Mosquito Eggs Without Eggshells Disrupt the Ability to Reproduce

Story by Roger L. Miesfeld and Jun Isoe | Special to the Arizona Daily Star

Up to 40 percent of the world’s population now lives in areas where Dengue virus-transmitting mosquitoes coexist with humans. Moreover, the Zika virus, also transmitted by Aedes aegypti mosquitoes, has emerged as a major infectious disease threat in most of South America, the Caribbean and parts of the United States, where nearly 150 locally transmitted cases have been reported.

Controlling mosquito populations in areas with high incidences of viral transmission is an effective means for decreasing human infection rates. However, it is important that control agents used in urban areas be environmentally safe and target processes that are mosquito selective. Alternatives to traditional approaches to mosquito control that include using mosquito traps and localized spraying have recently become available. These new methods rely on genetically-modified mosquitoes or mosquitoes that have been infected with a bacterial strain of bacteria called Wolbachia, which decreases rates of viral transmission. Clearly, multiple approaches are needed to stay one step ahead of mosquito-borne diseases that threaten human health.

One approach to mosquito control is to disrupt biochemical processes required for mosquito egg production as a means to decrease reproduction rates. Female mosquitoes feed on blood to obtain the nutrients they need to produce viable eggs. Aedes aegypti mosquitoes are capable of laying around 100 eggs three days after a blood meal. Shortly after the eggs are deposited in a moist area such as a flower pot or the edge of a pond, the eggshell hardens through a process called “tanning” in which the eggshell turns from white to brown during the maturation process. Because the drought-resistant larvae will hatch from the hardened mosquito eggs in the presence of water during the monsoon season in areas such as Tucson, the complete formation of the eggshell within a few hours of egg laying is essential to mosquito reproduction.

To discover mosquito genes that are uniquely required for mosquito eggshell synthesis in blood-fed female Aedes aegypti mosquitoes, we used a computer-based approach to identify genes that have evolved to be unique to mosquitoes and are not found in closely-related insects such as fruit flies and honeybees, nor in animals such as ourselves. Among the roughly 100 mosquito-specific genes we disrupted in blood-fed Aedes aegypti, we found one we call Eggshell Organizing Factor 1 (EOF1) that was absolutely required for completion of eggshell synthesis.

We discovered that 100 percent of the eggs laid by mosquitoes lacking the EOF1 protein were missing a tanned eggshell, and none of the larvae survived. The lethal effect of an EOF1 deficiency was in part because the eggs did not complete the tanning process required for eggshell maturation. Using a high-resolution imaging technique called scanning electron microscopy, assisted by Brooke Beam Massani at the CBC W.M. Keck Center for Nanoscale Imaging, we were able to show that the surface of the eggshell contained in the ovaries of EOF1 deficient mosquitoes was disorganized as compared with eggshells at the same time of development in control mosquitoes.

To be sure that these results were not due to genetic differences between laboratory strains of mosquitoes and mosquitoes native to Tucson, we compared the Rockefeller strain of Aedes aegypti, which have been reared in the laboratory since the 1930s, to Aedes aegypti that were captured during the 2016 monsoon season in Tucson by University of Arizona assistant professor Heidi Brown. Our experiments show that the EOF1 gene is also required for eggshell formation in Tucson mosquitoes, thereby confirming our earlier results with the Rockefeller strain of mosquitoes.

Our focus now is to discover the function of the EOF1 protein to better understand why it is required for completion of mosquito eggshell synthesis, and with this information, how it might be possible to develop bio-safe small-molecule inhibitors that selectively target blood-feeding mosquitoes. One clue has come from additional bioinformatics analysis we performed on the DNA sequence that encodes the EOF1 gene. This suggests that the EOF1 protein is required for the regulated decay of one or more proteins needed during the process of eggshell synthesis Importantly, in our initial screen for mosquito-specific genes required for the production of viable eggs in blood-fed mosquitoes, we discovered several other genes that appear to be functionally-related to EOF1 and are also under investigation as possible targets for mosquito control.

Our long-term goal is to limit the spread of mosquito-borne diseases in areas of high virus transmission to decrease the incidence of human disease. This does not mean that mosquitoes should be eliminated from our ecosystem, as that could have unknown consequences. Instead, we want to selectively reduce mosquito populations by decreasing reproductive rates at specific times of the year, such as the rainy season.

Photo Credit: Patrick O'Connor/for the Arizona Daily Star | Photo: Jun Isoe (left) and Roger L. Miesfeld

Release Date: 
Wednesday, February 1, 2017