BIOTIC INTERACTIONS OF THE COMMENSAL BACTERIUM ELIZABETHKINGIA ANOPHELIS (FLAVOBACTERIACEAE) WITH ANOPHELES STEPHENSI LISTON AND ANOPHELES GAMBIAE SENSO STRICTO GILES (DIPTERA: CULI CIDAE) By Veronica Talumba Uzalili A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Entomology - Master of Science 2015 ABSTRACT BIOTIC INTERACTIONS OF THE COMMENSAL BACTERIUM ELIZABETHKINGIA ANOPHELIS (FLAVOBACTERIACEAE) WITH ANOPHELES STEPHENSI LISTON AND ANOPHELES GAMBIAE SENSO STRICTO GILES (DIPTERA: CULI CIDAE) By Veronica Talumba Uzalili Vector - microbe interaction s can be viewed from the standpoint of mutual ist and commensalist partnerships or as antagonistic parasitism. Investi gating the roles of microbes living in association with arthropod vectors can provide insight s in to their influences, for example, on vectorial capacity. In this study the physiological effects on mosquito fecundity of the flavobacteri um Elizabethkingia an ophelis living in a gut - associated commensalism with two major malaria vector species , An. gambiae and An. stephensi , was investigated. Laboratory reared An. stephensi and An. gambiae were subjected to three treatments : (1) Adult, female mosquitoes were su pplied with the antibiotic erythromycin through sugar feeding in order to clear gut bacteria . After 7 days of antibiotic feeding , the mosquitoes were then supplemented with bacteria E. anophelis sugar meal. (2) Adult, female mosquitoes were treated similar ly to group 1 but with no bacterial feeding after antibi otic treatment. (3) Adult, female mosquitoes c onventionally reared. Additionally , hemolysin activity of E. anopheli s was determined first in vitro using culture on blood agar plates, and in vivo by di ssecting the midgut of blood fed mosquitoes from the three treatment groups and counting red blood cells. E. anophelis was found to augment fecundity in An. stephensi mosquitoes but not in An. gambiae . There was no significant effect of E. anophelis on egg viability, for both An. stephensi and An. gambiae . Hemolysin activity was demonstrated on blood agar as well as in An. stephensi . iii . To my late parents, Mr. Luke Emmanuel Uzalili and Mrs. Theresa Malikebu Uzalili . iv ACKNOWLEDGEME NTS I would like to express my sincere gratitude to all the people who helped me in one way or the other to make this thesis a success. My special gratitude goes to Dr. Edward Walker my advisor for his patience, motivation, expertise and mentorship during my two years of study. I also extend my gratitude to all the entire advisory committee members; Dr. Shicheng Kelvin Chen, Dr. Jim Miller and Dr. Zhiyong Xi for their advices, critics, directions and support during the entire study. I would like also to th ank Dr. Themba Mzilahowa of Malaria Alert center - Malawi for his continuous guidance and encouragement throughout the course of my study. I am indebtedly grateful to the MasterCard scholarship foundation for the financial support which enabled me to comple te my studies. To all my lab mates, thanks for sharing with me their expertise especially Julius Kuya, Abdullah Al - Omar, Seungeun Han, Rebecca Vini t and Samantha Hoyle, who assisted me with their insectary management skills, laboratory skills and computer skills. I wish also to thank Ms. Elizabeth Bandason for her assistance in data analysis and for her advices during this long journey. It would be unfair not to mention my roommate Chiwimbo Gwenambira, thanks for the encouragement and all the moments we sha red together. To my family, especially to my aunt Mrs. Prisca Polela, my sister Pilirani Uzalili and my cousins Charity Kalanje and Grace Malikebu thanks for the continuous moral and spiritual support. Last but not least I am forever grateful to the almi ghty God for the precious gift of life and his grace upon me, without him I am completely nothing. v TABLE OF CONTENTS LIST OF TABLES ................................ ................................ ................................ ........................ vii LIST OF FIGURES ................................ ................................ ................................ ..................... vii i KEY TO ABBREVIATIONS AND ACRONYMS ................................ ................................ ...... ix CHAPTER 1. INTRODUCTION ................................ ................................ ................................ ... 1 1.1 Background of the study ................................ ................................ ................................ .. 1 1.2 Rationale ................................ ................................ ................................ ........................... 3 1.3 Main objective ................................ ................................ ................................ .................. 4 1.3.1 Specific objectives ................................ ................................ ................................ .... 4 1.4 Hypothesis ................................ ................................ ................................ ........................ 4 CHAPTER 2. LITERATURE REVIEW ................................ ................................ ........................ 5 2.1 Overview of vectors and diseases ................................ ................................ ......................... 5 2.2 Mosquitoes ................................ ................................ ................................ ............................ 6 2.2.1 An. stephensi ................................ ................................ ................................ .................. 7 2.2.2 An. gambiae ................................ ................................ ................................ ................... 7 2.3 Malaria ................................ ................................ ................................ .............................. 8 2.3.1 Malaria parasite - life cycle ................................ ................................ ............................. 8 2.4 Anopheles mosquito biology and ecology ................................ ................................ .......... 11 2.4.1 Anopheles mosquito reproduction ................................ ................................ ............. 13 2.5 Mosquitoes and microbes ................................ ................................ ................................ ... 14 2.5.1 E. anophelis ................................ ................................ ................................ .................. 15 2.6 Bacteria hemolytic activity ................................ ................................ ................................ . 17 2.7 Malaria vector control ................................ ................................ ................................ ......... 17 CHAPTER 3. MATERIALS AND METHODS ................................ ................................ .......... 20 3.1 Mosquito rearing ................................ ................................ ................................ ................. 20 3.2. Microbial analysis ................................ ................................ ................................ .............. 20 3.2.1 Antibiotic treatment ................................ ................................ ................................ ..... 20 3.2.2 Bacterial cleara nce determination ................................ ................................ ................ 21 3.2.3 Agar medium preparation ................................ ................................ ............................ 21 3.2.4 Plate reading ................................ ................................ ................................ ................. 22 3.3 Mosquito infection with E. anophelis. ................................ ................................ ............ 22 3.3.1 Luria Bertani broth preparation ................................ ................................ ................... 22 3.4 16s rDNA pyrosequencing and da ta analysis ................................ ................................ . 23 3.5 Fecundity determination ................................ ................................ ................................ . 24 3.5.1 Blood feeding ................................ ................................ ................................ .......... 24 3.5.2 Egg laying and collection ................................ ................................ ........................ 24 3.5.3 Egg viability ................................ ................................ ................................ ............ 25 vi 3.5 E. anophelis hemolytic activity ................................ ................................ ...................... 25 3.5.3 In vitro experiment ................................ ................................ ................................ .. 25 3.6 2 In vivo experiment ................................ ................................ ................................ ... 25 3.7 Statistical methods ................................ ................................ ................................ .......... 26 CHAPTER 4. RESULTS, DISCUSSION AND CONCLUSION ................................ ................ 27 4.1 Microbial clearance ................................ ................................ ................................ ............. 27 4.1.1 Bacterial communities per treatment ................................ ................................ ........... 31 4.2 Fecundity determination ................................ ................................ ................................ ..... 33 4.2.1 Effect of E. anophelis on fecundity of An. stephensi ................................ ................... 33 4.2.2 An. stephensi egg viability results ................................ ................................ ................ 36 4.2.3 Effect of E. anophelis on An. gambiae fecundity ................................ ........................ 38 4.2.4 An. gambiae egg viability ................................ ................................ ............................ 42 4.3 E. anophelis hemolysin activity ................................ ................................ .......................... 44 4.3.1 In vitro ................................ ................................ ................................ .......................... 44 4.3.2 In vivo hemolysin activity An. stephensi ................................ ................................ ... 46 4.3.3 In vivo hemolysin activity An. gambiae ................................ ................................ .... 48 4.4 Discussion ................................ ................................ ................................ ........................... 50 4.5 Conclusion and recommendation ................................ ................................ ........................ 54 REFERENCES ................................ ................................ ................................ ............................. 55 vii LIST OF TABLES Table 1: The microbial diversity of the An. stephensi with OTU and their frequency percentages ................................ ................................ ................................ ................................ ....................... 30 Table 2 L SD statistical table on EA, EM and CO treatment groups. ................................ ........... 35 Table 3 : Number and percentage of eggs laid and dissected, number in parenthesis denote the total number of eggs (laid and dissected) ................................ ................................ ..................... 41 Table 4 An. gambiae egg viability statistical results. ................................ ................................ .... 43 Table 5 An. gambiae RBCs means after 24,36 and 48h post blood feedin g. ................................ 49 viii LIST OF FIGURES Figure 1. Malaria parasite life cycle inside the human body and in the Anopheles mosquito (www.cdc.gov) ................................ ................................ ................................ .............................. 10 Figure 2. Anopheles mosquito life cycle showing all the life stages (moodle.digital - campus.org) ................................ ................................ ................................ ................................ ....................... 12 Figure 3. Total CFU/ml in An. stephensi mid guts pulled from control and EM treated groups. . 28 Figure 4. OTU percentages and bacterial community variability at genus level with respect to treatment. ................................ ................................ ................................ ................................ ...... 32 Figure 5 . Mean number of eggs of An. stephensi when supplemented with E. anophelis bacteria (EA), erythromycin (EM), or unmanipulated as controls (CO). ................................ ................... 34 Figure 6 ................................ 37 Figure7 . Effects of E. anophelis on An. gambiae mean number of eggs produced. Each bar represents the mean number of eggs per treatment. Error bars denote the standard errors from the mean number of eggs. ................................ ................................ ................................ ................... 39 Figure 8. In vitro - MacConkey blood agar showing hemolytic activity, with alpha h emolysis activity on E. anopheli s and positive control, and gamma hemolysis on the control on blood media. ................................ ................................ ................................ ................................ ............ 45 Figure 9 .. 47 ix K EY TO ABBREVIATIONS AND ACRONYM S BP Base pair BTI Bacillus thuringiensis i sr ia elensis CDC Center for Disease Control and prevention CI Cytoplasmic Incompatibility CFU Colony forming unit CO Control group DDT Dichlorodiphenyltrichloroethane EA Eliz abethkingia anophelis treated group EEE Eastern equine encephalomyelitis EM Erythromycin treated group H H our IRS Indoor Residual Spraying ITN I nsecticide - treated net LB Luria Bertani LSM Larval source management PBS Phosphate buffered saline RBCs Red blood cells RDT Rapid diagnostic test TSA Tryptone soya Agar VBD Vector borne disease WHO World Health Organization % Percent x O C Degrees Celsius µl Micro litter 1 CHAPTER 1. INTRODUCTION 1.1 Background of the study Malaria is the third - most lethal hu man disease globally, as classified by World Health Organization (WHO, 2014) killing approximately 584 , 000 people in 2013 with 3200 million people being at risk in 97 countries mostly in Africa (WHO, 2015) . At least 3.4 billio n people live in malaria endemic areas of the tropical and subtropical regions (CDC, 2014) . Malaria r emains a challenge in most parts of the world and it is the leading cause of mortality and morbidity in developing countries throughout Africa, Asia and South America (Sachs, 2002 .) . Pregnant mothers and children under 5 years of age are particularly at high risk (CDC,2014) . Malaria is caused by a protozoan called Plasmodium which is transmitted by female mosquitoes of the A nopheles genus. Besides malaria, Anopheles mosquitoes are also known to transmit the nematode parasites that cause lymphatic filariasis . While filariasis tends not to be as fatal as malaria it incapacitates its victims. This disease is endemic to 80 count ries and about 1.1 billion people are estimated to be at risk (WHO, 2002). Together, the burden of malaria and filariasis has contributed not only to mortality and morbidity but also to the poor economic growth of families and governments due to absenteeis m from work (Sachs, 2002) . Even school attendance decreases because sick pupils cannot go to school, individuals cannot go to work and those who care for the sick are forced to stay at home. An estimated 12 billion US dollars is spent every year on the purchase of medicine, transportation to health clinics, burial ceremonies and preventive measures such as bed nets and insecticides (CDC, 2014) . Anopheles gambiae Giles and Anopheles stephensi Liston are among the most important vectors of malaria in Africa and the Indian sub - continent respectively (Valenzuela et al. , 2003) . Malaria 2 vectors are truly polyphagous. Adult mosquitoes feed on nectar and blood while the immature stages are aquatic and detritivores thereby acquiring microorganisms orally. These microbes p lay different roles such as helping in food digestion, nutrition and blood meal digestion (Gaio et al., 2011) . Anopheles mosquitoes require a blood meal for egg development, maturation and production (Clements, 1999) which facilitates vitellogenesis which is the y o l k deposition in to the oocytes. The red blood cells (RBCs) form a component of blood, and contain hemoglobin as well as a complement of amino acids, the building blocks of protein. In order for these proteins to be produced the RBCs have to be lysed, which has been shown to be facilitated by bacteria in Aedes aegypti mosquitoes (Gaio et al., 2011) . The female Anopheles mosquito acquires and transmits the Plasmodium parasite in the course of obtaining a blood meal. The malaria parasite undergoes two phases; the intrinsic phase which is asexual reproduction inside its vertebrate host in close association with RBCs, and then sexual reproduction and extrinsic incubation in the mosquito host. The microbiota of the Anopheles mosquito gut is predominantly composed of Flavobacteriaceae , Enterobacteraceae and Acetobacter aceae bacteria (Ricci et al., 2012b) . The gut micro bes in Anopheles have various functional roles, such as providing essential vitamins; and indeed the presence of some does increase mosquito fitness (Sharma et al., 2013) as evidenced by susceptibility increase to Plasmodium falciparum in antibiotic treated An. gambiae (Dong et al. , 2009) . Elizabethkingia anophelis is a gram - negative bacterium in a class of Flavobacteriaceae and is widely distributed in nature and can be found in fresh water, salt water and soil. Due to its close association with water, E. anophelis has been isolated from the midgut of several mosquito genera (Kämpfer et al., 2011) (Teo et al., 2014a) . On the other hand , E. anophelis caus es neonatal meningitis and nosocomial outbreaks in huma ns and can be passed from mother to infant (Lau et al., 2015) (Frank et al., 2013) . However, E. anophelis infection has been shown to reduce oocyst 3 load of Plasmodium parasites even with a low bact erial dose (10 cells/µl) (Bahia et al., 2014) . The presence of Elizabethkingia spp. in Anopheles and Aedes , vectors of malaria and dengue fever respectively (Wang et al. , 2011 )( Terenius et al. , 2012) and its ability to be transferred transstadially in the mosquitoes provide a great hope for the manipulation and paratransgenesis of the bacteria which could be turned into an effective tool in vector control (Chen et al. , 2015). In efforts to control vector borne diseases such as malaria and lymphatic filariasis, several vector control measures are commonly used including: use of insecticide treated bed nets (ITNs), indoor residual spraying (IRS), where insecticides are sprayed on the wall surfaces, environmental management and larviciding. Most of these measures are chemical based and their prolonged use has resulted in selection for resistant strains leading to the development of insecticide resistance among malaria vector popula tions . For instance, populations to An. stephensi have shown resistance to organophosphates, dichlorodiphenyltrichloroethane (DDT) and pyrethroids insecticides (Soltan et al. , 2015) (Fathian et al. , 2015), while populations of An. gambiae are also known to be resistant to pyrethroids, organophosphates, carbamates and DDT (Ochomo et al. , 2012), presenting a challenge for malaria control programs. 1.2 Rationale Insecticide resistance development by the malaria vectors poses a threat to the fight against malaria, hence the urgent need for novel malaria vector control methods which aim at reducing vectorial capacity using manipulation of the mosquito microbiome (Yee et al. , 2014). One potential method is the paratransgenesis of Anopheles gut microbe E. anophelis , b ut before this can be exploited further there is need to understand the physiological role of E. anophelis on the fitness of malaria Anopheles vectors hence the importance of this study. 4 1.3 Main objective The primary objective of this thesis was to evaluate the physiological role of E. anophelis in modulating fecundity in adult females of the malaria vector species An. gambiae s.s. and An. stephensi , which it naturally infects. 1.3.1 Specific objectives 1. To assess the effectiveness of antibiotic treatment in clea ring gut - microbes in Anopheles mosquitoes and whether the bacteria can be restored. 2. To assess the impact of E. anopheli s on fecundity and egg viability of An. stephen si and An. gambiae s.s. 3. To assess the role of E. anophelis bacteria on blood digestion th rough hemolysin activity. 1.4 Hypothesis Antibiotic treated An. stephensi and An. gambiae mosquitoes will harbor less bacteria than untreated (control) An. stephensi and An. gambiae mosquitoes, and the flavobacteria E. anophelis administered post antibiotic treatment will be able to colonize the mosquito gut. Fecundity in E. anophelis treated mosquitoes will increase than the fecundity in antibiotic treated mosquitoes and control mosquitoes. 5 CHAPTER 2. LITERATURE REVIEW 2.1 Overview of vectors and diseases Vector - borne diseases (VBDs) are diseases caused by pathogens and parasites, transmitted by arthropod vectors (WHO, 2014) . VBDs accou nt for one third of the persistent and emerging infectious diseases in humans (WHO, 2015) and according to one source caused more deaths in humans than all other causes combined in the early 20 th century (Kalluri et al., 2007) . VBDs are among the important diseases in the World Health Organization (WHO) dise ase ranking system, with malaria ranked third after human immunodeficiency virus/ acquired immune syndrome diseases ( Threats, 2008 ) and tuberculosis , killing at least 2.8 million, 1.6million and 1.3 million people per year respectively (Hill et al., 2005) . Arthropods involved in the spread of these pathogens may act as mechanical carriers, intermediate hosts or the primary hosts. Pathogens may propagate , cyclo - develop mentally or cyclo - preoperatively inside the bodies of individual vectors and then later be transmitted to humans through mechanical and biological modes of transmission. Pathogens can be transmitted from vector to vector either vertically through transovar ial processes, or by transtadial means. Vector to vector transmission can also be horizontal by venereal transmission or so - called co - feeding, when an uninfected individual feeding on blood near an infected individual on a vertebrate host becomes infected from that infected individual. Pathogens are horizontally transmitted from vectors to humans by salivation, regurgitation, stercorarian excretion, and ingestion by the host. There are several arthropods that are of medical importance such as: mosquitoes, ticks, deer flies, tsetse flies, black flies, lice, muscoid flies, sand flies, fleas and reduviid bugs (Kalluri et al., 2007) , (Stone et al,. 2012) . Some of the VBD apart from malaria that are also of medical impo rtance in the 21 st century include dengue, plague, leishmaniasis, 6 African trypanosomiasis, relapsing fever, yellow fever, West Nile encephalitis, Japanese encephalitis, Rift Valley fever, and chikungunya (Gubler, 2008). 2.2 Mosquitoes Mosquitoes are cons idered important vectors because they transmit virulent pathogens causing such diseases as malaria (Y. Chen & Xu, 2015) ( Chen et al . , 2015) besides other di seases like dengue, chikungunya, lymphatic filariasis, West Nile virus, yellow fever, western equine encephalitis and Rift Valley fever (Kalluri et al., 2007) . Different mosquito s pecies are carries of different disease agents such as protozoa, helminthes and viruses, with genus Aedes , Anopheles and Culex being the main disease vectors. For instance, Aedes aegypti and Ae. albopictus are known to transmit dengue and chikungunya virus es while Culex pipien is the vector for West Nile virus. Culiseta melanura transmits eastern equine encephalomyelitis (EEE) virus, while helminths for lymphatic filariasis can be vectored by species in four mosquito genera, i.e., Aedes , Culex , Mansonia and Anopheles (Kalluri et al., 2007) . The role of insects in disease transmission should not be undermined as evidenced by the efficiency in West Nile transmission which was found to be widely spread in the United S tates of America within four years after its discovery in 1999 (CDC, 20 14) . Anopheles mosquitoes are the only known vectors of malaria and are commonly found in the tropical and subtropical regions hence the disease distribution. Malaria is still a huge disease burden in Africa - south of the Sahara, the Indian sub - continent and parts of South America. An . gambiae s.l., An. funestus and An. stephensi are among some of the most important disease vectors in these areas (Valenzuela et al., 2003) (Gillies & Coetzee, 1987) (Gimnig et al., 2001) . 7 2.2.1 An. stephensi The geographical distribution of An. stephensi ranges from the Middle East through the Indian subcontinent and western China (Vector base, 2011) . Malaria is endemic to the Indian subcontinent where 70 100 million people are affected by this disease yearly (WHO, 2009) with An. stephensi being the major vector (Qi et al., 2012) . An. stephensi breeds in stream margins and irrigation canals. However, its larvae have also been found in peri - domestic water containers (Qi et al., 2012) and adults are highly ant hropophilic in that setting. As such it is responsible for malaria transmission both in Indian urban and suburban areas (Marinotti et al., 2013) . This endophilic and endophagic mosquito though present throughout the year is most abundant in the summer season which happens to be the peak season for malaria too. There are three forms of An. stephensi (Sharma, 2013) form respon sible for rural and peri - urban transmission and the mysorensis form which is zoophilic that has poor vectorial capacity and is known to be confined to rural areas (Vector base, 2011) . 2.2.2 An. gambiae An. gambiae species complex constitutes formerly seven and now nine morphologically indistinguishable sibling species namely, An. gambiae s.s., An . arabiensis, An. bwambae , An. merus, An. mel as , An. qudriannulatus A & B (Besansky et al., 1994) and Anopheles coluzzi Coetzee and Wilkerson and Anopheles amharicus Hunt , Wilkerson, and Coetzee (Coetzee et al., 2013) . Anopheles gambiae s.s. Giles is one of the primary vectors of P. falciparum in sub - Saharan Africa. Its larvae inhabit sunlit shallow pools, puddles, hoof prints and temporary rain water pool (Gillies & Coetzee, 1987) . An. gambiae is abundant during the wet season and decreases during the dry season but persists to the next wet season (Yaro et al., 201 2) . 8 2.3 Malaria Malaria is caused by a protozoan of the genus Plasmodia in the family of Plasmodiidae , order Haemasporida (Roberts and Janovy, 2005) and it is transmitted by female Anopheles mosquitoes from an i nfected person to non - infected person in the course of blood feeding. Blood is essential for egg development in anautogenous Anopheles mosquitoes. There are five species of Plasmodium that are known to cause malaria in human beings and these are; P. falcip arum , P. vivax , P. ovale , P. malariae and the recently discovered P. knowlesi which was considered to be a parasite for Old world monkeys (White, 2008) with P. falcipalum considered the most important causing >90% of malaria fatalities (Baird, J. K. 2007) . 2.3.1 Malaria p arasite - life cycle Plasmodium parasites undergo an intrinsic and extrinsic incubation periods inside vertebrate hosts and Anopheles mosquito`s bodies respectively. Before the parasite can be transmitted to a vertebrate, it must complete a biological dev elopmental process in the mosquito host (Sinden , 2002) called extrinsic period. The extrinsic form is the onset of gametogenesis where gametocytes are released from the blood (Attardo et al., 2005a) in the form of female and male gamet ocytes (Vlachou et al., 2006) . The female Anopheles picks up gametocytes during blood feeding into the midgut where they undergo gametogony cycle which is sexual reproduction that involves fertilization of exflagellated microgametocyte (microgametes) and macrogamete producing a diploid zygote. The zygote then matures and cyclo - propagates in to ooknite ( Mullen, 2009) . These ookinetes are then absorbed by the midgut epithelium cells by passing through a non - cuticle membrane known as a peritrophic matrix (PM). The PM is made up of a network of chitin microfibrils within a matrix of carbohy drate and protein (Klowden, 2010) (Vinetz, 2000 ) , though 9 the midgut is not cuticle lined, still more they need to produce chitinase that help them to penetrate the PM and then develop into oocysts (Klowden, 2010) . The oocysts undergo soma tic reduction to the haploid genetic condition, and later rupture to release thousands of motile, haploid sporozoites in the hemolymph which migrate to the median lobe of the salivary glands and penetrate them. Thereafter, when the mosquito salivates into human skin, the sporozoites are inoculated into the human being in the course of blood feeding again. Upon inoculation through subdermal capillaries into the human body, the sporozoites migrate to quickly infect the liver paranchymal cells in which they f orm primary tissue meront except for P. ovale and P. vivax , these will form hypnozoite ( Mullen, 2009) and then mature into schizoites then meroizoites rapture and move into the blood stream to invade the red blood cells (RBCs) where they now undergo asexual multiplication into trophozoites which is the onset of malaria, this cycle also takes at least 7 - 14 days (Eldridge & Edman, 2003) . A person suffering from malaria will have fever and general body pains and which can be diagnosed by taking a thick or thin blood film and stained then analyze under a microscope. Additionally, malar ia can also be detected using the rapid diagnostic test (RDT). 10 Figure 1 . Malaria parasite life cycle inside the human body and in the Anopheles mosquito ( www.cdc.gov ) 11 2.4 Anopheles mos quito biology and ecology Anopheles mosquitoes are in the order Diptera (two wings) and family Culicidae. They spend their life stages in two phases as immature and adults. They are known to be holometabolous that is they undergo a complete metamorphosis with the following stages ; egg, larva and pupa which is the immature stage and aquatic , lasting 5 - 14 days (CDC, 2012) and then they emerge into flying adults which are terrestrial. Anopheles mosquitoes lay their eggs singly which have lateral float ( CDC, 2012) on water surface and wet surface. The larval stage is divided into instars, and a larva has to undergo 4 instars before turning into a pupa. The larva has to molt to attain a next instar and on fourth instar the larva needs to metamorphose into pupa. Anopheles larvae lie parallel to the water surface as they lack a syphon for drawing atmospheric air for respiration (Foster and Walker 2009) and they do feed on microorganisms such as fungi, algae, bacteria and detritus found in water, while pupae stage do not feed at all. After 2 - 3 days as pupae, adult Anopheles emerge and these do feed on sucrose and nectar from plants for their energy. Temperature and larvae nutrition are crucial factors in stage development and adult population of Anopheles mos quitoes (Moodle.digital - campus.org ) . 12 Figure 2 . Anopheles mosquito life cycle showing all the life stages (moodle.digital - campus.org) 13 2.4.1 Anopheles mosquito reproduction Female Anopheles mosquitoes are known to be anautogenous, s ince they must obtain a blood meal for vitellogenesis to occur. Carbohydrates, protein and lipids are all essential for egg production (Chambers & Klowden, 1994) as such blood meal obtained from vertebrates is synthesized into proteins in the mid gut by gut microbes (Gaio et al., 2011) for protein provision to the mosquitoes. The proteins will then form yo lk which serves as a vital resource for egg development (Attardo et al., 2005b) . Olfactory cues play an important role in host seeking as the mosquitoes are attracted to body odors (Mboera et al., 1997) . This anautogenous behavior of mosquitoes is what caused the female mosquitoes to be disease transmitters as they move from person to person in seeking for blood meal (Attardo et al., 2005b) . Anopheles mosquitoes usually form swarms where female and males mate, they are attracted to these swarms by the male - female flight tone matching (Gibson et al.,2006) produced by the wing beats by the females (Belton, 1994) (Clements, 1999) , that acts as a form of species recognition too (Penneti er et al., 2010) . In some anopheline mosquitoes such as An. gambiae this usually happens at dusk and mating lasts for about 15 - 20 seconds (Shaw et al., 2015) . Mating is more successful for younger males than older males, with no respect to body sizes (Charlwood et a., 2002) . Some males such as An gambiae species do secrete seminal transglutaminase AgTA3 which he mating succession (Le et al., 2014) . Fertilization is equally important in embryo development, hence the importance of mating in Anopheles egg oviposition, just like in most animals (Clements, 1 999) . On average Anopheles mosquitoes can lay 50 - 200 eggs per oviposition (CDC, 2012), and this phenomenon of laying large number of eggs is what contributes the acceleration in vectorial capacity through vector density increase. 14 2.5 Mosquitoes and micro bes Microbes that include bacteria, protozoa, viruses and fungi are tiny organisms and are present everywhere, living and nonliving organism are known to harbor them. These microorganisms can either be symbionts, commensals or pathogens of the host (Kämpfer et al., 2015) . Among arthropods there are several examples of mutual microbes - insects symbiotic relationships (Chavshin et al., 2015) . For instance, termites have evolved together with its microbiota which help in cellulose digestion (Abe, 2000). Mosquitoes have not been spared from this microbe colonization. They do contain a diversity of microbiota (Gaio et al., 2011) . There are several bacterial families that reside inside mosquitoes such as , Enterobacteriaceae, Flavobacteriaceae, Acetobacteraceae, Rickettsiales and Proteobacterium. Adult mosquitoes emerge with already e stablished bacteria in their guts (Gusmão et al., 2010) which are acquired from their environment at larval stage , most gut bacteria do colonize the mosquito midgut , where also fertilization of malaria plasmodium microgametocytes and macrogametocytes take place to form a zygote known as ookinete (Bahia et al., 2014) . While in the midg ut, the plasmodium do face challenges such as the natural midgut microbial flora (Cirimotich . , 2011) which also contribute to the outcome of mosquito infections (Boissière et al., 2012) . It has been observed that the more the microbial load in the malaria vectors, result in the low infection of the plasmodium (Cirimotich et al., 2011) . The alpha - proteobacteria Wolbachia and Asaia (Ricci et al., 2012a) , have been discovered to reside inside some mosquitoes of the Anopheles genus (Rossi et al., 2 015) . Different microbes inhabit in different organs according to their functions. For examples, Wolbachia , an intracellular bacteria which is also known to be maternally transmitted (Joshi et al., 2014) has bee n isolated in the gut and reproductive tissue of Culex (Atyame et al., 2014) . W olbachia causes some 15 reproduction manipulation such as cytoplasmic incompatibility (CI), male killing and pathogenesis when introduced into other mosquitoes (Bourtzis & Thomas, 2006) . Some bacteria such as Serratia marcescens which is also one of the gut microbiota , have shown to reduce mosquito lifespan and also inhibit malari a plasmodium development (Bahia et al., 2014) . Another example is Asaia which is also an intracellular microbe localized in the reproductive tissue and can be transmitted tr ans - stadially (Fav ia et al., 2007) . The importance of microbes to their hosts ranges from beneficial as evidenced by the negative effect in host fecundity in antibiotic treated mosquitoes (Sharma et al., 2013) helping in digestion, providing macro nutrients and protection from other pathogens, while some enhance susceptibility to pathogens such as malaria parasites (Gendrin et al., 2015) , for example the remove of microbes such as Chryseobacterium , Enterobacter and Serratia species from vector gut by antibiotic treatment prior to infectious blood feed, enhances the susceptibility of An. gambiae to the plasmodium (Meister et al., 2009) . However there is still lack of knowledge on the biological role of various gut microbes such as E. anophelis that are found in mosquitoes (Sharma et al., 2013) . Therefore, a need for more studies like this and proper documentation of roles of microbes in mosquitoes, that may aid in application of paratransgenesis for vector control. 2.5.1 E. anophelis E. anophelis is a gram - negative extracellular bacterium in a family of Flavobactericeae of bacterioidetes and genus Elizabethkingia . Phylogenically it is closely related to Chryseobacterium . It has been found to predominantly reside in the midgut of An . gambiae (Kampfer et al 2011) (Wang et al., 2011) ), An. stephensi (Rani et al., 2009) and Aedes aegypti mosquitoes. The presence of E. a nophelis in these mosquitoes which are also of medical importance due to their efficiency in disease transmission, makes E. anophelis an important bacteria to study. Though E. 16 anophelis is also closely related (98.6%) RNA sequencing to E. meningoseptica b acteria (Kukutla et al., 2014a) , which is in a family of Flavobacteriaceae as well, and has been isolated from human beings, their only difference is the R26T (Kukutla et al., 2014b) . This difference in RNA sequencing placed E. anophelis as a novel bacterium species. E. anophelis has shown to be resistant to ampicillin, chloramphenicol, kanamycin, streptomycin and t etracycline (Kämpfer et al., 2011) . This emerging bacterium has been associated with neonatal meningitis and nosocomial outbreaks in Central African Republic (Frank et al., 2013) and in Singapore (Teo et al., 2014b) . E. anophelis is transmitted transstadially in mosquitoes and has been found to be transmitted from mother to child in humans like in one case in Thailand where the mother passed the bacterial to the baby. The combination of E. anophelis dominancy in Anopheles mosquitoes and its ability to be transmitted transstadially makes E. anophelis a good candidate for paratransg enic (S. Chen et al., 2015) . E. anophelis is characterized by producing yello w colonies if plated on Columbia agar plate (Frank et al. , 2013). Though these bacteria have been isolated from Anopheles mosquitoes, there are fears that they can cause life threatening infections in neonates, severely immunocompromised and post - operative patients (Li et al., 2015) . The role that mosquitoes play in the transmission of this bacterial is not known. However, there is evidence that the E. anophelis is being acquired from the environment by An. gambiae . Studi es have also revealed E. anophelis in the mosquito gut increases both in vivo and in vitro when vertebrate blood is provided indicating the possibility of E. anophelis being involved in erythrocytes lysing in order to acquire nutrients ( Chen et al., 2015) . 17 2.6 Bacteria hemolytic acti vity The relationship that exists between bacteria and their hosts are based on different roles. Some bacteria produce hemolytic enzymes into the extracellular space which help in the lysis of blood cells (Vogl et al., 2008) in order for the proteinases to be released. Hemolysis is the destruc tion of the cell membrane of RBCs caused by lipids and proteins. These will then aid in amino acids p roduction . In anautogenous insect vectors such as Anopheles mosquitoes, the efficient of blood hemolysis is an important activity since their reproduction effectiveness is largely determined by this phenomenon. Bacteria present three types of hemolysin activity which are Gamma, Alpha and Beta (Buxton, 2013) . This classification is based on color changes portrayed on the blood agar media where the bacteria have been cultured. No color change on the media represents Gamma hemolysin, a greenish or brownish color change represents alpha hemolysis while the transparent halos on the media indicate beta hemolysis (Buxton, 2013) . Understanding how blood is digested and how can this be regulated is important in the fight against vector borne diseases . 2.7 Malaria vector control There are several factors that affect malaria transmission suc h as, climate, host feeding preference and rainfall patterns. And in malaria control, all these factors need to be considered . As the war against mosquito - borne diseases such as malaria continues, there have been several measures which have been put in pl ace. Malaria control interventions do focus on reducing vector density, reducing human - vector interaction and finally killing of the Plasmodium . Control measures such as use of insecticide treated bed nets (ITN`s), indoor residual spraying (IRS), larv i cid ing and the general environmental management are t he common methods employed in vector density reduction. In the event that vector control is not effective enough such that a person ends up being 18 infected with the plasmodium, the use of drugs to kill the p arasite once it enters into the human body becomes the only option in order to cure malaria. Coartem ( a combination of artemether and lumefantrine) is now the recommended malaria drug (WHO, 2006) , as there is still no malaria vaccine up to date. However, though these measures have showed great achievements, morbidity and mortality of this disease is still high (Wilke & Marrelli, 2015) . Vector control in fighting malaria is facing quite a big challenge due to insecticide resistance development in Anopheles (Diabate et al., 2002) . Also some control measures such as IRS are expensive to maintain because of high cost resulting from low and short residual lifespan of the insecticides (Akogbéto et al., 2015) . The resistance may be as a result of gene modulation or vector behavior change, where the mosquitoes avoid resting on ITNs (Curtis et al., 2006) (Siegert et al., 2009) . Further, malaria control programs are potentially affected by the development of drug resistance by parasites . For example, r ecently the most deadly malaria parasite P. falciparum has bee n reported to be resistant to artemisnin which is the current malaria front line drug in south East Asia (Noedl et al., 2010) . On the other hand, the other common method of vector control is larv i ciding, where chemical and biol ogical insecticides are applied on vector breeding sites. This method has an advantage over bed net use as bed nets mainly target adult mosquitoes which are highly mobile and this proves to be difficult than larv i ciding which target the immature stage that is less mobile and less behavioral responsiveness (Killeen et al., 2002) . However the use of chemicals pose a threat to the environment as a result microbial application seems to be a better approach (Bhattacharya, 1998) . Microbes such as Bacillus thuringiensis var isria elensis and B. sphaericus which produce endotoxins once ingested by mosquitoes that destroys the gut lining of the insects, killing the insect eventually are used in larv i ciding (Suom et al . , 2008). 19 Larval source management (LSM) which is the management o f the vector breeding sites encompasses manipulation, modification and biological control of breeding sites (WHO, 2013) . In habitat manipulation the water level can be manipulated by flushing streams which might also be applied in irrigation farming. In habitat modification, land can be reclaimed to ensure that breeding site are being controlled. While biological control involves the introduction of other species into the larval habitants which are predators of the immature vectors (WHO, 2013) . However LSM is not an easy thing to do considering that most malaria endemic regions are in the tropics and are in developing regions, (www.malariac onsortium.org) . Furthermore, Anopheles mosquitoes are affected by environmental factors such as temperature, environmental sanitation (Murdock et al . , 2014) and rainfall. As such the hig h temperatures and lots of rainfall of the tropics and subtropical regions together create a conducive condition for mosquitoes. Also, impoundments are created for the Anopheles the rains (Vector base, 2011) . As such this intervention faces challenge as well. In view of all these, new control measures that look at manipulation of mosquito hosts and their microbial communities have been proposed (Ricci et al., 2012b) . The microbes can be manipulated to produce anti - pathogen molecules in their localized parts (Cirimotich et al., 2011) . These new measures will complement the already in use strategies to enhance the integrated malaria control measures. 20 CHAPTER 3. MATERIALS AND METHODS 3.1 Mosquito rearing An. stephensi Liston Johns Hopkins strain and An. gambiae Giles Mbita strain were reared in the MSU insectary chambers in 60 X 60 X 60 cm mosquito cages. The temperature in the incubators was main tained at 28 ±1 ºC and relative humidity between 50% 60% under a photo regime of 12 h light and 12 h darkness. Cotton wicks were soaked in 10% sucrose solution and placed inside the cages as a source of energy to the mosquitoes. After 3 - 5 days of sucrose feeding, mosquitoes were blood - fed with defibrinated bovine blood (Hemostat Lab, Dixon, CA) for their ovarian development by using an artificial feeder covered with a parafilm, presented to mosquitoes on tops of cages for 30 minutes and blood kept warm by circulating warm water (37ºC) from a water bath (Huang et al. 2005). After 3 days, Petri dishes containing soaked cotton balls and a filter paper on top were placed inside the cage for egg deposition. Two days later the filter paper with eggs was transfer red into the plastic pans containing distilled water for them to hatch. For the first instar larvae, first bite fish food (Kyron, Himeji, Japan) was provided and later kitten food (Purina cat chow Nestle, Switzerland) was provided for the second, third and fourth instar until pupation. 3.2. Microbial analysis 3.2.1 Antibiotic treatment The aim of this experiment was to clear the mosquito gut of microbiota and this was attempted using three independent set ups in cages (A, B and C) . Mosquito cages were wip ed with 70% alcohol and then a pan containing pupae (n=100) placed in each cage. After mosquitoes emerged, sterilized 10% sugar solution mixed with erythromycin antibiotic (200 µg/ ml) was provided to the mosquitoes through water wicks for 7 days, with fre sh antibiotic being administered daily. 21 Antibiotics were administered to the two cages (A and B) while mosquitoes in cage C were supplied 10% sucrose and no erythromycin as a control. 3.2.2 Bacterial clearance determination From day 2 of antibiotic treatm ent, a total of 5 mosquitoes were randomly collected from each cage daily for up to seven days for dissection. Each mosquito was immersed in 70% ethanol for one minute and then washed in sterile phosphate buffer solution (PBS). Thereafter the mosquito was placed on a clean slide containing sterile PBS. The midgut was pulled out under a microscope with the aid of a sterile pin and forceps. The midgut was washed three times in PBS and then placed into a 1.5 ml eppendorf tube containing 200 µl PBS. Using a p estle, the midgut was crushed in 200 µl PBS and then washed down the pestle with another 200 µl PBS to ensure maximum collection of the sample, then the volume was increased to 1000 µl and serial dilutions made accordingly. Aliquots of 100 µl of the homoge nate was then pipetted and plated onto agar media under sterile conditions (near a flame), using a grass rod. The results were inspected after 72h incubation of the plates at 28 ºC. 3.2.3 Agar medium preparation Agar medium was prepared by suspending 10 g bacto - tryptone, 5 g yeast extract and 10 g sodium chloride in 1000 ml distilled water and split into two 500 ml bottles, then 10 g agar added into each bottle and autoclaved. About 20 ml of the agar was then poured into each petri dish and let it to soli dify at room temperature. 22 3.2.4 Plate reading After 72 h of incubation, the plates were removed from the incubator, colonies were counted and colony forming units (CFU) were calculated and the colony colors recorded. CFU was calculated using the fo llowing formula below: CFU / ml= 3.3 Mos quito infection with E. anophelis. E. anophelis isolated from mosquito colonies reared at the Michigan State University insectaries as described in (Chen et al., 2015) were cultured in Luria - Bertani ( LB) broth. 3.3.1 Luria B erta n i broth preparation LB b roth was prepared by adding 10 g bacto - tryptone, 5 g yeast extract and 10 g NaCl into 800 ml distilled water with pH adjusted to 7.5 by adding sodium hydroxide, and then autoclaved. E. anophelis (100 µl) was added into 10 ml LB broth and left on a shaker ( 200 rpm) at 30 ºC for overnight. The following day, mosquitoes in cage A treated with EM were provided with 10 ml of 10% sterile sucrose to which 2.4 X 10 3 CFU/ml of cultured E.anophelis had been added. After 24 h, it was replaced with 10% sterile sucros e solution only. EM treated mosquitoes in cage B were supplied with 10% sterile sucrose for 2 days, post - antibiotic treatment. For the naïve cage C, 10 % sucrose was continuously supplied to the mosquitoes. 23 3.4 16s rDNA pyrosequencing and data analysis All DNA extractions were performed in a sterile environment (Biosafety II hood) to avoid contamination. Mosquitoes were surface - disinfected with 70% of ethanol and next rinsed with sterile water. Each individual was crushed using a sterilized pestle in an Eppe ndorf tube and re - suspended in 200 µl of extraction buffer. Next, the genomic DNA was extracted by DNeasy the polymerase chain reaction (PCR) amplification before t he samples were submitted for pyrosequencing. The PCR was carried out with Failsafe enzyme system and the following cycles were used: 94 ºC for 2mins, 30 cycles of 94 ºC for 15s, 50 ºC for 15s, and 72 º C for 1.5mins and a final extension at 72 ºC for 7min s. Amplicon tagging and pyrosequencing were carried out by Research Technology Support Facility (RTSF) at Michigan State University. A total of 12 rrs V4 amplicon libraries were prepared, barcoded, and sequenced by using the standard procedures (Reference ). A typical PCR FastStart High Fidelity Enzyme Blend (5U/ml). The cycles are: 95°C 2 min, 30 cycles of 95°C 20s, 55°C 15s and 72°C 5 min, and a final extension at 72°C 10 min. The samples were purified and loaded to Illumina Miseq sequencer for sequencing. Sequencing reads were processed and analyzed using mothur v.1.35.1 (3/31/2015 version) on the mothur wiki webpage (http://www.mothur.org). After denoising by using the PyroNoise, Uchime, and preclustering methods, the high quality sequence s (> 250 bp) without sequencing errors or chimeras were used for assigning OTUs using an average neighbor algorithm (97% similarity 24 cutoff). OTUs were classified at the genus level using the Bayesian method. Data were further analyzed by first trimming seq uence quality with different cutoffs using standard filtering tools estim ators (Chao, Jackknife, and Abundance Coverage Estimator), diversity indices (Simpson, Shannon) and Bray Curtis dissimilarity . 3.5 Fecundity determination 3.5.1 Blood feeding Mosquitoes were reared for 2 days after E. anophelis supplement in order to allow establ ishment of bacteria in the mosquito gut. On the third day female mosquitoes were starved for 12 h and then blood fed using bovine blood as described above. The unfed and partial fed mosquitoes were removed from the cages. 3.5.2 Egg laying and collection Three d ays post blood feeding, each gravid mosquito was transferred into respective 50 ml sterile tubes (n = 30) per treatment, covered with a netting material for easy access of sucrose. Each mosquito was provided with an autoclaved wet filter paper and cotton w ool for oviposition. Female mosquitoes were allotted 2 days for oviposition and then the filter papers were removed and number of eggs counted under a stereoscopic microscope. Both mosquitoes which laid eggs and those that did not lay eggs but were still g ravid, were dissected and mature eggs were removed and counted. 25 3.5.3 Egg viability For the determination of egg viability , each filter paper containing eggs was transferred into plastic pan with distilled water for the eggs to hatch and made sure that no eg gs were stuck on the sides of the pan to avoid drying up. Larval counts were then determined using the stereoscopic microscope after 24 h. 3.5 E. anophelis hemolytic activity 3.5.3 In vitro experiment For determination of hemolysin activity, E. anopheles was cult ured in LB broth overnight and the following day a 100 µl aliquot was spread on blood agar - Thermo scientific Remel blood agar (5% sheep blood Tryptone Soya Agar or TSA) Ma cC onkey, which was purchased from Fisher Scientific and incubated at 28 ºC for at least 48 h. Color change was used as an indicator for hemolysin activity by the bacteria. Positive and negative controls were also cultured in the same way. 3.6 2 In vivo experiment Hemolysin activity of E. anophelis was tested in vitro by first establishing the total number of RBC`s in Bovine blood (Hemostat Lab, Dixon, CA). Blood was diluted, 1:200 blood: isotonic solution (0.9% normal saline). 10 µl was placed on a hemocytometer covered with a clean glass slide. RBCs on the center square of the chamber wher e smaller squares have been drawn were counted (in five small squares) under a microscope. The total number of RBCs was deduced by using the formula below: Number of RBC`s = 26 Further, antibiotic treated mosquitoes were blood fed on bovine blood together with the control mosquitoes. Then RBC lysis was determined by pulling out midgut and diluting the blood meal into 0.9% no rmal saline ( 1:200) and the number of RBC`s counted using a hemocytometer and compared the total number of RBC in the midgut to the total number of RBC of the bovine blood, at 24 h, 36 h and 48 h after blood feeding (Gaio et al., 2011) . 3.7 Statistical methods Data were entered into Microsoft Exce l spread sheets, and later analyzed using SAS 9.3 for normality, then analysis of variance was used to compare means in fecundity, hatching rate and hemolysin activity; means were separated using Least signific 0.05). Graphs were made in SAS 9.3, SIGMA and Excel. 27 CHAPTER 4. RESULTS, DISCUSSION AND CONCLUSION The following abbreviations have been used in reporting the results: EA = E. anophelis , EM = erythromycin, and CO = control. 4.1 Microbial c learance The microbial density from the EM and control group was determined by pulling the mosquito midgut and plating them on agar media. A total of 30 mid guts from each group were used with 5 mid guts being pulled per day for 6 days an d plated. T he total CFU/ ml was calculated from each group and t he control group had a total of 6.7 x 10^5 CFU/ml which was higher as compared to EM treated group which had 2.2 x 10^5. 28 Figure 3 . Total CFU/ml in An. st ephensi mid guts pulled from control and EM treated groups . 0 1000000 2000000 3000000 4000000 5000000 6000000 7000000 8000000 EM Control Total CFU/ML Treatment 29 Microbial diversity of An. stephensi was determined using 16s rDNA sequencing, Amplicons were obtained from 12 mosquitoes and these were sampled from all three treatments (EA, EM and CO). The most dominant bacterial family was the gram - negative Enterobacteriaceae with the highest frequencies of OTU (275,296) and percentage of all OTUs (87.9%). Acetobacteraceae was the second most dominant family with a frequency of 32,376 OTUs, repr esenting 10.3%; while Flavobactericeae was the third with 2,812 OTUs, representing 0.89% of all OTUs. The fourth most common family was the Comamonadaceae which had a frequency of 554 OTUs (0.17%). Table 1 provides a complete list of the OTUs found in th e analysis. 30 Table 1 : The microbial diversity of the An. stephensi with OTU and their frequency percentages 31 4.1.1 Bacterial communities per treatment The composition of the bacterial community revealed in An. stephensi varied among the three treatment groups, when considering the top ten most abundant OTUs based on ranked frequency (figure 4 ). At least nine bacteria genera were found in samples from the control groups, with Enterobacteraceae being the most dom inant family with a percentage mean of 78.3% while the remaining 27.7% is distributed amongst 9 genera. Sequencing for EM antibiotic treated mosquitoes showed less variation in terms of bacterial species abundancy. Enterobacteraceae was the most dominant w ith a percentage mean 97.1% and the remaining 2.9% comprised of at least three different bacterial genera of Pseudomonas , Actinetobacter and Flavobacterium . In the EA treatment group, 91.2% mean percentage was the Enterobacteraceae and the rest which was 8 .8%included the following genera Shigella , Asaia , Pseudomonas Flavobacterium and Acinetobacter. 32 Figure 4 . OTU percentages and bacterial community variability at genus level with respect to treatment. 75% 80% 85% 90% 95% 100% EM treated EK supp Control OTU mean percentages Treatment Enterobactericecea -unclassified Shigella Asaia Pseudomonas Unclassified gammaproteobacteria-unclassfied Flavobacterium Proteobacteria-unclassified Incertae-sedis Acinetobacter 33 4.2 Fecundi ty determination 4.2.1 Effect of E. anophelis on fecundity of An. stephensi total of 90 gravid An. stephensi were used, with 30 mosquitoes assigned to each treatment (EA, EM and CO). The mean number of eggs was highest in mosquitoe s which had been cleared of gut microbiota and the EA supplemented back to them, lowest in mosquitoes treated with EM, and intermediate in mosquitoes treated as unmanipulated controls ( figure 5 ). Analysis of variance indicated that there was a significant effect of experimental treatments on the mean number of eggs (F = 15.93, df = 87, P = < 0.0001). Furthermore, the a posteriori comparison of means by the LSD mean separation method showed that all the treatment groups were significantly different from eac h other. 34 Figure 5 . Mean number of eggs of An. stephensi when supplemented with E. anophelis bacteria (EA), erythromycin (EM), or unmanipulated as controls (CO). 35 Table 2 LSD statistica l table on EA, EM and CO treatment groups. Differences of Least Squares Means Effect trt _ trt Estimate Standard Error DF t Value Pr > |t| Treatment CO EA - 7.999 3.0982 87 - 2.58 0.0115 Treatment CO EM 9.468 3.0982 87 3.06 0.003 Treatment EA EM 17.467 3.0982 87 5.64 <.0001 36 4.2.2 An. stephensi egg viability results 37 Figure 6 38 4.2.3 Effect of E. anophelis on An. gambi ae fecundit y In this experiment E. anophelis effectiveness in contributing to An. gambiae egg production was tested. A total of 84 mosquitoes were used in this experiment, with 25 in EA group, 32 EM group and 27 CO. In this regard, the results had no sign ificant effect in the mean number of eggs (laid and dissected) per treatment df = 81 (F = 2.22, P = 0.1151). Though no significant effect, the following mean number of eggs were observed EA =39, EM = 33 and CO = 40, with the CO having a highest mean, EM t he lowest and EA the intermediate. 39 Figure 7 . Effects of E. anophelis on An. gambiae mean number of eggs produced. Each bar represents the mean number of eggs per treatment. Error bars denote the standard error s from the mean number of eggs. 40 41 Table 3 : Number and percentage of eggs laid and dissected, number in parenthesis denote the total number of eggs (laid and dissected) Treatment Total number of eggs laid Total number of eggs dissected Percentage of eggs laid Percentage of eggs dissected EA 333 (990) 657(990) 33.63% 66.36% EM 359(1063) 704(1063) 33.77% 66.22% CO 114(1090) 976(1090) 10.45% 89.54% 42 4.2.4 An. gambiae egg viability A total number of 23 mosquitoes that laid eggs out of 84 mosquitoes representing 23.4%, were used in this experiment. The hatching rate of the An. gambiae eggs that were laid per treatment was deduced and 71.18% (n = 8) was the mean highest hatching rate in EA group, 56.21% in the EM group while CO had the least mean hatching rate of 51% .Th e egg viability was analyzed statistically and the results were no statistically significant df 20, (F=0.83, P= 0.4505). 43 Table 4 An. gambiae egg viability statistical results . Treatment Mean % hatching rate Std er ror P. value EA 71.18 9.4 0.4505 EM 56.21 8.6 CO 51 19.63 44 4.3 E. anophelis hemolysin activity 4.3.1 In vitro In determination of E. anophelis hemolysin activity on blood agar after a period of 48 h incubation, the results were indicated by change in color of the blood agar media. The 3 samples, negative control, positive control and test sample which was E. anophelis were plated on different sections of the blood agar plate. The test sample, in this regard E. anoph elis, presented a greenish/brownish color change from original color of media which was red, indicating an alpha hemolysis. The positive control also turned the media red color into greenish/brownish indicating alpha hemolysis as well while for the negativ e control, gamma hemolysis was observed, which was represented by no color change (F ig ure 8). 45 Figure 8 . In vitro - MacConkey blood agar showing hemolytic activity, with alpha hemolysis activity on E. anopheli s and positive control, and gamma hemolysis on the control on blood media. 46 4.3.2 In vivo hemolysin activity An. stephensi In order to assess the effect of E. anopheles on RBCs lysing, a total of 45 An. stephensi midgut were dissected ( with 5 midg uts assigned per treatment per time interval), and RBC`s number counted at three different time intervals (24 h, 36 h 1nd 48 h) 47 Figure 9 48 4.3.3 In vivo hemolysin activity An. gambiae Hemolysin activity of E. anophelis was ascertained in An. gambiae blood fed mosquitoes by comparing the number of RBCs as blood was being digested in mosquito midgut in the three treated groups EA, EM and C O. A total of 45 mosquitoes were sampled, with 5 samples per treatment per time interval. One way ANOVA on the mean number of RBC at three different time intervals ( 24 h, 36 h and 48 h) was conducted and there was no significant difference during all the time intervals P = 0.0764 , P = 0. 0574 and P = 0.1366 respectively. On the other hand the RBC means per treatment for each time interval were slightly different ( T able 5 ). 49 Table 5 An. gambiae RBCs means after 24,36 and 48h post blood feeding. Time (h) Treatment RBC mean P - value 24 EA 5,052,000 0.07644 EM 4,488,000 CO 4,214,000 36 EA 2,834,000 0.0574 EM 3,076,000 CO 2,748,000 48 EA 1,490,000 0.1366 EM 1,726,000 CO 1,4 02,000 50 4.4 Discussion 51 52 53 vitellogenesis to take place. This involves massive production of yolk protein precursors 54 4.5 Conclusion and recommendation 55 REFERENCES 56 REFERENCES Akhoua yri, I. G., Habtewold, T., & Christophides, G. K. (2013). Melanotic pathology and vertical transmission of the gut commensal Elizabethkingia meningoseptica in the major malaria vector Anopheles gambiae. 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