Marine Biotechnology (2020) 22:760–771 https://doi.org/10.1007/s10126-020-10001-8 ORIGINAL ARTICLE Genomic and Transcriptomic Analyses of Bioluminescence Genes in the Enope Squid Watasenia scintillans Masa-aki Yoshida1 & Junichi Imoto2,3 & Yuri Kawai4 & Satomi Funahashi4 & Ryuhei Minei5 & Yuki Akizuki5 & Atsushi Ogura5 & Kazuhiko Nakabayashi6 & Kei Yura4,7 & Kazuho Ikeo2 Received: 8 April 2020 /Accepted: 28 September 2020 / Published online: 24 October 2020 # The Author(s) 2020 Abstract Watasenia scintillans, a sparkling enope squid, has bioluminescence organs to illuminate its body with its own luciferase activity. To clarify the molecular mechanism underlying its scintillation, we analysed high-throughput sequencing data acquired previ- ously and obtained draft genome sequences accomplished with comparative genomic data among the cephalopods. The genome mapped by transcriptome data showed that (1) RNA editing contributed to transcriptome variation of lineage specific genes, such as W. scintillans luciferase, and (2) two types of luciferase enzymes were characterized with reasonable 3D models docked to a luciferin molecule. We report two different types of luciferase in one organism and possibly related to variety of colour types in the W. scintillans fluorescent organs. Keywords Firefly squid . Cephalopod . RNA editing . 3D modelling . Repetitive elements Introduction The squid also has dermal light organs producing ambient, greenish light. The dermal light organs are utilized for Around the Japanese sea, especially off Toyama, the appear- counter-illumination camouflage to match the brightness mak- ance of shoals of the sparkling enope squid, Watasenia ing it difficult for predators below to detect the squid (Young scintillans, heralds the beginning of spring. The W. scintillans and Roper 1977). Young et al. (1979) reported that is also known as the “firefly squid,” which is important for both Enoploteuthis, the closest relative of W. scintillans, has dermal tourism and the fishing industry in Japan. W. scintillans emits light organs similar to the ones of W. scintillans.Themembers light from three different types of photophores (Inamura et al. of the Enoploteuthidae seems to have a common mechanism of 1990). Five light organs are located around each eye, and a bioluminescence. Several researches in the past have cluster of three light organs is located at the tip of the arms. established the molecular mechanism of illumination that Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10126-020-10001-8) contains supplementary material, which is available to authorized users. * Masa-aki Yoshida 3 Present address: Fisheries Data Sciences Division, Fisheries [email protected] Resources Institute, Japan Fisheries Research and Education Agency, Fukuura 2-12-4, Kanazawa, Yokohama, Kanagawa 236-8648, Japan * Kei Yura [email protected] 4 Graduate School of Humanities and Sciences, Ochanomizu University, Tokyo, Japan 5 1 Marine Biological Science Section, Education and Research Center Department of Computer Bioscience, Nagahama Institute of for Biological Resources, Faculty of Life and Environmental Bio-Science and Technology, Nagahama, Japan Sciences, Shimane University, Oki, Japan 6 Department of Maternal-Fetal Biology, Research Institute, National Center for Child Health and Development, Tokyo, Japan 2 Department of Genomics and Evolutionary Biology, National 7 School of Advanced Science and Engineering, Waseda University, Institute of Genetics, Mishima, Japan Tokyo, Japan Mar Biotechnol (2020) 22:760–771 761 coelenterazine disulfate is the luciferin substrate, and the reac- (Oliver and Greene 2009). For instance, the inserts of REs tion requires ATP, Mg2+ and molecular oxygen (Tsuji 2002; were estimated as a cause of disruption of macrosynteny of Tsuji 2005; Teranishi and Shimomura 2008;Gotoetal.1974; the genome and resulted in the loss of the HOX gene cluster in Inoue et al. 1975; Inoue et al. 1976). octopus (Albertin et al. 2015). The question to be addressed is In addition to the study of luminescence, cephalopod mol- how REs arose and expanded across the cephalopod genomes luscs, especially squids, have fascinated many researchers during the evolution. Typically, types of causal transposable studying not only in the field of fishery science but also in elements (TEs) show the history that the species has followed the field of molecular biology, especially neuroscience, visual and are related to the characteristics of the organism. For ex- physiology and biophysics (Schwiening 2012;Seidouetal. ample, Alu-type short interspersed nuclear element (SINE), 1990; Hara and Hara 1980; Shichida and Matsuyama 2009; which is the most abundant repeat sequence found in human Murakami and Kouyama 2008). To fulfil the demand of the genome, occupies more than 10% of the genome sequence resources, the sparkling enope squid has played a decent role (Lander et al. 2001). Despite the abundance, a major burst of because collection of the squid is exceptionally stable com- the Alu amplification was estimated to have happened 25– pared with other deep-sea squids that are usually difficult to 50 Mya based on the human Alu subfamily sequence diversity collect. However, the recent advance genome biology has not (Shen et al. 1991). TEs are the major part of REs in the ceph- blessed this species. alopod genomes (da Fonseca et al. 2020) and undergo a rapid Lack of genome information had prevented us from increase in copy number in some animal clades, resulting in understanding the genetic basis of cephalopod biology. the increase of the genome size through TE amplification and, The report of octopus genome from Albertin et al. in some cases, in causing loci rearrangements through TE (2015) has finally advanced the understanding of insertions. The former was already observed in squid and in Cephalopoda class. The genome analysis revealed three octopus (Yoshida et al. 2011;Albertinetal.2015), and the notable characteristics of the octopus genome: (1) highly latter was suggested to have happened as large-scale genomic rearranged genome with transposable element expansion, rearrangements in the genome of California two-spot octopus (2) lineage specific duplication of certain types of genes Octopus bimaculoides (Albertin et al. 2015). It is considered and (3) whole transcript-wide adenosine to inosine (A-to- that the timing of its appearance is greatly related to the reor- I) RNA editing (Alon et al. 2015; Liscovitch-Brauer et al. ganization of the genome; however, burst timing of REs are 2017). These characteristics featured the octopus genome uncertain in the cephalopods even before/after squid-octopod as quite different from metazoan genomes. However, it is divergence. not clear whether these characteristics are specific to oc- Another characteristic of cephalopod genetic system is the topuses or to cephalopods including squids and cuttlefish. existence of extensive RNA editing (Liscovitch-Brauer et al. Squid and cuttlefish, different major lineage of the ceph- 2017). The events of RNA editing itself have been discovered alopods, show similar body plans and morphology to the in wide range of organisms including humans, Drosophila octopuses (Young 1971) but diversified over 270–284 and so forth. However, numerous RNA-editing sites were million years ago (Mya) (Kröger et al. 2011;Vinther conserved between squids and octopuses, especially in tran- et al. 2012). The timescale is comparable with the time scripts of nervous system, which is quite contrary to the situ- of the appearance of dinosaurs or mammals (324.7 Mya, ation in mammals and Drosophila. In humans, for instance, dosReisetal.2015). Hence, comparative genomic studies the locations of RNA editing in only 25 genes are conserved among octopus, squid and cuttlefish may accentuate ceph- across mammals (Pinto et al. 2014). In Drosophila, only about alopod commonalities with differences to other molluscs 65 editing sites are conserved across the Drosophila lineage and spotlight the specific features among them. (Yu et al. 2016). RNA editing is expected to be utilized for The average genome size of the cephalopods known to date enhancing transcriptome variation (Liscovitch-Brauer et al. is slightly bigger than that of other molluscan species 2017). (Gregory 2019). We have estimated the genome size of the Recently, the genome of Hawaiian bobtail squid Euprymna enope squid by comparing the mean copy number of mito- scolopes, a model cephalopod with symbiotic luminescence, chondria per cell and found that the haploid genome size was has been sequenced, and the highly repetitive nature of the about 4.78 giga base pair (Gbp) (Hayashi et al. 2016). The size genome was uncovered (Belcaid et al. 2019). To solidify the is bigger than the known genome sizes of squids (Gregory characteristics of cephalopod genomes and to uncover the 2019). Recent genome analysis has revealed that the regions commonality of RNA editing in this group of organisms, we with three giga base in total in the octopus genome have been obtained the draft genome sequences of the Japanese enope the result of enormous expansion of repetitive sequences, such squid, W. scintillans, which has several light organs different as transposons, contributing nearly 45% of the genome from the ones of E. scolopes. We further used a genomic (Albertin et al. 2015). Such repetitive elements (REs) often approach in combination with transcriptomic approach, name- cause genomic rearrangement across