Investigating changes in protein expression in an insect with a pleiotropic mutation
Dear Laidlaw scholars, I hope your research is going well! This summer and last, I have joined the Shuker laboratory in investigating a mutant strain of Lygaeus simulans (a species of seed bug). This mutation is of interest as the strain may serve as a new model species for the role of pleiotropy (a single gene influencing multiple seemingly unrelated traits) and supergenes in evolution. My previous blog entry (17/07/18) gave an explanation of the supergene concept and described some classical examples. This year I will I outline the experimental approaches used to investigate this mutation.
Our study species, Lygaeus simulans, is found throughout northern and central Europe, often in proximity to its sister species, the closely related Lygaeus equestris. L. simulans possesses chemical defenses and displays a red-black warning coloration to notify predators of such (this is called aposematism). In 2013 a mutant L. simulans strain arose in laboratory which, in contrast to the aposematism of the wild-type, has a pale coloration. As changes in coloration have historically been associated with supergene complexes, an investigation into the nature of the mutation began.
The first experiments were concerned with the inheritance patterns of the mutation and the life-history traits of the mutant strain. Traits can be divided into those controlled by a single genomic locus (this could be a supergene complex or a single gene) and those controlled by many loci (polygenic traits). The inheritance pattern of the mutation allows discrimination between these possibilities. The life-history of the mutant was also investigated to search for other traits alongside coloration which may differ between the mutant and wild-type.
Compared to the wild-type, mutant females have lower longevity and fecundity, while mutant males have a longer body length (of significance because there is some evidence that body length is correlated with mating success in males of this species). This suggests that the mutation may be sexually antagonistic (advantageous to one sex at the detriment of the other). Furthermore, later experiments identified differences in hybridization capability of mutant and wild-type with the sister species, Lygaeus equestris. The considerable portfolio of differences in life-history between mutant and wild-type indicates that the mutation is pleiotropic, i.e. lots of genes are affected, directly or indirectly. From the inheritance patterns of the mutation it was inferred that the trait is controlled by a single genomic locus. The combination of pleiotropy and control from a single locus leaves two possibilities for the nature of the mutation. Either the mutation is in a highly pleiotropic gene, or supergene formation has occurred.
After demonstrating the pleiotropy of the mutation, we are now interested in finding out whether the disparity in life-history traits extends to protein expression. This will not directly tells us whether the mutation is in a single pleiotropic gene or a supergene complex, but from this analysis we can identify the scale of differences in protein expression (are changes restricted to one or many cellular pathways for instance?) and the specific pathways which are altered. This will provide more information about the nature of the mutation. Furthermore, identification of altered pathways opens the door for their biochemical characterisation.
To analyze protein expression, we will be using a mass spectrometry technique called SWATH (Sequential-Window Acquisition of all Theoretical Mass Spectra). Mass spectrometry is a technique which identifies and measures the abundance of molecules in a sample. There are multiple different types of mass spectrometry which have advantages and disadvantages in different experimental contexts. We are using SWATH because it produces an unbiased scan of all proteins in the sample while maintaining a high resolution. With the proteomic information in hand the next step will be using genomic techniques to test for the presence of a supergene structure. Many thanks to my supervisor, Dr David Shuker, and his PhD student, Vicki Balfour, who very patiently taught me how things work in the lab and completed much of the research into the mutant. Also thanks to Dr Terry Smith for his expertise in mass spec.