Investigation of the anti-Plasmodium properties of the Yeast Candida oleophila in the

Malaria Mosquito, Anopheles gambiae

by

George Edward “Ned” Barringer III

A thesis submitted to Johns Hopkins University in conformity with the requirements for

the degree of Master of Science

Baltimore, Maryland

April,2014

II

Abstract

Plasmodium falciparum is a causative agent of malaria and a significant global

health burden. The primary vector of P. falciparum in sub-Saharan Africa is the

Anopheles gambiae species complex. Plasmodium species infect approximately 500

million people and cause nearly 1 million deaths annually. Current control efforts are

hampered by drug resistant P.falciparum parasites and insecticide resistant mosquitoes.

In the continuing effort to control malaria infection, microbes native to the midgut are

being studied for species that can inhibit P. falciparum. While the bacteria of the

mosquito microbiome have been well studied, few studies have explored fungi and yeast

in the mosquito microbiome. Presented here is research on a Candida oleophila yeast

isolated from the An. gambiae midgut that shows significant inhibitory effects on the P.

falciparum parasite in vivo. We have assessed the inhibition of P. falciparum

development in vivo and in vitro, as well as the impact of the presence of this yeast on the

midgut bacterial flora and vector survival. This yeast isolate inhibits P. falciparum in a

pre-oocyst stage through a secreted factor; numbers of oocysts are significantly reduced

in those An. gambiae harboring yeast or supernatant filtrate in their midguts. An. gambiae

harboring yeast in their midgut experience expansions in midgut bacteria populations, but

no change in cohort survival was observed.

Primary Reader: Dr. George Dimopoulos

Secondary Reader: Dr. Clive Shiff

III

Acknowledgements

First and foremost, I have to thank Dr. George Dimopoulos, for accepting me

into his dynamic and exciting laboratory. His encouragement and guidance were crucial

in helping me grow as a research scientist. I also want to thank my thesis reader, Dr.

Clive Shiff for excellent suggestions both on my experiments and my thesis even when

time was limited.

My thanks go to my colleagues in the Dimopoulos laboratory: Sarah Short, Pike,

Nathan Dennison, Yang Chen, Raul Saraiva, Tui, Yesseinia Anglero-Rodriguez,

Seokyoung Kang, Yuemei Dong, Simone Sandiford, and Alicia Majeau. I especially want

to thank Ben Blumberg for expert teaching of mosquito research techniques and many

interesting discussions which enriched my study. Additionally, I must thank Godfree

Mlambo and Anne Jedlicka, for use of the P. berghei ookinete system and training on

Quantitative real time PCR, respectively.

Last but not least, I give a special thanks to my family. My parents George E.

Barringer, Jr. and Nancy Franzen Barringer have offered continual support through my

education, and I would not be where I am today without them.

IV

Table of Contents

Abstract………………………………………...……………………………………..…..II

Acknowledgements…………….…………………….…………………………………..III

Table of Contents ……………………………….……………………………………….IV

Table of Figures ...……………………………….………………………………………VI

Chapter1: Introduction………...………………………………………………………..1

History………………………………………………………………………………...1

Plasmodium Species and Distribution………………………………………………...1

Plasmodium falciparum Biology……………………………………………………...2

Anopheles gambiae Species Complex ………………………………………………..5

Antimalarial Drugs and Vaccines ………………………………………………….....6

Plasmodium falciparum Control Using Genetically Modified Organisms…………..10

Mosquito Immune System ………………………………………………………….11

Mosquito Microbiome ……………………………………….……………………..13

Chapter 2:Methods……………………………………………………………………. 18

Rearing Anopheles gambiae ………………………………………….……………..18

Yeast Isolates……………………………………………………………………...…18

Sequencing…………………………………………………………………………...18

Mosquito Feeding with Yeast and Supernatant……………………………………...19

Plasmodium Challenge……………………………………………………………....19

Quantification of Midgut Bacteria……………………………………………….…..20

In Vitro P. berghei Ookinete Inhibition Assay……………………………………....20

V

Longevity Assay……………………………………………………………………..21

Data Analysis……………………………………………………………………...…22

Chapter 3:Results…………………………………………….………………………... 23

Isolation and Sequencing of Candida………………………………………………..23

The Presence of C. oleophila in the Mosquito Midgut Inhibits P. falciparum Infection

……………………………………………………………………………………......23

Oocyst Development is Inhibited in the Mosquito Midgut by a C. oleophila -Secreted

Factor………………………………………………...………………........................25

C. oleophila Does Not Inhibit Plasmodium berghei Ookinete Development in vitro

………………………………………………………………………………………..27

Presence of C. oleophila Results in an Increased Bacterial Load of the Mosquito

Midgut………………………………………………………………..……….……..28

The Presence of C. oleophila in the Midgut Does Not Influence Mosquito

Longevity…………………………………………………………………………….29

Chapter 4: Discussion……………………………..…………………..………………..31

References …………………………...………...………………………………………..38

VI

List of Figures

Figure 1.Global Distribution of Clinical Malaria Cases ……………….…………………2

Figure 2. Plasmodium Falciparum Lifecycle ….……………………...………………….4

Figure 3. Fold Change of Plasmodium Parasites at Various Stages in the Mosquito ...…..5

Figure 4. The Range of Potential and Current Malaria Vectors Worldwide ……………..6

Figure 5. The Impact of C. oleophila Presence in the Midgut on P. falciparum Oocyst

Development in vivo ……………………………………..…………...….………...…....25

Figure 6. C. oleophila Culture Supernatant Filtrate Inhibits Development of Oocysts in

An. gambiae ………………………………………………………….…………..……...26

Figure 7. There is no significant difference in ookinete development as concentration of

C. oleophila spores increases ….…………………………………………..…………….27

Figure 8. The Presence of C. oleophila in the An. gambiae Midgut Increases the Bacteria

Load of the Midgut ……………..………………………………...……………………..29

Figure 9. There is No Significant Difference Between Control and Yeast Fed Mosquitoes

Survival …………………………………………...………………………………….….30

1

Introduction

History

Malaria is one of the oldest recorded diseases, with medical practitioners from

ancient China, Greece, and Roman Empire writing about the disease. The oldest recorded

mention of the disease is in an ancient Chinese medical text. The Greek medical

practitioner Hippocrates recording the symptoms of the disease in detail: fever,

splenomegaly, and anemia [1]. Hippocrates was the first to note the connection between

the symptoms of malaria and the proximity to swamps[1]. The Romans, as well,

appreciated the relation of malarial symptoms to swamps and their knowledge led to

large scale swamp draining that prevented malaria from becoming a massive impediment

to Roman citizens [2]. Although the connection to swamps and warm weather was known

for millennia, the causative agent of malaria was thought to be miasma from swamps.

The term malaria derives “mal aria” or bad air [2]. It was thought that the foul air from

marshy areas would cause illness in those that lived nearby. It was only in the late 1800s

that Plasmodium falciparum was determined to be an etiological agent of malaria [2].

Plasmodium Species and Distribution

Malaria is a disease caused by protozoan parasites of the genus Plasmodium.

Plasmodium species infect a wide variety of macroorganisms including rodents, birds,

and primates. There are five species of Plasmodium that infect humans. The species

vary in range and vector and are P. falciparum, Plasmodium vivax, Plasmodium ovale,

and Plasmodium malariae. Plasmodium knowlesi is a simian parasite, but is also known

to infect humans [3]. P. falciparum is the most deadly of these five species, and is

2

currently endemic in South and Central America, Africa, and Asia (Fig 1) [3]. Grey areas

represent areas of unstable transmission (Fig 1). The large geographic area which P.

falciparum affects and the relative poverty of most of those endemic regions make the

elimination of the disease difficult [3].

Figure 1. The global distribution of clinical episodes of Plasmodium falciparum malaria

in 2007 [4].

Plasmodium falciparum Biology

Plasmodium species are transmitted in a cycle between an avian, reptilian, or

mammalian host and a vector mosquito. Vector mosquitoes of Plasmodium are from

several different genera; P. falciparum is transmitted by mosquitoes of the genus

Anopheles [5]. Humans are infected with P. falciparum sporozoites that are injected into

the skin with saliva by a feeding female mosquito. Sporozoites make their way through

the skin via capillaries to the circulatory system. The sporozoites rapidly migrate to the

liver where they infect liver cells [6]. Inside a liver cell, a sporozoite will begin asexual

3

multiplication of merozoite stage parasites. The cell ruptures following multiplication and

the merozoites enter the blood stream and infect red blood cells. When a merozoite

invades a red blood cell, it multiplies inside the cell [6], [7]. The blood cell will rupture

and release merozoites into the blood stream. The characteristic pathology seen in P.

falciparum malaria is a result of blood stage parasites rupturing red blood cells and

adhesion of parasitized red blood cells to the vasculature [8].

While many merozoites go on to infect other red blood cells, a number of

parasites differentiate into sexual cells called gametocytes. Gametocytes do not cause

human pathology, and will remain in the blood stream until taken up by a mosquito or

killed by either human immune system or drugs [7]. When a mosquito takes a blood

meal from the infected host, gametocytes are taken up into the female mosquito’s midgut.

The change in temperature from the mammalian host to the mosquito causes

microgametocyte exflagellation [7], [8]. While in the midgut, the now mobile

microgametes and the macrogametes fuse on contact. The result is a highly mobile

parasite called an ookinete. The ookinete will bind the midgut epithelium and penetrate to

the basal side of the midgut. The ookinete develops into an oocyst that remains attached

to the basal side of the midgut for several days [7], [8]. During the time the oocyst is

attached to the midgut, sporozoites develop inside the oocyst before rupturing the oocyst.

The sporozoites then migrate through the mosquito hemocoel to the salivary gland, from

which they can infect another human. [7], [8]. The length of time from the initial

infection with P. falciparum gametocytes to the time sporozoites migrate to the salivary

gland is approximately 14 days [7], [9].

4

This study focused on the mosquito stages and specifically on the parasites

infection of the midgut tissue (fig. 2). Inhibiting the parasite in the mosquito would offer

a novel approach to controlling malaria. Approaches to inhibiting the parasite in the

mosquito will be described later in this introduction.

Figure 2. The complete lifecycle of Plasmodium falciparum. The mosquito stages are

represented on the left hand side of the diagram, while the human stages are shown on the

right [8].

The numbers of live P. falciparum vary as the parasite migrates through the

mosquito. As the gametocytes enter the food bolus in the midgut and mate, about there is

a significant reduction in the number of viable parasites (fig 3). In vitro culture studies

indicate an over 300-fold decrease in parasites from the gametocyte stage to the ookinete

stage [10]. As these ookinetes traverse the midgut, many will be eliminated. On average,

only 1 to 5 will survive and form oocysts in a natural infection [7], [10]. However,

5

throusands of sporozoites will develop in each oocyst and become released in the

hemocoel when those few oocysts burst [10].

Figure 3. Fold Change of Plasmodium Parasites at Various Stages in the Mosquito [10].

Anopheles gambiae Species Complex

The Anopheles gambiae species complex is a group of closely related vectors of

P. falciparum in found in sub-Saharan Africa. An. gambiae and closely related species

predominate in the humid regions of sub-Saharan Africa, breeding in a variety of habitats

including rice fields and irrigation channels. (Fig 1) [11]. Knowledge of the Anopheles

malaria vector geographic prevalence is important for malaria control programs so that

limited public health budgets can be directed to save the greatest number of lives [12],

Gametocytes

ookinetes

oocysts

Sporozoites

≈300x

reduction

≈70x

Reduction

Undetermined

Proliferation

6

[13]. Additionally, with the changes in global climate the geographic range of the An.

gambiae complex may change significantly [3], [13]. According to climatological

modeling, increased humidity in currently arid locations would allow these potent vectors

to extend their geographic range [13].

Figure 4. The range of potential and current malaria vectors worldwide. An. gambiae is

found in a large geographic area across the middle of Africa south of the Sahara Desert

[15].

Antimalarial Drugs and Plasmodium falciparum Vaccines

The number of deaths attributed to malaria is a current area of controversy, as

studies by Lozano and Murray contend that previous work based on WHO World Malaria

Report numbers underestimates the actual burden of malaria [16],[17]. The WHO

7

Annual Malaria Report indicated that an estimated 135-287 million clinical episodes of

malaria and 627,000 deaths worldwide in 2012, with P. falciparum being the most lethal

of the Plasmodium species that infect humans [18]. While deaths due to malaria have

decreased since 2004 due to improved malaria control, the disease remains a major cause

of mortality especially for African children under the age of 5 [18].

Current malaria control comprises three main strategies: environmental

management, anti-Plasmodium therapeutic and transmission blocking drugs, and use of

insecticides. Environmental management refers to human made changes to the natural

and built environment to reduce vector interactions with people [19]. These interventions

are potentially effective, but often require large expenditures due to redesigning

infrastructure. Drugs have the advantage of treating individuals at the time of illness, and

some drugs such as artemisinin can inhibit gametocytes, reducing or preventing

transmission [20]. Insecticides and insecticide treated bednets are both highly effective in

preventing individuals from coming into contact with infected mosquitoes [21].

Unfortunately, with few exceptions resistance has developed to drugs as well as

insecticides [21]. Thus far, the most successful vaccine for P. falciparum has

approximately 25% efficacy.

Environmental management in the context of malaria refers to modifying the

environment to reduce the transmission of the disease. Various methods can be used

depending on the circumstances, including the swamp draining done by the Romans,

drains to avoid standing water, and keeping canals clear so that the water flows too fast

for mosquitoes to lay eggs in it [22]. These modifications have been shown to be

effective in reducing malaria incidence, but have distinct disadvantages. The large scale

8

environmental modification is costly and labor intense [19]. High initial expenses may be

difficult for some poorer countries to sustain [19]. Perhaps because of cost or because of

the successes of drugs including chloroquine and artemisinin, environmental management

has not been widely used for reducing transmission since the early 20th

century [22].

Chloroquine was used heavily after it was developed in 1934 by Bayer scientists,

and was distributed for clinical use in 1947 [23]. This drug was extremely effective in

curing P. falciparum malaria, but P. falciparum evolved resistance to the drug by the

1950s [23]. By the early 1990s, all sub-Saharan African countries had reported that there

was significant chloroquine resistance in local P. falciparum strains. In Malawi, for

example, treatment with chloroquine had a roughly 50% failure rate by 1993 [24]. The

high failure rate reduced the use of this drug, and the availability of other drugs reduced

usage of chloroquine greatly in most African countries by the mid-2000s [24].

The current WHO recommended therapy for P. falciparum malaria is artemisinin

combination therapy (ACT) [25]. Artemisinin is a compound that was isolated from the

traditional Chinese medicinal herb, Artemisia annua. This compound is well tolerated

and rapidly clears the malaria parasite from infected individuals [20]. It has been

determined that artemisinin acts on asexual stages of the P. falciparum parasite, and as a

result, it is useful in reducing the chance of clinical complications due to blood stage

parasites binding to the vascular epithelium [20]. Not only does arteminsinin reduce

symptoms by killing the asexual stages of the parasite, it also kills the gametocyte stage

of P. falciparum that is taken up by mosquitoes [20]. This is very important because this

drug can decrease the transmission of P. falciparum if there is sufficient distribution of

the drug to endemic areas [26]. Alone, artemisinin and its derivatives require a seven day

9

course for adequate treatment, and monotherapy has been discouraged by the WHO due

to the potential for development of resistance [27]. ACT combines an artemisinin

derivative with a drug such as lumefantrine, mefloquine, or sulfadoxine/pyrimethamine

that is slowly eliminated from the body [27]. The ACT treatment requires a three day

course, increasing compliance with the drug regimen compared with a seven day regimen

[20], [27].

While current treatments are effective in combatting malaria when used properly,

the current high rates of P. falciparum transmission and drug resistance to many drugs

such as chloroquine indicates that drugs alone will not be sufficient to meet targets of

malaria control [24]. Ideally, a vaccine could be developed to protect individuals from P.

falciparum malaria. Glaxo-Smith Kline and Walter Reed Army Institute of Research

have developed a vaccine known as RTS,S that is a promising tool to protect against

malaria. RTS,S contains circumsporozoite proteins from the sporozoite stage combined

with Hepatitis B virus surface antigens and a chemical adjuvant [28]. The makers intend

the vaccine to induce a strong immune response to the sporozoite stage with the goal of

preventing sporozoite liver infection [28]. The Phase III trials of this vaccine showed

25%-30% efficacy in infants in its most recent trial [28]. This vaccine can greatly reduce

the infection among children under 5, the age group suffering the greatest malaria

mortality [28].

While several classes of insecticide have been used to reduce An .gambiae

populations, pyrethroids are the most commonly used insecticides on insecticide treated

bednets [29]. Pyrethroids are attractive for malaria control inside homes because this

class of insecticide has a very low toxicity to mammals [29]. Since An. gambiae seeks

10

hosts for blood feeding during the night, the bednet reduces the likelihood of a mosquito

reaching a host and thereby reduces the transmission of malaria [30]. Coating the bednet

with insecticide has the additional benefit of killing those mosquitoes that come into

contact with the net. Unfortunately, mosquitoes have developed resistance to pyrethroids

in locations where they are heavily or indiscriminately used [31]. This insecticide

resistance jeopardizes future malaria control efforts using pyrethroid treated bednets [32],

[33].

Plasmodium falciparum Control in the Mosquito using Genetically Modified

Organisms

Thus far, there has not been a single control strategy that focuses on reducing the

burden of Plasmodium in the mosquito host deployed to the field. Within the mosquito,

the Plasmodium parasite has several vulnerabilities that can be exploited. One important

target for intervention is the bottleneck at the pre-oocyst stage of development where

ookinetes are traversing the midgut [7]. Parasite numbers decrease dramatically by the

oocyst stage; since there are so few oocysts compared to the other stages, there will be a

better chance of blocking infection at the early oocyst stage [7].

Examples of strategies to block Plasmodium infection of the mosquito are:

genetic modification of the mosquito itself to express anti-Plasmodium effector

molecules; genetic modification of a microbial species that will be introduced to the

mosquito, known as paratransgenesis; and introduction of an unmodified microbial

species to the midgut that inhibits Plasmodium development.

11

Research groups such as Jacobs- Lorena lab have developed both genetic and

paratransgenic tools to inhibit P. falciparum in the mosquito. A promising genetic

strategy developed by the Jacob-Lorena lab modifies the mosquito to produce the

Salivary and Midgut Peptide 1 (SM1). SM1 produced by the mosquito inhibits P.

falciparum by blocking a receptor in the midgut believed to be responsible for ookinete

binding, preventing ookinetes from traversing the midgut [34]. Other approaches

genetically modify microorganisms that live within the mosquito. These organisms could

secrete anti-Plasmodium factors to inhibit the parasite as it makes its way through the

mosquito. Two examples being researched by the Jacobs-Lorena lab are the natural

peptide Scorpine and the synthetic peptide Shiva-1 [35]. Both molecules show robust

killing of the parasite in vivo, and genes for the two peptides could be transformed into

microbes of the mosquito midgut to kill the malaria parasite [35]. These approaches are

promising, but not ready for field applications.

Mosquito Immune System

The immune system of An. gambiae is highly effective in reducing P. falciparum

numbers, and approaches to control P. falciparum should leverage the mosquito’s

immune response to enhance parasite killing. There are two broader types of mosquito

immune response; the first is a humoral response that activates production of small

peptides and other effector molecules. The second is cell-mediated, and is mediated by

the mosquito hemocytes. One well studied humoral response is the production of the

complement-like factor TEP-1, which is important in anti-Plasmodium defense [37].

TEP1 is a thioester containing protein that participates in the process of melanizing

12

certain Plasmodium species, including Plasmodium berghei and P. falciparum [36],

[37],[38]. An. gambiae strains refractory to Plasmodium have distinct TEP1 alleles

compared to susceptible strains, indicating that melanization is important in Plasmodium

control and lysis [37]. The cellular response is also important in the control of P.

falciparum. There are several types of hemocytes within the hemocoel, with the

phagocytosic granulocyte being the most common hemocyte cell type [38]. These

granulocytes can phagocytose harmful microorganisms in the circulatory system and

destroy them [38]. Not only do hemocytes function to phagocytose and lyse pathogens,

they also are believed to be responsible for immune priming in the mosquito [37] [39],

[40], [41]. Immune priming is a memory-like mechanism that increases killing of specific

pathogens including P. falciparum upon a second challenge. Whether humoral or cellular,

immunological control is mediated through four immune signaling pathways.

There are four main immune pathways that have been characterized in

Drosophila that also exist in Anopheles: Toll, IMD, JAK-STAT and JNK [42]. The Toll

pathway directs responses to fungal pathogens in addition to inhibiting gram positive

bacteria [43], [44]. JNK signaling has been demonstrated to induce molecules involved in

nitration of epithelial surfaces [45]. Nitration modifies that parasite and stimulates lysis

of the parasite by complement-like factors [45]. The 7 members of the STAT

transcription factor family regulate the production of Nitric Oxide Synthetase (NOS)

following STAT activation by a Janus Kinase (JAK) [46]. Gupta and colleagues

determined that the JAK-STAT pathway is important in defense against P. falciparum

infection by reducing oocyst survival [46]. The IMD pathway is canonically responsible

for defense against gram negative bacteria and P. falciparum in the midgut [40], [42].

13

In the mosquito, the IMD pathway functions to control the microbial load of the

mosquito midgut [42]. Components of the IMD pathway sense components of gram

negative bacteria and act through an NF-κB like transcription factor to modulate immune

responses [42]. When the mosquito takes a bloodmeal, midgut bacteria proliferate and the

IMD pathway is activated to control this bacterial population through anti-bacterial

effectors [42]. It may be that Plasmodium defense is a byproduct of maintaining the

midgut homeostasis [42]. If Plasmodium is present, effectors of the IMD pathway can act

against the pre-oocyst stages of P. falciparum [42]. Notable IMD regulated components

include LRRD7, FBN9, and the aforementioned TEP1 [37]. RNA silencing assays show

that knockdown of these components reduces Plasmodium inhibition in the mosquito

[37]. In addition to IMD effectors, Plasmodium infection is also inhibited by the

mosquito’s natural microbiome.

Mosquito Microbiome

Mosquitoes, like other insects, have a diverse microbial flora. There are high

concentrations of microbes in the mosquito midgut, with bacteria being majority of the

described organisms [47]. Just as the microbiome of higher organisms is located through

diverse organ systems and tissues, the insect microbiome is located across multiple

compartments [47].

Wolbachia pipientis is a commensal microorganism common in insects, including

several mosquito species [48]. Interestingly, naturally occurring strains of W. pipientis

can increase resistance to various insect borne pathogens such as West Nile virus [49].

While Anopheline mosquitoes are not naturally infected by Wolbachia, Bian and

14

colleagues reported the introduction of a stable Wolbachia infection in the malaria

mosquito An. stephensi, conferring resistance to P. falciparum [50]. Attempts to infect

An. gambiae with Wolbachia have developed transient somatic infections, but not stable

infections [51].

The natural midgut bacterial flora of An. gambiae have been demonstrated to

have a protective role against Plasmodium infections. Dong and colleagues determined

that mosquitoes cleared of bacteria with antibiotics are more permissive to Plasmodium

infection than those with an intact microbiome [52]. Average gut colonization of an An.

gambiae mosquito ranges from 104 to 10

5 bacteria. The Dimopoulos group have studied

the bacteria of the An. gambiae microbiome and found significant variation in the

quantity and species found in the midgut of mosquitoes deriving from the generation and

colony [52]. Nonetheless, certain species are common in an An. gambiae population.

Several of these bacteria could indirectly inhibit P. falciparum by activating the

mosquito IMD response as previously described [42]. Tchioffo and colleagues

determined that the magnitude of Plasmodium inhibition of certain bacterial isolates is

greater than other common isolates [53]. Escherichia coli, Serratia marcescens and

Pseudomonas stutzeri strains were noted to induce a marked reduction in infection

intensity compared with other isolates studied by Tchioffo [53]. The enhanced reduction

in infection intensity may be due to certain species being better adapted to colonizing the

midgut [53].

Although several bacteria inhibit P. falciparum through activation of the IMD

pathway, a strain of Enterobacter is known to actively inhibit P. falciparum [54]. This

15

strain of Enterobacter has been determined to directly inhibit Plasmodium through

production of reactive oxygen species. These reactive oxygen molecules inhibit

development of P. falciparum in the midgut [54]. In vitro culture of gametocytes with

this Enterobacter reduce development to the ookinete stage by approximately 90% [54].

While bacteria have been well studied, other organisms such as fungi and yeast are also

present within the midgut.

Many studies have focused on bacteria associated with insects, but a number of

studies suggest that there may be a large yeast and fungal component of the An. gambiae

microbiome. Drosophila, and other plant-associated insects such as leafhoppers have well

documented interactions with Candida [55], [56], [57], [58]. Some of these interactions

may be only transient, but studies have suggested yeasts may also be permanent

components of the respective insects’ microbiomes through interactions with flowers they

feed upon [58], [59], [60]. Research in Drosophila by Quintin and colleagues determined

that Candida glabrata can persistently infect Drosophila despite Toll immune pathway

activation, and it may be that other Candida can similarly avoid clearance by the immune

system of insects [61]. Other studies have explored fungi as a potential biocontrol agent.

Beauveria bassiana is primarily known as an entomopathogenic bio-control

agent. B. bassiana is frequently encountered by An. gambiae through contact with the

cuticle of the mosquito. When the fungi settle on the cuticle of a mosquito, they will

pierce the cuticle and grow inside the mosquito [62]. Following the death of the insect, B.

bassiana spores will be released into the environment from fungal outgrowth on the dead

mosquito [62].

16

This fungus shortens the lifespan of infected Anopheles mosquitoes; Blanford

exposed mosquitoes to B. bassiana after providing them a malaria infected bloodmeal,

and determined that B. bassiana significantly increases daily mortality in adult

mosquitoes over 10 days old. In a cohort of mosquitoes fed a malaria infected blood

meal and exposed to B. bassiana, mortality was 65 times higher than a control malaria

infected cohort at 11 days post infection [63]. Considering that a mosquito infected with

P. falciparum is only infectious to humans after 14 days post malaria infection, this

increased mortality reduces the number of mosquitoes that will survive long enough to

become infectious [7]. B. bassiana is lethal both to larval and adult An. gambiae, and can

be distributed in an oil-based solution for even distribution [64], [65]. Interestingly,

mosquitoes resistant to pyrethroid insecticides are more susceptible to B. bassiana [66].

Thus, B. bassiana could be used in place of insecticides, such as pyrethroids, in locations

where control is failing. While B. bassiana is a potent mosquito control tool, it must be

used carefully, because this fungus does not only infect mosquitoes, but has also a variety

of beneficial insects including honey bees and lady beetles [67], [68].

This paper will describe a yeast isolated out of Anopheles gambiae that has an

inhibitory effect on Plasmodium falciparum. The yeast is closely related to Candida

oleophila (also known as Candida rignihuensis) and Candida railenensis (also known as

Apiotrichum osvaldii) [69]. These two yeasts are closely related, and one study by Isaeva

posits that they represent varieties of the same anamorphic species with adaptations to

different environments [69].

Candida railenensis was isolated in the 1980s from decayed wood and dipterans,

and later from the acorns of the English Oak [69], [70], [71]. Considering the variety of

17

locations this yeast and related Candida has been found, including Russia and Chile, it is

likely to be ubiquitous across Europe and South America [60], [70], [71], [72]. If the

yeast is prevalent in an An. gambiae endemic region, it is likely that the mosquito would

have been exposed by landing on spore covered vegetation or by feeding on

contaminated plant nectar [73].

C. oleophila has been used as an agricultural biocontrol agent, to protect fruit

from harmful Penicillium species. The yeast has activity against fungi, such as

Penicillium digitatum, Penicillium italicum, and Geotrichum candidum that can infect

wounds on fruit [74]. C. oleophila is known to protect both the fruit as well as the tree

against post-harvest diseases [74]. One of the most studied strains is C. oleophila

Montrocher, used in the commercial pesticide Aspire [75]. C. oleophila produces lytic

enzymes which are highly effective at damaging Penicillium molds; degrading the

structural integrity of the fungal cell wall [74], [75]. Several examples of yeast from

different genera including C. oleophila are known to produce Exo-β-1, 3-glucanase and

chitinase [75], [76]. Other studies have described C. oleophila degrading phenolic

compounds and azo dyes found in wastewater [77], [78]. The variety of enzymes

secreted by C. oleophila indicates that this yeast can degrade a wide variety of organic

compounds. If our isolated species has similar enzymatic activity, it would be interesting

to determine whether it degrades protozoal or bacterial cell components.

18

Methods

Rearing Anopheles gambiae Mosquitoes

An. gambiae, Keele strain mosquitoes were raised and maintained in the Johns

Hopkins Bloomberg School of Public Health Insect Core Facility. Mosquitoes were kept

in temperature controlled chambers at 27°C and 70% humidity and light cycles inside the

chamber were set to 12 hours light/dark. Mosquito feeding was accomplished with a 10%

sucrose solution on sterile cotton. If required by the experiment, mosquitoes were

transferred to small sterile cups and covered with sterile netting using an aspirator.

Yeast Isolates

Midguts were dissected from twelve newly emerged female mosquitoes using

sterile technique: Prior to dissection, mosquitoes were placed in 100% ethanol for 10

seconds, and rinsed in two consecutive 1x Dulbecco’s PBS baths for a total of 10

seconds. All midgut dissections were done in 1x PBS, using forceps sterilized in 100%

ethanol. Dissected midguts were homogenized in 100µL 1x PBS. The homogenized

midgut contents were plated on potato glucose agar with 1µg/mL of amphotericin B

(Gibco, Grand Island, New York, USA) to select for yeasts and fungi. Potential yeasts

were visualized under bright field microscopy to determine their identity. Yeast colonies

were replated to derive pure cultures.

Sequencing

Once the yeast was grown in pure culture, yeast DNA was isolated using

chloramphenicol [79]. Isolated DNA was amplified in a Dyad Disciple PCR system

19

using 26S internal transcribed space sequence primers [80]. Aliquots of the isolated DNA

were sequenced by the JHMI Sequencing Core facility. Finch TV software (Geospiza,

Seattle, WA, USA) was used to view raw sequencing data and insure sequencing data

was not contaminated. DNA sequences were aligned with the NCBI BLAST tool.

Mosquito Feeding with Yeast and Supernatant

Yeast was grown on potato dextrose agar to develop a pure culture. Once a pure

culture was established, yeast were grown in potato dextrose broth or glucose-peptone

broth at 30°C for approximately two days before use. Before use, the yeast was spun in a

Sorvall Legend RT centrifuge (Thermo Scientific, Waltham, MA, USA) at 2000X for 10

minutes. In live yeast experiments, the supernatant is removed and the yeast washed at

2000X for 10 minutes in sterile PBS. The PBS was then removed and the yeast was

resuspended in 1mL of 10% sucrose, and fed to Anopheles gambiae on a sterile cotton

pad.

During supernatant feeding experiments, yeast was grown for three days in potato

dextrose broth, and 1.5 mL of the supernatant was passed through a 0.2 µm filter. The

resultant filtrate was then mixed with 10% sucrose solution and fed to mosquitoes 3-4

days old at initiation of the experiment on a sterile cotton pad. On day 2 of the

experiment, fresh sucrose-supernatant mixture was provided.

Plasmodium Challenge

The morning of infection with P. falciparum, female mosquitoes that had

undergone yeast or supernatant feeding for 2 days were fed on membrane feeders for 30

minutes with NF54W strain gametocytes at 37°C. The gametocyte culture was

20

centrifuged and mixed with red blood cells and serum to the required dilution. Within the

first day after feeding, mosquitoes were anesthetized and unfed mosquitoes removed.

Engorged mosquitoes were maintained to 8 days post infection. At 8 days post infection,

engorged mosquitoes were dissected and their midguts stained with 0.1%

mercurochrome. Oocyst numbers were ascertained using a light microscope (Olympus,

Center Valley, PA, USA), and the median was calculated.

Quantification of Midgut Bacteria

Quantification of midgut bacteria was assayed by the colony forming unit assay.

Mosquitoes were dissected using sterile technique, and their midguts homogenized. Serial

dilutions of midgut homogenate were carried out in sterile PBS and 75 µl of each dilution

were plated on LB media. Ten-fold dilutions were carried out to a final dilution of 1x10-7

.

The agar plates were incubated at 20°C for three days prior to counting. When assessing

which dilution to count, the dilution closest to the 20-250 colony forming unit range was

chosen. Plate counts were converted to total numbers of bacteria per midgut and log10

transformed using Microsoft Excel.

In- vitro P. berghei ookinete inhibition assay

To test the inhibition of ookinete development by C. oleophila, yeast were co-

cultured with P. berghei gametocytes in an in vitro system. Six to eight week old female

Swiss-Webster mice were infected with a transgenic P. berghei strain (PbwRL)

expressing a Renilla luciferase controlled by the WARP promoter, an ookinete specific

promoter [81]. The supernatant from C. oleophila was removed, and the yeast used in the

experiment were washed twice in sterile PBS for ten minutes at 2000 rpm. Yeast was

21

prepared at concentrations of 1x105, 1x10

7, and 1x10

9 yeast cells per 50 µL of sterile

PBS. Ookinetes were incubated at 19 C for 26-28 hours in culture with yeast solutions at

either 1x105, 1x107, or 1x109 yeast per 50 µL. The resultant culture was transferred to a

1.5 mL Eppendorf and spun at 1800 r.p.m. for 4 minutes [81]. The supernatant was

discarded following centrifugation [81]. The Renilla luciferase system (E2810, Promega)

was used to perform the luciferase assay, following the manufacturer’s instructions (G.

Mlambo and G. Dimopoulos, unpublished) [81]. To summarize, 100μl of 1X lysis buffer

was added the sample and vortexed gently [81]. The solution was incubated at 20°C for

15 min. The lysate was centrifuged at 14000 r.p.m. for 2 min, and the supernatant was

transferred to a 1.5 ml Eppendorf tube and placed on ice [81]. A total of 20 µL of

supernatant was mixed by pipette into 100μl of luciferase assay buffer [81]. The resultant

solution was immediately read on a 20/20n Luminometer (Turner Biosystems,

Sunnyvale, CA, USA) over a 10 sec integration period [81]. During each experiment,

three technical replicates were performed and the output rendered in arbitrary

luminescence units. Four biological replicates were performed.

Longevity Assay

Three to four day old female mosquitoes were sorted into two groups of at least

40, and fed either on a control 10% sucrose solution or on live yeast in 10% sucrose

solution prepared as described. Cohorts were kept separate in small, sterile wax-coated

cups in conditions as seen above in Rearing Mosquitoes section of the methods. The

second day, fresh yeast was provided to the test group. On the third day and afterward,

the mosquitoes were maintained on 10% sucrose solution. Counts of dead mosquitoes

22

were made each day at a fixed time, until all mosquitoes died. Six biological replicates

were performed.

Data analysis

All data analysis was done with Graphpad Prism 5 software (GraphPad Software,

La Jolla, CA, USA)

23

Results

Isolation and Sequencing of C. oleophila

A previous ScM student of the Dimopoulos group had isolated a yeast from the

Insect Core Facility An. gambiae colony. We determined that the same yeast is still

present in the Insect Core’s An. gambiae population through sequencing. The ribosomal

26S internal transcribed space sequence of yeasts is a region removed prior to the

production of mature rRNA in the yeast. Internally transcribed spacers are frequently

used in identification of fungal and yeast species, and we were able to amplify this region

and determine its nucleotide sequence identity [82]. No reference sequence completely

matched the amplified 26S sequence in NCBI database. BLAST sequence similarity

analysis indicate this yeast is closely related to Candida oleophila and Candida

railenensis, two yeasts which literature indicates are members of the same anamorphous

yeast species [69]. Based on BLAST results, we are considering this yeast a strain of C.

oleophila.

The Presence of C. oleophila in the Mosquito Midgut Inhibits P. falciparum

Infection.

To test whether our Candida isolate could influence An. gambiae susceptibility to

P. falciparum infection, we first introduced it into the mosquito midgut through sucrose

feeding and then fed the mosquitoes on a parasite gametocyte culture. Mosquitoes were

fed approximately 1x 109 live C. oleophila spores in a 10% sucrose solution for two

days, while control mosquitoes received a 10% sucrose solution devoid of yeast spores.

On the third day mosquitoes were fed on a P. falciparum gametocyte culture and

24

infection intensity and prevalence was determined 7 days later by counting oocyst on

dissected midguts. Those mosquitoes that harbored C. oleophila displayed a significant

reduction in the intensity of P. falciparum infection. The control group showed a median

infection intensity of 13 oocysts, while the yeast fed group had a median of 9 oocysts on

the midgut (Figure 5). The control group displayed a much greater range of oocysts on

the midgut than the yeast fed groups: the oocyst range of the control was 0-50 oocysts,

while the yeast group had a range of 0-34 oocysts per midgut. This indicates C. oleophila

inhibits a pre-oocyst stage of P. falciparum in the mosquito midgut.

Not only did the presence of the yeast reduce the quantity of oocysts in those

midguts that were infected, but also reduced infection prevalence. The control group had

a prevalence of approximately 97% which was reduced down to approximately 91% in

the Candida-fed group. We then wanted to determine whether the Plasmodium life cycle

was inhibited by some factor secreted by the yeast, or whether the yeast spore had to be

present for inhibition.

25

contr

ol

C. O

leophila

0

20

40

60

oo

cysts

/mid

gu

t

Figure 5. The impact of C. oleophila presence in the midgut on P. falciparum oocyst

development in vivo. Horizontal lines indicate the median infection intensity. A Mann-

Whitney test was used to determine statistically significant difference with a P value of

0.0076.

Oocyst Development is Inhibited in the Mosquito Midgut by a C. oleophila -Secreted

Factor

Once the C. oleophila was determined to inhibit the development of oocysts in the

midgut, we wanted to test whether inhibition depended on the presence of whole yeast

spores in the midgut or a yeast secreted factor. Literature indicates that C. oleophila

secretes multiple enzymes to degrade organic compounds in its environment [74], [77],

[78]. By filtering a yeast culture supernatant through a 0.2 micron filter, we could ensure

that the resultant filtrate would not contain whole yeast cells, but only yeast secreted

factors. After feeding mosquitoes for two days on the culture supernatant filtrate in a 10%

**

26

sucrose solution, we infected them with P. falciparum as described above and found

significantly reduced oocyst numbers compared to the control group that received a 10%

sucrose solution without the yeast supernatant filtrate. The range of oocysts on the midgut

was also reduced in those mosquitoes that had received filtered yeast supernatant. The

mosquitoes that had fed on the yeast culture filtrate contained a range of 0 to 21 oocysts

per midgut, while the control group contained 0 to 48 oocysts per midgut. The median

number of oocysts per midgut was 10 and 4 in the control and experimental groups,

respectively. The prevalence was also reduced from 89% to 83% in the yeast culture

filtrate fed group. This data suggests that some yeast-secreted factor is produced by the

yeast that inhibits the development of the P. falciparum.

contr

ol

C. O

leophila

0

20

40

60

oo

cysts

/mid

gu

t

Figure 6. C. oleophila culture supernatant filtrate inhibits development of oocysts in An.

gambiae. Horizontal lines indicate the median oocyst infection intensity. The asterisks

indicate statistical significance at a p value of 0.0035 when using the Mann-Whitney test.

**

27

C. oleophila Does Not Inhibit Plasmodium berghei Ookinete Development in vitro.

C. oleophila inhibits P. falciparum development in vivo, but the specific stage of the

parasite that was inhibited was unknown. To determine which stage of Plasmodium the

yeast acted on, we utilized an in vitro P. berghei ookinete culture assay in conjunction

with yeast exposure. Exposure of the ookinete culture to different concentrations of live

yeast would indicate whether the yeast would inhibit the development of a pre-ookinete

stage. Following three trials it was concluded that there is no significant difference in

development of ookinetes between groups exposed to sterile PBS and those exposed to

yeast. In each concentration of 105, 10

7 and 10

9 C. oleophila per 50 microliter ookinete

culture there was no significant difference between the treated and control groups.

Exposure of the ookinete culture to a 105 C. oleophila per 50 microliter ookinete culture

resulted in an increased development of ookinetes, compared to the control (fig 7).

ctrl

10^5

10^7

10^9

0.0

500000.0

1000000.0

1500000.0

Spores/50L

Flo

rescen

ce U

nit

s

Figure 7. There is no significant difference in ookinete development as concentration

of C. oleophila spores increases. There was no significant difference between groups

with a P value of 0.3916 according to the Kruskal-Wallace test.

28

Presence of C. oleophila Results in an Increased Bacterial Load of the Mosquito

Midgut

We were interested in assaying the influence of the yeast on the bacteria of the

mosquito midgut. The colony forming unit assay measures the number of live aerobic

bacteria present in a sample. In this experiment, we plated the contents of homogenized

midguts, from either yeast fed or control mosquitoes, on LB agar petri dish plates and

counted bacterial colonies on the plates after 3 days at room temperature. Mosquitoes fed

with Candida yeast show an increased aerobic bacterial load compared to the control

mosquitoes that had fed on a 10% sucrose solution. The log10 transformed median

number of bacteria per midgut in the control group was 3.5 whereas the log10 median

number of bacteria per midgut of the yeast-fed group was 4.6. Additionally, the

phenotypes of the bacteria plated from yeast harboring midguts suggests that the presence

of this yeast in the midgut may alter the prevalence of several bacterial species within the

midgut. The identity of those species has not yet been determined.

29

Ingesting C. Oleophila increasesthe number of midgut bacteria

Contr

ol

C. O

leophila

0

2

4

6

Lo

g1

0 C

FU

per

Mid

gu

t

Figure 8. The presence of C. oleophila in the An. gambiae midgut increases the

bacteria load of the midgut. Asterisks indicate statistical significance of P<0.01. The

two groups differ with a value of P =0.0081 according to the Mann-Whitney test.

The Presence of C. oleophila in the Midgut Does Not Influence Mosquito Longevity

We hypothesized that the increased bacterial load of the yeast-containing midgut

would result in an immune activation that could be costly to the mosquito and therefore

result in decreased longevity. The longevity assay is commonly used to assess the effects

of an experimental treatment on the mosquito lifespan. Groups of 40 mosquitoes were

fed on either a 10% sucrose solution or a 10% sucrose solution containing 1x109 C.

oleophila spores for two days. After two days, the mosquitoes were maintained on a 10%

sucrose solution and dead mosquitoes were counted at the end of each day. Throughout

the course of the longevity assay, mosquitoes of the two groups died at approximately the

same rate (fig 9).

**

30

0 5 10 150

20

40

60

80

100 control

yeast

Day

Perc

en

t su

rviv

al

Figure 9. There is no significant difference between control and yeast fed mosquitoes

survival. The Gehan-Breslow-Wilcoxen test determined a P value of 0.2483. Median

survival in the yeast fed group was 11 days compared to 10 days in the control group.

Discussion

31

This study has successfully isolated a yeast likely to be a subspecies of C. oleophila

from the midgut of the P. falciparum vector An. gambiae. C. oleophila is a biocontrol

organism used in agriculture to protect crops from Penicilium molds [74]. It is frequently

used post-harvest to reduce infection by P. digitatum among other fungi [74]. Our C.

oleophila isolate displays interesting properties in the malaria mosquito An. gambiae

when introduced through the oral route.

Because this yeast was originally isolated from the midgut of An. gambiae, we

wanted to assess its effect on the lifecycle of the mosquito and the malaria parasite P.

falciparum. We used a natural ingestion route in our assays; mosquitoes in the field are

most likely to encounter yeast through feeding on contaminated nectar sources [73].

When fed to An. gambiae two days prior to Plasmodium infection, the yeast significantly

inhibits P. falciparum oocyst development in the midgut. Both the intensity and

prevalence of oocysts were reduced in yeast fed mosquitoes compared to the control.

However, the yeast does not inhibit in vitro P. berghei ookinete development

significantly, suggesting that C. oleophila does not prevent development from the

gametocyte to ookinete. Inhibition most likely occurs at the pre-oocyst stage, either as the

ookinete binds to the midgut epithelium or as it traverses the midgut. Alternatively, the

yeast may induce a mosquito response that acts against the parasite [53], [54]. Another

possibility is that the presence of yeast induces an altered midgut bacterial microflora that

would reduce Plasmodium infection [53], [54].

The effects of this yeast in vivo are especially interesting when considering the

yeast’s potential negative effects on the ookinete and early oocysts. This Candida may be

secreting various glucanases and proteases that could degrade ookinete surface proteins.

32

The yeast in this study is related to C. oleophila, which is known to secrete a variety of

enzymes including proteases and glucanases and also produces enzymes that degrade

Azo dyes and phenolic compounds [74], [75], [76], [77], [78]. If the Candida species

produces proteases while in the midgut, those secreted enzymes may interact with the

ookinete and prevent the ookinete from continuing on to the oocyst stage. If ookinete

surface proteins are cleaved, an ookinete may not be able to bind to the midgut in order to

carry out the process of forming an oocyst. Alternatively, yeast could prevent the

ookinetes from reaching the midgut epithelium through steric hindrance.

In order to better understand the interactions of the yeast with An. gambiae,

quantification of C. oleophila through real time PCR using the 26S Internal Transcribed

Spacer sequence could be used whether it expands following ingestion or whether it is

cleared from the midgut [80]. An anti-C. oleophila antibody could give insight into the

interactions of the yeast within the midgut and malaria parasite through co-localization.

However, our assays with yeast culture supernatant filtrates suggest that a co-localization

is not necessary for Plasmodium inhibition.

Mosquito midguts harboring the Candida yeast show a significant increase in their

bacterial load compared to the controls. In vitro yeast and bacterial co-culture

experiments could provide insights on whether the yeast provides a bacterial growth

advantage by providing nutrients, or whether the increased bacterial load is because of a

mosquito-mediated process [37]. It has been shown that P. falciparum inhibition by

bacteria is dose-dependent, so if bacterial populations expand following introduction of

C. oleophila, this could be sufficient to inhibit P. falciparum [81]. If the mosquito is host

to bacteria that produce anti-parasitic compounds, the presence of the yeast may enhance

33

the effect of those organisms, thereby resulting in Plasmodium inhibition [54]. The

results suggest a bacteria-mediated mechanism of action is likely.

If the yeast-mediated bacterial expansion is proven to be responsible, the specific

bacterial species that proliferate in the presence of the yeast should be explored. This

could be performed through a metagenomic analysis of C. oleophila containing and

control mosquito midguts. Quantitative real-time PCR with primers specific to a given

bacterial species could also be used to determine whether any of the species in the midgut

are expanding [84].

The role of mosquito-associated fungi and yeast has to date not been thoroughly

studied. The Favia lab has studied the yeast Wickerhamomyces anomalus (formerly

Pichia anomala) in the mosquito Anopheles stephensi, and its potential for

paratransgenesis. Favia’s study could direct the future study of C. oleophila, since W.

anomalus is found in several compartments of the mosquito including the female

reproductive tract, suggesting that it may be useful to investigate whether Candida

species is present in the Malpighian tubules, ovaries, as well as the hemolymph [80].

Our study has shown the possibility of this yeast as a biocontrol agent, and more work

must be done to determine the nature of its interaction with Anopheles and Plasmodium.

The Johns Hopkins An. gambiae strain that has been kept in the laboratory

environment for hundreds of generations, and we can therefore not be certain whether its

interaction with C. oleophila reflects field mosquito-yeast interactions. The study of yeast

in natural field An. gambiae should therefore provide a better understanding of how the

yeast interacts with the mosquito. The magnitude of C. oleophila mediated P. falciparum

34

inhibition may differ in mosquitoes exposed to a natural environment as opposed to a

controlled laboratory environment. One could speculate that the inhibition may vary

between different An. gambiae strains due to genetic variations in immune pathways, as

well as differences of their microbiomes [85], [86]. However, our study represents an

important first step towards a better understanding of yeast mosquito interactions, and

how these can influence vector competence. Our C. oleophila strain inhibits P.

falciparum in vivo and alters the bacteria load of the midgut. Isolation and

characterization of C. oleophila strains from wild mosquitoes would provide insight into

the anti-Plasmodium properties of different strains of this yeast.

It is still unknown how the C. oleophila strain isolated in this study was

introduced into the mosquitoes of the insectary. While this particular yeast species has

not been reported in wild An. gambiae, it has been identified at locations in both South

America and Eurasia [69], [72]. It is possible that this yeast is a ubiquitous member of the

Anopheles microbiome across Africa, and that it influences Plasmodium transmission to

some degree. This is also true for a variety of other microorganisms that have been

shown in the laboratory to have an inhibitory effect on P. falciparum [52], [53], [54],

[81].

Most studies concerning malaria control using fungi have concentrated on

entomopathogenic properties but not in any great detail on Plasmodium inhibitory

properties [63], [64], [65], [66]. The role of fungi and yeast as commensal organisms

through the life span of the mosquito has not been explored in detail, but should be

investigated thoroughly to better understand the microbiome. Fungi are ubiquitous in the

environment where mosquitoes are present, and non-entomopathogenic fungi may come

35

into contact with mosquitoes through various routes [60]. Even if these organisms may

only interact transiently with the mosquito, it is still important to investigate whether they

cause mosquito immune modulation.

Further studies are also needed to better understand the anti-Plasmodium activity of

C. oleophila isolate in the context of vector competence. In this study, mosquitoes were

infected with P. falciparum two days after they had fed on the yeast, and it would be

interesting to determine how long this anti-Plasmodium effect lasts after the mosquito

ingests the Candida spores. The life span of an An. gambiae mosquito is relatively short,

with the majority of laboratory-reared mosquitoes surviving up to a month [9]. At field

conditions, mosquito lifespan is shorter due to limited food supply, predators, and

microbes such as entomopathogenic fungi [7]. Considering that a mosquito becomes

infectious two weeks after consuming P. falciparum gametocytes, most infectious

mosquitoes are close to reaching their life expectancy [5], [8], [9]. If a mosquito is

delayed by several days in becoming competent to support Plasmodium infection, the risk

of it transmitting the disease would be reduced. Even if the mosquito only feeds once on

C. oleophila in the field, it may still become protected from P. falciparum infection for

several days afterward. To further elucidate the duration of C. oleophila anti-Plasmodium

effects, mosquitoes could be yeast fed 3, 5, or 7 days prior to feeding on a gametocyte

culture.

While the approximately 5% reduction in infected mosquitoes compared to control

may not significantly impact mosquito vector competence in a lab setting, infection

intensity is much lower in nature than in a controlled laboratory infection assay. In

natural settings, less than 5 oocysts will typically develop after an infectious bloodmeal.

36

In a study done by Taylor, An. gambiae contained on average 3.38 oocysts in their

midgut, and only 11% of mosquitoes that had fed on infected blood had become infected

[86]. In our study, over half of mosquitoes fed to repletion on infected blood and the over

80% percent of the control group develop infections. It would be interesting to determine

whether the presence of C. oleophila in natural conditions described by Taylor could

clear An. gambiae of P. falciparum infection.

Guerra and colleagues studied the global intensity of transmission, and suggest that

outside of Africa most transmission is hypoendemic [87]. In the past decade, antimalarial

drugs and other control strategies have become widely available [88], [89], [90]. In areas

of low endemicity, a dedicated effort using multiple control strategies could realistically

eliminate P. falciparum [87].

This yeast is it is very inexpensive to culture and it could therefore be developed into

a low-cost malaria control strategy to supplement existing malaria control strategies.

While promising, this yeast requires additional study. We have not yet investigated

whether this C. oleophila strain can be vertically transmitted from parent to offspring, or

whether it would require continuous application in the field for mosquito exposure.

If this yeast is deployed to the field, it will take its place alongside other malaria

interventions. Current treatments have been very successful in reducing the morbidity and

mortality from P. falciparum infection, but Plasmodium infection remains a major public

health threat [18], [88], [89], [90]. Introducing an anti-Plasmodium microbe as part of an

integrated control strategy involving drug treatment and insecticide treated bednets would

37

leverage its effectiveness to make a significant reduction in malaria infection [54], [88],

[91].

This is the first study to investigate C. oleophila as a component of the An. gambiae

microbiome, and discovery of its anti-Plasmodium properties highlights the need to

explore the yeast and fungi populations of An. gambiae in greater depth.

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891342

George Edward Barringer III 50 Fox Run

Groton,MA 01450 g.edwardbarringer@gmail.com

45

Summary of Qualifications

Masters of Science in Molecular Microbiology and Immunology

Obtained Industry Experience through Groton Biosystems Quality Control Department

Proficient in Plasmodium falciparum culture handling, maintaining mosquito colonies

Extensive experience in culturing bacteria, yeast, and halophilic archaea

Education

Master’s of Science May 2014 Molecular Microbiology and Immunology. Johns Hopkins School of Public Health GPA: 3.71/4.00

Bachelor of Science May 2012 Biology,Minor in History Ursinus College GPA:3.71/4.00 Skills Analytical Technology: fraction collectors, cell counters, biochemistry analyzers, and Automated Reactor Sampling systems Techniques: Trizol & Chloramphenicol extraction, PCR, Quantitative Real time PCR, microscopy, florescent microscopy, UV-VIS spectroscopy, nano-injection, maintenance of transgenic mosquito colonies, sterile dissection Culturing: fastidious and non-fastidious bacteria, yeast, extremophile archaea

Academic Experience Johns Hopkins

University Bloomberg School of Public Health, Prof. George Dimopoulos Lab 2012-2014

Researched inhibition of Plasmodium falciparum in Anopheles gambiae by a Candida sp.

Isolated a novel Candida strain from the midgut of An. gambiae

Utilized nano-injection of dsRNA to investigate knockdown of mosquito immune pathway components

Studied bacteria and yeast interactions in the midgut with ex vivo and in vitro techniques

Determined Plasmodium inhibition occurs in the pre-oocyst stage

Determined the active anti-Plasmodium factor is secreted, increases bacteria load in the mosquito midgut

Ursinus College, Prof. Anthony Lobo Lab, Summer Fellows and Directed Research 2011-2012

Researched the molecular mechanism of resistance to sodium selenate of the halophile Haloferax mediterranei

Learned culturing techniques for fastidious organisms,

Produced and maintained a pure mutant strain of H. mediterranei through increasing exposures to sodium selenate

Developed biochemical tests to determine metabolic pathway activation

Investigated the nature of precipitate following H. mediterranei exposure to sodium selenate

Groton Biosystems, Quality Control Department Technician Summer 2009, 2010

Developed new methodologies for processing samples, filtration test methodology

Developed protocols to use viscous samples in the Groton Biosystems ARS reactor sampling system

Produced growth media and reagents for experiments