Another month, another paper to blog.
One of the most fascinating things about studying biology is finding out the numerous techniques living things use to survive through adversity. This month’s paper digs into an alliance between a tick species Ixodes scapularis and a bacterium Anaplasma phagocytophilum to help the pair survive through long winter months.
In places where winters can get extremely cold, people will oftentimes use antifreeze to help protect their car engines. Many cold-blooded animals (ectotherms) survive harsh winters in the same way. They produce antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) which are believed to bind to ice crystals and limit their growth and ability to damage the organism.
As Ixodes ticks are fairly active during winter months and are a known carrier of Anaplasma phagocytophilum which is a cause of human granulocytic anaplasmosis, a team of researchers from Yale Medical decided to investigate whether or not Anaplasma had any impact on the ability of Ixodes to survive cold weather.
Panel A of Figure 1 (below) is a survival curve. It shows what % of ticks which have Anaplasma (in dark black circles) and which don’t (in white circles) survived being placed in –20 degrees (Celsius, of course, not Farenheit: this is science after all!) for a given amount of time. While all the ticks died after 45 minutes, at any given timepoint more ticks with Anaplasma survived than the ticks without. While only ~50% of ticks without Anaplasma survived after ~25 minutes in the cold, over 80% of ticks with the bacterium survived!
What could explain this difference? The researchers suspected some sort of antifreeze protein, and, after combing through the tick’s genome, they were able to locate a protein which they called IAFGP which bore a striking resemblance to other antifreeze glycoproteins. But, was IAFGP the actual antifreeze mechanism which kept Ixodes alive? And did Anaplasma somehow increase its effectiveness?
Panel C of Figure 4 (above) shows the key findings of the experiments designed to answer those two questions. Along the vertical axis, the researchers measured the amount of IAFGP gene expression (relative to the gene expression of a control, actin [a structural protein which shouldn’t vary]). Along the horizontal, the researchers tested four different temperature states (23, 10, 4, and 0 Celsius) with Ixodes ticks that were carrying Anaplasma (dark circles) and those that were not (white circles). Each individual circle is an individual tick and the line is the average value of all the ticks in the experimental group (the reason its not in the middle is because the vertical axis is a log scale). This sort of chart is one of my favorites, as it packs in a lot of information in one small area but without generating too much noise:
- The lines get higher the further to the right we get: Translation: when temperatures go down, IAFGP levels go up – as you would expect if IAFGP was an antifreeze coping mechanism for Ixodes. (And if you could see Panel B of Figure 4, you’ll notice that IAFGP levels at 4 Celsius and 0 Celsius are statistically significantly higher than at 23 and 10)
- The black dots on average are higher than the white dots: Translation: just carrying Anaplasma seems to push Ixodes’s “natural” levels of antifreeze protein up. And, judging from the P value comparisons, the differences we are seeing are statistically significant.
So, it would seem that IAFGP is somehow related to the affect of Anaplasma on Ixodes, but, is that the only link? To test that, the researchers used an experimental technique called RNA interference (RNAi) which allows a researcher to shut down the expression of a particular protein. In this case, the researchers shut down IAFGP to see what would happen.
These results are interesting. Although, sadly, the charts (Panels B and F of Figure 5) are not on the same scale and are for different experiments, the numbers are striking. In Panel B, the researchers tested for the survival of ticks which were given a control RNAi (simulates the RNAi process except without what it takes to actually silence IAFGP, white circles) versus those which had IAFGP shut down via RNAi (white triangles). As you can see from the chart, after 25 min at –20 degrees Celsius, the control group hit 50% survival whereas the RNAi group’s survival rate plummeted to only 20%.
The researchers then repeated the experiment with Ixodes ticks which were given control RNAi (black circles) vs. the real thing (black triangles) and then allowed to feed on Anaplasma-infected mice for 48-hours. These ticks were then tested for survival after 50 min at –20 degrees Celsius. As you can see in Panel F, a 75% survival level amongst Anaplasma carrying ticks became less than 50% when IAFGP was shut down with RNAi.
All in all, a very simple positive-control, negative-control experiment showing a pretty clear linkage between Anaplasma, IAFGP gene expression levels, and the ability of Ixodes ticks to survive the cold. However, a few things still bug me about the study and stand out as clear next steps:
- Panels B and F of Figure 5 are fundamentally different experiments, but presented as comparable. At face value, its hard to tell if IAFGP is the primary mechanism for how Anaplasma alters tick response to the cold. The survival levels of Anaplasma-carrying ticks when IAFGP is shut down is still higher than the survival levels of Anaplasma-free ticks which also undergo the RNAi – but this could be a result of the different experimental conditions (feeding conditions and time). The paper text also reveals that the –20 degrees for 50 min condition was selected because it was supposed to be the point at which there was 50% survival for that particular experimental condition – but clearly, the control group was experiencing 75% survival (and the group with IAFGP shut off was at 50%). Something is off here… but I’m not sure what.
- Most of this research was conducted on a very abstract level – showing the impact of IAFGP expression levels on cold survival. While the RNAi experiments are very compelling, the lack of clear functional studies is problematic in my mind as I cannot tell from this data if IAFGP is directly responsible for cold survival or linked to other, potentially more important responses to cold.
- No mechanism was proposed for how Anaplasma increases IAFPG levels in Ixodes. Understanding that would be very powerful and could unveil a whole world of cross-species gene regulation which we were previously unaware of (and could reveal new potential targets for medical treatments of diseases borne by insects).
Regardless of my criticisms, though, this was an interesting study with a very cool result. However, its probably of no comfort to people who have to deal with ticks which can survive cold winter months…
(Image credit – tick) (Figures 1, 4, and 5 from paper)
Paper: Neelakanta, Grisih et al. “Anaplasma phagocytophilum induces Ixodes scapularis ticks to express an antifreeze glycoprotein gene that enhances their survival in the cold.” Journal of Clinical Investigations 120:9, 3179-3190 (Sep 2010) — doi:10.1172/JCI42868