Insects lay eggs, for most insects their role as parents stops there. The eggs have to fend for themselves as soon as they are laid. However, this is not without danger. They are exposed to the elements, predators and microorganisms. Nonetheless, insect eggs are not helpless. Early during development they form an extraembryonic membrane called the serosa (you can find more info about extraembryonic development here). This membrane is a single celled layer which separates the embryo from the dangerous world outside. So this membrane has the potential to protect against threats from the outside, such as infections. Although much is known about the immune response in insects (more info here), it remained unknown whether insect eggs are able to defend against pathogens. In a recent publication in the journal “Developmental and Comparative Immunology” we show that insect eggs are able to mount an impressive immune response against infection.
With or without serosa
The eggs of the flour beetle Tribolium castaneum produce the serosa early during development. However, the eggs of the fruit fly do not produce a serosa. Instead of a serosa they produce the amnioserosa, a membrane which will never enfold the embryo. This amnioserosa covers the top of the egg (see the figure below). We hypothesized that because the serosa enfolds the embryo completely, it is a prime candidate to protect against infection. This would also mean that the eggs of the fruit fly are not protected against infection. To verify that it is specifically the serosa which protects against infection (and not species specific adaptations) we made flour beetle eggs which do not have a serosa (egg in the middle, figure below). To obtain these serosa-less flour beetle eggs we performed RNAi on the gene Tc-zen1 (more info on RNAi here). In these serosa-less eggs, the amnion covers the top of the egg, much like the amnioserosa in the fruit fly egg. According to our hypothesis, these serosa-less flour beetle eggs should also not be protected against infection.
By infecting both the eggs of the fruit fly and the eggs of the flour beetle with bacteria, we stimulated the immune response in these eggs (as far as they were able to mount an immune response). To check whether we stimulated the immune response properly we also infected adult beetles and flies. To be sure we were looking at the response to bacteria we also pricked eggs and adults with a sterile needle. We already knew which genes are involved in the immune response (for more info about the insect immune response look here). So we wanted to measure the amount of RNA of some of the involved genes (generally, more RNA means more protein). To measure the amount of RNA we isolated RNA from infected and uninfected eggs and adults of both the flour beetle and the fruit fly. Than we measured the difference in the amount of RNA between uninfected and infected eggs and adults with a technique called qPCR. This technique provides us with a fold change, if the uninfected egg has 10 RNA molecules of a specific gene and the infected egg has a 100 copies of this same gene it is expressed 10x higher in infected eggs.
By measuring this fold change for several genes, we verified that the infection with bacteria induced higher expression of immune genes in adult flies and beetles. We also found higher expression of immune genes in flour beetle eggs. In contrast, the eggs of the fruit fly showed very little to no upregulation of any of the genes tested. I show the results for one of the tested genes below. This gene is called attacin and codes for an antimicrobial peptide. It is clearly visible that after infection (white bars) the RNA for attacin is present in much higher numbers than when uninfected (black bars). The exception is the fruit fly egg, in which both white and black bar are hardly visible (which means that the differences in the amount of RNA are small).
We measured the same genes in eggs with and without a serosa. In this way we could assess whether it is specifically the serosa which protects against infection. Like before we look at the example of the antimicrobial peptide attacin. In the figure below you can see that eggs with a serosa clearly have more attacin RNA after infection (white bar). Contrary to eggs with serosa, the amount of attacin RNA in eggs without a serosa is almost equal in infected and uninfected state! This clearly indicates an important role of the serosa in the protection against infection of the embryo.
In conclusion, the eggs of the flour beetle are well armed against infection. Very different from the flour beetle eggs, the fruit fly eggs show almost no immune response. By measuring the immune response in serosa-less flour beetle eggs we show that this immune response is dependent on the presence of the serosa. And so we see how the serosa helps flour beetle eggs survive embryogenesis!
Jacobs, C. G. C. and van der Zee, M. Immune competence in insect eggs depends on the extraembryonic serosa. Developmental and Comparative Immunology. http://dx.doi.org/10.1016/j.dci.2013.05.017