Unlike vertebrates, which have vast and adaptive responses to infection that can be combined for specific immune targets in countless ways, the immune response of insects is limited to the genes in their DNA to fight off pathogens, which is a finite and less adaptive system. One way insects fight off infection is with pattern recognition receptors (PRRs), which are proteins that search for molecular patterns that match known pathogens. These pathogen-associated molecular patterns (PAMPs) are present in pathogens, but not the host organism’s cells, so when PRRs find PAMPs, they bind to them, signaling the presence of a pathogen to the rest of the cell. This activates immune pathways that lead to the production of molecules that fight pathogens, defending the cell from dangerous invaders. One such PRR in mosquitoes is AgDscam, which has many forms, and exactly what forms are produced is determined by the IMD pathway. This is of particular interest because some forms of AgDscam are extremely efficient at fighting plasmodium infection, and can therefore be used to combat the spread of malaria.

To examine the effect of the IMD pathway on AgDscam production, researchers used a special microscope to observe the effects of activating or inhibiting the IMD pathway on the position of AgDscam and p. falciparum ookinetes and found that the number of times AgDscam interacted with the ookinetes was 6.4 times greater when the IMD pathway was activated than when it was inhibited.

The IMD (immune deficiency) pathway in mosquitoes plays a major role in killing off plasmodium falciparum before it can multiply and colonize in mosquitoes’ salivary glands and be spread to human hosts. This pathway, along with the Toll pathway that specializes in inhibiting p. berghei, which causes malaria in rodents, control the production of molecules that prevent plasmodium development, which they do by activating or inactivating the production of splicing factors. 

Splicing factors are proteins that help choose what kinds of proteins the cell will make by influencing the spliceosome, something that picks what parts of pre-mRNA will be deleted and what parts will turn into mRNA, which gets turned into protein. Pre-mRNA is made up of introns and exons, and introns don’t carry instructions for making proteins, so they are removed from the pre-mRNA with help from splicing factors. Splicing factors aid in picking which exons will be cut out and which will be joined together, and the final string of mRNA of all the connected exons determines the type of protein that will be produced. AgDscam’s ability to fight plasmodium infection depends on how it’s spliced, so what exons are chosen and connected from the pre-mRNA. The researchers hypothesized that the best splice forms of AgDscam at preventing plasmodium development are controlled by the Rel2 transcription factor in the IMD pathway, which regulates the activity of splicing factors that control how different forms of AgDscam are cut. To test this hypothesis, they found nine AgDscam splicing factors with activity that responded to infection and observed the results of inactivating the IMD pathway by silencing Rel2. As predicted by their hypothesis, the activity of the nine splicing factors was reversed when Rel2 was silenced.

Two of the nine splicing factors examined by the researchers are Caper and IRSF1, and to gather evidence that they create various splice forms of AgDscam, some of which are more effective than others at fighting plasmodium, researchers silenced both splicing factors and found that AgDscam was spliced differently than under normal circumstances. When Caper was silenced, AgDscam resembled the alternative splicing of when the IMD pathway was inhibited, which includes fewer forms of AgDscam known to fight plasmodium. When IRSF1 was silenced, there was actually an increase in the production of antimalarial forms of AgDscam. The research team applied this knowledge with actual mosquitoes and found further evidence; silencing Caper increased the medium numbers of p. falciparum oocysts in mosquitoes’ midguts by 174%, with a median oocyst count of 54 compared to 31 in the control, and silencing IRSF1 resulted in a median of just 6 oocysts, less than one fifth of the control.

To gather more support that Caper and IRSF1 play a role in plasmodium success, the researchers also measured the amounts of their transcript produced before and after injecting mosquitoes with p. falciparum. They did this 24 hours after the mosquito had taken an infected blood meal, and found that quantities of Caper transcripts multiplied by a factor of 1.8, and IRSF1 transcripts were cut by a factor of 2.1. To confirm that different forms of AgDscam have different effects on plasmodium resistance and investigate which splice forms are most effective, the researchers genetically modified a. stephensi mosquitoes to produce short and long forms of AgDscam, Pf-S for short and Pf-L for long. They then used the Cp-1 (carboxypeptidase 1) promoter to create an abundance of the AgDscam splice forms each time the mosquito took a blood meal. The researchers fed a parasite culture to GM and wt mosquitoes and found that compared to the wt mosquitoes, the Pf-S mosquitoes had 1.5 times fewer oocysts and the Pf-L mosquitoes had 2.4 fewer oocysts. These experiments fed the mosquitoes abnormally high levels of parasites, so they were repeated with lower concentrations to mimic natural levels and while the Pf-S mosquitoes did not exhibit significant resistance when compared to the wt mosquitoes, the median oocyst count in the Pf-L mosquitoes went from 4.5 to 0, making them almost entirely resistant to p. falciparum.

AgDscam plays a major role in mosquitoes’ ability to prevent plasmodium development, and its splice forms impact parasitic success in different ways, with the Pf-L form being one of the most effective. AgDscam is a PRR, which recognizes pathogenic patterns (PAMPs) on plasmodium parasites and signals presence of the foreign invader to the cell, activating immune pathways that prevent parasitic development. It can be spliced in different ways, notably by splicing factors Caper and IRSF1, which are regulated by Rel2, a transcription factor in the IMD pathway. AgDscam has been shown to drastically decrease the amount of oocysts that form on mosquitoes’ midguts, indicating its significance in immune response against malaria and the potential it holds to reduce cases in human populations as a result.

This week’s article is long and was a nightmare to get through, but the insight it provided on the role of AgDscam and how other immune factors affect its expression is really valuable as we’re analysing the genetic response to malaria in mosquitoes. I hope you learned as much as I did from it!

Do you remember IMD and Rel2? Or even Cp (carboxypeptidase)? They were key players in the first article I covered on GM mosquitoes, and I was very interested in how exactly they impacted plasmodium resistance. If you recall, that article included tests involving Cp, Vg, and Hybrid GM mosquitoes and their resistance to malaria, so it’s really cool to see the details of the Cp line’s interaction with the immune system. Next week we will be following a continuation of this line of GM mosquitoes, examining their fitness and other factors contributing to how well they will spread their anti-plasmodium genes to wild populations. Stay tuned to learn with me!

Dong, Yuemei, et al. “Anopheles NF-ΚB-Regulated Splicing Factors Direct Pathogen-Specific Repertoires of the Hypervariable Pattern Recognition Receptor AgDscam.” Cell Host & Microbe, U.S. National Library of Medicine, 18 Oct. 2012, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3614911/.