The Tibetan antelope (Pantholops hodgsonii) is endemic to the extremely inhospitable high-altitude environment of the Qinghai-Tibetan Plateau, a region that has a low partial pressure of oxygen and high ultraviolet radiation. Here we generate a draft genome of this artiodactyl and use it to detect the potential genetic bases of highland adaptation. Compared with other plain-dwelling mammals, the genome of the Tibetan antelope shows signals of adaptive evolution and gene-family expansion in genes associated with energy metabolism and oxygen transmission. Both the highland American pika, and the Tibetan antelope have signals of positive selection for genes involved in DNA repair and the production of ATPase. Genes associated with hypoxia seem to have experienced convergent evolution. Thus, our study suggests that common genetic mechanisms might have been utilized to enable high-altitude adaptation.

Manduca sexta, known as the tobacco hornworm or Carolina sphinx moth, is a lepidopteran insect that is used extensively as a model system for research in insect biochemistry, physiology, neurobiology, development, and immunity. One important benefit of this species as an experimental model is its extremely large size, reaching more than 10 g in the larval stage. M. sexta larvae feed on solanaceous plants and thus must tolerate a substantial challenge from plant allelochemicals, including nicotine. We report the sequence and annotation of the M. sexta genome, and a survey of gene expression in various tissues and developmental stages. The Msex_1.0 genome assembly resulted in a total genome size of 419.4 Mbp. Repetitive sequences accounted for 25.8% of the assembled genome. The official gene set is comprised of 15,451 protein-coding genes, of which 2498 were manually curated. Extensive RNA-seq data from many tissues and developmental stages were used to improve gene models and for insights into gene expression patterns. Genome wide synteny analysis indicated a high level of macrosynteny in the Lepidoptera. Annotation and analyses were carried out for gene families involved in a wide spectrum of biological processes, including apoptosis, vacuole sorting, growth and development, structures of exoskeleton, egg shells, and muscle, vision, chemosensation, ion channels, signal transduction, neuropeptide signaling, neurotransmitter synthesis and transport, nicotine tolerance, lipid metabolism, and immunity. This genome sequence, annotation, and analysis provide an important new resource from a well-studied model insect species and will facilitate further biochemical and mechanistic experimental studies of many biological systems in insects.

Serine protease (SP) and serine protease homolog (SPH) genes in insects encode a large family of proteins involved in digestion, development, immunity, and other processes. While 68 digestive SPs and their close homologs are reported in a companion paper (Kuwar et al., 2015), we have identified 125 other SPs/SPHs in Manduca sexta and studied their structure, evolution, and expression. Fifty-two of them contain cystine-stabilized structures for molecular recognition, including clip, LDLa, Sushi, Wonton, TSP, CUB, Frizzle, and SR domains. There are nineteen groups of genes evolved from relatively recent gene duplication and sequence divergence. Thirty-five SPs and seven SPHs contain 1, 2 or 5 clip domains. Multiple sequence alignment and molecular modeling of the 54 clip domains have revealed structural diversity of these regulatory modules. Sequence comparison with their homologs in Drosophila melanogaster, Anopheles gambiae and Tribolium castaneum allows us to classify them into five subfamilies: A are SPHs with 1 or 5 group-3 clip domains, B are SPs with 1 or 2 group-2 clip domains, C, D1 and D2 are SPs with a single clip domain in group-1a, 1b and 1c, respectively. We have classified into six categories the 125 expression profiles of SP-related proteins in fat body, brain, midgut, Malpighian tubule, testis, and ovary at different stages, suggesting that they participate in various physiological processes. Through RNA-Seq-based gene annotation and expression profiling, as well as intragenomic sequence comparisons, we have established a framework of information for future biochemical research of nondigestive SPs and SPHs in this model species.

Although Manduca sexta has significantly contributed to our knowledge on a variety of insect physiological processes, the lack of its genome sequence hampers the large-scale gene discovery, transcript profiling, and proteomic analysis in this biochemical model species. Here we report our implementation of the RNA-Seq cDNA sequencing approach based on massively parallel pyrosequencing, which allows us to categorize transcripts based on their relative abundances and to discover process- or tissue-specifically regulated genes simultaneously. We obtained 1,821,652 reads with an average length of 289 bp per read from fat body and hemocytes of naïve and microbe-injected M. sexta larvae. After almost all (92.1%) of these reads were assembled into 19,020 contigs, we identified 528 contigs whose relative abundances increased at least 5- and 8-fold in fat body and hemocytes, respectively, after the microbial challenge. Polypeptides encoded by these contigs include pathogen recognition receptors, extracellular and intracellular signal mediators and regulators, antimicrobial peptides, and proteins with no known sequence but likely participating in defense in novel ways. We also found 250 and 161 contigs that were preferentially expressed in fat body and hemocytes, respectively. Furthermore, we integrated data from our previous study and generated a sequence database to support future gene annotation and proteomic analysis in M. sexta. In summary, we have successfully established a combined approach for gene discovery and expression profiling in organisms lacking known genome sequences.

MicroRNAs (miRNAs) are a group of small RNAs involved in translation inhibition or mRNA degradation. Due to its large size, Manduca sexta has long been used as a model to study insect physiology and biochemistry. While transcriptome studies have greatly enriched our knowledge on M. sexta structural genes, little is known about posttranscriptional regulation by miRNAs in this lepidopteran species. We constructed four small RNA libraries from embryos, 4th instar feeding larvae, pupae, and adults, obtained 21 million reads of 18-31 nucleotides by Illumina sequencing, and found 163 conserved and 13 novel miRNAs. By searching the M. sexta genome assembly, we identified precursors of 82 conserved miRNAs, 76 of which had mapped reads in one or more of these libraries. After normalization, we compared numbers of miRNA and miRNA-star reads in these libraries and observed abundance changes during development. Interestingly, mse-miR-281-star, mse-miR-31-star, mse-miR-965-star, mse-miR-9a-star, msemiR-9b-star, mse-miR-2a-star, mse-miR-92b-star and mse-miR-279c-star are either more abundant or maintained at similar levels compared to respective mature miRNA strand. Expression profiling of the first set of miRNAs provided insights to their possible involvement in developmental regulation. This study will aid in the annotation of miRNA genes in the genome.