Invertebrate zoology is in many ways the most comprehensive survey course on biology, encompassing most multicellular life on the planet. (Indeed, the study of the Order Coleoptera alone, comprising the beetles, would satisfy that criterion: J.B.S. Haldane once remarked that what his study of biology had taught him about God was that the Creator had an inordinate fondness for beetles.) At the apex of the invertebrates are the coleoid cephalopods (octopus, cuttlefish and squid), which show a degree of development and specialization unmatched in any other Class. For example, octopus brain comprises half a billion neurons, more than six times the number in a mouse brain, and these animals exhibit complex behaviors, including "complex problem solving, task-dependent conditional discrimination, observational learning and spectacular displays of camouflage." And recently, a collection of scientists* has elucidated the genomic sequence of these creatures, finding genetic evidence for the basis of some of these unique traits (including the largest nervous system among invertebrates, "camera" eyes, prehensile arms and adaptive coloration).
These researchers performed whole-genome sequencing of a single male individual of the California two-spot octopus, Octopus bimaculoides. The octopus was estimated as having a haploid genome size of about 2.7 gigabases (Gb) based on fluorescence (2.66–2.68 Gb) and k-mer (2.86 Gb) measurements that was confirmed by sequencing data. Genomic sequencing was performed to 6-fold redundant coverage using a contig N50-length of 5.4 kb and a scaffold N50-length of 470 kb. The longest scaffold contains 99 genes and half of all predicted genes are on scaffolds with 8 or more genes. Nearly 45% of the assembled genome was found to be composed of repetitive elements, associated with two bursts of transposon activity occurring ~25-million and ~56-million years ago. These resulted in large-scale genomic rearrangements that are closely associated with transposable element expansions.
At the level of the whole genome, the researchers reported that chromosome number increased by fragmentation not duplication, and was associated with the "expansion of existing gene families, evolution of novel genes, modification of gene regulatory networks, and reorganization of the genome through transposon activity." However, there was no evidence for whole genome duplication (which had previously been posited as the basis for cephalopod genome structure). At finer resolution, the sequencing data revealed 33,638 protein-coding genes (more than found in the human genome), with alternate splicing at 2,819 loci. There were a total of 1,424,497 single nucleotide polymorphisms (SNPs) found at 1,740,621,467 eligible sites. The search for specific gene families was performed in deposited genome and transcriptome databases for L. gigantea, A. californica, C. gigas, C. teleta, T. castaneum, D. melanogaster, C. elegans, N. vectensis, A. queenslandica, S. kowalevskii, B. floridae, C. intestinalis, D. rerio, M. musculus and H. sapiens. The results of these searches disclosed hundreds of coleoid- and octopus-specific genes, many of which were expressed in tissues containing novel structures, including the chromatophore-laden skin, the suckers and the nervous system.
Looking at gene families, the intron-exon structure and "domain architecture" resembled other bilateral invertebrates, including he limpet Lottia gigantea, the polychaete annelid Capitella teleta and the cephalochordate Branchiostoma floridae. "Toolkit" genes (including "developmentally important transcription factors and signaling pathway genes") showed no significant expansion. However, there were several "notable" gene families showing expanded copy number in octopus, including protocadherins ("168 multi-exonic protocadherin genes, nearly three-quarters of which are found in tandem clusters on the genome"; for comparison, other bilateral invertebrates like Lottia contain 17-25 such genes and these genes are absent in Drosophila melanogaster and C. elegans), C2H2 zinc-finger proteins (C2H2 ZNFs), interleukin-17-like genes (IL17-like), 328 G-protein-coupled receptor (GPCRs) genes, as well as chitinases and sialins. Expansion of the protocadherins was particularly noted, first because the researchers characterized this expansion as "massive." Also, protocadherins are homophilic cell adhesion molecules whose function has been primarily studied in mammals, where they are required for neuronal development and survival, as well as synaptic specificity; this gene family was previously thought to be uniquely enlarged in vertebrates.
The other gene family found to be expanded in the octopus genome was the C2H2 ZNF transcription factor genes. These were found in multiple clusters, comprising nearly 1,800 multi-exonic C2H2-containing genes, more than the 200–400 C2H2 ZNFs found in other lophotrochozoans and the 500–700 found in eutherian mammals. The expansion of the O. bimaculoides C2H2 ZNF genes was estimated to coincide with a burst of transposable element activity approximately 25 million years ago. The flanking regions of these genes showed a "significant enrichment" in a 70–90 base pair (bp) tandem repeat (this discovery was said to parallel linkage of C2H2 gene expansions to β-satellite repeats in humans).
Also detected were variations in the sizes of neurotransmission gene families between human and lophotrochozoans, but there was no evidence for systematic expansion of these gene families in vertebrates relative to octopus or other lophotrochozoans; neurotransmission gene family sizes in the octopus were very similar to those seen in other lophotrochozoans.
The researchers also termed "remarkable" the structure of the developmentally important Hox genes, which were found not to be organized into clusters (as in most other genomes from animals having a bilateral body plan), but are found distributed throughout the genome ("completely atomized").
The structure of repeated elements was also assessed, particularly with regard to a class of octopus-specific short interspersed nuclear element sequences (SINEs), which accounts for 4% of the octopus genome. These repeats were associated with changes in genetic linkage (when compared with the genome structure of related animals): the "transposon-rich" octopus genome showed a "substantial" loss of linkages purported to have arisen in ancestral bilaterian species that are conserved in other species, and these genes unlinked in the octopus genome were found to be flanked by sequences enriched in these SINE repeats. Specifically, the researchers reported 484 genes for retained synteny and 1,193 genes in lost synteny for all transposable element (TE) classes; 440 and 1,107, respectively, for SINEs; and 116 and 290, respectively, for another type of repeat termed Mariner.
In addition, these researchers also sequenced multiple transcriptomes from a wide variety of tissues to understand gene expression patterns. Twelve transcriptomes were sequenced from RNA isolated from ova, testes, viscera, posterior salivary gland (PSG), suckers, skin, developmental stage 15 (St15), retina, optic lobe (OL), supraesophageal brain (Supra), subesophageal brain (Sub), and axial nerve cord (ANC). There are 3,557 putative octopus-specific protein-coding genes expressed in the transcriptomes, with 1,520 expressed in a tissue-specific manner. Transcription patterns of certain genes were suggestive of their biological roles; for example, the majority of C2H2 ZNF genes transcripts were found expressed in embryonic and nervous tissues, consistent with roles for C2H2 ZNFs in cell fate determination, early development and transposon silencing. There is also the possibility of sensory receptors in suckers containing atypical nicotinic acetylcholine receptor-like genes, most of which are tandemly arrayed in clusters, and don't bind acetylcholine (because they lack amino acid residues at particular positions in the protein that have been associated with acetylcholine binding). Differential expression of 31 IL17-like genes was found in suckers and skin, and skin was also found to express reflectins, which the researchers posited were involved in the octopus's ability to change skin color.
In addition to the specific results disclosed by these scientists, these results illustrate the diversity in the mechanisms exploited by evolution to produce modern species. The researchers were able to estimate that the octopus and squid lineages diverged about 270 million years ago (for those paying attention, this split would have occurred in the middle of the Permian geologic era). In view of the mass extinction that occurred during the Permian, octopus genome structure (combined with other genomes that arose during that time, as they are elucidated) may in future shed additional light on the biological processes that occur during mass extinctions (or that help species survive them). This may become more and more relevant to current concerns if the developing pattern of mass extinction occurring in the present day continues.
*The scientists involved in this effort are: Caroline Albertin and Clifton Ragsdale (Department of Organismal Biology and Anatomy, University of Chicago); Oleg Simakov (Centre for Organismal Studies, University of Heidelberg), Eric Edsinger-Gonzales, Sydney Brenner, and Daniel Rokhsar (Okinawa Institute of Science and Technology Graduate University, Onna, Okinawa), Therese Mitros (Department of Molecular and Cell Biology, University of California, Berkeley, California), Z. Yan Wang and Judit Pungor (Department of Neurobiology, University of Chicago).
