Pangolins are bizarre creatures that do not seem to attract a lot of attention, but when they do, they hit the headlines big time. And usually not in a very positive way. After being labeled “the most trafficked mammal you’ve never heard of”, pangolin’s unfortunate reputation has been recently reinforced by the news suggesting that pangolins might be involved in the coronavirus outbreak as an intermediate host.
Since this finding was announced in the form of a press release, let’s not jump to conclusions and let’s wait for the publication that comes out of the peer-review. Nevertheless, this Saturday, February 15, is World Pangolin Day, and thus, it is a good time to do some PR for these fascinating animals.
In 2016, researchers published genome assemblies for two out of eight pangolin species, the Malayan (Manis javanica) and Chinese (M. pentadactyla) pangolins. Choo et al. used whole-genome shotgun sequencing of short-read Illumina libraries to generate 145X and 59X genomes of the Malayan and Chinese pangolins, respectively.
The team, which consisted primarily of researchers from Malaysia, Russia, and the US, focused on looking for loss-of-function genes that have been pseudogenized. Considering that pangolins are toothless creatures with poor eyesight, one can imagine that there’s a lot to find.
Pangolins have a highly specialized diet consisting of ants and termites, and similarly as anteaters, they have no teeth because they can just slurp up the ants and termites using their tongues. Hence, screening 107 tooth-development-related genes, Choo et al. identified a number of potential pseudogenization candidates. Specifically, they found mutations in three genes encoding proteins related to tooth development – enamelin (ENAM), amelogenin (AMELX), and ameloblastin (AMBN). Sanger sequencing was used to validate these pseudogenes and check that they are shared with the African pangolins, suggesting that the mutations occurred early on in the pangolin lineage.
“Loss of function of these genes through pseudogenization has also been reported in other edentulous vertebrates such as toothless baleen whales, birds, and turtles.” (Choo et al. 2016)
Note to myself: Add “edentulous” on the list of words that you should know to sound smart.
In a different study on the Malayan pangolin, Ma et al. (2017) performed a transcriptomic analysis focusing on the digestive system. The authors found transcripts potentially connected to various pathways related to the pangolin’s special diet, myrmecophagy. “Pangolins favor high-protein, high-fat, and high-calorie food,” and who doesn’t, really, “and they have the notable ability to digest and absorb chitin in the digestive system.”
“The chitin-degrading enzyme acidic mammalian chitinase (CHIA), which is involved in the degradation of the chitin in the insect cuticle and the peritrophic membrane of the dietary ant, was found in the amino sugar and nucleotide sugar metabolism pathway(KEGG: 00520), thus suggesting that this pathway may be directly involved in ant digestion by M. javanica.” (Ma et al. 2017)
“Ants are a protein-rich food. They contain more than 50% crude protein, according to a nutritional value evaluation, and contain more than 20 amino acids; various microelements; special chemicals, such as formic acid and herbaceous acetaldehyde, which are triterpenoid compounds, and several vitamins. Many pangolin genes are likely to be involved in the digestion of these materials because 27 of the transcripts related to the terpenoid backbone biosynthesis, and this might be one of the biological basis for their adaptions to myrmecophagy.” (Ma et al. 2017)
Talking about food, pangolins find the ants and termites using their smell, and it must be quite efficient, because a single pangolin apparently consumes more than 70 million insects per year (Ma et al. 2017). Contrary to reductions in tooth development and poor vision, olfactory receptor (OR) genes are among the most significantly expanded gene families, “possibly helping locate prey and counterbalancing poor vision” (Choo et al. 2016). Olfactory transduction was also found to be the most represented pathway in the Ma et al. (2017) Malayan pangolin study, with a total of 942 transcripts being annotated as various kinds of olfactory receptors.
Choo et al. also screened 217 vision-related genes and observed frameshift mutations and premature stop codons in two vision-related genes, BFSP2 and GUCA1C, suggesting that pangolins suffered from a loss of optical clarity and reduced rates of phototransduction. Pseudogenization of BFSP2 and GUCA1C genes has been previously identified in mice with reduced vision.
You win some, you lose some.
This is a tough one. Let me evade the topic with a fun fact: Pangolins can close their ears and nostrils to protect them from the agitated ants.
Finally, we are getting to the ground of this, to the thing that makes pangolins pangolins – their scales. Pangolins are the only mammals that are fully covered in scales. Choo et al. used a branch site test on a set of 8250 protein-coding orthologs shared among a number of model and non-model organisms in order to trace signals of positive selection. The authors identified candidate genes related to hair formation, for instance keratins, which are essential compounds in hair and scales.
Choo et al. hypothesized that the scaly armour can be a compensation for deficits in the pangolin’s immune system. They discovered loss-of-function mutations in a single-copy intronless gene IFNE, which is “a unique interferon exclusively expressed in skin epithelial cells and inner mucosa-protected tissues” and acts as a defense against pathogens.
“We propose that pangolin scales may be an important morphological innovation to compensate for the decrease in immunity normally provided by the skin. These hard and overlapping scales may act as defensive armor to protect pangolins against injuries (or stress) which would make pangolins even more vulnerable to infection or the invasion of pathogens. Moreover, pangolins curl into near impregnable balls, covering their scaleless abdomen using their well-developed neuromuscular system, during sleep or when threatened, which would also support the hypothesis that these adaptations serve to protect pangolins from skin injuries.” (Choo et al. 2016)
Choo, S. W., Rayko, M., Tan, T. K., Hari, R., Komissarov, A., Wee, W. Y., … & Wilson, R. K. (2016). Pangolin genomes and the evolution of mammalian scales and immunity. Genome research, 26(10), 1312-1322. doi:10.1101/gr.203521.115
Ma, J. E., Li, L. M., Jiang, H. Y., Zhang, X. J., Li, J., Li, G. Y., … & Chen, J. P. (2017). Transcriptomic analysis identifies genes and pathways related to myrmecophagy in the Malayan pangolin (Manis javanica). PeerJ, 5, e4140. doi: 10.7717/peerj.4140