bioRxiv preprint doi: https://doi.org/10.1101/588889; this version posted March 25, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 De novo transcriptome assembly of the African bullfrog Pyxicephalus 2 adspersus for molecular analysis of aestivation 3 4 Naoki Yoshida1*, Chikara Kaito1,2 5 1Laboratory of Immunology and Microbiology, Graduate School of Pharmaceutical 6 Sciences, The University of Tokyo 7 2Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama 8 University 9 10 Running Head: De novo transcriptome assembly of the African bullfrog 11 12 *Corresponding author 13 E-mail: [email protected] 14 1 bioRxiv preprint doi: https://doi.org/10.1101/588889; this version posted March 25, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 15 Abstract 16 The molecular mechanisms of aestivation, a state of dormancy that occurs under dry 17 conditions at ordinary temperature, are not yet clarified. Here, we report the first de 18 novo transcriptome assembly of the African bullfrog Pyxicephalus adspersus, which 19 aestivates for 6-10 months during the hot and dry season. Polyadenylated RNA from 20 tissues was sequenced to 75,320,390 paired-end reads, and the de novo assembly 21 generated 101,682 transcripts. Of these, 100,093 transcripts had open reading frames 22 encoding more than 25 amino acids. BLASTx analysis against the Uniprot Xenopus 23 tropicalis protein database revealed 64,963 transcripts having little homology with an E 24 value higher than 1E-5 and 8147 transcripts having no homology, indicating that the 25 African bullfrog has many novel genes that are absent in X. tropicalis. The other 28,570 26 transcripts had homology with an E value lower than 1E-5 for which molecular 27 functions were estimated by gene ontology (GO) analysis and found to contain the 28 aestivation-related genes conserved among other aestivating organisms, including the 29 African lungfish. This study is the first to identify a comprehensive set of genes 30 expressed in the African bullfrog, thus providing basic information for molecular level 31 analysis of aestivation. 32 2 bioRxiv preprint doi: https://doi.org/10.1101/588889; this version posted March 25, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 33 Introduction 34 Dormancy is a phenomenon by which organisms temporarily decrease their physiologic 35 activity under conditions such as low temperature, drying, and depletion of energy 36 sources, and is observed in many organisms from microorganisms to mammals. In 37 vertebrates, hibernation and aestivation are two common types of dormancies performed 38 under either a low temperature condition or a drying and ordinary temperature 39 condition, respectively. Molecular biologic and physiologic analyses of vertebrate 40 hibernation have been performed for the Siberian chipmunk and Northern leopard frog. 41 In Siberian chipmunks, hibernation is initiated by two cues: a seasonal change in the 42 photoperiod depending on the melatonin level, and a hibernation-specific protein 43 complex induced by cold temperature [1,2]. Energy-related genes are regulated to 44 decrease body temperature and to suppress the contractile activity of cardiac myocytes. 45 In the Northern leopard frog, glucose and glycerol concentrations in the body are 46 increased to confer freeze tolerance, and the mammalian target of rapamycin (mTOR) is 47 activated to prevent muscle atrophy during hibernation [3,4,5]. These molecular and 48 physiologic aspects of hibernation have attracted attention as the potential basis for 49 novel therapeutics against malignant tumors or tissue necrosis. 50 Aestivation in vertebrates is observed in many amphibian and fish species. 51 Aestivation is similar to hibernation in terms of the reduced metabolic activity, but it is 52 clearly distinguished from hibernation in terms of the environmental conditions; 53 aestivation is performed under dry conditions at ordinary temperatures, whereas 54 hibernation is performed under aqueous conditions at low temperatures [6]. Therefore, 55 the mechanisms of aestivation are assumed to differ from those of hibernation, such as 3 bioRxiv preprint doi: https://doi.org/10.1101/588889; this version posted March 25, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 56 suppression of energy metabolism at an ordinary temperature or tolerance against 57 dehydration. Molecular level analyses of aestivation are important to uncover 58 mechanisms of dormancy that have not been clarified in studies of hibernation, but such 59 studies are scarce at present. 60 So far, three species that undergo aestivation – the Green striped burrowing frog 61 (Cyclorana alboguttata), Couch’s spadefoot toad (Scaphiopus couchii), and African 62 lungfish (Protopterus annectens) – have been analyzed at the transcriptional level to 63 identify genes whose expression is induced or suppressed during aestivation [7,8,9]. 64 Very little is known about the molecular mechanisms of aestivation, however, including 65 how energy metabolism is suppressed or how tissue damage is minimized in the 66 aestivating organisms. 67 In the present study, we aimed to identify genes expressed in the African bullfrog, 68 P. adspersus using de novo transcriptome assembly. The African bullfrog, a species 69 belonging to the family of Pyxicephalidae, is a large frog – body size of males is larger 70 than 20 cm and that of females is ~14 cm. This frog inhabits a savanna area of east 71 Africa, south Africa, and the southern part of central Africa, where the air temperature 72 ranges from 20-30˚C throughout the year, and there are pronounced dry and rainy 73 seasons. During the dry season, the frogs burrow underground and form a tough cocoon 74 to reduce evaporative water loss and decrease the respiration rate to less than 10% that 75 during the active stage [10,11]. The aestivation of the frog continues for 6-10 months. 76 Because aestivation of the frog can be artificially induced and breeding methods are 77 established for breeding the frogs as pets, the African bullfrog is a suitable research 78 model animal to investigate the molecular mechanisms of aestivation. Furthermore, the 79 African bullfrog is phylogenetically and geographically distant from the other two 4 bioRxiv preprint doi: https://doi.org/10.1101/588889; this version posted March 25, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 80 aestivating frogs: the Green striped burrowing frog and Couch’s spadefoot toad, which 81 inhabit Australia and North America, respectively. Therefore, studies of the aestivation 82 of the African bullfrog will help to elucidate the molecular mechanisms of aestivation 83 that are conserved among different aestivating frog species. In this study, we identified 84 and characterized the genes of the African bullfrog to provide basic information for 85 investigating the aestivation mechanism. 86 87 Materials and Methods 88 Ethics statement 89 All protocols and procedures conformed to the Regulations for Animal Care and Use of 90 the University of Tokyo. No formal approval is required by the University of Tokyo to 91 perform experiments using amphibians. 92 93 Experimental animal 94 A captive bred African bullfrog was purchased from a specialty reptile and amphibian 95 store (Hachurui Club, Nakano, Japan). The purchased frog was maintained in a plastic 96 container with coarse sand (5-7 mm diameter) and water. The frog was fed every day 97 with house crickets (Acheta domestica) or wax worms (the larvae of the Galleria 98 mellonella) for the first 3 weeks, and then fed every day with artificial diets 99 (Samuraijapan, Ibaraki, Japan) for 2 months. 100 101 5 bioRxiv preprint doi: https://doi.org/10.1101/588889; this version posted March 25, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 102 Isolation of genomic DNA and total RNA 103 The young adult frog (15 g body mass) was kept without feeding for 36 h. The frog was 104 anesthetized by placing it into crushed ice for 10 min and then dissected on ice. The 105 intestines were separated, the intestinal contents removed in phosphate buffered saline 106 (pH7.4), and the intestines frozen in liquid nitrogen. The other tissues, including inner 107 organs, muscle, and skin, were quickly excised to 3 - 5 mm3 (0.1 - 0.3 g) and frozen in 108 liquid nitrogen. The frozen samples were maintained at -80˚C. 109 Genomic DNA was isolated by a standard phenol/chloroform extraction method 110 [12]. Frozen muscle tissues (5 mm3, 0.2 g) were submerged in a mixture of 5 ml of 111 phenol/chloroform/isoamyl alcohol (25:24:1) and 5 ml of TE Buffer (10 mM Tris-HCl 112 [pH 8.0], 1mM EDTA). Then, the tissues were homogenized at 10,000 rpm three times 113 for 30 s each with a Polytron benchtop homogenizer (Kinematica, Switzerland).