Weaponized fungus takes on the mosquito

P Rajendran

A mosquito infected with a Metarhizium fungus that was labeled with a green fluorescent protein gene

An Anopheles mosquito infected with Metarhizium anisopliae labeled with a gene for green fluorescent protein. Photo: Weiguo Fang, University of Maryland

Fungi could be one answer to the world’s mosquito problem, or so suggests recent research done by researchers from different parts of the globe.

Mosquitoes have had the dubious distinction of carting around the germs for some of the most unpleasant diseases around: malaria, dengue, zika, chikungunya, yellow fever and a host of other diseases – caused by viruses and protozoa (the latter being single-celled organisms). Bacteria, though not well-represented in the mosquito’s cargo, does have a representative in Francisella tularensis, which causes tularemia, a disease that can claim 30 percent of those infected.

Malaria, the best-known of these diseases, infects over 200 million people worldwide every year with Plasmodium, a protozoan that kills over 400,000 of those affected. While the number of deaths have been falling over the years, the malaria parasite has been developing immunity to many drugs and, perhaps more importantly, the mosquito is evolving resistance to the insecticides.

One way to deal with this is to kill the messenger, in this case by returning the favor – handing the mosquito a problem it is not equipped to handle.

These efforts have included infecting them with Wolbachia and Bacillus thuringiensis israelensis, and using of mosquito larvicides such as BinAB, a toxic bacteria-generated crystal whose structure was recently revealed. BinAB is effective against Culex and Anopheles, two of the three species that carry the most prominent mosquito-borne diseases, but not Aedes (carrier of the delightful trio of zika, chikungunya and dengue viruses).

Antibiotics have also been shown to make mosquitoes more likely to die of malaria. The story goes that the Plasmodium normally weakens the insect’s food canal thus letting out gut bacteria. The presence of these bacteria spur the mosquito’s granulocytes to fight off the infection and so be less likely to pass it on to humans.

Mycosis for mosquitoes

And so we come back to fungi. One variety of Metarhizium had often been shown to infect many insects, including ecologically beneficial species as moths, beetles, ants, and even plants such as cowpea and cucumber.

But, according to Brian Lovett, a graduate student in the University of Maryland Department of Entomology and co-author of the paper, things are a wee bit more nuanced.

While different Metarhizium species are known to infect a wide array of insects, the team is working a strain of Metarhizium pingshaense that specifically infects mosquitoes, he said. M. pinghaense does latch on to mosquitoes though not other possible hosts, such as bees – which is why the researchers are working with this strain for mosquito control.

In general, Metarhizium spores, called conidia, stick to the cuticle, the outer surface of the mosquito or another suitable insect host, and germinate there. It then generates units called appressoria that can work its way into the mosquito or a wide variety of susceptible insects. This process is aided by proteins that weaken and break down the cuticle. The conidia are protected from sunlight by special pigments, enzyme versions of sunglasses – keeping out both heat and harmful ultraviolet rays.

Once the fungus is in the hemocoel cavity, home to hemolymph, that sluggish, insect equivalent of blood, it can develop at leisure, protected possibly by a gelatin-like material that offers no purchase to the host immune system. Finally, the well-fed parasite fling branches called hyphae out of the dead insect to make spores that go forth and multiply anew.

While this may seem bad enough treatment for even a mosquito, the research team was not quite satisfied. So into the fungal cells were pumped some genes for venom from a variety of spiders and one species of scorpion. They included a promoter (a gene fragment that triggers the activation of these genes) that would let the toxins be produced only when the fungus is in the hemocoel, through which flows the hemolymph, the insect equivalent of blood. They also tossed in a gene for a red fluorescent protein (one that glows red under suitable light) in the wild-type mosquito (shortened to Met-RFP), and a green fluorescent protein in one (Met-Hybrid) that incorporated genes for venom from the Blue Mountains funnel-web spider, Hadronyche versuta. This venom targets potassium and calcium channels.

A control group of mosquitoes were exposed to the non-mutant, and correspondingly less deadly, M. pinghanense.

Mortality and transmission of fungus-exposed mosquitoes

a) Interest in blood feeding was measured in time that mosquitoes exposed to the wild-type and two mutant varieties of M. pinghaense spent near a guinea pig host just out of reach. This measure of interest was quantified as the percentage of mosquitoes remaining closest to the host. The“*” denotes no significant differences in mosquito choices with or without a host.
b) Light areas represents the percentage of mosquitoes surviving each treatment, while the dark ones represent the percentage of mosquitoes in each treatment that are alive and would seek a host to blood feed on (and thus be capable of transmitting malaria). The upper dashed line represents the LT50 while the lower dashed line represents the 80% control threshold suggested by the World Health Organization for a successful vector control agent.
Photo: Scientific Reports, CC NY

AaIT, the sole toxin in the arsenal representing the scorpion class of chemical weapons, attacks sodium channels, but is particularly effective against mosquitoes that are resistant to the insecticide group, pyrethroids.

According to the paper, “Since AaIT and pyrethroids bind to different sites on insect [sodium voltage] channels…, mutations that confer resistance to pyrethroids actually increase binding of AaIT.”

Knowing the post-prandial habits of mosquitoes, the researchers left strategically placed fungi-coated sheets that mosquitoes could rest after a blood meal. They exposed the insects to a maximum of an hour with M. pinghaense.

A BBC video describes the use of Metarhizium fungus in biocontrol:

The summing up

After testing for 14 days, the researchers gauged the resultant infections in human-reared Anopheles gambiae mosquitoes and in wild-caught insecticide-resistant An. coluzzii and An. gambiae.

“Our most potent fungal strains engineered to express multiple toxins, are able to kill mosquitoes with a single spore,” Brian Lovett said in a UMD press release. Met-RFP did not do too badly either.

Reports of Metarhizium infection in humans are rare. TC found two reports describing infection in vertebrates. Both from the 1990s, they documented the case of three patients and a cat that were infection and none with compromised immune systems.

As Lovett put it, “Anything is possible in biology, but it is exceedingly improbable that Metarhizium would switch hosts to vertebrates. The human body temperature is considerably higher than the preferred growing temperature of Metarhizium fungi.” He pointed out that vertebrates lack the chemicals and topology on insect cuticles that the fungi target and that Metarhizium fungi, abundant in soils worldwide, are routinely applied in agriculture with no reported adverse effects on people.

According to Lovett, “Metarhizium fungi are among the most thoroughly vetted and safest biocontrol agents, but understanding and mitigating their risks remains an important focus of our research.”

Their research was published in the June 13 issue of Scientific Reports. The original report is available

Here is an external link to video of Prof. St.Leger’s talk on his lab’s work at the NASA Goddard Space Flight Center Scientific Colloquia Series

Featured image: The growth of a modified Metarhizium (anisopliae in this case) on the body of a diamondback moth (Plutella xylostella). Photo: National Taiwan University, CC NY

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