquitoes 20 2.7 Effect of blood types and feeding methods on reproduction in female Anopheles mosquitoes 21 2.8 Use of anaesthetics in live animal feeding of adult female Anopheles mosquitoes 23 CHAPTER THREE 25 3.0 MATERIALS AND METHODS 25 3.1 Study site 25 3.2 Mosquito colony and rearing conditions 26 3.3 Sources and handling of adult mosquito sugar diets 27 3.4 Sources and handling of adult female mosquito blood diets 28 3.5 Evaluation of the effect of different sugar types on adult mosquito survival 29 3.6 Evaluation of the effect of different sugar types on adult male mosquito mating potential 30 vi University of Ghana http://ugspace.ug.edu.gh 3. Response of female Anopheles gambiae to different blood feeding methods 32 3.8 Evaluation of the effect of different blood feeding methods on egg production of female mosquitoes 34 3.9 Evaluation of the effect of different anaesthetic agents on blood feeding response of female mosquitoes 36 3.10 Evaluation of the effect of different anaesthetic agents on egg production of female mosquitoes 38 3.11 Experimental design and data analysis 39 CHAPTER FOUR 41 4.0 RESULTS AND OBSERVATIONS 41 4.1 Percentage survival of male An. gambiae on 6 % concentration of sugars 41 4.2 Percentage survival of male An. gambiae on 10% concentration of sugars 42 4.3 Percentage survival of female An. gambiae on 6% concentrations of sugars 43 4.4 Percentage survival of female An. gambiae on 10 % concentrations of sugars 44 4.5 Relationship between percentage survival of male and female An. gambiae mosquitoes 45 4.6 Potential of male mosquitoes to inseminate female mosquitoes 48 vii University of Ghana http://ugspace.ug.edu.gh 4.7 Percentage blood feeding of female An. gambiae fed using different Blood feeding methods and time regimens 49 4.8 Percentage egg production in female An. gambiae fed using different blood feeding methods 51 4.9 Percentage blood feeding response of female An. gambiae fed using Guinea pigs anaesthetised with different anaesthetics and time regimens 52 4.10 Percentage egg production in female An. gambiae fed using Guinea pigs anaesthetised with different anaesthetics 54 CHAPTER FIVE 55 5.0 DISCUSSION 55 CHAPTER SIX 62 6.0 CONCLUSIONS AND RECOMMENDATIONS 62 6.1 Conclusions 62 6.2 Recommendations 64 REFERENCES 66 APPENDICES 91 viii University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES Table Page 1. Relationship between concentration and feeding material on % survival of male and female An. gambiae mosquitoes 47 ix University of Ghana http://ugspace.ug.edu.gh LIST OF PLATES Plate Page 1. An. gambiae eggs set up in egg bowl 27 2. Larval rearing in trays filled with 1000 larvae in 1000 ml water 27 3. Mesh-covered trays 27 4. An. gambiae pupae in a plastic cup 27 5. 6 % and10 % concentrated solutions of sugar types in glass and plastic Bottles 28 6. Adult male and female mosquitoes in holding cages for data collection 30 7. Dissection of females for spermathecae and examination of spermathecae for presence of sperm 31 8. Human arm feeding (HAF) of adult female An. gambiae mosquitoes 33 9. Restrained Guinea pig feeding (RGF) of female An. gambiae mosquitoes 33 10. Anaesthetised Guinea pig feeding (AGF) of female An. gambiae mosquitoes 34 11. Iso-set up for females that blood fed on human arm 35 12. Iso-set up for females that blood fed on restrained Guinea pig 35 13. Iso-set up for females that blood fed on anaesthetised Guinea pig 36 14. Ketamine/Xylazine anaesthetised Guinea pig feeding of adult female mosquitoes 37 x University of Ghana http://ugspace.ug.edu.gh 15. Ketamine/Diazepam anaesthetised Guinea pig feeding of adult female mosquitoes 38 16. Iso-set up for females fed on Ketamine/Xylazine anaesthetised Guinea Pig 39 17. Iso-set up for females fed on Ketamine/Diazepam anaesthetised Guinea Pig 39 xi University of Ghana http://ugspace.ug.edu.gh LIST OF FIGURES Figure Page 1. Map of Ghana 25 2. Map showing location of GAEC in Accra, Ghana 25 3. Survival of Adult male An. gambiae on 6 % glucose, sucrose and honey solutions 42 4. Survival of Adult male An. gambiae on 10 % glucose, sucrose and honey solutions 43 5. Survival of Adult female An. gambiae on 6 % glucose, sucrose and honey solutions 44 6. Survival of Adult female An. gambiae on 10 % glucose, sucrose and honey solutions 45 7. Inseminated spermatheca with bundles of sperm 48 8. Spermatheca of uninseminated female 48 9: Effect of 10 % concentration of sugar types on the potential of male mosquitoes to inseminate female mosquitoes 49 10. Effect of blood feeding methods on percentage blood feeding response of An. Gambiae 50 11. Effect of feeding times on percentage blood feeding response of An. gambiae 51 xii University of Ghana http://ugspace.ug.edu.gh 12. Effect of blood feeding methods on percentage egg production of An. Gambiae 52 13. Effect of anaesthetics on percentage blood feeding response of An. Gambiae 53 14. Effect of feeding times on percentage blood feeding response of An. Gambiae 53 15. Effect of anaesthetics on percentage egg production of An. gambiae 54 xiii University of Ghana http://ugspace.ug.edu.gh LIST OF APPENDICES Appendix Page 1: Analysis of variance (ANOVA) for weekly effect of sugar types and concentrations on adult mosquitoes survival 91 2: Regression and correlation analysis for sugar types and concentrations 97 3: Analysis of variance (ANOVA) for effect of sugar types on male potential to inseminate females 101 4: Analysis of variance (ANOVA) for the effect of blood feeding methods on adult female mosquitoes productivity 101 5: Analysis of variance (ANOVA) for the effect of anaesthetics on adult female mosquitoes productivity 103 xiv University of Ghana http://ugspace.ug.edu.gh ABSTRACT The sterile insect technique (SIT) is being developed for the control of malaria transmitting mosquitoes. Critical to the success of applying the SIT is the establishment of standardized mass production systems for the target species. As part of efforts to develop standardised mass production systems for malaria vectors, this project sought to optimize adult blood and sugar feeding in a mass production system. Different sugar types (glucose, sucrose and honey) were evaluated at 6 % and 10 % concentrations in water to determine the best sugar diet and concentration for feeding adult An. gambiae. Different blood feeding methods, restrained Guinea pig, anaesthetised Guinea pig and human arm feeding were evaluated. Adult survival, female insemination and egg production were used as criteria to determine optimum sugar and blood feeding. The effect of anaesthetics on blood feeding response and egg production of female An. gambiae was determined by comparing feeding response and egg production of females fed with anaesthetised Guinea pigs as against physically restrained Guinea pigs (Control). The specific effect of different anaesthetic agents on blood feeding response and egg production of female mosquitoes was also determined by comparing the feeding response and egg production of females fed with either Ketamine/Xylazine anaesthetised Guinea pigs or Ketamine/Diazepam anaesthetised Guinea pigs. Effects due to sugar types and concentrations on percentage survival of male and female mosquitoes were observed to be significant at (p < 0.05). Honey at 10 % concentration recorded the highest survival of 84.3 % after 1 week and 12.7 % after 6 weeks whilst glucose and sucrose recorded 81.0 % and 78.0 % respectively after 1 week and 1.3 % for both after 6 weeks. Survival up to 35 and 49 days for males and females xv University of Ghana http://ugspace.ug.edu.gh respectively occurred only in adults fed with honey. The combined effects of blood feeding methods, anaesthetic types and feeding times on the propensity of female An. gambiae to feed and produce eggs were not significantly different (P > 0.05). However, human arm feeding (HAF) method and Ketamine/Xylazine (KX) anaesthetics fed for 25 minutes recorded higher percentage feeding (76.0 % and 68.0 % respectively) and egg production of 19.0 % and 20.8 % respectively. Anaesthetised Guinea pig feeding (AGF) of adults for 15 minutes followed closely with 60.0 % and 15.1 % blood feeding and egg production respectively whilst restrained Guinea pig feeding (RGF) method and Ketamine/Xylazine (KD) anaesthetic agents recorded the least values. It can be concluded that 10 % honey solution resulted in optimum feeding, survival and female insemination in adult An. gambiae compared to other treatments. Although both human arm feeding and Ketamine/Xylaxine anaesthetised Guinea pig feeding resulted in optimum productivity, anaesthetised Guinea pig feeding of adults for 15 minutes is the ideal feeding method for mass production systems. We recommend that 10 % honey solution, anaesthetised Guinea pig feeding method using 0.05 ml Ketamine/Xylazine anaesthetics and 15 minutes feeding time be employed for adult feeding in mass production of this species. xvi University of Ghana http://ugspace.ug.edu.gh CHAPTER ONE 1.0 INTRODUCTION 1.1 BACKGROUND Anopheline mosquitoes are known vector species responsible for the transmission of Plasmodium parasites to humans in many parts of the world. These vector species vary from region to region but in Africa the parasite is transmitted mainly by members of the Anopheles gambiae complex and Anopheles funestus group (Kelly-Hope, 2009; Annan et al., 2007; Appawu et al., 2004; Craig et al., 1999). Of these, the An. gambiae is found to be the most important and efficient vector species for the parasite in Sub-Saharan Africa (Rodrigues et al., 2012; Obbard et al., 2009; Alavi et al., 2003). Only the female anophelines are capable of transmitting the malaria parasite from an infected person to a non-infected person or to an already infected person (Cox, 2010; Rhodes, 2008). This is because only females require blood which sometimes do not only contain important protein sources they need to develop their eggs, but Plasmodium parasites that equally prefer such blood to mature exceptionally well (Lavazec and Bourgouin, 2008). Even though male Anopheline mosquitoes lack this feature and therefore do not take blood or transmit the parasite, they still play an indirect but very vital role by providing the needed insemination of females with fertile sperm (Gouagna et al., 2010; Jiang, 2008; Nayar and Van Handel, 1971; Clements, 1955). A phenomenon which forms an essential part of the reproductive 1 University of Ghana http://ugspace.ug.edu.gh process that gives rise to progenies that eventually continue to transmit the parasites to millions of people each year. This phenomenon is likely to continue if better and more effective strategies are not put in place to significantly reduce or eliminate the vector. Currently, though several prevention and control interventions from mechanical, biological through to largely chemical (use of burning marts, smoking coils, indoor residual insecticide sprays, insecticide-treated bed nets and anti-malarial drugs) are in place, malaria infection continues to remain high in many parts of Africa for several reasons (Griffin, 2010). Major among these is the development of multiple insecticide resistance among anopheline mosquitoes, continuous development of Plasmodium parasite resistance against anti-malaria drugs, and the short reproductive life cycle of anopheline mosquitoes that enables them to reproduce large numbers at very short periods (Edi et al., 2012; WHO, 1995; Briegel & Hörler, 1993). In addition, the continuous application of insecticides in vector control also tends to cause significant reduction in the population of natural control agents needed to naturally keep vector numbers below destructive thresholds (Marrelli, 2012). The need to incorporate novel control strategies into existing methods to augment their effectiveness or as complementary measures in vector prevention and control becomes imperative (Brelsfoard and Dobson, 2009). One such promising novel control strategy is the Sterile Insect Technique (SIT); an autocidal approach that provide an area-wide vector control without the accompanying problems of toxicity and the emerging vector/parasite resistance 2 University of Ghana http://ugspace.ug.edu.gh development associated with the widely used chemical approach (Kala, 2012; Parker and Mehta, 2007). Central to the success of this strategy for area-wide mosquito control is the production and release of large numbers of laboratory-reared, genetically-modified sterile male mosquitoes that can successfully mate with the wild females, cause reproductive failure in the females and in so doing stop them from reproducing (Kala, 2012; Dyck et al., 2005; Knipling, 1955). In this way, the wild vector population will collapse (Morrison et al., 2010) and malaria infection cases reduced considerably. A sustainable production of these large numbers in the laboratory requires well- maintained brood stock colonies that are readily available for regular and timely supply of sterile adult males for field releases when the need arise. Maintaining the stock colony also requires feeding the colony as well as the adults to be released with appropriate sugar and blood sources, using properly standardised feeding methods and feeding regimens (Clements, 1992). Sugar is considered the major food resource for all adult mosquitoes (Woodbridge, 1995) as it provides the essential nutrition adults need to generate the energy needed to survive long enough, maintain fitness and reproduce (Gu mail et al., 2011). Adult males for example, require this energy to effectively compete among themselves for females and successfully inseminate females (Gouagna et al., 2010). In the wild, adult male mosquitoes depend on plant juices and exudates (e.g. floral 3 University of Ghana http://ugspace.ug.edu.gh nectar, honeydew, damaged fruits and vegetative tissues etc) for these essential sugars (Woodbridge, 1995). From these different sources, they obtain sucrose, fructose, and glucose among others depending on the source from which the sugar is taken (Benedict, 1997; Clements, 1992; Briegel and Kaiser, 1973, Gillett et al., 1962). Similarly, females also require these sugars for survival throughout their mating, eggs production and oviposition periods and beyond. To this effect, sugar feeding constitutes an indispensable aspect of the life of adult mosquitoes (Woodbridge, 1995). In a controlled environment such as the laboratory, adult mosquitoes are equally reared on different synthetic sugar sources including glucose, sucrose and honey (artificially extracted) as means of providing food sources similar to what exist in the wild (Benedict, 1997; Clements, 1992; Briegel and Kaiser, 1973; Gillett et al., 1962). Thus, there is the need to evaluate the different synthetic sugar sources and the amounts currently employed in laboratory feeding of mosquitoes in order to determine the specific type and quantity that can result in optimum feeding, survivability and productivity in mass rearing systems. Though natural sugar sources contain protein needed by females to develop their eggs, the concentrations are not high enough for their reproductive role (Clements, 1992). Blood feeding in female anopheline mosquitoes therefore forms an integral part of their reproductive cycle (Olayemi et al., 2011; Briegel, 1990). In addition, it is a very important component in their mass rearing towards the successful implementation of SIT for area-wide control of the vector. In the wild, female mosquitoes depending on host preference may obtain blood meals from live 4 University of Ghana http://ugspace.ug.edu.gh mammals, reptiles, birds and amphibians (Means, 1968). In controlled environments such as the laboratory, mosquitoes are mostly fed on processed blood meal using artificial delivery systems such as the membrane feeder or by using natural delivery systems such as direct human arm feeding, restrained, and anaesthetised animal feeding methods using human beings, rabbits, Guinea pigs, mice, rats etc (Benedict et al., 2007). The source and the size of blood meal females ingest determines the number of females that develops eggs and the number of eggs developed per female. These variations can be attributed to differences in the type of proteins and their constituent amino acids contained in such blood meals (Prasad, 1987). Thus, blood of superior nutritional quality is necessary for optimum feeding and egg production in mass production systems (Olayemi et al., 2011). LaFlamme (2011) stated that though female Anopheles mosquitoes in most cases prefer human blood to other hosts, this may be practically impossible where very large numbers are to be fed. Similarly, though direct feeding of live mammals (Guinea pigs, rabbits etc) to vector mosquitoes may provide a suitable alternative, its use may require sedating the live mammal with anaesthetic agents in order to immobilise and restrain its movement during feeding. This is because any movement in the live mammal during feeding can disrupt feeding and seriously affect the extent to which the mosquitoes feed. (Gerberg, 1970). Over the years, different anaesthetic agents have been employed for this purpose, however, the presence of an anaesthetic agent in the blood of mammals used as host may affect feeding response and productivity in female mosquitoes fed in a number of ways. For instance, the amount of blood they imbibe, how long they survive to lay eggs and the number of eggs that they lay during the oviposition period may be affected 5 University of Ghana http://ugspace.ug.edu.gh by these anaesthetic agents (Murrieta et al., 2010). It is therefore important to evaluate the effect of these anaesthetic agents on females‟ blood feeding response and egg production in order to determine the appropriate anaesthetics and the combinations that are optimum for their productivity in mass rearing systems. 1.2 STATEMENT OF THE PROBLEM Effective vector control and more specifically mosquito control, is a complex and difficult problem, as illustrated by the continuing prevalence (and spread) of mosquito-transmitted diseases (Alphey et al., 2010). Though the sterile insect technique (SIT) has been reiterated as the much-needed novel approach to stop malaria parasite/disease transmissions, the success of its application for area-wide mosquito control heavily requires inducing reproductive failure in females through unviable matings between laboratory-reared sterile males and wild females. By so doing the reproductive capacity of the wild females will be reduced considerably and the vector will be eliminated over time (Alphey et al., 2010). This outcome can only be achieved if adult males are reared to numbers that can match or exceed the males population in the wild (Feldmann and Hendrichs, 2001) and if the large number of adult males required for field release can be readily obtained at regular time intervals when the need arise. To this effect, it has become necessary to maintain adult stock colonies in mass production systems on the right sugar and blood sources using appropriate feeding methods and feeding time regimens that give optimum feeding and in effect increase the fitness, longevity and productivity of the laboratory stock in mass rearing systems. 6 University of Ghana http://ugspace.ug.edu.gh Several sugar types, feeding concentrations as well as blood sources, blood feeding methods and time regimens have been identified and explored by researchers over the years to attain the production of these large numbers in the laboratory (Syoziro, 1964). However, these sugars and blood sources and feeding methods/regimens have not been properly developed and standardized for optimum feeding in mass rearing systems (Fabrizio, 2012). This has over the years led to reduced survival, fitness and low productivity among laboratory-reared stocks. Thus, makes it very difficult to turn out the large numbers needed to successfully use the sterile insect technique to control vector mosquitoes especially in sub-Saharan Africa where the vector remains endemic and malaria prevalence is still very high. 1.3 RELEVANCE AND JUSTIFICATIONS There is very clear evidence that current conventional methods, largely chemical applications have very serious limitations in controlling vector mosquitoes/malaria parasites in the sub-region. Moreover, the deleterious effect of these chemicals on human life and the environment as a result of over dependence on chemical methods to control mosquitoes and malaria parasites are becoming increasingly visible by the day (Marrelli, 2012). These have led to the impetus to include other novel control strategies such as SIT in the effort to eliminate the vector and the parasite. The Sterile Insect Technique (SIT) which is one such novel methods has the capacity to successfully synergise with good aspects of existing conventional methods (WHO, 2004, 2008) to make vector/malaria control more effective 7 University of Ghana http://ugspace.ug.edu.gh (Brelsfoard and Dobson, 2009). It is a species-specific, environment-benign, clean (leaving no residues) and sustainable vector control strategy that can prevent, suppress, contain or eliminate particular insect pest or vector populations. The technique as part of area-wide integrated pest management (AW-IPM) programmes has celebrated many successes in eliminating and suppressing several economically important insect pest populations as well as preventing vector establishment in new areas without the accompanying limitations associated with existing methods (Knipling, 1959; Knipling, 1968; Dyck et al., 2005). For example, it was successfully mounted to control important insect pests and vectors of agricultural and medical importance including tropical fruit flies, Bactrocera spp, some species of tsetse flies, Glossina spp., the pink bollworm, Pectinophora gossypiella (Saunders), and the codling moth, Cydia pomonella (Joint FAO/IAEA, 2012; Dyck et al., 2005; Krafsur, 1998; Knipling, 1998, 1979, 1955). There is therefore renewed interest in using sterile insects for managing endemic, as well as emerging or re-emerging vector-borne diseases (Alphey et al., 2010). However, the development of SIT for use in mosquito AW-IPM programmes is in its infancy, and many fundamental components of the technique still need to be developed, standardised and optimised. These components include aspects of the mass-rearing of the vector and the quality of the sterile males produced in the laboratoryIt has been established that nutrition is one of the important factors that has huge impact on the survival, fitness and productivity of adult vector Anopheline mosquitoes in mass rearing systems (LaFlamme, 201l; Gu mail et al., 2011; Dame et al., 2009; Woodbridge, 1995; Nayar and Sauerman, 1975). 8 University of Ghana http://ugspace.ug.edu.gh In exploiting the huge prospects of this technique for area-wide vector mosquito control, it has become imperative to thoroughly investigate sugar and blood feeding (an essential aspect of mass rearing of the vector) which constitute an indispensable part of the adults‟ life, but have not been fully developed and standardised for optimum and sustainable mass production of the insects in the laboratory. This study therefore seeks to evaluate different sugar types, blood feeding methods and feeding time regimens employed in feeding Anopheles gambiae mosquitoes in the laboratory so as to properly develop standardised sugar type, blood feeding method and time regimen that will give optimum feeding, survival and productivity in a mosquito mass rearing system. 9 University of Ghana http://ugspace.ug.edu.gh 1.4. Objectives 1.4.1 General objective The general objective of this study is to investigate the effects of three different sugar types and three blood feeding methods on the biology and productivity of Anopheles gambiae mosquitoes in a mass production system. 1.4.2 Specific objectives i. To evaluate the effects of 6 % and 10 % concentrations of three sugar types (glucose, sucrose and honey) on the survival and productivity of adult Anopheles gambiae mosquitoes. ii. To evaluate the effects of three different blood feeding methods on feeding response and productivity of female Anopheles gambiae mosquitoes. iii. To determine the effects of Ketamine/Xylazine and Ketamine/Diazepam anaesthetics used to immobilise live animals to feed female mosquitoes on feeding response and productivity of Anopheles gambiae mosquitoes. 10 University of Ghana http://ugspace.ug.edu.gh CHAPTER TWO 2.0 LITERATURE REVIEW 2.1 Brief biology of Anopheles mosquitoes Anopheline mosquitoes have four stages in their life cycle (McCafferty, 1983). According to Miller (2012) their life cycle starts with females laying 50 to 200 boat- shaped eggs singly on the surface of water. The eggs under optimum conditions of temperature and relative humidity may hatch into larvae within 24 hours or more depending on species. The larvae develop through four instars or larval stages with a final moult that give rise to the pupae (Clements, 1992). Adequate nutrition in the larval stage is very crucial to the insects as nutrient reserves from this stage are needed to sustain the insects during their pupal stage where the insect stop feeding completely and enters into a state of structural modifications with the immature tissues breaking down to form adult structures and therefore rely on these reserves for survival (Harzsch and Hafner, 2006; Clements, 1992). Unavailability or inadequate amounts of these nutrient reserves from the larval stage can adversely affect the insects‟ body size, fitness, survivability and productivity in the adult stage (Araújo et al., 2012; Wheeler, 1996). According to Helinski et al. (2006b) the pupal stage is also an important stage of the insect as it lends itself as a key stage for SIT development and application in the control of Anopheline vector mosquitoes. This is because, apart from the adult stage, sexual sterility in male mosquitoes which is needed to cause reproductive failure in 11 University of Ghana http://ugspace.ug.edu.gh the wild female population can also be induced in the pupal stage of mosquitoes prior to their emergence into adults. The adult male Anopheline mosquito recognised by its feathery antenna and long palpi usually lives up to one week in nature and has the sole responsibility of inseminating adult females. The females which have a lifespan of 2 to 3 weeks in nature but can survive up to a month or more in the laboratory mate only once in their lifetime. Upon insemination, female Anopheline mosquitoes seek their host in search of blood meals to develop eggs that are laid singly on the surface of water (Cross, 2004). Soon after oviposition, the female Anopheline mosquito resumes the search for a new blood meal from same or different hosts to lay new batch of eggs after three to four days if conditions are favourable. This provides the link between the human host and the parasite, and accounts for their short reproductive cycle as well as the usually high vector prevalence and malaria infection rates. However, their short reproductive cycle can also be exploited for mass production of male mosquitoes at short time intervals that will enable regular and sustainable supply of sterile males for vector mosquito AW-IPM programmes. According to the Centres for Disease Control and Prevention (CDC) (2010), the first three stages (egg, larva and pupa) which form the immature stages of the insect are aquatic and last from 5 to 14 days depending on species and ambient temperature. Unlike the adult (female) stage that transmits the malaria parasites, the other stages of 12 University of Ghana http://ugspace.ug.edu.gh the insect do not play a direct role in the transmission of the parasite to humans (WHO, 2009). 2.2 Role of Anopheline mosquitoes as vectors in malaria transmission Mosquitoes play very important role as vectors in the transmission of disease- causing parasites that account for several diseases and millions of death each year (Rhodes, 2008, Molavi, 2003). According to Molavi (2003), mosquito-borne diseases infect about 700 million people in Africa, South America, Central America and much of Asia yearly and kill over 3 million people globally every year. From this number, over one million deaths are reported to be caused by the infective bites of Anopheline mosquitoes, the main vector responsible for transmitting Plasmodium parasites that cause malaria infections especially in Sub-Saharan regions of Africa (Fortin et al., 2002). In Ghana, over 3.5 million clinical cases of malaria and 3,000 deaths are reported each year, with 8,200 clinical cases occurring daily (Sodzi-Tettey, 2011). According to The Global Poverty Information Bank 2012 report on mosquito/malaria prevention and control in Ghana, the country spends about 6 % of its GDP on malaria prevention and control annually. Aside these huge government spending, residents also spend substantial part of their livelihoods on repellents, insecticides, screens and other anti-mosquito/malaria products in attempts to prevent and or control mosquitoes and malaria infections (Woodbridge, 1995). Despite the huge financial expenditure and vigorous application of existing interventions to manage the vector problem, mosquito/malaria infection cases however continue to show high prevalence rates (Marrelli, 2012). 13 University of Ghana http://ugspace.ug.edu.gh Evidence provided by the work of Dash et al. (2008), revealed that though malaria was nearly eradicated from some parts of the world over the years, the disease in recent times has re-emerged with new features which were not witnessed during the pre-eradication days due to vector resistance to insecticide(s) and the resistance of Plasmodium parasites to chloroquine and other anti-malarial drugs. These new features in both the vector and the parasite rendered current control strategies less effective in dealing with the problem. 2.3 Need for novel control strategies With increasing international attention and effort to bring the situation under control, vector control strategies including the use of insecticide-treated nets (ITNs) and indoor residual spraying (IRS) (as part of available chemical control methods in place) were introduced with the aim to suppress the transmission intensity and the disease burden (WHO, 2007, 2008). However, scale-up applications of these interventions inevitably do not suffice to sustain long-term control efforts (Read et al., 2009; Chambers et al., 2008). Controlling the disease has therefore become a complex enterprise, and its management will require incorporating other novel control strategies into current control strategies in order to effectively manage the vector problem (Marrelli, 2012; Alphey et al., 2010; Baker et al., 1986). One such important novel control strategy gaining much approval is the Sterile Insect Technique (SIT) which is more of a biological (birth control) method used in several areas to control other key insect pests and vectors of agricultural and medical importance (Klassen, 2005). 14 University of Ghana http://ugspace.ug.edu.gh According to WHO (2008, 2004), Dyck et al. (2005), Krafsur (1998) and Knipling (1998, 1979, 1955), the SIT which exhibited great tendency to effectively synergize with the useful aspects of current prevention/control methods is species-specific, environmental-friendly, clean (leaving no residues) and sustainable vector control tool. It employs mating between released laboratory-reared sexually sterilised males and the native females to reduce or eliminate the reproductive potential of the wild females through production of infertile eggs. This is done by releasing the laboratory-reared sterile males at overflooding ratios over a sufficient period of time. Over the years, the Sterile Insect Technique has proven to be a safe and effective method to prevent, suppress, contain or eliminate insect pest and vector populations and has been mounted successfully against many vectors by using large numbers of the laboratory-reared sterile males (Helinski et al., 2006a). Thus any effort at using SIT for area-wide management of vector mosquitoes must include the mass production of males. According to Dyck et al. (2005) the chances of obtaining such large numbers from the laboratory depend on the fitness, survival (longevity) and productivity of the laboratory-reared population. However, some research findings in this area have shown that mosquitoes reared in the insectary tend to have reduced fitness, survivability and productivity thus making their mass production in the laboratory almost impossible. For example, the work of Kija et al. (2005) attributed the general failure of mosquito control programmes launched in the 1970s to poor fitness, survivability and productivity in the laboratory-reared population. Clements 15 University of Ghana http://ugspace.ug.edu.gh (1955) also attributed this failure largely to poor nutrition among the laboratory- reared stock. These findings were further buttressed by the work of Gouagna et al. (2010), Nayar and Sauerman (1975) and House (1961), who indicated that the fitness, survival and reproduction of insects depend significantly on nutrition. Thus, developing optimal adult diet as well as appropriate blood feeding method/feeding time regimen in adult females is very crucial to optimising feeding among the laboratory reared stocks in order to compensate for these survival, fitness and productivity deficits. 2.4 Sugar feeding in adult Anopheles mosquitoes Takken and Verhulst (2012), Gouli et al. (2004) and Harrington et al. (2001) reported that sugar (carbohydrates) and mammalian blood are critical sources of nutrition among adult mosquitoes. Of these two, sugar is regarded as their basic food since it is commonly and frequently ingested by both sexes to derive the energy needed to maintain fitness, survive long enough and reproduce (Woodbridge, 1995; Nayar and Sauerman, 1975). Foster and Takken (2004) in their findings also stated the importance of the mosquitoes‟ need for sugar by reporting that both sexes emerge into adults with little available energy and hence are strongly attracted to nectar-related volatiles which they often prefer to host-related volatiles when they newly emerged under laboratory conditions. This preference for sugar in their early stages indicates that sugar feeding is not only fundamental for maintaining vital activities of mosquitoes in laboratory but an early priority due to the risk of starvation (Magnarelli, 1986). This dependency on sugar is further illustrated by their behavioural, structural and 16 University of Ghana http://ugspace.ug.edu.gh physiological specialisations for finding, feeding and processing these sugars (Woodbridge, 1995). Foster (1995) and Nayar and Sauerman (1975) further stated that though hematophagous females generally utilize protein from blood meals to develop eggs, they still utilize sugar to help meet their metabolic needs and increase survivorship. In addition, sugar also provides females with a ready source of flight energy (Nayar and Van Handel, 1971; Clements, 1955) and can, in some cases, improve fecundity, both by helping to develop follicles to the resting stage in small females (Magnarelli, 1978, Nayar and Sauerman, 1975) and by increasing the number of follicles undergoing vitellogenesis (Mostowy and Foster, 2004). According to Gary (2005), experiments focusing on the first gonotrophic cycle also suggest that sugar feeding increases fecundity more than blood feeding alone. Gary and Foster (2001) and Straif and Beier (1996) again reported that it is common laboratory practice to maintain female mosquitoes on sugar between blood meals and that sugar in combination with daily blood meals did increase laboratory life span over that of daily blood meals alone, suggesting that though exclusive blood feeding is observed in some mosquitoes it is only a function of sugar scarcity. In buttressing this finding, Morrison et al. (1999); Costero et al. (1998); Scott et al. (1997), Gibson (1996); Foster (1995); Van Handel et al. (1994) and Macfie, (1915) reported that though some mosquitoes species seem to have a fitness advantage feeding on blood alone, this is only due to their anthropophilic and endophilic nature that enable them to adapt and live in domestic environments where blood is readily available and sugar is scarce. 17 University of Ghana http://ugspace.ug.edu.gh Briegel et al. (2002), Gary and Foster (2001), Nayar and Pierce (1980), Nayar and Sauerman (1971, 1975) and Briegel and Kaiser (1973), also presented further evidence that many anautogenous species survive longer and/or produce more eggs with sugar as well as vertebrate blood in their diet. Other field studies also presented evidence that sugar-feeding forms part of their normal feeding behaviour and that the continued existence of sugar feeding both in the laboratory and in the field, indicates that sugar meals strongly provide a fitness and productivity advantage to this species (Laarman, 1968). 2.5 Sugars as nutritive food resource for adult mosquitoes In nature, mosquitoes obtain sugar meals primarily from plant sources such as floral and extra-floral nectaries, damaged fruit, and honeydew (Foster, 1995). Sugars from these sources consist mostly of fructose, glucose, and sucrose (Van Handel, 1972). Other sugars and amino acids may also be present, but usually in trace amounts (Baker and Baker, 1983; Van Handel, 1972, Auclair, 1963). In the insectary, Anopheline mosquitoes can be fed on different synthetic sugar sources such as glucose, sucrose (granulated table sugar) and honey (Benedict et al., 2007). However, these different sources contain specific carbohydrates with varying amounts of energy yield per calories (Tan, 2013). Glucose as energy source provides approximately 3.75 kilocalories per gram for cellular activities and serves as precursors for the production of more complex biological molecules but lack essential supplemental elements such as proteins, minerals and vitamins (Wikipedia, 2012). 18 University of Ghana http://ugspace.ug.edu.gh Fructose, though contains 3.89 kilocalories per gram and releases energy more readily, it also lacks essential vitamins, minerals and proteins like the glucose units, (Sefcik, 2010). On the other hand, sucrose (complex sugar units as found in refined sugars such as Table sugar) contains relatively higher caloric value (3.94 kilocalories per gram) than the simple sugars, but yield their energy more gradually due to chemical bonds between their constituent glucose and fructose units from which they are formed (Lustig, 2013; Wikipedia, 2011). Therefore, in utilizing sucrose, adult mosquitoes tend to expend more energy in hydrolysing these bonds, hence are unable to keep sufficient amount of the energy generated for future use, and in severe cases may deplete their glycogen reserves quickly to satisfy their immediate energy needs. This creates energy deficits that reduce their fitness and longevity. In addition, supplemental elements such as vitamins and minerals are destroyed during their refining processing rendering them deficient of minerals, protein and vitamins (Tan, 2013). Honey is a mixture of sugars (mostly fructose 38.5 %, glucose 31.0 %, smaller quantities of maltose, sucrose, and other complex carbohydrates) and other compounds such as vitamins (B6, thiamin, niacin, riboflavin and pantothenic acid) (Bogdanov et al., 2008), proteins (trace amounts of all essential amino acid except methionine and histidine, and non-essential amino acids e.g. proline) (Mckenzie, 2011), and minerals (calcium, copper, iron, magnesium, manganese, phosphorus, potassium, sodium and zinc). It also contains tiny amounts of several compounds 19 University of Ghana http://ugspace.ug.edu.gh thought to function as antioxidants including chrysin, pinobanksin, vitamin C, catalase and pinocembrin, and possess antimicrobial, antifungal and antiviral properties (Molan, 1997 and White, 1975). Unlike sucrose, it digests easily and is absorbed rapidly into the bloodstream providing quick supply of energy (Bogdanov et al., 2008). One gram of honey contains twice the amount of calories (8.2 kilocalories) found in sucrose. According to Clements (1992), selecting the appropriate sugar type is therefore very critical if feeding is to be optimised to increase fitness, longevity and productivity of the laboratory reared stock. 2.6 Blood feeding in female Anopheles mosquitoes According to Richards et al. (2013) and Clements (1992) blood feeding is exclusive to adult female mosquitoes since unlike the males, females have a gonotrophic cycle that begins with a blood meal and ends with oviposition. Thus, blood feeding is not only an essential activity for colonisation and maintenance of female mosquitoes for research work on vector-borne diseases but also for possible production of large numbers of adult mosquitoes needed for AW-IPM programmes (Deng et al., 2011). According to LaFlamme (2011), adult females apart from the sugars they require to maintain fitness and survive long enough, also take blood meals for supplemental substances such as protein (more importantly threonine) to mature their eggs and enhance reproduction. Takken et al. (1998) in their work also confirmed that sugar feeding was not sufficient to bring follicles of An. gambiae mosquitoes to resting 20 University of Ghana http://ugspace.ug.edu.gh stage and blood meal was required to help build protein reserves, develops follicles to the resting stage, and initiate vitellogenesis and yolk uptake for oocytes to develop completely. Takken et al. (1998) findings was further buttressed by the work of Clements (1992), who stated that primary ovarian follicles of undersized female mosquitoes are arrested in the previtellogenic phase if a meal of blood is not taken. This is because blood has some connection to juvenile hormone suppression, a phenomenon required to stimulate development of follicles to the previtellogenic stage (Clements, 1992). In satisfying this incessant need for blood, female mosquitoes also ingest Plasmodium parasites (agents of malaria infections) from parasite-infected hosts and eventually transmit them to other hosts (either uninfected or already infected) during subsequent blood feeding (Olayemi et al., 2011). 2.7 Effect of blood types and feeding methods on reproduction in female Anopheles mosquitoes Reproduction in adult mosquitoes forms an essential part of their life and influenced by a number of factors (Clements, 1992; Briegel, 1990). According to Billingsley and Hecker (1991) one such factor which is of immense importance is how much blood females ingest during feeding. This according to Billingsley and Hecker (1991) this is very important because it determines how much proteins/amino acids are made available to synthesise egg yolk proteins during reproduction. This was further reiterated by Taylor and Hurd (2003), Lounibos and Conn (1991), Foster and Eischen (1987), who stated that the blood source used and the feeding method and feeding time regimen employed in feeding the mosquitoes 21 University of Ghana http://ugspace.ug.edu.gh is a strong determinant of how females respond to feeding and therefore the amount of blood they eventually ingest to concentrate these essential proteins/amino acids for reproduction. According to Zimmerman et al. (2006) and Burkot (1988), the choice of blood meal in Anopheline mosquitoes is also influenced by several factors including host availability and preference , nutritional requirements and vector density. Laflamme (2011), Lacey and Lacey (1990) and Chandler and Highton (1975) reported that though Anopheline mosquitoes exhibit a wide range of host preferences including preference for humans, livestock, birds, and reptiles, the Anopheles gambiae mosquito tend to show greater preference not only for human hosts but for direct ingestion of blood from the skin of the host rather than from other alternative blood sources and feeding methods. This according to Meijerink, et al. (2000), Roberts and Janovy, Jr. (2000), Konate, et al. (1999), Cork and Park (1996), Knols and De Jong (1996), Kline et al. (1991) and Takken and Kline (1989) is because the female Anopheles gambiae (which rely on two inherent odorant-binding proteins (OBP) to aid host search) is more attracted to volatiles and microflora present on human skin compared to other hosts. However, the use of humans as host in mosquito production systems is not practicable where rearing involves large scale production of the insects. Feeding the insects on such scale from time to time will therefore require the use of other alternative blood hosts and or delivery systems other than the human host. Gerberg (1970) stated that Guinea pigs, rats, mice, rabbits etc are alternative hosts that can be used to mass feed vector mosquitoes in the insectary. Coluzzi (1964) also reported that although the use 22 University of Ghana http://ugspace.ug.edu.gh of human arm is the ideal method to feed adult mosquitoes when maximum adult vitality is required, Anopheles gambiae mosquitoes can also be induced to feed on Guinea pigs in the laboratory. 2.8 Use of anaesthetics in live animal feeding of adult female Anopheles mosquitoes Various methods including mechanical restraining, and immobilising mammals (sedating with anaesthetic agents) to feed adult mosquitoes are being used in several insectaries as substitute for the human arm feeding method. However, immobilising live mammals with anaesthetic agents prior to blood feeding the mosquitoes can greatly affect their feeding response and ultimately their productivity (Gerberg, 1970). For example, the use of anaesthetic agents in live mammals was found to cause profound physiological changes such as altered peripheral circulation and decreased body surface temperatures that can adversely alter experimental results (Murrieta et al., 2010). Thus, it is important that live mammals are anaesthetised with the right anaesthetics in order to minimise as much as possible the negative impact of these anaesthetics on the productivity of the insects in the laboratory. Among the several anaesthetics employed to immobilise live mammals to feed adult female mosquitoes are Xylaxine, Ketamine and Diazepam (Green et al. 1981). According to Mohammed et al. (2012), Murrieta et al. (2010), Gonza´ lez Gil et al. (2003), the quantity of anaesthetics administered to induce anaesthesia in live animals determines how much anaesthetic is eventually introduced into their bloodstream hence the extent to which normal physiological processes such as blood circulation 23 University of Ghana http://ugspace.ug.edu.gh and body temperature of the live host are altered. Kingsolver (1987) also stated that the body temperature of host animals can affect blood feeding response in female Anopheline mosquitoes. According to Khan and Maibach (1971) female mosquitoes tend to show increased response to blood feed when body temperature of the host is within a range of 34 °C to 36 °C than at lower temperatures. Findings from the work of VanDyk (2007) also revealed that mosquitoes were attracted three times more to hosts with skin temperature of 36.7°C than at a lower skin temperature of 18.3°C or below. Therefore the effect of anaesthetics on blood circulation and body temperature of the host organism can adversely affect feeding response and productivity in Anopheline mosquitoes. The need to evaluate the effect of these anaesthetic agents or their combinations on the feeding response and productivity of vector mosquitoes is very crucial if productivity is to be optimised among laboratory reared mosquito stocks. 24 University of Ghana http://ugspace.ug.edu.gh CHAPTER THREE 3.0 MATERIALS AND METHODS 3.1 Study site The study was carried out at the Mosquito Insectary of the Radiation Entomology and Pest Management Centre (REPMC) of the Biotechnology and Nuclear Agriculture Research Institute (BNARI), Ghana Atomic Energy Commission, Ghana. The insectary is a four compartments facility consisting of a cleaning room, an experiment room, larval and adult rearing rooms. It is located 20 km north of Accra at an altitude of 76 m above sea level (Figs. 1 and 2). Fig. 1. Map of Ghana Fig. 2. Map showing location of GAEC in Accra, Ghana. 25 University of Ghana http://ugspace.ug.edu.gh 3.2 Mosquito colony and rearing conditions The Redco strain of An. gambiae established from larval collections around REDCO Flats in Madina near Accra, Ghana were used throughout this study. Adults were maintained under standard rearing conditions of 27 ± 1 °C temperature, and 65 – 80 % relative humidity in a 12 hr day and 12 hr night photoperiod in the insectary. With characteristics similar to the species used in the work of Clements (1955), all adult mosquitoes were sugar feeders and the females which were largely anautogenous (i.e. requiring a blood meal for ovary development), stenogamous (i.e. able to copulate in a small space), human-biting and non-diapausing could easily be cultured in the laboratory. th Eggs obtained from the 66 generation of the laboratory stock colony were used to raise the initial colony for this study. The eggs were set-up in plastic bowls filled with 1.5 L of distilled water to hatch (Plate 1) and the first instar larvae from all egg bowls were mixed and transferred into larval trays at a rearing density of 1000 3 larvae per tray (40 x 30 x 7 cm ) containing 1000 ml of water (Plate 2). Larvae in st rd each larval tray were given 0.25 g Baker‟s yeast on the 1 and 3 days and 0.25 g Dog biscuit from day five (5) until pupation. Trays were covered with fine polyester mesh after feeding to prevent larvae that pupate and subsequently emerge into adults in the tray prior to collection from escaping into the insectary (Plate 3). Pupae were collected into plastic cups partly filled with water (Plate 4) and placed in 6.6 litre plastic bucket (Instawares, Kennesaw, GA) cages to emerge into adults. Un-emerged pupae were removed from the cage after 24 hours in order to generate same age individuals for the experiments. Mosquitoes were maintained under standard rearing conditions throughout the experiment. 26 University of Ghana http://ugspace.ug.edu.gh Plate 1: An. gambiae eggs set up in Plate 2: Larval rearing in trays filled with plastic bowl 1000 larvae in 1000 ml water Plate 3: Mesh-covered trays Plate 4: An. gambiae pupae in a plastic cup 3.3 Sources and handling of adult mosquito sugar diets Artificially synthesised glucose, sucrose (Table sugar) and honey obtained from the food section of the Accra Shopping Mall, Ghana, made up the adult sugar diet. Each sugar type was prepared to 6 % and 10 % concentrations (current standard 27 University of Ghana http://ugspace.ug.edu.gh concentrations in use in mosquito insectaries) by dispensing 6 and 10 grams respectively of the sugar types into glass reagent bottles (Plate 5). One hundred millilitres of water was added in all cases to obtain the required concentrations. Plate 5: 6 % and 10 % conc. solutions of sugar types in glass and plastic bottles 3.4 Sources and handling of adult female mosquito blood diets The blood diets for the blood feeding experiments comprised of fresh human blood fed from human forearm (human arm feeding method) or fresh Guinea pig blood fed from either an anaesthetised guinea pig (anaesthetised Guinea pig feeding method) or restrained live guinea pig (restrained Guinea pig feeding method). Same hosts and time of day were used throughout the study to eliminate errors that might come from variations in blood composition when different hosts and time of day are used. Guinea pigs of same age (2 years), body weight (250 g) and sex (males) were also used in the experiments to ensure uniformity and minimise experimental errors. Animals were clinically examined at the Ghana Atomic Energy Commission Veterinary Clinic to certify their health status prior to their use in the experiments. 28 University of Ghana http://ugspace.ug.edu.gh For the human arm feeding, each generation of female mosquitoes were blood fed only once in all cases to protect the human host from contracting Plasmodium parasites during feeding. In addition, Cages used to blood feed females were thoroughly washed with detergents (e.g. bleach) and sun dried to eliminate any possible chance of the human host contracting other infections. 3.5 Evaluation of the effect of different sugar types on adult mosquito survival One hundred (100) adult mosquitoes of 50 males and 50 females each were aspirated with hand-held aspirator into 3.3 litre plastic bucket cages with fine mesh screen tops. Sugar feeding was done ad libitum in all cases with adults per cage fed using either 6 % or 10 % concentration of one particular sugar type in cotton balls (replaced daily) placed in sugar tubes that were placed inside the cages. In no particular order, each adult cage was fed with 6 % concentration of a particular sugar type in solution. That is, 6 % glucose solution, sucrose solution or honey dilution. Adults in the last cage were given only water to serve as control (Plate 6). The same protocol was followed to feed adults with same sugar types at 10 % concentration. Each experiment was replicated three (3) times using different generations of adults. Data collection was done similar to the procedure described by (Manda et al., 2007). To determine the effect of the different sugar types and different concentrations fed on adult survival (longevity), dead adult mosquitoes per cage per day were removed into holding cups and the number of adults that survive per cage per day (based on sugar types, concentrations and sex) were counted and recorded. This was continued daily until the last death was recorded in all cases. The sugar 29 University of Ghana http://ugspace.ug.edu.gh type and percentage concentration that optimised adult survival were determined based on the type and concentration that recorded the highest number of days that adults survived. Plate 6: Adult male and female mosquitoes in holding cages for data collection 3.6 Evaluation of the effect of different sugar types on adult male mosquito mating potential Hundred adults consisting of 50 male and 50 female mosquitoes each were collected with hand-held aspirator into 3.3 plastic bucket cages with mesh screen tops. Adults per cage were sugar fed ad libitum on 10 % concentrations of honey, glucose or sucrose solution in cotton balls and kept for a period of one week in all cases to ensure mating had taken place. Using a procedure similar to the protocol described by Vrzal et al. (2010) Thirty (30) females per sugar type were collected o into adult holding cups and immobilised in a chiller at 4 C for 2 minutes and ® dissected under dissecting microscope (Radical , Germany) to remove the 30 University of Ghana http://ugspace.ug.edu.gh spermathecae. The spermatheca from each female was observed under stereoscope (Novel, Holland) to determine the presence or absence of spermatozoa (Plate 7). Females with spermatozoa present in spermatheca were considered inseminated females and those with transparent spermatheca were termed as uninseminated females. The females inseminated per sugar type were counted and recorded. Each experiment was replicated three (3) times. The sugar type that optimised male mating potential was determined based on the type that gave the highest number of females with sperms present in spermatheca after the one week mating period. Plate 7: Dissection of females for spermathecae and examination of spermathecae for presence of spermatozoa 31 University of Ghana http://ugspace.ug.edu.gh 3.7 Response of female Anopheles gambiae to different blood feeding methods One thousand (1000) adult mosquitoes of 500 males and 500 females were collected with hand-held aspirator into a 6.6 litre plastic bucket cage with a mesh screen top. The mosquitoes were fed ad libitum on glucose solution prepared at 10 % concentration, soaked in cotton ball and placed in sugar tubes that were placed inside the cage for a one week mating period. Fifty (50) females each from this stock were collected into 3.3 litre plastic bucket cages and allowed to blood feed for 15, 20 and 25 minutes using Human arm feeding (HAF) method (human arm was placed on top of the fine mesh covering of the cage for mosquitoes to feed) or anaesthetised Guinea pig feeding (AGF) method ( Guinea pig anaesthetised with Ketamine/Xylazine and placed on top of the fine mesh covering of the cage for the mosquitoes to feed) or by restrained Guinea pig feeding (RGF) method (restrained Guinea pig placed on top of the mesh covering of the cage for mosquitoes to feed). The RGF method served as control and was used as such to determine whether or not the use of anaesthetics in live animal feeding of mosquitoes affect blood feeding response and egg production capacity in An. gambiae (Plates 8, 9 and 10). Each experiment was replicated three (3) times. Following the protocol described by Solarte et al. (2007), females with blood present in their abdomen for each of the different blood feeding methods (HAF, AGF and RGF) after the 15, 20 and 25 feeding times were counted as females that blood fed. The number of females that blood fed per feeding method and feeding time were numerically counted and recorded. The feeding method and feeding time that recorded the highest number of female blood feeding was considered as the 32 University of Ghana http://ugspace.ug.edu.gh feeding method and time regimen that optimised blood feeding response in An. gambiae. Plate 8: Human arm feeding (HAF) of adult female An. gambiae mosquitoes Plate 9: Restrained Guinea pig feeding (RGF) of female An.gambiae mosquitoes 33 University of Ghana http://ugspace.ug.edu.gh Plate 10: Anaesthetised Guinea pig feeding (AGF) of female An. gambiae mosquitoes 3.8 Evaluation of the effect of different blood feeding methods on egg production of female mosquitoes For the egg production experiment, twenty females each were randomly collected from females that blood fed per human arm, restrained Guinea pig and Ketamine/Xylazine anaesthetised Guinea pig feeding methods used in the blood feeding response experiments. Each female was Iso-set up in separate oviposition cups to lay eggs for a period of fourteen (14) days. Females in each cup were provided daily with 10 % concentration of glucose solution in cotton pads placed on the mesh screen top of the cups in order to ensure that females survive during the oviposition period (Plates 11, 12 and 13). The Iso-female cups were observed daily for presence of eggs and eggs laid per cup per day were counted and recorded. Each experiment was replicated three (3) times. The feeding method which had females producing the highest number of eggs during the fourteen days period was scored as the method that optimised egg production. 34 University of Ghana http://ugspace.ug.edu.gh Plate 11: Iso-set up for females that blood fed on human arm Plate 12: Iso-set up for females that blood fed on restrained Guinea pig 35 University of Ghana http://ugspace.ug.edu.gh Plate 13: Iso-set up for females blood fed on anaesthetised Guinea pig 3.9 Evaluation of the effect of different anaesthetic agents on blood feeding response of female mosquitoes One thousand (1000) adult mosquitoes of 500 males and 500 females each were collected with hand-held aspirator into a 6.6 litre plastic bucket cage with a mesh screen top. 10 % Glucose solution soaked in cotton ball was fed ad libitum to the mosquitoes for a period of one week. Fifty females each (a week old) from this stock were collected into 3.3 litre plastic bucket cages and fed for 15, 20 and 25 minutes using Guinea pig anaesthetised with 0.05 ml Ketamine/Xylazine (KX) anaesthetic agents (Prepared in the ratio 2:1) and placed on top of the mesh screen cover of the cage in one case and Guinea pig anaesthetised with 2 ml Ketamine/Diazepam (KD) anaesthetic agents in another case (Plates 14 and 15). Variations in the volumes of the different anaesthetics administered was due to the fact that anaesthesia was induced in Ketamine/Xylazine anaesthetised Guinea pigs at relatively lower dose of 0.05 ml whilst in the case of Ketamine/Diazepam, 36 University of Ghana http://ugspace.ug.edu.gh anaesthesia was induced in Guinea pigs at relatively higher doses of 2 ml. This variation in the Guinea pig anaesthesia induction time was similarly reported by Ozkan et al. (2010) for same combinations of anaesthetics and attributed to differences in anaesthesia induction properties of the two different anaesthetics. A third cage of female mosquitoes was fed using restrained Guinea pig (RGF) as control. Fed females were observed for presence or absence of blood in their abdomen to determine the blood feeding method and feeding time that optimised blood feeding response. Females with blood present in the abdomen after feeding on either Ketamine/Xylazine, Ketamine/Diazepam or restrained Guinea pigs per 15, 20 and 25 minutes feeding times were considered as females that blood fed. The number of females that blood fed per feeding method and feeding time were counted and recorded. The feeding method and feeding time that recorded the highest number of females blood feeding was considered as the feeding method and time regimen that optimised blood feeding response in An. gambiae. Plate 14: Ketamine/Xylaxine anaesthetised Guinea pig feeding of adult female mosquitoes. 37 University of Ghana http://ugspace.ug.edu.gh Plate 15: Ketamine/Diazepam anaesthetised Guinea pig feeding of adult female mosquitoes 3.10 Evaluation of the effect of different anaesthetic agents on egg production in female mosquitoes Twenty (20) females each from the females that blood fed in the Ketamine/Xylazine (KX) and Ketamine/Diazepam (KD) anaesthetised Guinea pig feeding experiments were randomly collected for the experiment. The females collected were Iso-set up in separate oviposition cups to lay eggs and each cup provided with 10 % concentration of glucose solution in cotton pad (replaced daily) placed on top of the mesh screen cover of the oviposition cups to ensure females‟ survive during the oviposition period (Plates 16 and 17). The Iso-female cups were observed daily for presence of eggs and the eggs laid per cup per day were counted and recorded. Each experiment was replicated three (3) times. The anaesthetic agent used to immobilise guinea pigs to feed the mosquitoes in which female mosquitoes produced the highest number of eggs during the fourteen days period was counted as the method that optimised egg production. 38 University of Ghana http://ugspace.ug.edu.gh Plate 16: Iso-set up of females fed on Ketamine/Xylazine anaesthetised Guinea pig Plate 17: Iso-set up of females fed on Ketamine/Diazepam anaesthetised Guinea pig 3.11 Experimental design and data analysis The various factors studied in these experiments were arranged in a factorial fashion using the complete randomised block design as a base. Data collected for the various parameters determined in the experiment were analysed by following th the two way ANOVA procedure using GENSTATS (12 edition). Data collected 39 University of Ghana http://ugspace.ug.edu.gh on adult survival, female blood feeding response and productivity (egg production) were arc sine transformed prior to analysis. Means for the various parameters studied were separated by the LSD method at 5 % probability level when F-test proves significant at 5 %. Regression analysis was performed to establish a relationship between the % survival values obtained by male and female mosquitoes for the different sugar types fed at different concentration levels. This was done to give an account of variations within the two data sets. 40 University of Ghana http://ugspace.ug.edu.gh CHAPTER FOUR 4.0 RESULTS AND OBSERVATIONS 4.1 Percentage survival of male An. gambiae on 6 % concentration of sugars From the graph (Fig. 3) it was observed that the different sugars evaluated showed varying effects on percentage survival of adult male An. gambiae mosquito for the period monitored. For 60 % survival used as reference point for males, 60 % of the honey fed males survived up to 18 days to mate with females, whiles males fed on glucose recorded same percentage survival at day 11. 60 % of sucrose fed males survived to day 10 with males fed on water (control) recording same percentage survival as early as day 3. For the entire period monitored, honey fed adult males recorded the longest percentage survival period of 28 days followed by glucose and sucrose with 21 and 18 days respectively whiles the control recorded an overall survival period of 4 days. Generally, all sugars recorded a decreasing percentage survival of adult males as the number of days increases with the control showing a relatively sharper decline in percentage survival over the entire period monitored (Fig.1). 41 University of Ghana http://ugspace.ug.edu.gh 120.0 100.0 6 % 80.0 GLUCOSE 6 % SUCROSE 60.0 6 % HONEY 40.0 WATER (CONTROL) 20.0 0.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Number of days of sugar feeding FIG. 3: Survival of adult male An. gambiae on 6 % glucose, sucrose and honey solutions 4.2 Percentage survival of male An. gambiae on 10 % concentration of sugars 10 % concentration of all three sugars and the control were also observed to produce different effects on the percentage survival of the adult male An. gambiae. 60 % of males fed on 10 % honey solution survived to day 18 to mate with females with an overall survival period of 30 days whiles glucose fed males recorded the same 60 % survival at day 14 and survived to 26 days throughout the entire period. 60 % of sucrose fed males survived to 12 day with an entire survival period of 21 days to mate and inseminate females. Adult males fed with water had 60 % of males survived to day 3 with zero % survival recorded as early as day 4 (Fig. 2). A general decreasing trend was observed in the percentage survival of adult males for all the sugar types as the number of days increases. A sharp decline in survival was observed for the control (Fig. 2). 42 % survival University of Ghana http://ugspace.ug.edu.gh 120.0 100.0 80.0 10 % GLUOCSE 10 % 60.0 SUCROSE % SURVIVAL 40.0 WATER (CONTROL) 20.0 0.0 1 2 3 4 5 6 7 8 9 101112 131415 1617 181920 212223 242526 272829 30 Number of days of sugar feeding FIG.4: Survival of adult male An. gambiae on 10 % glucose, sucrose and honey solutions 4.3 Percentage survival of female An. gambiae on 6 % concentration of sugars The percentage survival of female An. gambiae mosquitoes on 10 % concentration of the sugars was observed to be different for all sugar types and the control. For 60 % adults survival, 60 % of sucrose and glucose fed females survived to day 12 and 18 respectively to mate and lay eggs whiles honey recorded a relatively longer survival period of day 21with the control recording the same 60 % survival at day 2. Throughout the entire period monitored, honey fed females recorded the longest percentage survival period of 36 days with glucose and sucrose fed females recording lower values of 29 and 22 days respectively. Survival occurred up to only 4 days in the control. A decreasing trend in percentage survival of females for all sugars as the number of days increased together with a sharp decline in percentage survival of the control were observed over the period (Fig.3). 43 % survival University of Ghana http://ugspace.ug.edu.gh 120.0 100.0 6 % 80.0 GLUCOSE 6 % 60.0 SUCROSE 6 % HONEY 40.0 WATER (CONTROL) 20.0 0.0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 Number of days of sugar feeding FIG. 5: Survival of adult female An. gambiae on 6 % glucose, sucrose and honey solutions 4.4 Percentage survival of female An. gambiae on 10 % concentration of sugars The effects of the 10 % concentration of the sugars on the percentage survival of adult female mosquitoes were also varied. For the same 60 % survival mark, adult females fed on honey survived relatively longer to 24 days to mate with males and lay eggs. 60 % of glucose fed females only survived to 16 days whiles the same percentage of sucrose fed females survived to day 13 with the control recording same 60 % survival as early as day 3. For the entire duration monitored, honey fed females recorded the longest percentage survival period of 45 days whilst glucose and sucrose fed females recorded 34 and 23 days respectively. Survival occurred up to only 4 days in the control. A decreasing trend in percentage survival of females for all sugars as the number of days increased was observed. A sharp decline in percentage survival of the control also occurred over the period (Fig.4). 44 % survival University of Ghana http://ugspace.ug.edu.gh 120.0 100.0 10 % GLUCOSE 80.0 10 % SUCROSE 60.0 10 % HONEY 40.0 WATER (CONTROL) 20.0 0.0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 Number of days of sugar feeding FIG. 6: Survival of adult female An. gambiae on 10 % glucose, sucrose and honey solutions 4.5 Relationship between percentage survival of male and female An. gambiae mosquitoes The regression coefficients established between the percentage survival of male and female mosquitoes were found to be extremely significant (p ≤ 0.001) with the exception of glucose fed at 6 % with p-value of 0.001 (Table 7). The regression coefficients obtained from the correlation between percentage survival of male and female mosquitoes were in the range of 80 to 100 % (Table 7). 2 Among the regression coefficients (R ), the correlation between percentage survival of male and female for glucose fed at 6 % produced the least values compared to 45 % Survival University of Ghana http://ugspace.ug.edu.gh 2 2 sucrose and, honey with R values in the range of 90 to 100 %. Similar range of R values were obtained by the control treatment (water). 46 University of Ghana http://ugspace.ug.edu.gh Table 1: Relationship between concentration and feeding material on % survival of male and female An. gambiae mosquitoes S/C C RM RC p-value (s) a 6 % FM = 0.74 MM + 0.67 0.9993 ≤ 0.001 Water a 10 % FM = 0.78 MM + 0.63 0.9993 ≤ 0.001 a 6 % FM = 1.07 MM + 2.86 0.9894 ≤ 0.001 Sucrose a 10 % FM = 0.97 MM + 3.96 0.9829 ≤ 0.001 a 6 % FM = 0.98 MM + 7.08 0.9740 ≤ 0.001 Honey a 10 % FM = 0.90 MM + 13.82 0.9384 ≤ 0.001 a G l u c o s e 6 % FM = 1.08 MM + 9.11 0.8957 0.001 Glucose a 10 % FM = 0.91 MM + 9.28 0.9472 ≤ 0.001 Means of % survival should be compared in columns with respect to the days and concentration levels. Means followed by the same letters in columns were not significantly different. *** = extremely significant ns= not significant. S/C = sugar types/control C = Concentrations RM = Regression models RC = Regression coefficients aFM = Female mosquitoes MM = Male mosquitoes. 47 University of Ghana http://ugspace.ug.edu.gh 4.6 Potential of male mosquitoes to inseminate females The spermathecae of inseminated female mosquitoes were found to contain long thread- like spermatozoa which appear as fine concentric threads (Figure 3). Uninseminated females were observed to have a fairly transparent spermathecae (Figure 4) Bundles of sperm Spermathecae Fig. 7: Inseminated spermatheca with Fig. 8: Spermatheca of uninseminated bundles of sperm female No statistical significance was observed among the three sugar types regarding the potential of male mosquitoes to inseminate females, however, adult males fed with honey showed higher potential to inseminate females (Figure 5). 48 University of Ghana http://ugspace.ug.edu.gh 50 43 40 34 31 30 20 10 0 Glucose Honey Sucrose Sugars Types Figure 9: Effect of 10 % concentration of sugar types on the potential of male mosquitoes to inseminate female mosquitoes 4.7 Percentage blood feeding of female An. gambiae fed using different blood feeding methods and time regimens The effects of blood feeding methods on blood feeding response in female An. gambiae mosquitoes were observed to be significant (p = 0.1). The Human arm feeding (HAF) method recorded the highest percentage feeding response of 70.6 %, followed by 50.1 % for anaesthetised guinea pig feeding (AGF) and 33.1 % for restrained guinea pig feeding (RGF) method. The control (RGF) recorded the least percentage feeding response of 33.1 % (Fig. 1). For the effect of feeding time on percentage blood feeding response in females , 57.6 %, 48.3 % and 49.0 % blood feeding response were recorded at 15, 20 and 25 minutes feeding times respectively. The highest percentage blood feeding of 57.6 % 49 % Insemination University of Ghana http://ugspace.ug.edu.gh was recorded when adults were fed for 15 minutes whiles the least percentage of 48.3 % was obtained at 20 minutes feeding time(Fig. 2). a 80 70.6 70 b 51.2 60 50 c HAF 33.1 40 AGF RGF 30 20 10 0 Blood feeding methods Fig 10: Effect of blood feeding methods on percentage blood feeding response of An. gambiae 50 % blood feeding University of Ghana http://ugspace.ug.edu.gh a 57.6 58 56 54 15 minutes b 52 b 49.0 48.3 20 minutes 50 48 25 minutes 46 44 42 Feeding times Fig 11: Effect of feeding times on percentage blood feeding response of An. gambiae 4.8 Percentage egg production in female An. gambiae fed using different blood feeding methods Significant differences (p ≤ 0.001) were observed for the effect of blood feeding methods on % egg production of female An. gambiae over the period monitored. HAF recorded percentage egg production of 28.3 % whiles AGF and RGF (control) recorded 23.8 % and 14.6 % respectively. The highest percentage egg production of 28.3 % was obtained using the HAF method whilst the least percentage egg production of 14.6 % was recorded for the control (RGF) (Fig. 3). 51 % blood feeding University of Ghana http://ugspace.ug.edu.gh a 28.3 30 b 23.8 25 20 c HAF 14.6 AGF 15 RGF 10 5 0 Blood feeding methods Fig. 12: effect of blood feeding methods on percentage egg production of female An. gambiae 4.9 Percentage blood feeding response of female An. gambiae fed using Guinea pigs anaesthetised with different anaesthetics and time regimens The effects of anaesthetics on the percentage blood feeding of female An. gambiae mosquitoes were observed to be significant (p ≤ 0.001). Ketamine/Xylazine (KX) anaesthetics recorded the highest percentage feeding of 66.3 % followed by 50.1 % for Ketamine/Diazepam (KD) anaesthetics and 31.6 % for the control (RGF) (Fig: 4). For the effect of feeding time on percentage blood feeding response of females, 15 minutes feeding time recorded 55.6 % whiles 20 and 25 minutes obtaining 41.5 % and 44.2 % respectively. The highest percentage blood feeding of 55.6 % was therefore obtained at 15 minutes whiles the least percentage of 41.5 % was recorded when 20 minutes feeding time was allowed (Fig. 5). 52 % egg prodcution University of Ghana http://ugspace.ug.edu.gh a 66.3 70 b 60 50.1 KX 50 c KD 31.6 40 RGF 30 20 10 0 Anaesthetic types Fig. 13: effect of anaesthetics on percentage blood feeding of An. gambiae a 55.6 60 b b 44.2 50 41.5 15 minutes 40 30 20 minutes 20 25 minutes 10 0 Feeding times Fig. 14: effect of feeding times on percentage blood feeding response of An. gambiae 53 % blood feeding % blood feeding University of Ghana http://ugspace.ug.edu.gh 4.10 Percentage egg production in female An. gambiae fed using Guinea pigs anaesthetised with different anaesthetics The effects of the different anaesthetic types on percentage egg production were observed to be significantly different (p ≤ 0.001). Percentage egg production of 21.2 %, 29.4 % and 16.9 % were recorded for Ketamine/Diazepam, Ketamine/Xylazine and Restrained guinea pig (RGF) (control) respectively (Fig. 6). The highest percentage egg production of 29.4 % obtained over the period was recorded when females were fed Guinea pigs anaesthetised with the KX anaesthetics whiles the least percentage egg production of 16.9 % was obtained with the use of the RGF (control). a 29.4 30 b 25 21.2 c 16.9 20 KX KD 15 RGF 10 5 0 Anaesthetic types Fig. 15: effect of anaesthetics on percentage egg production of An. gambiae 54 % egg prduction University of Ghana http://ugspace.ug.edu.gh CHAPTER FIVE 5.0 DISCUSSION The type of sugar as well as amount obtained by adult Anophelines determines how long they survive (Coluzzi, 1964). Findings from this study similarly revealed that under optimum laboratory conditions, the survival of the adult An. gambiae is dependent on sugar type as well as the concentration used to feed adult Anopheles mosquitoes. For the different sugars used in the study, honey-fed males and females survived longer than those fed on glucose and sucrose solutions. These findings agree with the work of Coluzzi, (1964) who reported that honey has superior natural dietary substances such as amino acids, vitamins and trace mineral elements absent in glucose, sucrose and water (control) and therefore might be responsible for the increased survival and productivity of honey fed adults in the laboratory as compared to glucose and sucrose. Bogdanov et al. (2008) and Molan (1997) further reported that honey apart from possessing antimicrobial, antifungal and antiviral properties that prevent its rapid deterioration in quality, it also contains relatively higher caloric content (about twice the amount found in sucrose and glucose) and other compounds including antioxidants, enzymes and metabolites that enhance their digestion and utilisation. Even though Coluzzi (1964) stated that 10 % sucrose solution can be successfully used as a suitable substitute for honey to sustain survival of adult mosquitoes in the laboratory, 55 University of Ghana http://ugspace.ug.edu.gh findings from this work have shown that sucrose at both 6 and 10 % concentrations proved least suitable in sustaining the survival of both sexes of the species for a longer period as compared to honey and glucose. According to Ashley (2011) this might be due to the fact that although artificially synthesised sucrose like glucose contains high calorific content vital in the survival of adult mosquitoes, it lacks all other essential compounds such as proteins, vitamins and minerals equally needed to enhance survival. For the two concentrations (6 % and 10 %) evaluated, the 10 % concentration of the sugars in solution sustained the survival of both male and female An. gambiae for relatively longer period as compared to the 6% concentration. This outcome might be largely attributed to differences in the quantity of sugar each concentration contains as different grams of the sugars were used to obtain the two different concentrations. Rudloff (2013) and Robergs and Kravitz (2000) also reported that the quantity of sugar present in solution determines its calorific content hence its energy yield. It can therefore be deduced from this premise that the 10 % sugar solutions tend to contain higher sugar content (grams), calorific content and energy yield that sustained adults for relatively longer period as compared to the 6 % concentration. Variations seen in the longevity of both sexes of mosquitoes fed with the same 10 % honey solution can be attributed to sex differences among same species of organisms rather than the concentrations as male mosquitoes by nature survive for relatively fewer days as compared to females (Styer et al., 2007; Carey et al., 1995). 56 University of Ghana http://ugspace.ug.edu.gh Similar adult survival rate seen in the first 3 to 5 days for all sugars and concentrations used, and the steady decline in survival as the monitoring period increases was similarly reported in the work of Manda et al. (2007). It is reported that nutrient reserves carried from the larval stage to the adult stage might have provided sufficient nourishment for adults in the first few days, thus, the distinct effects of the sugars and concentrations used might have been masked during that period (Telang et al., 2005). However, the effects of the sugars and concentrations on the adult survival of adult Anopheles mosquitoes became apparent as the larval reserves depleted with time. Although, results obtained on the potential of male An. gambiae to inseminate females showed no significant differences for the sugars, honey fed males showed greater potential to inseminate females compared to glucose and sucrose fed males. According to Rehan et al. (1975) this might be due to the fact that presence of proteins and other compounds in honey might have produced some positive effects on sperm production in honey fed males hence the relatively greater female insemination recorded for females that were mated with honey-fed males. For the blood feeding methods evaluated in this work, the study revealed that the effect of the different blood feeding methods and feeding times on female An. gambiae blood feeding response and egg production varied significantly (p ≤ 0.001). As evident in the result obtained in this study, female anophelines showed relatively high inclination to blood feed when feeding was done using the human arm (HAF) method as compared to the anaesthetised Guinea pig feeding (AGF). Feeding was however higher in females fed 57 University of Ghana http://ugspace.ug.edu.gh with the AGF method when the AGF was used against the RGF (restrained Guinea pig feeding) method. These findings were similarly reported by Coluzzi (1964) who stated that adult An. gambiae mosquitoes fed with the HAF method recorded higher percentage blood feeding as compared to those fed using other live hosts. This might be due partly to the fact that odorant proteins (proteins that aid host search) found in female An. gambiae mosquitoes tend to show stronger affinity for certain volatiles and microflora present on the human skin, making humans the natural host for the vector and partly due to its greater preference for large size hosts (Takken and Verhulst, 2012; Kweka et al., 2010; Cork and Park, 1996; Coluzzi, 1964).. Again, Murrieta et al. (2010) reported that anaesthetics tend to alter peripheral blood circulation, decrease body surface temperatures and cause physiological changes that could adversely alter experimental results. These possible changes in the body temperature and general physiology of anaesthetised hosts might have also accounted for the relatively low percentage blood feeding and egg production recorded by AGF as against the HAF method. The relatively low percentage blood feeding and egg production obtained in the control (RGF) might be attributed to feeding disruptions that might have occurred from body movements (wriggling) of live hosts during feeding. Thus, females were unable to ingest sufficient blood to mature enough eggs as compared to HAF and AGF. 58 University of Ghana http://ugspace.ug.edu.gh For the effect of feeding times on percentage blood feeding, feeding response was optimum when females were exposed to their host for15 minutes as compared to 20 and 25 minutes feeding times. This might be due to fact that as feeding time increases, the anaesthetic agents severely affected the hosts‟ physiology and body temperature, affecting feeding response negatively in the case of the AGF method. For the HAF method, this might be attributed to the fact that feeding response was not significantly different among all three feeding times used and therefore there is no need to exceed the 15 minutes feeding time. This was similarly reported in the work of Turley et al. (2009) and might confirmed why the 15 minutes feeding time is used as the standard feeding time by most mosquito insectaries to feed adult female mosquitoes. For percentage egg production in females, the human arm feeding method (HAF) again recorded higher egg production of 28.3 % as against 23.8 % and 14.6 % scored for AGF and RGF respectively emphasising a positive relationship between feeding method and egg production. This means that the method used to blood feed female An. gambiae mosquitoes can significantly affect the number of females that will blood feed, the amounts of blood ingested, the number of females that lay eggs and the number of eggs eventually laid by females (Taylor and Hurd, 2001; Billingsley and Hecker, 1991; Lounibos and Conn, 1991; Foster and Eischen, 1987; Roitberg and Prasad, 1987). 59 University of Ghana http://ugspace.ug.edu.gh Although findings from this work and the work of other researchers including Coluzzi (1964) established that the HAF method which uses human beings as live hosts is the ideal method to blood feed female mosquitoes in the insectary, this cannot be the case for a mass production system where mass feeding is required and therefore becomes practically impossible to use human as host where large scale rearing is required. On this premise, AGF with an added advantage of being used when same generation of An. gambiae are to be fed more than once (without any risk of malaria infections as possible with human hosts) becomes a suitable method to blood feed adult Anopheles gambiae mosquitoes as similarly reported by Coluzzi (1964). For the experiments carried out on the effect of anaesthetic agents on blood feeding response and productivity of An. gambiae fed with live animals immobilised with anaesthetics, significant differences were observed due to the effect of the anaesthetics. A positive correlation was again established between the type of anaesthetic used to immobilise the live animals used to feed the mosquitoes and the amount of blood they ingest as well as the number of eggs produced. This is because the relatively high percentage blood feeding response recorded for females that were blood fed with Guinea pigs anaesthetised with Ketamine/Xylazine (KX) anaesthetics as against the Ketamine/Diazepam (KD) anaesthetics was again recorded in the egg production of the females fed with KX anaesthetised Guinea pigs as compared to KD and the control. These results therefore established that aside the fact that anaesthetic agents produce effects on blood feeding and egg production in female An. gambiae mosquitoes, these effects are varied depending on the anaesthetic used. The relatively lower feeding 60 University of Ghana http://ugspace.ug.edu.gh response and egg production recorded for KD anaesthetics might be attributed to a greater effect of the KD anaesthetics on the physiology of the host as compared to the KX anaesthetics which might have equally affected blood feeding and egg production in the vector as reported by Murrieta et al. (2010). 61 University of Ghana http://ugspace.ug.edu.gh CHAPTER SIX 6.0 CONCLUSIONS AND RECOMMENDATIONS 6.1 Conclusions The number of An. gambiae mosquitoes that survive and how long they survive in a mass production system is primarily dependent on sugar type and concentration. Although 10 % glucose and sucrose solutions performed relatively well, their suitability as sugar sources to feed adult mosquitoes in the laboratory as compared to honey may be greatly influenced by other factors such as availability and cost other than merely nutritional composition which is very critical and indispensable to the utmost survival of the An. gambiae mosquito in the insectary. Honey solution which contains several nutritional elements and properties absent in glucose, sucrose and water (control) increased the survival and longevity of both sexes of An. gambiae mosquitoes in the laboratory against all the sugar types as well as the control (water) evaluated in this study. Furthermore, the potential of male An. gambiae mosquitoes to inseminate females was relatively greater when male mosquitoes fed on 10 % honey solution as compared to 10 % glucose and sucrose solutions. 62 University of Ghana http://ugspace.ug.edu.gh It was concluded that 10 % honey solution resulted in optimum feeding, survival and productivity (male potential to inseminate females) of adult An. gambiae mosquitoes in the insectary. For blood feeding, it was established that the amount of blood meal female An. gambiae mosquitoes obtain to mature eggs and the number of eggs they produce in a mass production system is also strongly dependent on the type of feeding method, time regimen and anaesthetic agents used to immobilise live animals. For the three methods evaluated in this study, the HAF method is well preferred by female An. gambiae mosquitoes as evident in the relatively higher percentage blood feeding response and egg production recorded for females that were blood fed using the HAF method as compared to the AGF and RGF methods. The RGF which recorded the least number of females that blood fed also recorded the least number of females that laid eggs and the number of eggs laid for all three methods. Although the HAF method was determined as the ideal method to blood feed adult female An. gambiae mosquitoes in the insectary, its suitability in a production system is restricted to small scale production units because of the dependence of the method on human beings as live hosts to feed adult female mosquitoes. On the other hand, The AGF method which uses anaesthetised Guinea pigs instead of humans as live host in feeding female mosquitoes has the capacity to support large scale and sustainable feeding of female An. gambiae mosquitoes in mass production systems. This method also obtained relatively higher percentage blood feeding and egg production in female An. gambiae mosquitoes as compared to the RGF. We therefore conclude that the 63 University of Ghana http://ugspace.ug.edu.gh AGF method is a more suitable blood feeding method for adult female An. gambiae mosquitoes in a mass production system as compared to the HAF and the RGF. For the two different combinations of anaesthetic agents and blood feeding regimen evaluated in this study, KX anaesthetics administered to immobilise live animals (Guinea pigs) to feed female An. gambiae mosquitoes at 15 minutes feeding time recorded the highest number of females that blood fed and produced eggs as against the other treatments and feeding time regimens used. We also conclude that KX anaesthetics and 15 minutes feeding time resulted in optimum blood feeding and productivity of adult female An. gambiae mosquitoes in a mass production system. 6.2 Recommendations We recommend that 10 % honey solution provided ad-libitum,, anaesthetised Guinea pig feeding (AGF) method using Ketamine/Xylazine (KX) anaesthetics and 15 minutes feeding time be used in mass rearing of this species. Further studies should be carried out to evaluate the potential of other sugar sources (propolis and juice from riped fruits such as oranges) to optimise feeding, survival, and the potential of male An. gambiae to inseminate females. 64 University of Ghana http://ugspace.ug.edu.gh Further work should be done to evaluate the effects of other blood feeding methods and anaesthetic agents used in live animal feeding on An. gambiae productivity in a mass production system. The outcome of this study and other future works could be useful in the mass production of the An. gambiae and consequently facilitate the speedy implementation of the sterile insect technique programme for area-wide control of the mosquito vectors in the near future. 65 University of Ghana http://ugspace.ug.edu.gh REFERENCES Alavi, Y., Arai, M., Mendoza, J., Tufet-Bayona, M. and Sinha, R. (2003). 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R 2 59.37 29.69 4.76 C 1 20.39 20.39 3.27 0.092 S 3 1351.81 450.60 72.21 ≤ 0.001 C x S 3 59.27 19.76 3.17 0.058 E 14 87.36 6.24 Total 23 1578.19 CV= 3.3 % R = Replication C = Concentration S = Sugar types E = Error Table 2: ANOVA for adult males in week 2 (14 days) Source Degree Sum of of of Mean Variance Variation Freedom Square square ratio F.pr. R 2 303.07 151.53 6.23 C 1 237.51 237.51 9.77 0.007 S 3 5858.92 5286.31 217.45 <.001 C x S 3 218.92 72.97 3.00 0.066 E 14 340.35 24.31 Total 23 16958.76 CV= 11.1 % R = Replication C = Concentration S = Sugar types E = Error 91 University of Ghana http://ugspace.ug.edu.gh Table 3: ANOVA for adult males in week 3 (21 days) Source Degree Sum of of of Mean Variance Variation Freedom Square square ratio F.pr. R 2 342.45 171.23 5.25 C 1 94.64 94.64 2.90 0.111 S 3 7118.17 2372.72 72.73 <.001 C x S 3 109.35 36.45 1.12 0.375 E 14 456.71 32.62 Total 23 8121.33 CV= 23.2 % R = Replication C = Concentration S = Sugar types E = Error Table 4: ANOVA for adult males in week 4 (28 days) Source Degree Sum of of of Mean Variance Variation Freedom Square square ratio F.pr. R 2 18.776 9.388 2.99 C 1 54.180 54.180 17.27 <.001 S 3 2013.573 671.191 213.90 <.001 C x S 3 126.701 42.234 13.46 <.001 E 14 43.930 3.138 Total 23 2257.160 CV= 20.4 % R = Replication C = Concentration S = Sugar types E = Error 92 University of Ghana http://ugspace.ug.edu.gh Table 5: ANOVA for adult males in week 5 (35 days) Source Degree Sum of of of Mean Variance Variation Freedom Square square ratio F.pr. R 2 9.054E-05 4.527E-05 2.01 C 1 1.244E+01 1.244E+01 5.537E+05 <.001 S 3 3.751E+01 1.250E+01 5.564E+05 <.001 C x S 3 3.733E+01 1.244E+01 5.537E+05 <.001 E 14 3.146E-04 2.247E-05 Total 23 8.728E+01 CV= 0.2 % R = Replication C = Concentration S = Sugar types E = Error Table : 6 ANOVA for adult females in week 1 (7 days) Source Degree Sum of of of Mean Variance Variation Freedom Square square ratio F.pr. R 2 11.777 5.889 2.00 C 1 5058.673 1686.224 571.70 <.001 S 3 0.653 0.653 0.22 0.645 C x S 3 19.432 6.477 2.20 0.134 E 14 41.293 2.949 Total 23 5131.829 CV= 2.4 % R = Replication C = Concentration S = Sugar types E = Error 93 University of Ghana http://ugspace.ug.edu.gh Table : 7 ANOVA for adult females in week 2 (14 days) Source Degree Sum of of of Mean Variance Variation Freedom Square square ratio F.pr. R 2 99.556 49.778 5.43 C 1 20273.050 6757.683 737.53 <.001 S 3 0.001 0.001 0.00 0.993 C x S 3 85.122 28.374 3.10 0.061 E 14 128.277 9.163 Total 23 20586.005 CV= 6.0 % R = Replication C = Concentration S = Sugar types E = Error Table : 8 ANOVA for adult females in week 3 (21 days) Source Degree Sum of of of Mean Variance Variation Freedom Square square ratio F.pr. R 2 170.32 85.16 6.19 C 1 12410.04 4136.68 300.62 <.001 S 3 8.70 8.70 0.63 0.440 C x S 3 100.71 33.57 2.44 0.108 E 14 192.65 13.76 Total 23 12882.41 CV= 10.6 % R = Replication C = Concentration S = Sugar types E = Error 94 University of Ghana http://ugspace.ug.edu.gh Table : 9 ANOVA for adult females in week 4 (28 days) Source Degree Sum of of of Mean Variance Variation Freedom Square square ratio F.pr. R 2 222.63 111.32 10.58 C 1 6217.57 2072.52 196.93 <.001 S 3 175.45 175.45 16.67 0.001 C x S 3 70.91 23.64 2.25 0.128 E 14 147.34 10.52 Total 23 6833.90 CV= 16.8 % R = Replication C = Concentration S = Sugar types E = Error Table : 10 ANOVA for adult females in week 5 (35 days) Source Degree Sum of of of Mean Variance Variation Freedom Square square ratio F.pr. R 2 0.18968 0.09484 4.91 C 1 1455.12255 485.04085 25087.29 <.001 S 3 274.97032 274.97032 14222.02 <.001 C x S 3 275.37219 91.79073 4747.60 <.001 E 14 0.27068 0.01933 Total 23 2005.92542 CV= 1.8 % R = Replication C = Concentration S = Sugar types E = Error 95 University of Ghana http://ugspace.ug.edu.gh Table : 11 ANOVA for adult females in week 6 (42 days) Source Degree Sum of of of Mean Variance Variation Freedom Square square ratio F.pr. R 2 6.659E-06 3.329E-06 1.28 C 1 4.888E+01 4.888E+01 1.873E+07 <.001 S 3 1.468E+02 4.892E+01 1.875E+07 <.001 C x S 3 1.466E+02 4.888E+01 1.873E+07 <.001 E 14 3.653E-05 2.609E-06 Total 23 Table : 12 ANOVA for adult females in week 7 (49 days) Source Degree Sum of of of Mean Variance Variation Freedom Square square ratio F.pr. R 2 0.002611 0.001305 1.00 C 1 0.001305 0.001305 1.00 0.334 S 3 0.003916 0.001305 1.00 0.422 C x S 3 0.003916 0.001305 1.00 0.422 E 14 0.018275 0.001305 Total 23 0.030024 CV= 2.8 % R = Replication C = Concentration S = Sugar types E = Error 96 University of Ghana http://ugspace.ug.edu.gh Appendix 2: Regression and correlation analysis for sugar types and concentrations Table : 1 Summary of analysis for 6 % concentration of glucose Source Degree Sum of of of Mean Variance Variation Freedom Square square ratio F.pr. Regression 1 6624.7 6624.7 42.92 0.001 Residual 5 771.8 154.4 Total 6 7396.5 1232.7 Percentage variance accounted for 87.5 Standard error of observations is estimated to be 12.4 Table : 2 Summary of analysis for 10 % concentration of glucose Source Degree Sum of of of Mean Variance Variation Freedom Square square ratio F.pr. Regression 1 5645.7 5645.74 89.72 <.001 Residual 5 314.6 62.93 Total 6 5960.4 993.40 Percentage variance accounted for 93.7 Standard error of observations is estimated to be 7.93 97 University of Ghana http://ugspace.ug.edu.gh Table : 3 Summary of analysis for 6 % concentration of honey Source Degree Sum of of of Mean Variance Variation Freedom Square square ratio F.pr. Regression 1 6007.54 6007.54 468.78 <.001 Residual 5 64.08 12.82 Total 6 6071.61 1011.94 Percentage variance accounted for 98.7 Standard error of observations is estimated to be 3.58 Table : 4 Summary of analysis for 10 % concentration of honey Source Degree Sum of of of Mean Variance Variation Freedom Square square ratio F.pr. Regression 1 5785.7 5785.6 76.20 <.001 Residual 5 379.6 75.93 Total 6 6165.3 1027.55 Percentage variance accounted for 92.6 Standard error of observations is estimated to be 8.71. 98 University of Ghana http://ugspace.ug.edu.gh Table : 5 Summary of analysis for 6 % concentration of sucrose Source Degree Sum of of of Mean Variance Variation Freedom Square square ratio F.pr. Regression 1 6007.54 6007.54 468.78 <.001 Residual 5 64.08 12.82 Total 6 6071.61 1011.94 Percentage variance accounted for 98.7 Standard error of observations is estimated to be 3.58 Table : 6 Summary of analysis for 10 % concentration of sucrose Source Degree Sum of of of Mean Variance Variation Freedom Square square ratio F.pr. Regression 1 5713.15 5713.15 288.14 <.001 Residual 5 99.14 19.83 Total 6 5812.29 968.71 Percentage variance accounted for 98.0 Standard error of observations is estimated to be 4.45 99 University of Ghana http://ugspace.ug.edu.gh Table : 7 Summary of analysis for 6 % concentration (Control) Source Degree Sum of of of Mean Variance Variation Freedom Square square ratio F.pr. Regression 1 1868.114 1868.1138 7653.02 <.001 Residual 5 1.221 0.2441 Total 6 1869.334 311.5557 Percentage variance accounted for 99.9 Standard error of observations is estimated to be 0.494. Table : 8 Summary of analysis for 10 % concentration (Control) Source Degree Sum of of of Mean Variance Variation Freedom Square square ratio F.pr. Regression 1 1852.002 1852.0017 6831.79 <.001 Residual 5 1.355 0.2711 Total 6 1853.357 308.8929 Percentage variance accounted for 99.9 Standard error of observations is estimated to be 0.521. 100 University of Ghana http://ugspace.ug.edu.gh Appendix 3: Analysis of variance (ANOVA) for the effect of sugar types on male potential to inseminate females Table : 1 ANOVA for percentage male insemination of females Source Degree Sum of of of Mean Variance Variation Freedom Square square ratio F.pr. R 2 28.40 14.20 0.56 S 2 242.97 121.48 4.78 0.087 E 4 101.65 25.41 Total 8 373.02 CV=14.0 % R = Replication S = Sugar types E = Error Appendix 4: Analysis of variance (ANOVA) for the effect of blood feeding methods on adult female mosquitoes productivity. Table : 1 ANOVA for adult females blood feeding response Source Degree Sum of of of Mean Variance Variation Freedom Square square ratio F.pr. R 2 212.52 106.26 7.43 FM 2 6311.63 3155.81 220.67 <.001 TR 2 476.07 238.04 16.64 <.001 FM x TR 4 73.26 18.31 1.28 0.319 E 16 228.81 14.30 Total 26 302.30 CV= 2.8 % R = Replication FM = Feeding method TR =Feeding regimen FM x TR = Feeding method x Time regimen E = Error 101 University of Ghana http://ugspace.ug.edu.gh Table : 2 ANOVA for adult females egg production in week 1 Source Degree Sum of of of Mean Variance Variation Freedom Square square ratio F.pr. R 2 4.993 2.497 1.24 FM 2 484.968 242.484 120.50 <.001 TR 2 3.009 1.505 0.75 0.489 FM x TR 4 10.294 2.573 1.28 0.319 E 16 32.197 2.012 Total 26 535.460 CV= 11.2 % R = Replication FM = Feeding method TR =Feeding regimen FM x TR = Feeding method x Time regimen E = Error Table : 3 ANOVA for adult females egg production in week 2 Source Degree Sum of of of Mean Variance Variation Freedom Square square ratio F.pr. R 2 6.597 3.299 0.72 FM 2 161.123 80.562 17.63 <.001 TR 2 46.224 23.112 5.06 0.020 FM x TR 4 45.004 11.251 2.46 0.087 E 16 73.123 4.570 Total 26 332.072 CV= 22.4 % R = Replication F = Feeding method TR =Feeding regimen FM x TR = Feeding method x Time regimen E = Error 102 University of Ghana http://ugspace.ug.edu.gh Appendix 5: Analysis of variance (ANOVA) for the effect of anaesthetics on adult female mosquitoes productivity. Table : 1 ANOVA for adult females blood feeding response Source Degree Sum of of of Mean Variance Variation Freedom Square square ratio F.pr. R 2 75.47 37.73 1.72 FM 2 5443.78 2721.89 123.81 <.001 TR 2 26.54 13.27 0.60 0.559 FM x TR 4 220.76 55.19 2.51 0.083 E 16 351.76 21.98 Total 26 6118.31 CV= 9.5 % R = Replication AT= Anaesthetic types TR = Time regimen AT x TR = Anaesthetic types x Time regimen E = Error Table : 2 ANOVA for adult females egg production in week 1 Source Degree Sum of of of Mean Variance Variation Freedom Square square ratio F.pr. R 2 2.004 1.002 0.12 FM 2 434.530 217.265 25.92 <.001 TR 2 18.584 9.292 1.11 0.354 FM x TR 4 32.859 8.215 0.98 0.446 E 16 134.131 8.383 Total 26 622.107 CV= 22.8 % R = Replication AT= Anaesthetic types TR = Time regimen AT x TR = Anaesthetic types x Time regimen E = Error 103 University of Ghana http://ugspace.ug.edu.gh Table : 3 ANOVA for adult females egg production in week 2 Source Degree Sum of of of Mean Variance Variation Freedom Square square ratio F.pr. R 2 2.6955 1.3477 1.52 AT 2 57.1904 28.5952 32.24 <.001 TR 2 23.3420 11.6710 13.16 <.001 AT x TR 4 100.3805 25.0951 28.29 <.001 E 16 14.1931 0.8871 Total 26 197.8015 CV= 9.6 % R = Replication AT= Anaesthetic types TR = Time regimen AT x TR = Anaesthetic types x Time regimen E = Error 104