Some 400 million years ago, the first insects inhabited the Earth. Their descendants colonized the planet, adopting different shapes and sizes, giving rise to millions of distinct species. This immense diversity stems from changes in insect genomes that allowed them to adapt to new environments over time. Many of these genetic changes, known as mutations, occurred through duplication—an error in DNA replication responsible for many evolutionary innovations in the animal kingdom, such as insect flight. The insulin receptor (InR) gene is one example of gene duplication.

“The insulin receptor (InR) pathway is a cellular signaling route that regulates key processes such as growth, cell proliferation, longevity, sugar levels, and reproduction,” explains David Pujal, a PhD student at IBE and co-first author of the study.

Currently, all winged insects have two copies of the InR gene due to a duplication that occurred approximately 400 million years ago. Additionally, recent studies have revealed a second duplication of this gene in the common ancestor of cockroaches, termites, mantises, and stick insects about 350 million years ago. However, it remained unclear why this error persisted in these insects to the present day.

Now, a study led by the Institute of Evolutionary Biology (IBE)—a joint center of the Spanish National Research Council (CSIC) and Pompeu Fabra University (UPF)—reveals that the two most recent copies of the InR gene may reinforce the function of the ancestral insulin receptor, which is essential for the survival of cockroaches and closely related groups. The team suggests this error could have provided an evolutionary advantage for these insects and sheds light on mechanisms of evolutionary innovation in animals.

Gene duplications: a source of evolutionary innovation

Gene duplications are frequent and can range from duplicating a few DNA bases to copying an entire genome. These are random errors that, depending on their effect on the individual, will either be eliminated or persist in the species’ genome over generations.

“Mutations occur constantly and randomly in the genome. When they affect a gene of vital importance, the individual often doesn’t survive, and the mutation is not inherited. But if the mutation provides a benefit, allowing the individual to produce more offspring compared to others in its species, the mutation, inherited by descendants, is likely to become fixed,” explains José Luis Maestro, leader of the study and principal investigator of the Nutritional Signals in Insects group at IBE.

Generally, mutations that accumulate in a duplicated gene progressively blur its original function until it becomes useless. However, these changes sometimes offer an evolutionary advantage to the individual. The gene might develop a new function (neo-functionalization) or reinforce a vital function for survival (sub-functionalization). In this way, evolution turns error into virtue: gene duplication creates an opportunity for change and even improvement.

With this premise, the IBE team sought to determine why cockroaches and their closest relatives have preserved three copies of the same gene for more than 350 million years. To do so, they conducted functional studies on the reproduction of the cockroach Blattella germanica. Their analysis found no signs of neo-functionalization in any case. However, the vital role of the insulin pathway held the answer.

Female of Blattella germanica with ootheca. Credit to Cristina Olivella.

“The insulin receptor is essential in animals. That’s why we believe having an extra copy of this gene may have reinforced its function, which was key to the persistence of the new copies,” notes Maestro. “The redundancy of genes that perform the same functions can provide stability in critical processes,” says Rosa Fernández, principal investigator of the Metazoa Phylogenomics Lab at IBE, who participated in the study.

Evolution has strengthened insulin signaling in cockroaches

To identify the functions of the InR gene copies, the team reduced the activity of each in adult Blattella germanica specimens. They used RNA interference, a molecule capable of specifically binding to the different messenger RNA copies of the insulin receptor and deactivating them. Through these tests, they discovered that reducing the levels of InR1 and InR3 had no noticeable effects on Blattella. However, blocking InR2 caused significant inhibition of the insulin receptor pathway. This revealed InR2 to be the most active copy—or the “hardest worker of the trio,” as the group describes it—with the highest expression levels in all tissues studied.

“InR2 is the copy that retains the gene’s primary activity, while the other two act as reinforcements. This is called genetic redundancy. InR2 would be the lead singer of a musical group with two backup singers, capable of providing support and continuing the song when needed,” explains Xavier Bellés, principal investigator of the Insect Metamorphosis Evolution group at IBE, who also participated in the study.

Thus, the study determined that the three InR receptor copies have been conserved across generations to reinforce the vital function of insulin in cockroaches. This process is, therefore, an example of sub-functionalization. However, the team does not rule out the possibility that specific functions for the different InR gene copies could be identified in other processes or species with this gene triplication.

“We have conducted functional studies on Blattella germanica, but many aspects of its physiology—and that of other related insect groups—remain to be explored and could reveal new functions for one of the InR gene copies,” concludes Maestro.

Referenced Article:

Pujal, D., Escudero, J., Cabrera, P., Bos, L., Vargas-Chávez, C., Fernández, R., Bellés, X., & Maestro, J.L. (2024). Functional redundancy of the three insulin receptors of cockroaches. Insect Biochemistry and Molecular Biology, 172, 104161. https://doi.org/10.1016/j.ibmb.2024.104161