Project description:Antistasin, first identified as a potent inhibitor of the blood coagulation factor Xa, is a novel family of serine protease inhibitors. In this study, we purified a novel antistasin-type inhibitor from leech Poecilobdella manillensis called poecistasin. Amino acid sequencing of this 48-amino-acid protein revealed that poecistasin was an antistasin-type inhibitor known to consist of only one domain. Poecistasin inhibited factor XIIa, kallikrein, trypsin, and elastase, but had no inhibitory effect on factor Xa and thrombin. Poecistasin showed anticoagulant activities. It prolonged the activated partial thromboplastin time and inhibited FeCl₃-induced carotid artery thrombus formation, implying its potent function in helping Poecilobdella manillensis to take a blood meal from the host by inhibiting coagulation. Poecistasin also suppressed ischemic stroke symptoms in transient middle cerebral artery occlusion mice model. Our results suggest that poecistasin from the leech Poecilobdella manillensis plays a crucial role in blood-sucking and may be an excellent candidate for the development of clinical anti-thrombosis and anti-ischemic stroke medicines.
Project description:The study of non-model organisms stands to benefit greatly from genetic and genomic data. For a better understanding of the molecular mechanisms driving neuronal development, and to characterize the entire leech Hirudo medicinalis central nervous system (CNS) transcriptome we combined Trinity for de-novo assembly and Illumina HiSeq2000 for RNA-Seq. We present a set of 73,493 de-novo assembled transcripts for the leech, reconstructed from RNA collected, at a single ganglion resolution, from the CNS. This set of transcripts greatly enriches the available data for the leech. Here, we share two databases, such that each dataset allows a different type of search for candidate homologues. The first is the raw set of assembled transcripts. This set allows a sequence-based search. A comprehensive analysis of which revealed 22,604 contigs with high e-values, aligned versus the Swiss-Prot database. This analysis enabled the production of the second database, which includes correlated sequences to annotated transcript names, with the confidence of BLAST best hit.
Project description:The European medicinal leech has been used for medicinal purposes for millennia, and continues to be used today in modern hospital settings. Its utility is granted by the extremely potent anticoagulation factors that the leech secretes into the incision wound during feeding and, although a handful of studies have targeted certain anticoagulants, the full range of anticoagulation factors expressed by this species remains unknown. Here, we present the first draft genome of the European medicinal leech, Hirudo medicinalis, and estimate that we have sequenced between 79-94% of the full genome. Leveraging these data, we searched for anticoagulation factors across the genome of H. medicinalis. Following orthology determination through a series of BLAST searches, as well as phylogenetic analyses, we estimate that fully 15 different known anticoagulation factors are utilized by the species, and that 17 other proteins that have been linked to antihemostasis are also present in the genome. We underscore the utility of the draft genome for comparative studies of leeches and discuss our results in an evolutionary context.
Project description:BACKGROUND:Salivary cell secretion (SCS) plays a critical role in blood feeding by medicinal leeches, making them of use for certain medical purposes even today. RESULTS:We annotated the Hirudo medicinalis genome and performed RNA-seq on salivary cells isolated from three closely related leech species, H. medicinalis, Hirudo orientalis, and Hirudo verbana. Differential expression analysis verified by proteomics identified salivary cell-specific gene expression, many of which encode previously unknown salivary components. However, the genes encoding known anticoagulants have been found to be expressed not only in salivary cells. The function-related analysis of the unique salivary cell genes enabled an update of the concept of interactions between salivary proteins and components of haemostasis. CONCLUSIONS:Here we report a genome draft of Hirudo medicinalis and describe identification of novel salivary proteins and new homologs of genes encoding known anticoagulants in transcriptomes of three medicinal leech species. Our data provide new insights in genetics of blood-feeding lifestyle in leeches.
Project description:Many monophagous animals have coevolutionary relationships with bacteria that provide unavailable nutrients to the host. Frequently, these microbial partners are vertically inherited and reside in specialized structures or tissues. Here we report three new lineages of bacterial symbionts of blood-feeding leeches, one from the giant Amazonian leech, Haementeria ghilianii, and two others from Placobdelloides species. These hosts each possess a different mycetome or esophageal organ morphology where the bacterial cells are located. DNA sequencing of the bacterial 16S rRNA genes and fluorescent in situ hybridization placed these symbionts in two separate clades in the class Gammaproteobacteria. We also conducted a broad phylogenetic analysis of the herein-reported DNA sequences as well as others from bacterial symbionts reported elsewhere in the literature, including alphaproteobacterial symbionts from the leech genus Placobdella as well as Aeromonas veronii from the medicinal leech, Hirudo medicinalis, and a Rickettsia sp. detected in Hemiclepsis marginata. Combined, these results indicate that blood-feeding leeches have forged bacterial partnerships at least five times during their evolutionary history.
Project description:The medicinal leech is one of the most venerated model systems for the study of fundamental nervous system principles, ranging from single-cell excitability to complex sensorimotor integration. Yet, molecular analyses have yet to be extensively applied to complement the rich history of electrophysiological study that this animal has received. Here, we generated the first de novo transcriptome assembly from the entire central nervous system of Hirudo verbana, with the goal of providing a molecular resource, as well as to lay the foundation for a comprehensive discovery of genes fundamentally important for neural function. Our assembly generated 107,704 contigs from over 900 million raw reads. Of these 107,704 contigs, 39,047 (36%) were annotated using NCBI's validated RefSeq sequence database. From this annotated central nervous system transcriptome, we began the process of curating genes related to nervous system function by identifying and characterizing 126 unique ion channel, receptor, transporter, and enzyme contigs. Additionally, we generated sequence counts to estimate the relative abundance of each identified ion channel and receptor contig in the transcriptome through Kallisto mapping. This transcriptome will serve as a valuable community resource for studies investigating the molecular underpinnings of neural function in leech and provide a reference for comparative analyses.
Project description:Background:The pangolin is a Pholidota mammal with large keratin scales protecting its skin. Two pangolin species ( Manis pentadactyla and Manis javanica ) have been recorded as critically endangered on the International Union for Conservation of Nature Red List of Threatened Species. Optical mapping constructs high-resolution restriction maps from single DNA molecules for genome analysis at the megabase scale and to assist genome assembly. Here, we constructed restriction maps of M. pentadactyla and M. javanica using optical mapping to assist with genome assembly and analysis of these species. Findings:Genomic DNA was nicked with Nt.BspQI and then labeled using fluorescently labeled bases that were detected by the Irys optical mapping system. In total, 3,313,734 DNA molecules (517.847 Gb) for M. pentadactyla and 3,439,885 DNA molecules (504.743 Gb) for M. javanica were obtained, which corresponded to approximately 178X and 177X genome coverage, respectively. Qualified molecules (?150 kb with a label density of >6 sites per 100 kb) were analyzed using the de novo assembly program embedded in the IrysView pipeline. We obtained two maps that were 2.91 Gb and 2.85 Gb in size with N50s of 1.88?Mb and 1.97?Mb, respectively. Conclusions:Optical mapping reveals large-scale structural information that is especially important for non-model genomes that lack a good reference. The approach has the potential to guide de novo assembly of genomes sequenced using next-generation sequencing. Our data provide a resource for Manidae genome analysis and references for de novo assembly. This note also provides new insights into Manidae evolutionary analysis at the genome structure level.
Project description:In this study we used Illumina RNA-seq to identify genes expressed by A. veronii in mid-log phase growth in a rich medium and within the digestive tract of the medicinal leech. Our results shed light on the physiology of A. veronii during colonization of the leech gut. A comparison of Illumina RNA-seq of A. veronii in vivo versus in vitro.
Project description:The medicinal leech has established a long-term mutualistic association with Aeromonas veronii, a versatile bacterium which can also display free-living waterborne and fish- or human-pathogenic lifestyles. Here, we investigated the role of antibiotics in the dynamics of interaction between the leech and its gut symbiont Aeromonas. By combining biochemical and molecular approaches, we isolated and identified for the first time the antimicrobial peptides (AMPs) produced by the leech digestive tract and by its symbiont Aeromonas. Immunohistochemistry data and PCR analyses evidenced that leech AMP genes are induced in the gut epithelial cells when Aeromonas load is low (starved animals), while repressed when Aeromonas abundance is the highest (post blood feeding). The asynchronous production of AMPs by both partners suggests that these antibiotic substances (i) provide them with reciprocal protection against invasive bacteria and (ii) contribute to the unusual simplicity of the gut microflora of the leech. This immune benefit substantially reinforces the evidence of an evolutionarily stable association between H. verbana and A. veronii. Altogether these data may provide insights into the processes making the association with an Aeromonas species in the digestive tract either deleterious or beneficial.
Project description:In this study we used Illumina RNA-seq to identify genes expressed by A. veronii in mid-log phase growth in a rich medium and within the digestive tract of the medicinal leech. Our results shed light on the physiology of A. veronii during colonization of the leech gut. Overall design: A comparison of Illumina RNA-seq of A. veronii in vivo versus in vitro.