In 2013, it had been known that mosquitoes’ microbiome affects their susceptibility to disease, as studies had shown that the presence of certain bacteria affected the development of some diseases. One example is the Wolbachia bacterium, which is vertically transmitted from female mosquitoes to their offspring, which was the focus of several studies due to its ability to increase resistance to many diseases in the mosquitoes it infected. For the two decades prior, scientists had been trying to see how Wolbachia infection could affect plasmodium resistance in anophelines, but anopheles mosquitoes aren’t naturally infected by the bacterium, and researchers were not able to induce and maintain Wolbachia infection. However, one research team was eventually identified a Wolbachia strain called wAlbB, which came from Aedes mosquitoes, that could persist in a. stephensi mosquitoes.

Researchers injected A. stephensi embryos with wAlbB amplified the DNA from the females that developed from the embryos. They created a line of mosquitoes that descended from one infected female and measured infection frequency for 34 generations, and found that every descendant was infected with the wAlbB strain. This showcases the stability of the infection caused by the mechanism used, supporting its potential for implementation in wild populations. Transmission efficiency was tested by randomly selecting 60 total offspring in generations 9, 10, and 11 and performing diagnostic PCR, all of which were shown to carry wAlbB, along with using FISH (fluorescent in situ hybridization), to visualize the site of infection inside cells. FISH utilizes fluorescent probes that mark specific DNA sequences, such as ones found in wAlbB. Observing the location of the fluorescent probes showed researchers how many offspring were infected, which was 100%, along with allowing them to compare bacterial colonization sites with other mosquito species, which was consistent with A. albopictus and A. aegypti mosquitoes. After ensuring that infection would persist over several generations, researchers introduced varying ratios (5, 10, and 20%) of infected females to cages of 100 uninfected (LIS) mosquitoes of equal proportions of males and females, along with 100 infected males at every generation. By the eighth generation, the entire population was infected regardless of the ratio of infected females and stayed that way for subsequent generations. The ability for infection to overtake and persist uninfected populations show the potential of wAlbB as a means for decreasing malaria transmission, but this mechanism requires a large amount of infected males to be regularly released, so mechanisms should be investigated to isolate mass amounts of male mosquitoes.

To test if wAlbB infection suppresses the development of plasmodium, researchers fed a highly concentrated gametocyte culture of the parasite to infected mosquitoes, LIS mosquitoes, and infected mosquitoes that had been treated with tetracycline, an antibiotic that kills wAlbB. They recorded plasmodium development at various stages and found that the quantity of oocysts that formed in the infected mosquitoes was significantly smaller than in both of the controls. The quantity of sporozoites in the salivary glands of infected mosquitoes was 3.4 times smaller than the LIS mosquitoes, which is crucial because sporozoites in the salivary glands are what are transmitted to human hosts. To identify the way wAlbB inhibits plasmodium development, researchers monitored levels of hydrogen peroxide in infected and LIS mosquitoes. Hydrogen peroxide is a ROS (reactive oxygen species), which has been shown to impair plasmodium development in anophelines. Previous studies have shown that Wolbachia infection increases the production of ROS in Aedes, so detecting enhanced levels of ROS would indicate the mechanism of wAlbB for decreasing plasmodium success. Researchers found that ROS levels were heightened throughout the fatbody, midgut, and the entire body in infected mosquitoes in comparison to LIS mosquitoes, supporting the concept that it is responsible for suppressing plasmodium development.

However, use as an antimalarial tool only works if the presence of infected mosquitoes can persist in wild populations, so impacted fitness could jeopardize success. Researchers monitored fitness by recording what percent of laid eggs hatched and found that descendants of the original infected female had impaired hatching rates. While LIS mosquitoes experienced a hatch rate of 91.0%, only 52.4% of eggs from two infected parents hatched. However, when infected males mated with LIS females, only 1.2% of eggs hatched, which is ideal for inhibiting malaria transmission. Since infection is only vertically transmitted by the mother, a low hatch rate is ideal for LIS offspring, as it reduces the reproductive rates of mosquitoes that don’t carry wAlbB. Hatch rate rose to 85.9% when infected mosquitoes were treated with tetracycline, indicating that the reduced hatch rate was a result of Wolbachia infection. wAlbB’s cost to fitness could potentially be the demise of its prospective role as a means to prevent malaria transmission, because infected mosquitoes could be outcompeted by wild mosquitoes. The research team noted that reduced hatch rate is not observed in A. aegypti mosquitoes infected with the same strain, and proposed that the reduced rate might be due to using mice to feed the mosquitoes. This is because mice are not a natural host for A. stephensi, and reduced hatch rates have been observed during a study in which Aedes mosquitoes infected with a Wolbachia strain were fed with nonhuman blood in comparison to human blood.

Wolbachia certainly has potential for use as an antimalarial tool, but its impact on reproductive success might prevent it from being implemented in malaria control strategies. Research beyond this paper is warranted to see if impairments to fitness could be mitigated, or if other methods should be investigated. Either way, knowledge about resistance to plasmodium due to biotic factors is valuable and contributes to future research about malaria control.

Okay, so I really should have covered this article before the other one on symbiotic bacteria that inhibit malaria development (see: A Gut Feeling)… but I just found this one last week! What’s a girl to do?

This one will go before the other one in my honors project, because that one is more recent, seems like it would be more successful, and actually involves genetic modification, which is kind of a focus of my project. I think putting this one first will be a nice introduction to how a mosquito’s microbiome can affect disease success before we move into refinement through genetic modification.

It’s really interesting that last week’s article was about producing large quantities of males and was immediately followed by one that warrants the mass release of males. It will also go before the Ifegenia article in my project, which is nice because readers will see exactly how something like Ifegenia can be applied. Also, it’s nice that this paper mentioned using FISH, which is something we recently learned about in my biology class! I love it when I can apply my class knowledge in a deeper scientific context.

This was the last skeeter article! I don’t know whether to be sad or elated. I’m definitely relieved that I don’t have to break down dense scientific papers (although this one wasn’t bad at all), but I did really enjoy learning about this topic, and I know you guys did too! It’s pretty likely that I’ll revisit this in the future, because malaria isn’t going away any time soon, but in the meantime, I’m going to enjoy the break. I think I’ll post my entire honors project at some point so you can read the comprehensive compilation, because although there won’t be anything new, it will be slightly edited so it fits the criteria of the project. Plus, you’ll be able to see it all in its full glory! But next week will probably be the last section of my English paper. Stay tuned to learn with me!

Guowu Bian et al., Wolbachia Invades Anopheles stephensi Populations and Induces Refractoriness to Plasmodium Infection.Science340,748-751(2013).DOI:10.1126/science.1236192