Title: A review of progress toward field application of transgenic mosquitocidal

entomopathogenic fungi

Authors: Brian Lovett1, Etienne Bilgo2, Abdoulaye Diabate2, Raymond St. Leger1*
1
Department of Entomology, University of Maryland, College Park, Maryland, 20742,
USA
2
Institut de Recherche en Sciences de la Santé/Centre Muraz, Bobo-Dioulasso, Burkina
Faso
*
Correspondence to: stleger@umd.edu

Abstract

In Africa, adult mosquito populations are primarily controlled with insecticide-

impregnated bed nets and residual insecticide sprays. This coupled with widespread
applications of pesticides in agriculture has led to increasing insecticide resistance in
mosquito populations. We have developed multiple alternative strategies for exploiting
transgenic Metarhizium spp. directed at: 1) shortening the lifespan of adult mosquitoes
2) reducing transmission potential of Plasmodium spp.; 3) reducing vector competence
via pre-lethal effects. The present challenge is to convert this promising strategy into a
validated public health intervention by resolving outstanding issues related to the
release of genetically modified organisms.

Keywords: Entomopathogenic fungi, genetically modified organisms, Metarhizium,

vector control, mosquitoes, malaria

1 Introduction

Approximately 100 species of anopheline mosquitoes transmit the five species of

Plasmodium capable of causing human malaria1. In sub-Saharan Africa, where annual

burdens of malaria-induced morbidity and mortality are greatest, Plasmodium

falciparum causes the majority of deaths2. In Africa, this parasite is transmitted by

mosquitoes of the Gambiae complex (comprising An. gambiae, An. coluzzii, and An.

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arabiensis) and An. funestus3. Several inextricable factors have contributed to the

continued burden of vector-borne diseases: operational obstacles, climate change,

vector and pathogen resistance to interventions, slow progress in vaccine development,

the increasing human population, progressive urbanization, globalization of trade and

travel and a shift in emphasis from prevention to emergency response present

interlinking challenges 4–7. A single method of control will not address the rising

emergence of mosquito vectors. However, given the enormity of the mosquito burden,

any new approach that causes even a 1% reduction in malaria incidence could spare

five million people from the burden of disease8.

Many millions of dollars have been spent on developing novel chemical

insecticides, antimalarial drugs, vaccines and transgenic mosquito technology, but even

though many technical challenges have been overcome, operational applications are at

least 5 to 10 years away. As an example, researchers have pondered for over half a

century how gene drives found in nature could be repurposed to combat mosquito-

borne illnesses9. These early attempts were impeded by difficulties in gene editing and

the requirement that the antimalarial genes are “driven” into populations instead of just

dying out. The CRISPR (clustered regularly interspaced short palindromic repeats) gene

editing technique has enabled biologists to overcome historic gene editing limitations

and precisely change the genetic makeup of a species. This development ushered in a

proliferation of studies developing “gene drive” techniques that, in theory, could allow a

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small number of released An. gambiae, An. coluzzii and An. arabiensis to quickly

replace entire natural populations of these species. The aim being to limit mosquito

populations (e.g., by engineering all male offspring) or to spread genes that make the

mosquitoes resistant to Plasmodium spp.10.

Necessary precautions before a release requires clearly formulating the

protection goals and considering the potential negative impact of the release well in

advance11,12. To date, CRISPR gene drives have been studied in laboratory settings

only, but as a precedent building exercise, genetically modified (GM) “male-sterile”

mosquitoes went through the regulatory process for release in Burkina Faso in 2018,

with the Target Malaria research consortium as the sponsor. As of writing the release

has not yet occurred as discussions continue with interested parties in the Burkina Faso

government. Notably, the mosquitoes proposed for release do not have a gene drive

mechanism, but the release is intended to build infrastructure, technical capacity and

experience of dealing with GM mosquitoes, and is a necessary transitional step to

prepare and challenge the regulatory and political system for release of gene drive

mosquitoes in the future.

Nevertheless, CRISPR gene drives produce an unusual set of challenges that

are hard to model. Given that a drive released in one part of Africa could eventually

cross borders as it spreads throughout the mosquito population, some have suggested

testing these technologies on islands with local mosquito populations. There may of

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course be the potential for dispersal via boats and planes travelling between islands and

the mainland, but this model has worked for pilot Wolbachia spp. studies, that seek to

exploit this bacterial mutualist of insects as a natural, microbial gene drive13.

Considering the rate of development of gene drives, the timeline for release of all these

proposed transgenic technologies for vector control will likely be determined by

regulatory and social hurdles, as these hurdles may increase with each border that a

gene drive might potentially cross. This highlights the need for proactive and

commensurate investment in regulatory and social anthropological capacity in countries

where these technologies would be applied.

The WHO Global Technical Strategy for Malaria 2016–2030 aims to reduce

malaria transmission by at least 90% over the next 15 years14. Similar ambitious targets

are set out in the Aspiration to Action document prepared by the Bill and Melinda Gates

Foundation. The requirement for supplementary tools to be implemented at scale within

the next 5 years to avert an anticipated rebound in malaria (due to waning natural

immunity and potential impacts of insecticide resistance) puts an emphasis on

approaches that are close to field-ready, and limits the immediate utility of prospective

tools that are still far from operational15. An alternative to waiting for gene drive systems

to be perfected and approved is to genetically engineer microbes that associate with

mosquitoes. Pantoea agglomerans bacteria have been engineered to express an anti-

malarial transgene; this species occurs naturally in mosquitoes and is passed from

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female to offspring16. A similar paratransgenetic system has been established using a

Serratia species which has been demonstrated to spread both horizontally and vertically

through mosquito populations17. These attributes greatly facilitate introducing a GM

bacterium into mosquitoes in the field. However, like the transgenic insect approach,

they require that transmission-blocking molecules be expressed by symbionts without

compromising their fitness or that of the insect host, and in addition the availability of

gene driving mechanisms to replace field populations18. These considerations do not

apply to a transgenic pathogen that is not designed to recycle, and the principal

consideration will be effectiveness of the engineered trait. Overall, therefore, field

release of a pathogen is much easier to navigate compared to mosquito germline

transformations or commensals. Furthermore, transgenic pathogens would not

necessarily be specific to one or a few species of anophelines, thus preventing other

species from filling the niche left by a suppressed mosquito species - an inherent

weakness of mating-based techniques.

2 Use of entomopathogenic fungi to control insect pests

Fungi are probably the most common pathogens of mosquitoes in nature19,20,

and Metarhizium spp. have a variety of properties useful in vector control; they

recognize various species of insect, having evolved enzymatic systems for invading

each, while at the same time avoiding an immune response21.

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 Metarhizium spp. are already deployed as a biological control agent against crop

pests (EPA and EU approved) in the USA, China, Australia, the Philippines and several

African and South American countries, with a very long track record of safety 22. The

devastating plagues of locusts in the mid-1980’s provided a sense of urgency to apply

Metarhizum spp., and spurred the development of standardized products that advanced

our knowledge in formulation, quality control, storage, application, environmental

impact, safety testing and host-pathogen ecology23. Industrial production of Metarhizium

spp. products is now highly efficient and automated, allowing them to be competitively

priced compared with chemical? insecticides24.

Whether working with insect commensals or pathogens, the biggest stumbling

block can be exposing wild mosquitoes to sufficient numbers of microbes16. However,

as contact bioinsecticides, fungal pathogens lend themselves to strategies currently

used for delivery of chemical insecticides. For example, fungal entomopathogens have

been successfully sprayed on indoor surfaces of houses, cotton ceiling hangings,

curtains and bed nets23, or used in eave tubes or outdoor odor baited traps25,26. Several

studies have highlighted the promising use of insect fungal pathogens for controlling

adult malaria mosquitoes, and reducing malaria transmission rates 23,27–29. Scholte et

al.28 took the idea of mosquito-killing fungi to Tanzania. They hung black cotton sheets

impregnated with M. anisopliae spores from the ceiling of traditional houses. Black is

attractive to bloodfed mosquitoes, and these sheets provided a resting area for

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mosquitoes to digest bloodmeals taken in houses. Nearly a quarter of the mosquitoes

collected from these target sheets were infected with the fungus, and they died

considerably faster than control mosquitoes maintained in the laboratory. They

estimated that, under the conditions of their field experiments, the intensity of malaria

transmission could be reduced by 75% from 262 to 64 infective bites per person per

year28.

Natural Metarhizium strains fall short as stand-alone mosquito control agents due

to their slow speed of kill (usually one to two weeks to reach >90% mortality) and

unreliability, the latter in large part steming from susceptibility to abiotic stresses. As

such, unmodified Metarhizium spp. have not met WHO Prequalification Vector Control

(WHOPES) standards for a vector control product30. This is consistent with intensive

efforts to use these fungi to control other pests; historically biocontrol agents have not

met expectations because of low virulence31, and many Metarhizium spp. products are

not widely used due to their unpredictable efficacy22.

We adopted a transgenic approach because, once inside the mosquito, it takes

10-14 days for malaria parasites to mature through the gut, travel through the

hemolymph and translocate into the salivary glands, where they can be transmitted to

another host (Figure 1). It takes several days for wild-type (WT) fungal spores to infect

mosquitoes through the cuticle and kill them. If the mosquitoes are infected with fungi

soon after they pick up Plasmodium spp., these mosquitoes will die before they can

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pass them on, but if they are infected with Metarhizium spp. any later, which is likely,

they could still spread malaria before they die32. The level of adoption by stakeholders

that is required for early infection of most mosquitoes in a population is operationally

hard to achieve, as it requires a high proportion of houses be treated and that there be a

high probability of infection with the fungus before each bloodmeal33. The requirement

for high inoculum means that only a minority of mosquitoes landing on a treated surface

may pick up a lethal spore dose. Compounding this, the viability of the fungus

decreases over time, reducing infection rates23 and increasing the cost of the product

relative to chemicals that have longer residual activity. Persistence of spores on treated

surfaces is thus a key factor in determining the ultimate success of any biopesticide

approach34.

Metarhizium spp. provide very tractable model systems for biotechnology with

many available tools for their genomic, genetic and biochemical characterization35. The

prospects of using transgenic pathogens have rapidly gained strength, owing to the

standardization of rapid high throughput transformation techniques36, the

characterization of fungal promoters that can drive the expression of transgenes in an

insect tissue- and stage-specific manner37 and the identification and

characterization of effector molecules that can rapidly kill the mosquito or interfere with

the development of parasites in the mosquito. Effector molecules include naturally

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occurring or synthetic antibodies against parasite proteins and antimicrobial or

insecticidal peptides35,38.

To increase fungal virulence for mosquito control, we combined the natural ability

of Metarhizium spp. to penetrate into insects with insecticidal peptides. The peptides we

tested were derived from arthropod venoms. These show complete specificity to insects

and rapidly degrade in the environment, and thus lend themselves to environmentally

benign pest management strategies39. Their chief limitation is that they do not penetrate

the insect gut and so require a means of delivery into the insect hemocoel. Reviews

detail how and why the ca. 1 million arachnid toxins will be a major resource for

biotechnology if provided with suitable delivery systems39–41. Metarhizium spp. are

routine and efficient expression systems, but with the added advantage of also

providing an intrahemocoel delivery mechanism for the many toxins not considered

practical insecticides because they cannot cross the insect gut or cuticle. Contrasting

sharply with insect transgenesis, transformation protocols with Metarhizium spp. are

inexpensive, rapid and require only basic microbiological training. Given the ease of

engineering Metarhizium spp. and its ability to correctly process and express very

different proteins, it provides a platform for conveniently testing multiple transmission

blocking strategies and insecticidal toxins.

3 The potential of entomopathogenic fungi against disease vectors

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 The strong and highly hemolymph-specific Mcl1 promoter allows a strategy of

engineering Metarhizium pingshaense (Mp) to express a toxin only in the mosquito

stages of fungal development, without compromising host-specificity or contaminating

the environment42. Using this promoter, we initially tested the 70 AA Androctonus

australis insect toxin AaIT (a sodium channel blocker) because it is very potent and

provides a benchmark for efficacy. Arthropod toxins include defensive toxins that may

toxicity to vertebrates, and offensive toxins such as AaIT that are highly selective to

insects. Modified fungus expressing AaIT achieved the same mortality rates in

mosquitoes at 9-fold lower spore doses than the WT, and survival times at some doses

were reduced by 40%43. Following this, we compared genes from arthropod predators

encoding insect specific sodium, potassium and calcium channel blockers for their

ability to improve the efficacy of Mp against wild-caught, insecticide-resistant

anophelines44. As one of the most potent toxins, we characterized the pre-lethal effects

generated by expression of Hybrid (Ca++/K+ channel blocker). Due to reduced blood

feeding, nearly all insects infected with ~6 conidia were unable to transmit malaria five

days following infection with transgenic Mp, surpassing the WHO successful vector

control agent threshold45. Moreover, compared to strains expressing single toxins,

strains co-expressing both Hybrid and AaIT required 45% fewer spores to achieve a 5-

day LT50, and provided lethality to mosquitoes at doses as low as a single spore per

mosquito45. This large reduction in effective spore dose could be crucial for successful

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control because the percent of mosquitoes picking up a lethal infection should be

greatly increased compared to the 25% reported by Scholte et al.28. They calculated

that if 50% of mosquitoes were infected, the rate of malaria transmission could be

lowered by 96%28. Also, the effective persistence of the biopesticide on treated surfaces

increases because even with an unaltered rate of spore decay, the lower inoculum

requirement of the genetically modified fungi will ensure that the inoculum threshold will

be met for a longer period of time.

Furthermore, as well as being equally effective on insecticide-susceptible and

insecticide-resistant mosquitoes, toxin-expressing strains work synergistically with

chemical insecticides-restoring the knockdown phenotype in insecticide-resistant

mosquitoes five days following fungal infection46. These results highlight the

compatibility of transgenic entomopathogens with widely used chemical control

methods.

Following the WHO framework for GM mosquitoes47,48 (Figure 2), the first phase

towards deploying transgenic Mp was the production and laboratory testing of

transgenic, hypervirulent Mp strains. Phase 2, recently completed, demonstrated that

Mp-Hybrid was as effective in a “MosquitoSphere” malaria endemic environment (i.e., a

compartmentalized facility of mosquito netting) in Burkina Faso as we had anticipated

from laboratory investigations. Phase 3 will involve a series of confined and open field

trials of increasing size, duration and complexity, to truly assess the efficacy,

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survivability, and environmental risk posed by genetically engineered insect pathogens,

and phase 4 will entail large-scale deployment and monitoring. The transition from one

phase to the next is subject to “go/no-go” decision criteria, incorporating efficacy and

safety endpoints, regulatory approvals and social acceptance. Progress therefore

requires active coordination across these scientific, regulatory and social domains.

4 Efficacy and safety considerations of Metarhizium pingshaense deployment in a

MosquitoSphere, and subsequent phase 3 and phase 4 trials

The safety of biocontrol agents based on Metarhizium spp. has been clearly

established through several decades of high quality research49. M. robertsii is

composed of asexual clonal populations that persist over time and space without

sexually reproducing 50–52. The strain of Mp currently under development was originally

isolated from Aedes aegypti. It shows sustainable properties that do not vary

considerably between habitats and formulations and is stable at the high (34oC)

temperatures pertaining to household conditions. These were promising foundations for

further development. In addition, Mp does not infect a panel of insects [cockroaches,

honey bees (Apis mellifera) caterpillars (Manduca spp.) and others], thus limiting the

possibility of a threat to non-target insects44. We have confirmed that expression of

neurotoxins Hybrid and AaIT by Mp under control of the Mcl1 promoter does not

broaden host range, even to include another fly (Calliphora). Studies with several other

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strains have also shown that host range is not altered by expression of toxins (i.e., the

host range is determined by the fungus, not by the engineered toxin)53. This is probably

because specificity is principally determined before penetration of the insect cuticle54.

A particular advantage of using Metarhizium spp. for groundbreaking trials of a

transgenic product is that it produces large, sticky spores that attach to one another.

Dispersal of Metarhizium spp. would not passively move through a double layer of

mosquito netting because it is not naturally present in air samples49,55,56, so the semi-

field site contains the fungus as well as the mosquitoes. Metarhizium spp. spores are

too large and sticky to fit between the cilia of the lungs, and thus do not cause inhalation

allergies.However, even in an open-field application, given the presumptive residential

use sites, application methods with reduced opportunity for offsite movement of

Metarhizium spp. (e.g., attachment to sheets) and the very limited ability of Metarhizium

spp. to survive solar radiation, it is not likely that use of Metarhizium spp. products will

result in significant fungal spore dispersal. An additional characteristic that will limit the

survivability of recombinant fungi is the reduced ability to form progeny43. The yield of

fungal conidia from a fungal infection is a key factor controlling fungal population

dynamics. The slow killing speed imparted by wild type fungi is adaptive, as it allows the

fungus to fully exploit host tissues and maximize fitness and yield of conidia. Changes

made to the fungal genome to make it a more effective bioinsecticide may therefore

also drive recombinant fungi to extinction due to their limited ability to propagate57.

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 It has also been argued that propogation by secondary transfer of fungi from

mosquitoes is very unlikely since fungal spores are only produced once the insect is

dead, and many cadavers are scavenged before sporulation. In this regard, the spread

of fungal transgenes would be much easier to manage than mosquito transgenes58.

Nevertheless, methods to help reduce transmission further have been developed to

address any concerns raised by stakeholders. For example, we have knocked out

genes for environmental stress resistance, producing strains with greatly reduced ability

to survive in soil or other environments, but that maintain wild-type virulence59.

5 The potential for mosquitoes to evolve resistance to transgenic fungi

A potential downside to the increased effectiveness of transgenic Mp is

increased selective pressure on the mosquitoes to evolve resistance. The wild-type

fungus kills mosquitoes slowly enough that they can still achieve part of their lifetime

reproductive output. As a result, there is little pressure on them to evolve resistance23,33.

Extensive genetic variation in individual resistance from the same geographical

population could set the stage for the evolution of resistance with major implications for

the sustainability of wild-type or transgenic fungi. In contrast to mosquitoes, Drosophila

is a very tractable model system. Individual fruit flies vary extensively in their ability to

control and tolerate fungal and bacterial pathogens during infection, and resistance

correlated with several unexpected phenotypes, including sleep60. However, there is

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reason for optimism that resistance will not evolve against toxin-expressing strains for

many years because in every realistic scenario, genetically modified (GM) Metarhizium

spp. would be used in conjunction with existing technologies, thereby contributing to an

overall greater reduction in malaria over the next ten years. Transgenic Metarhizium

spp. are as infectious to insecticide-resistant mosquitoes as they are to susceptible

mosquitoes46. Insecticides and biologicals could be applied concurrently or alternated

as part of an integrated vector management program to maximize mosquitocidal impact,

and to manage current resistance to chemicals and potential resistance to biologicals.

We and others have identified and engineered a suitable array of genes to potentially

provide sustainable depletion of anopheline populations and/or Plasmodium spp. Aside

obtaining very useful additive or synergistic effects, using multiple transgenes minimizes

the potential for cross resistance, and reduces the probability of emergence of insect

resistance to one mechanism31. Furthermore, the calcium and potassium channels

targeted by the Hybrid toxin are previously unexploited insecticide targets61, reducing

the likelihood of pre-existing resistance. Most widely used chemical insecticides target

sodium channels. Interestingly, since AaIT binds to a site that is distinct from those of

pyrethroids in insect sodium channels, and channel mutations that confer resistance to

pyrethroids actually increase binding of AaIT, the simultaneous application of

pyrethroids and AaIT could minimize the possibility resistance will develop62.

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 Furthermore, while resistance is almost always a problem with chemical

insecticides, it has not yet become a problem with larvicidal Bacilllus thuringiensis

bacteria, and it has yet to be shown that resistance could develop against a fungal

pathogen. Pathogens have “built-in” resistance management properties, and there are

many natural examples of introduced pathogen strains that provide sustainable

depletion of genetically diverse insect populations over very wide areas (e.g., the fungi

Entomophaga maimaga controlling gypsy moth and Zoophthora radicans controlling

aphids63). Likewise, a single application of the bacterium Bacillus popilliae to grasslands

can suppress Japanese beetle populations for as long as ten years64. The purpose of

citing these examples is to demonstrate that insect pathogenic fungi have evolved

genetic systems that can provide long-term suppression of insect populations. This

highlights the major advantage biopesticides have over chemical pesticides: biological

systems have an inherent vested interest in the successful infection of the insect, while

chemical pesticides are inert. In one study, this interest has been challenged in

experiments aiming to “force” resistance to entomopathogenic fungi in Galleria

mellonella (waxworms): the authors reported that intensive investment in cuticular

defences could reduce infection by Beauveria bassiana, but resistance to M. anisopliae

could not be achieved after 25 generations of selection65. Even in the case of B.

bassiana, these authors concluded that the shunting of resources to the cuticle would

cripple the fitness of these moths in nature.

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 We have tested a broad array of engineering strategies (Figure 3) to be

consistent with the highly exploratory approach required for optimizing the biocontrol

potential of a pathogen, and also with a need to be flexible in following up the most

promising lines of research. For example, we have developed a fungus that kills

mosquitoes as slowly as the natural isolates, but that, in the meantime, stops them from

passing on the malaria parasite. It does this by expressing molecules that kill the

Plasmodium parasites (i.e., scorpine or the single-chain, sporozoite-agglutinating

antibody PfNPNA-1) or prevent it from translocating into salivary glands (i.e., salivary

gland and midgut peptide 1- SM1)66. Multiple effectors expressed by the fungus worked

synergistically to inhibit sporozoite invasion of salivary glands, and the best

combinations (scorpine/SM1 fused with scorpine and scorpine/PfNPNA-1) reduced the

sporozoite intensity approximate 98%66. Relying on antimalarial effects could in the long

run lead to the evolution of resistant malaria parasites. However, the single-chain

antibody (PfNPNA-1) specifically recognizes the repeat region (Asn-Pro-Asn-Ala) of the

P. falciparum surface circumsporozoite protein67, so a single mutation would not be

sufficient to achieve resistance. Exploiting Metarhizium spp. to express diverse anti-

plasmodials or insecticidal proteins demonstrates that fungal pathogens can efficiently

deliver bioactive molecules for alternative strategies.

We are interested in advancing both strategies outlined above: using GM

Metarhizium spp. expressing insecticidal toxins to quickly reduce An. gambiae

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populations and using GM Metarhizium spp. to produce mosquitoes with a lower level of

malaria infection. The population suppression strategy should have more rapid and

obvious effect, and its impact can readily be tested in field trials against existing

chemical pesticide methods. However, the disease reduction strategy may better

circumvent development of insect resistance, reducing the possibility of non-target

effects. It is also likely that employing a transgenic fungus that differs from the wild-type

only in expression of a specific anti-plasmodium gene would be less likely to raise

concern since Plasmodium spp. would only be present in anophelines.

We currently have strong community support for the work we are proposing,

particularly for the toxin-expressing strain, as the Burkinabe (Burkina Faso) government

and people would prefer a product that can be seen to be effective at alleviating

mosquitoes right away. However, there is likely to be no single strategy that is best

under all conditions, and our goal is to make available a wide array of options and

resources that different communities can tailor to their vector control challenges and

specific circumstances.

6 Social acceptability of transgenic entomopathogenic fungi

An important challenge related to the release of genetically modified organisms is the

resolution of regulatory, ethical, and social issues. Significant numbers of transgenic

microbial pest control agents have already been marketed without fanfare68, providing

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crucial precedents for our work. In addition, the U.S. EPA has approved the Hybrid

peptide (also known as Versitude) in an oral formulation for control of cabbage loopers

(Trichoplusia ni). The EPA has also already sanctioned outdoor field trials of genetically

engineered Metarhizium spp.69,70. In these and subsequent studies, we used easily

identifiable marker genes (expressing green fluorescent and red fluorescent proteins),

functional genomic tools for identifying genetic changes, and genes that are implicated

in pathogenicity to provide detailed perspective on persistence and potential modes of

adaptation in transgenic fungi released into the field52. Deployment against mosquito

populations could (subject to approval by African communities) be rapid, with products

integrating readily into existing control strategies.

Social acceptability presents a challenge for many technologies under

consideration. These issues may to a large extent be resolved by the properties of the

technology under development. However, new technologies may not change the

atitudes of vocal anti-science groups that categorically oppose gene modification on

principle, by appealing to emotion and confirmation bias71. A recent House of Commons

Science and Technology Committee report by the British government offered several

recommendations for communicating science72. They stated that scientists need

support to become more competent communicators, and aid in developing strategies for

engaging the media and general public to ensure that science benefits everyone. Still, it

is questionable whether many people can be reasoned out of positions if these are

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based on emotion, not evidence. Furthermore, attributing public concerns to lack of

comprehension of the science ignores evidence from many fields that ideological

differences may strengthen as people become better informed because they have more

information for confirmation bias to work with73. To responsibly attain informed consent,

attention to communication is critical with rural communities in Africa in order to

sufficiently explain concepts that are often difficult to fully articulate through multiple

languages.

Nevertheless, the concerns many scientists have that regulatory and social

contraints will prevent the public benefiting from GMO vector control technology could

be a prime example of ‘synbiophobia-phobia’, a term coined to describe fears among

scientists that emerging fields with great potential will be susceptible to subversion by a

reflexively fearful public74. In fact, the experience of many groups helping to introduce

new technologies is that African communities expect to form their own opinions on

benefits after experiencing these technologies. A good recent example is mobile

banking (Orange money, Nana-Express, Telecel-Flash), which has been

enthusiastically adopted by people in villages where brick and mortar banking systems

are difficult to come by.

This was also the case with Burkinabe GM cotton-farmers, who discounted

“noise” made by anti-GMO groups. Burkina Faso is one of the most proactive African

countries regarding biotechnology, and one of only three to have commercialized GM

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crops (South Africa and Egypt are the other two). National research institutes in Ghana,

Nigeria and Burkina Faso have sought support from the African Agricultural Technology

Foundation (AATF) to move the Bt trait into local cowpea varieties in these countries as

a way to boost cowpea production and lower chemical pesticide use. Setting a

precedent, GM Bt-cotton was quickly accepted by Burkinabe farmers and produced the

expected beneficial results75. However, a controversy erupted over the shorter fiber

length produced by Bt-cotton, prompting a temporary withdrawal of the seeds in 2015

after they had been in use for eight years. This was much to the displeasure of farmers

who report suffering significant crop loss growing non-GMO cotton, despite using more

pesticides: the overwhelming consensus is that shorter fibers are better than no fibers76.

Monsanto, now owned by Bayer, attributed the problem to the lack of an ongoing

Burikinabe-based breeding program to improve upon the cultivar, which allowed

undesired traits like short fiber length to re-emerge77, while anti-GMO groups have

accused Monsanto of striving to achieve a monopoly over seed supply. This controversy

exemplifies how the GMO debate has moved on, with good-faith concern tending to

focus on who will benefit rather than safety. However, according to Dr. Oumar Traore,

the director of the National Biosafety Agency (NBA) in Burkina Faso, lessons that arose

from the Bt-cotton case included avoiding mistakes when it comes to biosafety matters.

If the issue with Bt-cotton had been a biosafety issue, the whole technology could have

been forfeited. In his role as director of the NBA laboratory, he has learned these

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lessons through consultation with local stakeholders firsthand. Dr. Traore also predicted

Burkinabes will welcome GMO technologies for fighting malaria: “When it comes to

health, people don’t have any issues with genetic engineering”78.

7 Future approaches to engineering pathogens.

To date, entomopathogenic fungi have been employed either as one component of an

integrated pest management strategy or as inundative mycoinsecticides79. It has been

axiomatic that a recombinant pathogen of any kind would be applied as an inundative

insecticide that might last a single season. Products based on these strains would

require repeated production and application. The costs of these repeated applications

for transgenic microbes may be lower because of greater potency per unit weight of

fermentation medium. These savings could be passed either to the consumer or

increase profits. The extent to which cost savings were passed to the consumer could

have an outsized impact on their utility in developing countries. The best way to ensure

costs that would be appropriate to use in the developing world would be to create free-

living microbes that will reproduce under field conditions at a level that would provide

effective control. Even a control agent that had to be applied occasionally, for instance

once per year, would still likely be cheaper than a new chemical insecticide.

Advances in genetic engineering now make it feasible to develop recombinant

microbes that will show narrow specificity for the target pest and persist in the

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environment, providing sustainable control for much longer periods than existing

chemicals80,81. Highly specific viruses, bacteria and fungi already exist, and some

provide sustainable depletion of insect populations providing natural models for

sustainable biocontrol. Many studies have shown that these specialist strains offer

several levels of specificity, including loss of many genes for opportunism82. This

reduces the chances that engineering could alter host range. Genes also exist in nature

that target specific sites in insects for producing highly virulent, but targeted pathogens.

Expression of host molecules (e.g., Trypsin modulating oostatic factor, hormones, and

neuropeptides) has been shown to yield Beauveria strains with insect-specific increases

in virulence83–85. These enable manipulation of host behaviors, and, in theory, raise the

bar considerably for resistance to develop in the target insect compared to other

approaches38. Additional important achievements have been made in the engineering

of Beauveria spp. and Metarhizium spp., including improved resistance to all of the field-

relevant, environmental abiotic stresses (e.g., UV and temperature extremes) that have

limited application of these fungi in the past 59,80,86. The state of knowledge and

technology for modifying entomopathogenic fungi reveals a field ripe for tailored

development and application for sustainable large-scale depletion of a target pest.

Scientifically, the development of transgenic entomopathogenic fungi for vector control

has progressed sufficiently for field application; however this technology needs an

informed community ready to usher in a new era of transgenic microbial control.

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Acknowledgements

This work was supported by NIH-NIAID under award RO1-AI106998 to R.J.S. and A.D.

This paper was given at the workshop on Natural Products in Pest Management:

Innovative approaches for increasing their use which took place in Bellagio, Italy on 25-

29 September 2018, and which was sponsored by the OECD Co-operative Research

Programme: Biological Resource Management for Sustainable Agricultural Systems

whose financial support made it possible for the author to participate in the workshop.

The opinions expressed and arguments employed in this paper are the sole

responsibility of the authors and do not necessarily reflect those of the OECD or of the

governments of its Member countries.

This article is protected by copyright. All rights reserved.

References

1. Raghavendra K, Barik TK, Reddy BPN, Sharma P, Dash AP, Malaria vector
control: from past to future. Parasitol. Res. 108(4):757–79 (2011).
2. WHO | World malaria report 2017. WHO (2018).
3. Emami SN, Ranford-Cartwright LC, Ferguson HM, The transmission potential of
malaria-infected mosquitoes (An.gambiae-Keele, An.arabiensis-Ifakara) is altered
by the vertebrate blood type they consume during parasite development. Sci.
Rep. 7:40520 (2017).
4. Snounou G, Rénia L, The vaccine is dead – long live the vaccine. Trends
Parasitol. 23(4):129–32 (2007).
5. Enayati A, Hemingway J, Malaria Management: Past, Present, and Future. Annu.
Rev. Entomol. 55(1):569–91 (2010).
6. Trape J-F, Tall A, Diagne N, Ndiath O, Ly AB, Faye J, et al., Malaria morbidity and
pyrethroid resistance after the introduction of insecticide-treated bednets and
artemisinin-based combination therapies: a longitudinal study. Lancet Infect. Dis.
11(12):925–32 (2011).
7. Caminade C, Kovats S, Rocklov J, Tompkins AM, Morse AP, Colón-González FJ,
et al., Impact of climate change on global malaria distribution. Proc. Natl. Acad.
Sci. U. S. A. 111(9):3286–91 (2014).
8. van der Goes van Naters W, Carlson JR, Insects as chemosensors of humans
and crops. Nature 444(7117):302–7 (2006).
9. CRAIG GB, HICKEY WA, VANDEHEY RC, An inherited male-producing factor in
Aedes aegypti. Science 132(3443):1887–9 (1960).
10. Eckhoff PA, Wenger EA, Godfray HCJ, Burt A, Impact of mosquito gene drive on
malaria elimination in a computational model with explicit spatial and temporal
dynamics. Proc. Natl. Acad. Sci. U. S. A. 114(2):E255–64 (2017).
11. Okumu F, Andrade PP de, Savadogo M, James S, Roberts A, Quemada H, et al.,
Results from the Workshop “Problem Formulation for the Use of Gene Drive in
Mosquitoes.” Am. J. Trop. Med. Hyg. 96(3):530–3 (2017).
12. Conko G, Kershen DL, Miller H, Parrott WA, A risk-based approach to the
regulation of genetically engineered organisms. Nat. Biotechnol. 34(5):493–503
(2016).
13. O’Connor L, Plichart C, Sang AC, Brelsfoard CL, Bossin HC, Dobson SL, Open
Release of Male Mosquitoes Infected with a Wolbachia Biopesticide: Field
Performance and Infection Containment. PLoS Negl. Trop. Dis. 6(11):e1797
(2012).
14. Organization WH, Global Technical Strategy for Malaria 2016-2030. WHO
(2017).
15. Barreaux P, Barreaux AMG, Sternberg ED, Suh E, Waite JL, Whitehead SA, et

This article is protected by copyright. All rights reserved.

 al., Priorities for Broadening the Malaria Vector Control Tool Kit. Trends Parasitol.
33(10):763–74 (2017).
16. Wang S, Ghosh AK, Bongio N, Stebbings KA, Lampe DJ, Jacobs-Lorena M,
Fighting malaria with engineered symbiotic bacteria from vector mosquitoes. Proc.
Natl. Acad. Sci. U. S. A. 109(31):12734–9 (2012).
17. Wang S, Dos-Santos ALA, Huang W, Liu KC, Oshaghi MA, Wei G, et al., Driving
mosquito refractoriness to Plasmodium falciparum with engineered symbiotic
bacteria. Science 357(6358):1399–402 (2017).
18. Rio RVM, Hu Y, Aksoy S, Strategies of the home-team: symbioses exploited for
vector-borne disease control. Trends Microbiol. 12(7):325–36 (2004).
19. Roberts DW, Fungal parasites of mosquitoes. Impact Use Microorg. Aquat.
Environ. Proc. 13:49 (1975).
20. Scholte E-J, Knols BGJ, Samson RA, Takken W, Entomopathogenic fungi for
mosquito control: A review. J. Insect Sci. 4(1) (2004).
21. Pal S, St. Leger RJ, Wu LP, Fungal peptide Destruxin A plays a specific role in
suppressing the innate immune response in Drosophila melanogaster. J. Biol.
Chem. 282(12):8969–77 (2007).
22. Faria MR de, Wraight SP, Mycoinsecticides and Mycoacaricides: A
comprehensive list with worldwide coverage and international classification of
formulation types. Biol. Control 43(3):237–56 (2007).
23. Thomas MB, Read AF, Can fungal biopesticides control malaria? Nat. Rev.
Microbiol. 5(5):377–83 (2007).
24. Langewald J, Kooyman C, Green muscle, a fungal biopesticide for control of
grasshoppers and locusts in Africa. Biol. Control. a Glob. Perspect. CABI,
Oxfordsh. :311–27 (2007).
25. Lwetoijera DW, Sumaye RD, Madumla EP, Kavishe DR, Mnyone LL, Russell TL,
et al., An extra-domiciliary method of delivering entomopathogenic fungus,
Metharizium anisopliae IP 46 for controlling adult populations of the malaria
vector, Anopheles arabiensis. Parasit. Vectors 3(1):18 (2010).
26. Knols BGJ, Farenhorst M, Andriessen R, Snetselaar J, Suer RA, Osinga AJ, et
al., Eave tubes for malaria control in Africa: an introduction. Malar. J. 15(1):404
(2016).
27. Hancock PA, Snow R, Guerra C, Noor A, Myint H, Hay S, et al., Combining
Fungal Biopesticides and Insecticide-Treated Bednets to Enhance Malaria
Control. PLoS Comput. Biol. 5(10):e1000525 (2009).
28. Scholte E-J, Ng’habi K, Kihonda J, Takken W, Paaijmans K, Abdulla S, et al., An
entomopathogenic fungus for control of adult African malaria mosquitoes. Science
308(5728):1641–2 (2005).
29. Scholte E-J, Knols BGJ, Takken W, Infection of the malaria mosquito Anopheles
gambiae with the entomopathogenic fungus Metarhizium anisopliae reduces

This article is protected by copyright. All rights reserved.

 blood feeding and fecundity. J. Invertebr. Pathol. 91(1):43–9 (2006).
30. Aw KMS, Hue SM, Mode of Infection of Metarhizium spp. Fungus and Their
Potential as Biological Control Agents. J. fungi (Basel, Switzerland) 3(2) (2017).
31. Gressel J, Novel Biotechnologies for Biocontrol Agent Enhancement and
Management: Failsafe mechanisms for preventing gene flow and organism
dispersal of enhanced microbial biocontrol agentsSpringer Netherlands,
Dordrecht, pp. 353–62 (2007).
32. St. Leger RJ, Wang C, Genetic engineering of fungal biocontrol agents to achieve
greater efficacy against insect pests. Appl. Microbiol. Biotechnol. 85(4):901–7
(2010).
33. Read AF, Lynch PA, Thomas MB, How to Make Evolution-Proof Insecticides for
Malaria Control. PLoS Biol. 7(4):e1000058 (2009).
34. Enserink M, Microbiology. Mosquito-killing fungi may join the battle against
malaria. Science 308(5728):1531–3 (2005).
35. Zhao H, Lovett B, Fang W, Advances in Genetics: Genetically Engineering
Entomopathogenic Fungi, ed. by Lovett B, St. Leger RJ., Elsevier, London, pp.
137–63 (2016).
36. Xu C, Zhang X, Qian Y, Chen X, Liu R, Zeng G, et al., A High-Throughput Gene
Disruption Methodology for the Entomopathogenic Fungus Metarhizium robertsii.
PLoS One 9(9):e107657 (2014).
37. Zhao H, Xu C, Lu H-L, Chen X, St. Leger RJ, Fang W, Host-to-pathogen gene
transfer facilitated infection of insects by a pathogenic fungus. PLoS Pathog.
10(4):e1004009 (2014).
38. Ortiz-Urquiza A, Luo Z, Keyhani NO, Improving mycoinsecticides for insect
biological control. Appl. Microbiol. Biotechnol. 99(3):1057–68 (2015).
39. Edwards MG, Gatehouse AMR, Novel biotechnologies for biocontrol agent
enhancement and management: Biotechnology in crop protection: towards
sustainable insect controlSpringer, pp. 1–23 (2007).
40. Whetstone PA, Hammock BD, Delivery methods for peptide and protein toxins in
insect control. Toxicon 49(4):576–96 (2007).
41. King GF, Hardy MC, Spider-Venom Peptides: Structure, Pharmacology, and
Potential for Control of Insect Pests. Annu. Rev. Entomol. 58(1):475–96 (2013).
42. Wang C, St. Leger RJ, A collagenous protective coat enables Metarhizium
anisopliae to evade insect immune responses. Proc. Natl. Acad. Sci. U. S. A.
103(17):6647–52 (2006).
43. Wang C, St. Leger RJ, A scorpion neurotoxin increases the potency of a fungal
insecticide. Nat. Biotechnol. 25(12):1455–6 (2007).
44. Bilgo E, Lovett B, Fang W, Bende N, King GF, Diabate A, et al., Improved efficacy
of an arthropod toxin expressing fungus against insecticide-resistant malaria-
vector mosquitoes. Sci. Rep. 7(1):3433 (2017).

This article is protected by copyright. All rights reserved.

45. Bilgo E, Lovett B, Fang W, Bende N, King GF, Diabate A, et al., Improved efficacy
of an arthropod toxin expressing fungus against insecticide-resistant malaria-
vector mosquitoes. Sci. Rep. 7(1) (2017).
46. Bilgo E, Lovett B, Bayili K, Millogo AS, Saré I, Dabiré RK, et al., Transgenic
Metarhizium pingshaense synergistically ameliorates pyrethroid-resistance in
wild-caught, malaria-vector mosquitoes. PLoS One 13(9):e0203529 (2018).
47. World Health Organization, Geneva, Switzerland: World Health Organization:
Guidance framework for testing of genetically modified mosquitoes. (2014).
48. Vontas J, Moore S, Kleinschmidt I, Ranson H, Lindsay S, Lengeler C, et al.,
Framework for rapid assessment and adoption of new vector control tools. Trends
Parasitol. 30(4):191–204 (2014).
49. Zimmerman G, Review on safety of the entomopathogenic fungi Beauveria
bassiana and Beauveria brongniartii. Biocontrol Sci. Technol. 17(6):553–96
(2007).
50. St. Leger RJ, May B, Allee LL, Frank DC, Staples RC, Roberts DW, Genetic
differences in allozymes and in formation of infection structures among isolates of
the entomopathogenic fungus Metarhizium anisopliae. J. Invertebr. Pathol.
60(1):89–101 (1992).
51. Gao Q, Jin K, Ying S-H, Zhang Y, Xiao G, Shang Y, et al., Genome sequencing
and comparative transcriptomics of the model entomopathogenic fungi
Metarhizium anisopliae and M. acridum. PLoS Genet. 7(1):e1001264 (2011).
52. Wang S, O’Brien TR, Pava-Ripoll M, St. Leger RJ, Local adaptation of an
introduced transgenic insect fungal pathogen due to new beneficial mutations.
Proc. Natl. Acad. Sci. U. S. A. 108(51):20449–54 (2011).
53. Fang W, Lu H-L, King GF, St. Leger RJ, Construction of a hypervirulent and
specific mycoinsecticide for locust control. Sci. Rep. 4:7345 (2014).
54. St Leger RJ, Screen S, Fungal Biocontrol Agents--Progress Problems and
Potential: Genetic improvement of fungi for insect and weed control. CABI
Publishing, Wallingford, UK, (2001).
55. Carmichael JW, Fungi from Alberta rodents. Mycopathol. Mycol. Appl. 14(2):129–
35 (1961).
56. Thomas MB, Darbro JM, Spore Persistence and Likelihood of Aeroallergenicity of
Entomopathogenic Fungi Used for Mosquito Control. Am. J. Trop. Med. Hyg.
80(6):992–7 (2009).
57. Pava-Ripoll M, Posada FJ, Momen B, Wang C, St. Leger R, Increased
pathogenicity against coffee berry borer, Hypothenemus hampei (Coleoptera:
Curculionidae) by Metarhizium anisopliae expressing the scorpion toxin (AaIT)
gene. J. Invertebr. Pathol. 99(2):220–6 (2008).
58. Blanford S, Chan BHK, Jenkins N, Sim D, Turner RJ, Read AF, et al., Fungal
pathogen reduces potential for malaria transmission. Science 308(5728):1638–41

This article is protected by copyright. All rights reserved.

 (2005).
59. Fang W, St. Leger RJ, Enhanced UV resistance and improved killing of malaria
mosquitoes by photolyase transgenic entomopathogenic fungi. PLoS One
7(8):e43069 (2012).
60. Wang JB, Lu H-L, St. Leger RJ, The genetic basis for variation in resistance to
infection in the Drosophila melanogaster genetic reference panel. PLOS Pathog.
13(3):e1006260 (2017).
61. Tedford HW, Sollod BL, Maggio F, King GF, Australian funnel-web spiders:
master insecticide chemists. Toxicon 43(5):601–18 (2004).
62. Gordon D, Karbat I, Ilan N, Cohen L, Kahn R, Gilles N, et al., The differential
preference of scorpion α-toxins for insect or mammalian sodium channels:
Implications for improved insect control. Toxicon 49(4):452–72 (2007).
63. Vega FE, Goettel MS, Blackwell M, Chandler D, Jackson MA, Keller S, et al.,
Fungal entomopathogens: new insights on their ecology. Fungal Ecol. 2(4):149–
59 (2009).
64. Dutky SR, The milky diseases. Insect Pathol. an Adv. treatise 2:75–115 (1963).
65. Dubovskiy IM, Whitten MMA, Yaroslavtseva ON, Greig C, Kryukov VY, Grizanova
E V., et al., Can Insects Develop Resistance to Insect Pathogenic Fungi? PLoS
One 8(4):e60248 (2013).
66. Fang W, Vega-Rodríguez J, Ghosh AK, Jacobs-Lorena M, Kang A, St. Leger RJ,
Development of transgenic fungi that kill human malaria parasites in mosquitoes.
Science 331(6020):1074–7 (2011).
67. Chappel JA, Rogers WO, Hoffman SL, Kang AS, Molecular dissection of the
human antibody response to the structural repeat epitope of Plasmodium
falciparum sporozoite from a protected donor. Malar. J. 3(1):28 (2004).
68. Wozniak CA, McClung G, Gagliardi J, Segal M, Matthews K, Regulation of
Agricultural Biotechnology: The United States and Canada: Regulation of
Genetically Engineered Microorganisms Under FIFRA, FFDCA and
TSCASpringer Netherlands, Dordrecht, pp. 57–94 (2012).
69. Hu G, St. Leger RJ, Field Studies Using a Recombinant Mycoinsecticide
(Metarhizium anisopliae) Reveal that It Is Rhizosphere Competent. Appl. Environ.
Microbiol. 68(12):6383–7 (2002).
70. Liao X, O’Brien TR, Fang W, St. Leger RJ, The plant beneficial effects of
Metarhizium species correlate with their association with roots. Appl. Microbiol.
Biotechnol. 98(16):7089–96 (2014).
71. Blancke S, Van Breusegem F, De Jaeger G, Braeckman J, Van Montagu M, Fatal
attraction: the intuitive appeal of GMO opposition. Trends Plant Sci. 20(7):414–8
(2015).
72. Creasy S, Dowd J, Stringer G, HC 162 Science communication and engagement
Eleventh Report of Session 2016-17 Report, together with formal minutes relating

This article is protected by copyright. All rights reserved.

 to the report Stephen Metcalfe MP (Conservative, South Basildon and East
Thurrock) (Chair) Victoria Borwick MP (Conservative, Kensington). (2017).
73. Kahan DM, Peters E, Wittlin M, Slovic P, Ouellette LL, Braman D, et al., The
polarizing impact of science literacy and numeracy on perceived climate change
risks. Nat. Clim. Chang. 2(10):732–5 (2012).
74. Marris C, The Construction of Imaginaries of the Public as a Threat to Synthetic
Biology. Sci. Cult. (Lond). 24(1):83–98 (2015).
75. Vitale J, Vognan G, Ouattarra M, Traore O, The commercial application of GMO
crops in Africa: Burkina Faso’s decade of experience with Bt cotton. (2010).
76. Gakpo JO, Burkina Faso cotton industry wants to bring back GMO seeds -
Alliance for Science. https://allianceforscience.cornell.edu/blog/2018/02/burkina-
faso-cotton-industry-wants-bring-back-gmo-seeds/ [accessed 2018 Sep 18]
77. Gakpo JO, How African nations expect to learn from Burkina Faso’s GMO Bt
cotton breeding problems - Genetic Literacy Project.
https://geneticliteracyproject.org/2018/01/05/african-nations-expect-learn-burkina-
fasos-gmo-bt-cotton-breeding-problems/ [accessed 2018 Sep 18]
78. Gakpo JO, Burkina Faso is moving forward with GMO research - Alliance for
Science. https://allianceforscience.cornell.edu/blog/2018/01/burkina-faso-is-
moving-forward-with-gmo-research/ [accessed 2018 Sep 18]
79. Milner RJ, Metarhizium flavoviride (FI985) as a promising mycoinsecticide for
Australian acridids. Mem. Entomol. Soc. Canada 129(S171):287–300 (1997).
80. Liao X, Lu H-L, Fang W, St. Leger RJ, Overexpression of a Metarhizium robertsii
HSP25 gene increases thermotolerance and survival in soil. Appl. Microbiol.
Biotechnol. 98(2):777–83 (2014).
81. Fang W, St. Leger RJ, RNA binding proteins mediate the ability of a fungus to
adapt to the cold. Environ. Microbiol. 12(3):810–20 (2010).
82. Hu X, Xiao G, Zheng P, Shang Y, Su Y, Zhang X, et al., Trajectory and genomic
determinants of fungal-pathogen speciation and host adaptation. Proc. Natl. Acad.
Sci. U. S. A. 111(47):16796–801 (2014).
83. Kamareddine L, Fan Y, Osta MA, Keyhani NO, Expression of trypsin modulating
oostatic factor (TMOF) in an entomopathogenic fungus increases its virulence
towards Anopheles gambiae and reduces fecundity in the target mosquito.
Parasit. Vectors 6(1):22 (2013).
84. Fan Y, Borovsky D, Hawkings C, Ortiz-Urquiza A, Keyhani NO, Exploiting host
molecules to augment mycoinsecticide virulence. Nat. Biotechnol. 30(1):35–7
(2012).
85. Fan Y, Pereira RM, Kilic E, Casella G, Keyhani NO, Pyrokinin β-Neuropeptide
Affects Necrophoretic Behavior in Fire Ants (S. invicta), and Expression of β-NP in
a Mycoinsecticide Increases Its Virulence. PLoS One 7(1):e26924 (2012).
86. Chantasingh D, Kitikhun S, Keyhani NO, Boonyapakron K, Thoetkiattikul H,

This article is protected by copyright. All rights reserved.

Pootanakit K, et al., Identification of catalase as an early up-regulated gene in
Beauveria bassiana and its role in entomopathogenic fungal virulence. Biol.
Control 67(2):85–93 (2013).

This article is protected by copyright. All rights reserved.

Accepted Article

Figure 1 Legend: Life history schematic of a typical adult female An. gambiae in a malaria endemic region.

122x30mm (300 x 300 DPI)

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Accepted Article

Figure 2 Legend: Four-phase development and deployment of a new vector control strategy; green, yellow
and red dots represent progress of transgenic entomopathogenic fungi for mosquito control (adapted
from47,48).

329x183mm (300 x 300 DPI)

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Accepted Article
Fo
rP
ee
rR
ev
iew

Figure 3 Legend: Strategies for modifying Metarhizium pingshaense to better prevent Plasmodium
transmission by anopheline mosquitoes in the open field. A) Pathogen induced increased susceptibility to
insecticide on a mosquito net; B) spores engineered to resist abiotic stresses (green bars in inset); C)
Penetrant Metarhizium releasing insecticidal and plasmocidal agents into hemocoel; D) Blood fed mosquito
resting on a black sheet impregnated with Metarhizium.

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