The Stuff of Insect Nightmares: Genetically Engineered Entomopathogenic Fungi

June 14, 2019

Insects are a high protein food source with a low environmental footprint. This has made insects an attractive dish in many cultures, and the practice of eating insects (entomophagy) is on the rise in the United States. Though it may be some time before insects start regularly appearing on our plates, live insects have been on the menu for fungi for millions of years. Entomopathogenic fungi are any fungi that kill insects. They span the entire fungal kingdom, and they have evolved some awe-inspiring ways to play with their food. Fungal strategies range from turning cicadas into living spore “salt-shakers” to sending their ant prey on a macabre climb upwards to rain spores upon her unlucky sisters (David Attenborough’s description of this process is my favorite).
 
These fungi, while there are exceptions, infect insects on contact and burrow their way inside. Some fungi land on the cuticle with large spores with everything they need to drill through the insect cuticle. This chitin-protein matrix surrounds insects forming their exoskeleton with a waxy outer layer. Other fungi produce cocktails of enzymes that degrade this matrix and harvest these raw materials from the cuticle itself to drive further penetration
 
A generalized infection diagram for Metarhizium species fungi. First, a spore (also called a conidium) lands on the outside of an insect, then the fungus produces a sticky holdfast structure called an appressorium. Appresoria produce pressure and secrete enzymes to allow the fungus to penetrate through the chitin-protein matrix of the insect cuticle. When the fungus reaches the insect blood (hemolymph) it buds off producing yeast-like structures called blastospores. These blastospores proliferate through the insect blood covered in a collagen-like protein coat to avoid the insect immune system. Modified from original source.
A generalized infection diagram for Metarhizium species fungi. First, a spore (also called a conidium) lands on the outside of an insect, then the fungus produces a sticky holdfast structure called an appressorium. Appresoria produce pressure and secrete enzymes to allow the fungus to penetrate through the chitin-protein matrix of the insect cuticle. When the fungus reaches the insect blood (hemolymph) it buds off producing yeast-like structures called blastospores. These blastospores proliferate through the insect blood covered in a collagen-like protein coat to avoid the insect immune system. Modified from original source.

It’s clear that as insects evolve resistance strategies, fungi counteract with new virulence means. In the course of this race, fungi have evolved some incredibly useful natural abilities. These fungi are naturally phenomenal resources for fundamental research and application as natural living pesticides. However, we are no longer limited by their natural abilities, and it is now possible to engineer these fungi to be even more useful to us as applied biotechnologies.

Harvesting Entomopathogenic Fungi: Developing Applications

Some species infect a range of insects as generalists, while others, like the so-called “zombie fungi” (Ophiocordyceps species), have evolved to kill a preferred species of ant. While selectivity to a single species of ant is a nice party-trick, this can be a burden because they require the ant to reproduce. This means that these fungi are limited by the range of their preferred ant host, making them difficult to culture and of little use as an applied technology for insect control.
 
Ants biting the underside of leaves as a result of infection by Ophiocordyceps unilateralis.
Ants biting the underside of leaves as a result of infection by Ophiocordyceps unilateralis.

As early as 1880, generalist fungi were proposed to control insect pests. This was when the green spores of the Metarhizium genus on insect cadavers first caught the attention of immunologist Elie Metchnikof. The genus has been under study since then, changing names multiple times, as fungi do.
 
Because Metarhizium reproduce as molds, they don’t generate fruiting bodies and will happily sporulate on artificial media. While some of them can be quite selective about which insects they kill, they will grow on plates in the lab making them more cooperative to study and genetically engineer.
 
Scientists have learned an enormous amount about the biology of these fungi: scientists have sequenced genomes of multiple species, characterized enzymes they use to exploit insects in delicious detail and manipulated their genomes to identify genes that are critical for their ability to kill insects.
 
These studies have revealed that Metarhizium fungi are ubiquitous in the environment. Recently, scientists have learned that these fungi produce plant hormones that boost plant growth, and evidence suggests that for some species, their ability to kill nitrogen-rich insects is a trick to gain the upper hand in the economy of the soil. Plants trade sugar for these insect-derived nutrients, like nitrogen, which are a limited resource in the soil.
 
Due to their inherent abilities to kill insects, their ubiquity, and their indifference to humans as hosts (Metarhizium species do not infect humans), these fungi have been successfully applied to millions of hectares of land worldwide to control agricultural pests. Already, society has benefited from this partnership with fungi, but the benefit has been limited by fungal self-preservation strategies. After entering into the insect hemolymph, these fungi take their time to kill their insect hosts, absorbing resources in the insect blood and proliferating, in order to extract as many nutrients as possible. This works fine for some agricultural products: sugarcane, for example, does not have to be in pristine condition to be valuable. However, other crops where the damaged leaves or produce lower the price considerably do not have this luxury.

Killing More Efficiently: How to Improve Fungal Applications

This is where our careful characterization of these fungi over decades of research comes in handy: scientists can leverage decades of discoveries with modern molecular biology techniques to design insect pathogens that work on our time-frame. Developing these fungi as biotechnology tools is a natural extension of our long partnership with these insect pathogens.
 
One example is scientists’ ability to time expression of specific genes. Metarhizium species surround themselves in a collagenous coat to evade the insect immune system; this high-cost change in structure only occurs in insect blood. Dissecting this gene regulation has provided scientists with a promoter that is only activated in insect blood. Scientists can now use this on/off switch to program expression of any gene.
 
What types of applications does this genetic programmability offer? This genetic switch has been used to generate fungi that can produce human antibodies and antimicrobial proteins in mosquito blood. When these fungi infect a mosquito carrying malaria parasites, they then cure the mosquitos of malaria. This same promoter has also been used to generate a fungus that produces an insect-specific neurotoxin normally made by spiders. The transgene increased mosquito-killing efficacy, and these transgenic fungi were also highly effective at controlling mosquito populations in contained field conditions.
 
An Anopheles coluzzii cadaver with green fluorescent Metarhizium pingshaense sporulating on the surface. Courtesy B. Lovett.
An Anopheles coluzzii cadaver with green fluorescent Metarhizium pingshaense sporulating on the surface. Courtesy B. Lovett.

Two major hurdles to the success of these fungi in field application are UV light and heat, which inactivate the fungal spores. Scientists could search for naturally UV-resistant fungal strains (and they should), or they could engineer tolerance to environmental stresses. If you add a UV-photolyase from an archaean to the Metarhizium genome, it becomes much more resistant to UV stress. Likewise, over-expressing a native heat shock protein grants increased heat resistance to these fungi. Similar strategies could render these fungi more susceptible to environmental stresses. These genetic manipulations will allow us to determine where these fungi could spread (or not spread).

As our understanding of the genetic underpinnings of these fungi grows, so does our ability to tailor them to the unique needs of those burdened by insect-transmitted diseases or insects devouring their crops. Scientists have only scratched the surface of potential biotechnologies using these fungi as a chassis. This is a brave new world of fungal biotechnology. This world is within our reach today, and with requisite regulation and community acceptance, we could help these fungi win their evolutionary battle against insects. Our society will be better for it.
 

Author: Brian Lovett, Ph.D.

Brian Lovett, Ph.D.
Brian Lovett is a postdoctoral researcher working on fungal biology and biotechnology at West Virginia University.