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Proteomics and Deep Sequencing Comparison of Seasonally Active Venom Glands in the Platypus Reveals Novel Venom Peptides and Distinct Expression Profiles*

Open AccessPublished:August 16, 2012DOI:https://doi.org/10.1074/mcp.M112.017491
      The platypus is a venomous monotreme. Male platypuses possess a spur on their hind legs that is connected to glands in the pelvic region. They produce venom only during the breeding season, presumably to fight off conspecifics. We have taken advantage of this unique seasonal production of venom to compare the transcriptomes of in- and out-of-season venom glands, in conjunction with proteomic analysis, to identify previously undiscovered venom genes. Comparison of the venom glands revealed distinct gene expression profiles that are consistent with changes in venom gland morphology and venom volumes in and out of the breeding season. Venom proteins were identified through shot-gun sequenced venom proteomes of three animals using RNA-seq-derived transcripts for peptide-spectral matching. 5,157 genes were expressed in the venom glands, 1,821 genes were up-regulated in the in-season gland, and 10 proteins were identified in the venom. New classes of platypus-venom proteins identified included antimicrobials, amide oxidase, serpin protease inhibitor, proteins associated with the mammalian stress response pathway, cytokines, and other immune molecules. Five putative toxins have only been identified in platypus venom: growth differentiation factor 15, nucleobindin-2, CD55, a CXC-chemokine, and corticotropin-releasing factor-binding protein. These novel venom proteins have potential biomedical and therapeutic applications and provide insights into venom evolution.
      The platypus inhabits the river systems of eastern Australia and is one of 12 extant species of venomous mammals. Male platypuses bear erectable keratinous spurs on their hind limbs that are each connected, via a duct, to a kidney-shaped venom gland. Males produce venom in significant quantities only during the breeding season (
      • Temple-Smith P.
      ). The venom glands increase in size during the breeding period from June to August but regress thereafter, accompanied by a loss of secretory granules and shrinkage of secretory tubules (
      • Temple-Smith P.
      ,
      • Krause W.J.
      Morphological and histochemical observations on the crural gland-spur apparatus of the echidna (Tachyglossus aculeatus) together with comparative observations on the femoral gland-spur apparatus of the duckbilled platypus (Ornithorhyncus anatinus).
      ). Thus, it is believed that platypus venom functions in mate competition and is used against male rivals (
      • Temple-Smith P.
      ). Envenomated humans report immediate and severe local pain; hyperalgesia (increased sensitivity to pain); fever; significant swelling that persists for up to three months, contributing to long-term loss of function of the envenomated limb; spreading pain and inflammation; cold sweats; and stomach pains (
      • Martin C.J.
      • Tidswell F.
      Observations on the femoral gland of Ornithorhynchus and its secretion; together with an experimental enquiry concerning its supposed toxic action.
      ,
      • Fenner P.J.
      • Williamson J.A.
      • Myers D.
      Platypus envenomation—a painful learning experience.
      ). Clinical pathology includes an increased erythrocyte sedimentation rate, which is an indication of inflammation, and increased blood pressure (
      • Fenner P.J.
      • Williamson J.A.
      • Myers D.
      Platypus envenomation—a painful learning experience.
      ).
      The availability of the platypus genome sequence and recent advances in sequencing technologies have facilitated the identification of putative platypus toxins based on sequencing of a venom-gland transcriptome (
      • Whittington C.M.
      • Papenfuss A.T.
      • Locke D.P.
      • Mardis E.R.
      • Wilson R.K.
      • Abubucker S.
      • Mitreva M.
      • Wong E.S.W.
      • Hsu A.L.
      • Kuchel P.W.
      • Belov K.
      • Warren W.C.
      Novel venom gene discovery in the platypus.
      ,
      • Wong E.S.W.
      • Papenfuss A.T.
      • Whittington C.M.
      • Warren W.C.
      • Belov K.
      A limited role for gene duplications in the evolution of platypus venom.
      ). Prior to this, the contents of platypus venom were relatively poorly studied, as the result of a lack of captive colonies for experimental research, and difficulty in obtaining venom samples. In addition, platypus envenomation is rare, and no human fatalities have been reported. Early experiments on rabbits and guinea pigs revealed extensive edema spreading from the site of venom injection, with higher doses of venom causing widespread intravascular coagulation and death (
      • Martin C.J.
      • Tidswell F.
      Observations on the femoral gland of Ornithorhynchus and its secretion; together with an experimental enquiry concerning its supposed toxic action.
      ,
      • Kellaway C.
      • LeMessurier D.
      The venom of the platypus (Ornithorhynchus anatinus).
      ). The reported coagulation is consistent with anecdotal evidence that little bleeding can be seen from the puncture site after the spur is forcibly removed (
      • Fenner P.J.
      • Williamson J.A.
      • Myers D.
      Platypus envenomation—a painful learning experience.
      ). Lowered blood pressure was also reported in experimental animals and was attributed to vasodilation. In vitro studies show conflicting actions; the venom caused smooth muscle relaxation in rat uterus (
      • de Plater G.
      • Martin R.L.
      • Milburn P.J.
      A pharmacological and biochemical investigation of the venom from the platypus (Ornithorhynchus anatinus).
      ) but muscle contraction in guinea pig uterus and rabbit bowel (
      • Kellaway C.
      • LeMessurier D.
      The venom of the platypus (Ornithorhynchus anatinus).
      ). Platypus venom possesses some cytolytic activity (
      • Kellaway C.
      • LeMessurier D.
      The venom of the platypus (Ornithorhynchus anatinus).
      ) and stimulates calcium-dependent current from intracellular calcium release in cultured dorsal root ganglion cells (
      • de Plater G.M.
      • Milburn P.J.
      • Martin R.L.
      Venom from the platypus, Ornithorhynchus anatinus, induces a calcium-dependent current in cultured dorsal root ganglion cells.
      ). The venom also has protease activity and induces mast cell histamine release that may contribute to pain, vasodilation, and edema (
      • de Plater G.
      • Martin R.L.
      • Milburn P.J.
      A pharmacological and biochemical investigation of the venom from the platypus (Ornithorhynchus anatinus).
      ,
      • de Plater G.M.
      • Martin R.L.
      • Milburn P.J.
      A C-type natriuretic peptide from the venom of the platypus (Ornithorhynchus anatinus): structure and pharmacology.
      ).
      Early proteomics studies identified at least 19 classes of peptides in platypus venom (
      • de Plater G.
      • Martin R.L.
      • Milburn P.J.
      A pharmacological and biochemical investigation of the venom from the platypus (Ornithorhynchus anatinus).
      ,
      • de Plater G.M.
      • Martin R.L.
      • Milburn P.J.
      A C-type natriuretic peptide from the venom of the platypus (Ornithorhynchus anatinus): structure and pharmacology.
      ,
      • de Plater G.
      ,
      • Torres A.M.
      • Wang X.
      • Fletcher J.I.
      • Alewood D.
      • Alewood P.F.
      • Smith R.
      • Simpson R.J.
      • Nicholson G.M.
      • Sutherland S.K.
      • Gallagher C.H.
      • King G.F.
      • Kuchel P.W.
      Solution structure of a defensin-like peptide from platypus venom.
      ,
      • Torres A.M.
      • Tsampazi M.
      • Kennett E.C.
      • Belov K.
      • Geraghty D.P.
      • Bansal P.S.
      • Alewood P.F.
      • Kuchel P.W.
      Characterization and isolation of L-to-D-amino-acid-residue isomerase from platypus venom.
      ). Five different types of molecules were identified: hyaluronidase, C-type natriuretic peptides, nerve growth factor (OvNGF), l-to-d-amino acid-residue isomerase, and defensin-like peptides. Only two of these components have been fully sequenced: natriuretic peptides and defensin-like peptides (
      • de Plater G.M.
      • Martin R.L.
      • Milburn P.J.
      A C-type natriuretic peptide from the venom of the platypus (Ornithorhynchus anatinus): structure and pharmacology.
      ,
      • Torres A.M.
      • de Plater G.M.
      • Doverskog M.
      • Birinyi-Strachan L.C.
      • Nicholson G.M.
      • Gallagher C.H.
      • Kuchel P.W.
      Defensin-like peptide-2 from platypus venom: member of a class of peptides with a distinct structural fold.
      ,
      • Kita M.
      • Black D.S.
      • Ohno O.
      • Yamada K.
      • Kigoshi H.
      • Uemura D.
      Duck-billed platypus venom peptides induce Ca2+ influx in neuroblastoma cells.
      ). These polypeptides and enzymes likely work together to cause swelling, lowered blood pressure, and pain. C-type natriuretic mRNA is posttranslationally cleaved to produce peptides that form cation channels in lipid bilayer membranes (
      • Kourie J.I.
      Characterization of a C-type natriuretic peptide (CNP-39)-formed cation-selective channel from platypus (Ornithorhynchus anatinus) venom.
      ), relax smooth muscles, release mast cell histamine (
      • de Plater G.M.
      • Martin R.L.
      • Milburn P.J.
      A C-type natriuretic peptide from the venom of the platypus (Ornithorhynchus anatinus): structure and pharmacology.
      ), and cause calcium influx into neuroblastoma cells (
      • Kita M.
      • Black D.S.
      • Ohno O.
      • Yamada K.
      • Kigoshi H.
      • Uemura D.
      Duck-billed platypus venom peptides induce Ca2+ influx in neuroblastoma cells.
      ). The function of venom defensin-like peptides is unclear; although they have a three-dimensional structure similar to those of sea anemone sodium channel neurotoxins and antimicrobial peptides (defensins), they do not modulate sodium channel function or display antimicrobial activity (
      • Torres A.M.
      • de Plater G.M.
      • Doverskog M.
      • Birinyi-Strachan L.C.
      • Nicholson G.M.
      • Gallagher C.H.
      • Kuchel P.W.
      Defensin-like peptide-2 from platypus venom: member of a class of peptides with a distinct structural fold.
      ,
      • Torres A.M.
      • Wang X.
      • Fletcher J.I.
      • Alewood D.
      • Alewood P.F.
      • Smith R.
      • Simpson R.J.
      • Nicholson G.M.
      • Sutherland S.K.
      • Gallagher C.H.
      • King G.F.
      • Kuchel P.W.
      Solution structure of a defensin-like peptide from platypus venom.
      ).
      Previously, we used the platypus genome sequence and next-generation transcriptome sequencing to identify putative toxins in the platypus venom gland (
      • Whittington C.M.
      • Papenfuss A.T.
      • Locke D.P.
      • Mardis E.R.
      • Wilson R.K.
      • Abubucker S.
      • Mitreva M.
      • Wong E.S.W.
      • Hsu A.L.
      • Kuchel P.W.
      • Belov K.
      • Warren W.C.
      Novel venom gene discovery in the platypus.
      ). However, to distinguish toxin proteins from the thousands of non-venom-related proteins in a venom gland, we had to rely on sequence similarity to known venom peptides from other species. Given the independent evolution of venom in the platypus and the evolutionary divergence of platypus and other venomous lineages, this prevented the identification of truly novel venom genes. Here, we have combined proteomic analysis on three samples of whole platypus venom with a comparison between the transcriptomes of an in-season and out-of-season venom gland to identify platypus-venom peptides and compare their expression during the breeding cycle. This approach has the advantage of being able to identify completely novel toxins that do not possess any similarity to known toxins and allows good coverage for the largest proteins. The genome assembly was used to quantify gene expression and to ensure specificity in gene identification. This is the first time such an integrated -omics approach has been used to identify venom peptide in a venomous mammal. Based on our proteomic and transcriptomic results, we have amplified and sequenced full-length cDNAs of several novel venom peptides. Herein we examine their evolution and speculate on the potential roles of these peptides in producing the known physiological effects of platypus envenomation.

      RESULTS

      Fragment ion spectra from mass spectrometry of trypsin-digested whole venom were matched to de novo assembled Illumina sequence data from a normalized platypus venom cDNA library (
      • Whittington C.M.
      • Papenfuss A.T.
      • Locke D.P.
      • Mardis E.R.
      • Wilson R.K.
      • Abubucker S.
      • Mitreva M.
      • Wong E.S.W.
      • Hsu A.L.
      • Kuchel P.W.
      • Belov K.
      • Warren W.C.
      Novel venom gene discovery in the platypus.
      ) (Fig. 1). The assembly contained 87,343 transcripts with an N50 size of 295 nt. High-scoring transcripts were annotated by searching against a public protein database. Full open reading frames of venom genes were amplified from venom-gland cDNA. We then used RNA-seq to compare the expression of venom transcripts in venom glands in and out of breeding season (in-season and out-of-season, respectively) obtained from two opportunistically acquired animals from Tasmania (Fig. 1).
      Figure thumbnail gr1
      Fig. 1Overview of methods used for gene identification. Whole venom was digested and was separated via liquid chromatography. Eluted peptides were subjected to tandem mass spectrometry. Fragment ions were assigned to protein sequences derived from two sources: an assembled database of transcripts derived from a normalized cDNA library of a venom gland, and a database that contained mostly predicted proteins based on the genome assembly. Annotation of high-scoring transcripts was performed by searching against the UniProt database and matching sequence data back to the platypus genome using BLAT on the UCSC genome browser. Full cDNA sequences were amplified using molecular strategies, and venom genes were checked for differential expression between breeding cycles.
      We performed shotgun proteomics analysis on independent venom samples from three platypuses collected from the Murrumbidgee River (Childowla, NSW) in September and October of 2008. The proteomes of the venom samples from the three animals were similar. A large subset of peptides was routinely detected in venom from different individuals, suggesting that the platypus venom proteome is highly similar among individuals within a population. Up to 20 transcripts were identified from each venom sample (Table I). Many of these transcripts were found in all of the samples tested, but levels fell below our conservative score cut-off in some cases. Because of the fragmentation of the de novo assembly, some transcripts were too short to be confidently annotated, and many transcripts were derived from the same protein. All putative toxins were identified using the transcriptomic spectra-matching database. A list of all peptide sequences assigned, including precursor charge and mass/charge, a list of all modifications observed, and scores are in supplemental File 2. All spectra and search results can be found in supplemental Files 3–8 and can be viewed using ProteinPilot.
      Table IThe number of transcripts, the number of distinct peptides, and number of spectra corresponding to each venom sample following LC-MS/MS (detected protein threshold score of 1.3 (95% confidence))
      Number of identified database sequences (from assembled transcriptome database)Number of distinct peptides identifiedNumber of identified spectra
      Sample 1529195
      Sample 22064335
      Sample 31280374
      The in- and out-of-season venom gland libraries were sequenced on an Illumina GAIIx producing 56,614,944 and 63,158,862 reads, respectively (accession: SRP003465). 5,157 platypus Ensembl genes were expressed. After trimming of low quality sequences, the average read length was 41 nt (see supplemental Table S1 for mapping statistics). We did not have biological replicates, but assumed Poisson variation of count data for differential expression analysis using the Bioconductor package edgeR (
      • Robinson M.D.
      • McCarthy D.J.
      • Smyth G.K.
      edgeR: a Bioconductor package for differential expression analysis of digital gene expression data.
      ). We identified 2,587 genes that were up-regulated in the in-season gland and 1,821 genes that were up-regulated in the out-of-season gland (FDR < 0.01). Fold change between in- and out-of-season counts and proteomics data were used to infer the regulation of toxin genes during the breeding cycle (supplemental Table S2 and supplemental Fig. S1). Enriched functional terms from the top 200 genes showing the highest fold change in the in-season and out-of-season glands are visualized in Figs. 2A and 2B, respectively. These terms have been derived from 3,667 human orthologues of platypus genes expressed in the venom glands.
      Figure thumbnail gr2
      Fig. 2Visualization of gene ontology (GO) terms of the top 200 genes based on fold change of genes. A, in-season gland. Enriched GO terms of p < 0.01 were used to construct the map, and similar functional terms are summarized using the same color. The size of the box corresponds to the number of terms. B, out-of-season gland. Enriched GO terms of p < 0.01 were used to construct the map, and similar functional terms are summarized using the same color. The size of the box corresponds to the number of terms.
      We identified 10 venom proteins: two enzymes and eight other polypeptides (Table II; Genbank accessions: JQ350810, JQ350811, JQ350812, JQ350813, JQ350814, JQ350815, JQ350816, JQ350817, JQ350818, JQ350819, JQ350820). Five of these have not been identified in the venom of any other species. We also identified nerve growth factor, C-type natriuretic peptides, and venom defensin-like peptides, which had been characterized previously. As is expected of secreted venom proteins, signal peptides were identified in all sequences. We note that mass spectral analysis of a fraction of protein spots from a two-dimensional gel of the venom confirmed our findings and did not result in additional protein information supporting the comprehensiveness of our shotgun strategy for canvassing the venom proteome (data not shown). Comparisons between in- and out-of-season venom glands showed a pattern of marked up-regulation of venom proteins during the breeding season (supplemental Table S2). Six of the ten proteins—serpin, chemokine, hyaluronidase, amide oxidase, GDF15, and CRFBP—were among the top 200 most highly up-regulated genes during the breeding season. One protein (cysteine-rich secretory protein) was not significantly up- or down-regulated between the glands. We provide details of the identified proteins below and speculate upon their role in venom.
      Table IIInformation on identified venom genes
      Name (accession)Ensembl IDPeriod in which higher expression was observedDifferentially expressed at adjusted p < 0.01Identified in proteome?Signal peptide present?
      Hyaluonidase (JQ350818)ENSOANG00000000407In-seasonYesYesYes
      Amide oxidase (JQ350817)ENSOANG00000005712In-seasonYesYesYes
      Whey acidic protein (ABL67638)ENSOANG00000012145Out-of-seasonYesYesYes
      Peptidoglycan recognition protein-1 (JQ350813)YesYes
      Serpin (JQ350816)In-seasonYesYesYes
      Kunitz-domain containing serine protease inhibitor (JQ350815)YesYes
      Corticotropin-releasing factor-binding protein (JQ350810)In-seasonYesYesYes
      Nucleobindin (JQ350819)ENSOANG00000007683In-seasonYesYesYes
      Differentiation factor-15 (JQ350812)ENSOANG00000001923In-seasonYesYesYes
      Complement decay-acceleration factor (JQ350811)ENSOANG00000020509 (partial)In-seasonYesYesYes
      CXC-chemokine (JQ350820)ENSOANG00000006911In-seasonYesYesYes
      Cysteine-rich secretory protein (JQ350814)In-seasonNoYesYes
      Genes without Ensembl annotation are denoted by “—.” The Kunitz-domain protein was not identified in the genome, and thus expression information is not available (denoted by “—”). Peptidoglycan recognition protein-1 was partially identified in the genome, which might account for its low counts (less than 20). As a result of the low values, its expression was not analyzed and is also denoted by “—.”

      Enzymes

      We have sequenced the mRNA of hyaluronidase and amide oxidase in platypus venom. Both these enzymes are also found in snake venoms. Hyaluronidase and amide oxidase are not produced in the ancestral glands of platypuses or snakes. Therefore, independent changes to the regulatory mechanisms of these enzymes have evolved independently in each lineage.
      Hyaluronidase has been identified in the venoms of vertebrates, arthropods, and mollusks (
      • Fry B.G.
      • Roelants K.
      • Champagne D.E.
      • Scheib H.
      • Tyndall J.D.A.
      • King G.F.
      • Nevalainen T.J.
      • Norman J.A.
      • Lewis R.J.
      • Norton R.S.
      • Renjifo C.
      • Rodríguez de la Vega R.C.
      The toxicogenomic multiverse: convergent recruitment of proteins into animal venoms.
      ). Platypus hyaluronidase, a 140 kDa disulfide-linked trimeric protein, was first detected in platypus venom by de Plater et al. (
      • de Plater G.
      • Martin R.L.
      • Milburn P.J.
      A pharmacological and biochemical investigation of the venom from the platypus (Ornithorhynchus anatinus).
      ). The breakdown of hyaluronic acid, widely distributed in connective, epithelial, and neural tissues, has been postulated to play a role in accelerating the spread of toxins and hemostatic factors, thereby potentiating their noxious actions (
      • Fry B.G.
      • Roelants K.
      • Champagne D.E.
      • Scheib H.
      • Tyndall J.D.A.
      • King G.F.
      • Nevalainen T.J.
      • Norman J.A.
      • Lewis R.J.
      • Norton R.S.
      • Renjifo C.
      • Rodríguez de la Vega R.C.
      The toxicogenomic multiverse: convergent recruitment of proteins into animal venoms.
      ). As expected, the expression of venom-gland hyaluronidase expression was five-fold up-regulated during the breeding season.
      Amine oxidase, a nonhydrolytic protein, was identified in the venoms of all three animals. The gene is located on contig7479 in the genome assembly. Platypus amine oxidase is more similar to copper-containing amine oxidase than the flavin-containing l-amino acid oxidases found in snake venoms (
      • Du X.-Y.
      • Clemetson K.J.
      Snake venom-amino acid oxidases.
      ). Copper-containing amine oxidases degrade primary amines such as histidine to aldehydes, releasing ammonia and hydrogen peroxide. In viperid, crotalid, and elapid snakes, amide oxidases have high enzymatic activities that are believed to contribute to toxicity upon envenomation through the production of hydrogen peroxide (
      • Du X.-Y.
      • Clemetson K.J.
      Snake venom-amino acid oxidases.
      ). They have been shown to induce human platelet aggregation (
      • Suhr S.-M.
      • Kim D.-S.
      Purification and characterisation of L-amino acid oxidance from king cobra (Ophiophagus hannah) venom and its effects on human platelet aggregation.
      ), assist in hemorrhage (
      • Du X.-Y.
      • Clemetson K.J.
      Snake venom-amino acid oxidases.
      ) and apoptosis (
      • Suhr S.-M.
      • Kim D.-S.
      Identification of the snake venom substance that induces apoptosis.
      ), and cause edema and hemolysis (
      • Ali S.A.
      • Stoeva S.
      • Abbasi A.
      • Alam J.M.
      • Kayed R.
      • Faigle M.
      • Neumeister B.
      • Voelter W.
      Isolation, structural, and functional characterization of an apoptosis-inducing l-amino acid oxidase from leaf-nosed viper (Eristocophis macmahoni) snake venom.
      ). We predict that the platypus protein would cause similar symptoms. Consistent with a toxin role, expression of amine oxidase was 21-fold up-regulated in the breeding-season venom gland.
      Polypeptides
      Antimicrobials

      Peptidoglycan Recognition Protein-1

      A putative antibacterial peptidoglycan recognition protein-1 peptide was found to be expressed in all three platypus venoms studied. This is the first time a member of the peptidoglycan family has been identified in vertebrate venom. Platypus peptidoglycan recognition protein-1 (PGLYRP1) is partially located on contig17172, with the 5′ end of the gene not detected in the genome assembly. Given the short length of the sequence that aligned to the genome (∼1 kb), and also perhaps because of comparatively low expression in the venom gland, we were unable to detect changes in the level of expression of the gene in and out of the breeding season. PGLYRP1 is a leukocyte-secreted disulfide-linked pattern receptor that hydrolyzes bacterial cell wall peptidoglycan and exerts direct bactericidal activity against both Gram-positive and Gram-negative bacteria. PGLYRP1 expression has also been detected in another modified sweat gland, the mammary gland, suggesting PGLYRP1 was a component of the common ancestral sweat gland (
      • Kappeler S.R.
      • Heuberger C.
      • Farah Z.
      • Puhan Z.
      Expression of the peptidoglycan recognition protein, PGRP, in the lactating mammary gland.
      ). Peptidoglycan recognition family proteins are known to play a role in wasp venoms. However, it is likely that the wasp and platypus proteins target different pathways and exert distinct physiological effects as insect PGYLRPs are not bactericidal-it is believed they are used to evade the host immune response through suppression of the phenoloxidase cascade, a major insect host defense pathway.

      WAP

      Notably, a WAP peptide, WFDC2 (WAP four-disulfide core domain 2), identical to a WAP peptide expressed in the platypus lactating mammary gland (EMBL:ABL67638) (
      • Sharp J.A.
      • Lefèvre C.
      • Nicholas K.R.
      Molecular evolution of monotreme and marsupial whey acidic protein genes.
      ) was identified in the venom using the Ensembl database. WAP-domain-containing peptides have diverse functions, including protease inhibitory and antimicrobial activities (
      • Sallenave J.M.
      Antimicrobial activity of antiproteinases.
      ). Two snake venom WAP peptides, nawaprin and omwaprin, have been isolated (
      • Nair D.G.
      • Fry B.G.
      • Alewood P.
      • Kumar P.P.
      • Kini R.M.
      Antimicrobial activity of omwaprin, a new member of the waprin family of snake venom proteins.
      ,
      • Torres A.M.
      • Wong H.Y.
      • Desai M.
      • Moochhala S.
      • Kuchel P.W.
      • Kini R.M.
      Identification of a novel family of proteins in snake venoms. Purification and structural characterization of nawaprin from Naja nigricollis snake venom.
      ). The biological function of nawaprin is unknown, but omwaprin has been found to exhibit antimicrobial activity against Gram-positive bacteria with no protease inhibitory activity or in vivo toxicity in mice (
      • Nair D.G.
      • Fry B.G.
      • Alewood P.
      • Kumar P.P.
      • Kini R.M.
      Antimicrobial activity of omwaprin, a new member of the waprin family of snake venom proteins.
      ). The convergent evolution of WAP domain peptides in the venom of platypuses and snakes suggests that the platypus peptide might perform a toxin-related role, but its biological significance is unclear. Antimicrobial peptides have been identified in both digestive glands and sweat glands, which gave rise to the venom glands of snakes and platypuses, respectively (
      • Temple-Smith P.
      ,
      • Mathews M.
      • Jia H.P.
      • Guthmiller J.M.
      • Losh G.
      • Graham S.
      • Johnson G.K.
      • Tack B.F.
      • McCray P.B.
      Production of beta-defensin antimicrobial peptides by the oral mucosa and salivary glands.
      ,
      • Schittek B.
      • Hipfel R.
      • Sauer B.
      • Bauer J.
      • Kalbacher H.
      • Stevanovic S.
      • Schirle M.
      • Schroeder K.
      • Blin N.
      • Meier F.
      • Rassner G.
      • Garbe C.
      Dermcidin: a novel human antibiotic peptide secreted by sweat glands.
      ,
      • Fry B.G.
      • Vidal N.
      • Norman J.A.
      • Vonk F.J.
      • Scheib H.
      • Ramjan S.F.R.
      • Kuruppu S.
      • Fung K.
      • Blair Hedges S.
      • Richardson M.K.
      • Hodgson W.C.
      • Ignjatovic V.
      • Summerhayes R.
      • Kochva E.
      Early evolution of the venom system in lizards and snakes.
      ). Thus, the platypus and snake WAP-domain venom peptides might be remnants of the respective venom glands' ancestral functions, and might also function in protecting the venom glands themselves. Consistent with this, the gene was five times as highly expressed in the out-of-season venom gland.
      Protease Inhibitors

      Serpin

      We amplified a kallistatin-like venom mRNA using a primer designed from a 31-residue fragment identified using mass spectrometry. The 5′ end of the gene aligned to the middle of the scaffold Ultra378 (1,065,020–1,065,532) in the genome assembly, whereas the 3′ end aligned to contig14450 in the genome assembly, indicative of misassembly of this region. The gene was one of the most highly up-regulated genes during the breeding season (7,642-fold), suggesting a key role in venom function. The predicted peptides are most similar to therian serine protease inhibitors SERPINA4 and SERPINA11. Serine protease inhibitors are found in the venoms of snakes, where they play key roles in blood coagulation (
      • Filippovich I.
      • Sorokina N.
      • Pierre L.S.
      • Flight S.
      • de Jersey J.
      • Perry N.
      • Masci P.P.
      • Lavin M.F.
      Cloning and functional expression of venom prothrombin activator protease from Pseudonaja textilis with whole blood procoagulant activity.
      ,
      • Zupunski V.
      • Kordis D.
      • Gubensek F.
      Adaptive evolution in the snake venom Kunitz/BPTI protein family.
      ). They have also been identified in sea anemone (
      • Schweitz H.
      • Heurteaux C.
      • Bois P.
      • Moinier D.
      • Romey G.
      • Lazdunski M.
      Calcicludine, a venom peptide of the Kunitz-type protease inhibitor family, is a potent blocker of high-threshold Ca2+ channels with a high affinity for L-type channels in cerebellar granule neurons.
      ) and wasp (
      • Colinet D.
      • Dubuffet A.
      • Cazes D.
      • Moreau S.
      • Drezen J.-M.
      • Poirié M.
      A serpin from the parasitoid wasp Leptopilina boulardi targets the Drosophila phenoloxidase cascade.
      ) venoms. The platypus serpin does not belong to the Kunitz family of serine protease inhibitors found in snake and anemone venoms, but instead is a member of MEROPS inhibitor family 14, clan ID. These proteins function in proteolytic cascades, including blood clotting and inflammation (
      • Lwaleed B.A.
      • Bass P.S.
      Tissue factor pathway inhibitor: structure, biology and involvement in disease.
      ), and cause irreversible “suicide inhibition” when they bind to their protease substrate (
      • Whisstock J.C.
      • Bottomley S.P.
      Molecular gymnastics: serpin structure, folding and misfolding.
      ).
      The function of platypus serpin is unclear. Its apparent homology to eutherian SERPINA4 and SERPINA11 suggests that it might induce hypertension via the inhibition of kallikreins that cause blood vessel dilation (
      • Zhou G.X.
      • Chao L.
      • Chao J.
      Kallistatin: a novel human tissue kallikrein inhibitor. Purification, characterization, and reactive center sequence.
      ). As such, the protein might contribute to increased blood pressure following envenomation (
      • Fenner P.J.
      • Williamson J.A.
      • Myers D.
      Platypus envenomation—a painful learning experience.
      ). Serpins share sequence homology with snake neurotoxins that modulate ion channel function (
      • Zupunski V.
      • Kordis D.
      • Gubensek F.
      Adaptive evolution in the snake venom Kunitz/BPTI protein family.
      ,
      • Lu J.
      • Yang H.
      • Yu H.
      • Gao W.
      • Lai R.
      • Liu J.
      • Liang X.
      A novel serine protease inhibitor from Bungarus fasciatus venom.
      ), but we do not know whether the platypus serpin has this activity. The irreversible termination of proteolytic function attributed to SERPINA4 suggests that this platypus serpin is unlikely to function in a protective capacity in the venom gland to allow storage of the protease component of venom, as suggested for other types of protease inhibitors (
      • Whittington C.M.
      • Papenfuss A.T.
      • Locke D.P.
      • Mardis E.R.
      • Wilson R.K.
      • Abubucker S.
      • Mitreva M.
      • Wong E.S.W.
      • Hsu A.L.
      • Kuchel P.W.
      • Belov K.
      • Warren W.C.
      Novel venom gene discovery in the platypus.
      ).

      Kunitz-domain-containing Serine Protease Inhibitor

      We have identified a Kunitz-type protease inhibitor in platypus venom that does not align to the genome assembly, suggesting that it was not sequenced in the genome. Other Kunitz-type protease inhibitors were previously identified in the platypus venom gland (
      • Whittington C.M.
      • Papenfuss A.T.
      • Locke D.P.
      • Mardis E.R.
      • Wilson R.K.
      • Abubucker S.
      • Mitreva M.
      • Wong E.S.W.
      • Hsu A.L.
      • Kuchel P.W.
      • Belov K.
      • Warren W.C.
      Novel venom gene discovery in the platypus.
      ). The ancestral function of these proteins is the inhibition of serine proteases, such as those involved in hemostasis, but some venom Kunitz-domain proteins have evolved ion-channel blocking activity (
      • Schweitz H.
      • Heurteaux C.
      • Bois P.
      • Moinier D.
      • Romey G.
      • Lazdunski M.
      Calcicludine, a venom peptide of the Kunitz-type protease inhibitor family, is a potent blocker of high-threshold Ca2+ channels with a high affinity for L-type channels in cerebellar granule neurons.
      ,
      • Bayrhuber M.
      • Vijayan V.
      • Ferber M.
      • Graf R.
      • Korukottu J.
      • Imperial J.
      • Garrett J.E.
      • Olivera B.M.
      • Terlau H.
      • Zweckstetter M.
      • Becker S.
      Conkunitzin-S1 is the first member of a new Kunitz-type neurotoxin family. Structural and functional characterization.
      ,
      • Schweitz H.
      • Bruhn T.
      • Guillemare E.
      • Moinier D.
      • Lancelin J.M.
      • Béress L.
      • Lazdunski M.
      Kalicludines and kaliseptine. Two different classes of sea anemone toxins for voltage sensitive K+ channels.
      ). These toxin proteins are well documented in snakes but are also found in the venoms of spiders, cone snails, and sea anemones (
      • Schweitz H.
      • Heurteaux C.
      • Bois P.
      • Moinier D.
      • Romey G.
      • Lazdunski M.
      Calcicludine, a venom peptide of the Kunitz-type protease inhibitor family, is a potent blocker of high-threshold Ca2+ channels with a high affinity for L-type channels in cerebellar granule neurons.
      ,
      • Bayrhuber M.
      • Vijayan V.
      • Ferber M.
      • Graf R.
      • Korukottu J.
      • Imperial J.
      • Garrett J.E.
      • Olivera B.M.
      • Terlau H.
      • Zweckstetter M.
      • Becker S.
      Conkunitzin-S1 is the first member of a new Kunitz-type neurotoxin family. Structural and functional characterization.
      ,
      • Schweitz H.
      • Bruhn T.
      • Guillemare E.
      • Moinier D.
      • Lancelin J.M.
      • Béress L.
      • Lazdunski M.
      Kalicludines and kaliseptine. Two different classes of sea anemone toxins for voltage sensitive K+ channels.
      ).
      Stress Response

      Corticotropin-releasing Factor-binding Protein

      Corticotropin-releasing factor-binding protein (CRF-BP) competitively inhibits corticotropin-releasing factor (CRF), a neuropeptide secreted in response to stress (
      • Flik G.
      • Klaren P.H.M.
      • Van den Burg E.H.
      • Metz J.R.
      • Huising M.O.
      CRF and stress in fish.
      ). The gene was up-regulated 44-fold in the in-season venom gland, consistent with a toxin role. CRF-BP expression in mammals has been linked to changes in psychological and behavioral states. Exposure of rats to a predator increases CRF-BP expression in the amygdala, which correlates with behavioral inhibition and submissive posturing (
      • Roseboom P.H.
      • Nanda S.A.
      • Bakshi V.P.
      • Trentani A.
      • Newman S.M.
      • Kalin N.H.
      Predator threat induces behavioral inhibition, pituitary-adrenal activation and changes in amygdala CRF-binding protein gene expression.
      ). Platypuses are purported to use venom during intraspecific competition for females, and as such, increased submissive behavior in the spurred individual might increase the reproductive success of the spurrer. CRF-BP can also trigger synaptic transmission when coupled with CRF in mice, suggesting that it might also function as a neurotoxin (
      • Ungless M.A.
      • Singh V.
      • Crowder T.L.
      • Yaka R.
      • Ron D.
      • Bonci A.
      Corticotropin-releasing factor requires CRF binding protein to potentiate NMDA receptors via CRF receptor 2 in dopamine neurons.
      ). Furthermore, CRF-BP might have a proinflammatory effect by limiting CRF. CRF stimulates the release of anti-inflammatory glucocorticoids through adrenocorticotropic hormone secretion. Indeed, administration of corticosteroids to patients following platypus envenomation has led to decreased pain and swelling (
      • Fenner P.J.
      • Williamson J.A.
      • Myers D.
      Platypus envenomation—a painful learning experience.
      ).

      Nucleobindin

      Platypus nucleobindin 2 (ENSOANP00000012244) is the first nucleobindin molecule identified in any animal venom. Nucleobindin 2 is the precursor of a secreted calcium-binding protein, nesfatin-1, that influences the excitability of neurons in a part of the brain associated with stress and physiological changes (
      • Yoshida N.
      • Maejima Y.
      • Sedbazar U.
      • Ando A.
      • Kurita H.
      • Damdindorj B.
      • Takano E.
      • Gantulga D.
      • Iwasaki Y.
      • Kurashina T.
      • Onaka T.
      • Dezaki K.
      • Nakata M.
      • Mori M.
      • Yada T.
      Stressor-responsive central nesfatin-1 activates corticotropin-releasing hormone, noradrenaline and serotonin neurons and evokes hypothalamic-pituitary-adrenal axis.
      ). It stimulates autonomic nervous system activity, increases blood pressure (
      • Yosten G.L.C.
      • Samson W.K.
      Nesfatin-1 exerts cardiovascular actions in brain: possible interaction with the central melanocortin system.
      ), and has been associated with a range of brain functions, including the regulation of feeding. Nucleobindin induces fear and anxiety in rats (
      • Merali Z.
      • Cayer C.
      • Kent P.
      • Anisman H.
      Nesfatin-1 increases anxiety- and fear-related behaviors in the rat.
      ). Nesfatin-1 also elevates the intracellular concentration of Ca2+ in mouse neurons and causes calcium signaling via calcium influx through CaV2.2 calcium channels (
      • Iwasaki Y.
      • Nakabayashi H.
      • Kakei M.
      • Shimizu H.
      • Mori M.
      • Yada T.
      Nesfatin-1 evokes Ca2+ signaling in isolated vagal afferent neurons via Ca2+ influx through N-type channels.
      ). Although platypus venom also elicits calcium signaling, this is believed to be due to the release of calcium from intracellular stores rather than the activation of calcium channels on the plasma membrane (
      • de Plater G.M.
      • Milburn P.J.
      • Martin R.L.
      Venom from the platypus, Ornithorhynchus anatinus, induces a calcium-dependent current in cultured dorsal root ganglion cells.
      ). Nesfatin-1 and CRF-BP might act synergistically. Nesfatin-1 activates CRF-responsive neurons in rats (
      • Yoshida N.
      • Maejima Y.
      • Sedbazar U.
      • Ando A.
      • Kurita H.
      • Damdindorj B.
      • Takano E.
      • Gantulga D.
      • Iwasaki Y.
      • Kurashina T.
      • Onaka T.
      • Dezaki K.
      • Nakata M.
      • Mori M.
      • Yada T.
      Stressor-responsive central nesfatin-1 activates corticotropin-releasing hormone, noradrenaline and serotonin neurons and evokes hypothalamic-pituitary-adrenal axis.
      ). Induction of CRF expression by venom nesfatin-1 in envenomated animals could result in the downstream interaction of CRF and venom CRF-BP. Thus, nucleobindin 2/nesfatin-1 might function to potentiate the effects of CRF-BP. As expected, nucleobindin-2 was up-regulated six-fold during the breeding season.
      Other Venom Proteins

      Differentiation Factor 15

      Differentiation factor 15 (GDF15) (ENSOANG00000001923) was identified in the venom proteome and up-regulated 48-fold during the breeding season. This was the first time GDF15 had been identified in venom. The platypus peptide contains the seven conserved cysteine residues in the C-terminal region that are required for the formation of a cysteine knot (
      • Zimmers T.A.
      • Jin X.
      • Hsiao E.C.
      • McGrath S.A.
      • Esquela A.F.
      • Koniaris L.G.
      Growth differentiation factor-15/macrophage inhibitory cytokine-1 induction after kidney and lung injury.
      ). GDF15 is a growth differentiation factor that is a divergent member of the transforming growth factor β (TGFB) superfamily of immune molecules. GDF15 is a regulator of the inflammatory response and is required for apoptosis (
      • Ago T.
      • Sadoshima J.
      GDF15, a cardioprotective TGF-β superfamily protein.
      ); it is triggered in response to injury and stress conditions in humans and mice (
      • Zimmers T.A.
      • Jin X.
      • Hsiao E.C.
      • McGrath S.A.
      • Esquela A.F.
      • Koniaris L.G.
      Growth differentiation factor-15/macrophage inhibitory cytokine-1 induction after kidney and lung injury.
      ).
      Platypus venom triggers nociceptor excitation via the activation of a serine or tyrosine kinase (
      • de Plater G.M.
      • Milburn P.J.
      • Martin R.L.
      Venom from the platypus, Ornithorhynchus anatinus, induces a calcium-dependent current in cultured dorsal root ganglion cells.
      ). Because GDF15, like other TGFB superfamily ligands, binds to and activates a serine/threonine receptor kinase as a first step in the signaling cascade (
      • Subramaniam S.
      • Strelau J.
      • Unsicker K.
      Growth differentiation factor-15 prevents low potassium-induced cell death of cerebellar granule neurons by differential regulation of Akt and ERK pathways.
      ), we suggest that GD15 might function in platypus venom to cause pain and hyperalgesia. Based on accounts of spreading pain in envenomated patients (
      • Fenner P.J.
      • Williamson J.A.
      • Myers D.
      Platypus envenomation—a painful learning experience.
      ), it is possible that the protein might exert both systemic and local effects on nerve cells at the site of envenomation.

      CD55

      Complement decay-accelerating factor (CD55) was sequenced from venom-gland cDNA based on the identification of the peptide in venom. CD55, along with CD59 (protectin), is a potent inhibitor of complement-mediated lysis in innate immunity. This was the first time CD55 was identified in the venom of any species, and its putative function is uncertain. Interestingly, CD59, a protein with an analogous function, is structurally related to snake venom neurotoxins, suggesting that toxin CD55 might have a similar neurotoxic role in the platypus (
      • Kieffer B.
      • Driscoll P.C.
      • Campbell I.D.
      • Willis A.C.
      • van der Merwe P.A.
      • Davis S.J.
      Three-dimensional solution structure of the extracellular region of the complement regulatory protein CD59, a new cell-surface protein domain related to snake venom neurotoxins.
      ). Consistent with this, CD55 was ∼0.3-fold up-regulated during the breeding season.

      CRISP

      A cysteine-rich secretory protein (CRISP) peptide, corresponding to a gene that was previously identified in the venom-gland transcriptome (
      • Whittington C.M.
      • Papenfuss A.T.
      • Locke D.P.
      • Mardis E.R.
      • Wilson R.K.
      • Abubucker S.
      • Mitreva M.
      • Wong E.S.W.
      • Hsu A.L.
      • Kuchel P.W.
      • Belov K.
      • Warren W.C.
      Novel venom gene discovery in the platypus.
      ), was also identified and sequenced. CRISPs contain an SCP domain (an extracellular domain also found in plant defense and mammalian testis-specific proteins) and a C-terminal cysteine-rich region. CRISPs are found in snake and reptile venoms, where they act as ion channel neurotoxins (
      • Yamazaki Y.
      • Morita T.
      Structure and function of snake venom cysteine-rich secretory proteins.
      ) or myotoxins (
      • Peichoto M.E.
      • Mackessy S.P.
      • Teibler P.
      • Tavares F.L.
      • Burckhardt P.L.
      • Breno M.C.
      • Acosta O.
      • Santoro M.L.
      Purification and characterization of a cysteine-rich secretory protein from Philodryas patagoniensis snake venom.
      ); and in cone snail venoms, where they have proteolytic activity (
      • Milne T.J.
      • Abbenante G.
      • Tyndall J.D.A.
      • Halliday J.
      • Lewis R.J.
      Isolation and characterization of a cone snail protease with homology to CRISP proteins of the pathogenesis-related protein superfamily.
      ). They are also found in the venoms of hymenopterans, arachnids, and cephalopods and in the feeding secretions of various hematophagous taxa (
      • Fry B.G.
      • Roelants K.
      • Champagne D.E.
      • Scheib H.
      • Tyndall J.D.A.
      • King G.F.
      • Nevalainen T.J.
      • Norman J.A.
      • Lewis R.J.
      • Norton R.S.
      • Renjifo C.
      • Rodríguez de la Vega R.C.
      The toxicogenomic multiverse: convergent recruitment of proteins into animal venoms.
      ,
      • Lu G.
      • Villalba M.
      • Coscia M.R.
      • Hoffman D.R.
      • King T.P.
      Sequence analysis and antigenic cross-reactivity of a venom allergen, antigen 5, from hornets, wasps, and yellow jackets.
      ). Thus, platypus CRISP might play a role in producing the symptoms of muscle atrophy seen in envenomated patients through toxic action on muscle and nerve tissues (
      • Whittington C.M.
      • Papenfuss A.T.
      • Locke D.P.
      • Mardis E.R.
      • Wilson R.K.
      • Abubucker S.
      • Mitreva M.
      • Wong E.S.W.
      • Hsu A.L.
      • Kuchel P.W.
      • Belov K.
      • Warren W.C.
      Novel venom gene discovery in the platypus.
      ). It might also form the protease component of platypus venom (
      • de Plater G.
      • Martin R.L.
      • Milburn P.J.
      A pharmacological and biochemical investigation of the venom from the platypus (Ornithorhynchus anatinus).
      ). Platypus CRISP shared the highest sequence similarity (75%, based upon an alignment using the BLOSUM62 scoring matrix) with an Anguimorpha lizard venom CRISP (Gerrhonotus infernalis), and it is also similar to helothermine, a toxin from the Mexican beaded lizard that blocks ryanodine receptors, intracellular calcium channels found in some excitable tissues (
      • Morrissette J.
      • Krätzschmar J.
      • Haendler B.
      • el-Hayek R.
      • Mochca-Morales J.
      • Martin B.M.
      • Patel J.R.
      • Moss R.L.
      • Schleuning W.D.
      • Coronado R.
      Primary structure and properties of helothermine, a peptide toxin that blocks ryanodine receptors.
      ). Yet, despite its sequence similarity to known toxins, CRISP is expressed at similar levels in both in- and out-of-season venom glands (0.1-fold difference). It is possible that the protein possesses both toxic and nontoxic roles in venom. Nontoxic functions of eutherian CRISP proteins include antimicrobial activity, sperm maturation, and gamete fusion.

      Chemokine

      A CXC chemokine was identified in the venom proteome (ENSOANG00000006911) and was three-fold up-regulated during the breeding season. Chemokines in this group are chemotactic, mediate cell growth, and trigger an inflammatory response. The platypus chemokine is a lineage-specific gene duplicate (122 residues) that is most similar to mammalian macrophage inflammatory protein-2. Cytokines have important roles in the regulation of the immune system, and platypus venom chemokine might serve to disrupt immune homeostasis in envenomated animals.

      DISCUSSION

      Using an integrated genomic, transcriptomic, and proteomic approach, we have uncovered ten putative toxins in the venom of the platypus. The use of this -omics approach has the benefit of detecting toxins that share little or no homology with known toxins. Accordingly, five of the venom proteins we identified are novel and had not been found in any other animal venom prior to this work. It is important to note that the sensitivity of protein identification is affected by protein stability, ionization biases, and abundance (
      • Liu H.
      • Sadygov R.G.
      • Yates 3rd, J.R.
      A model for random sampling and estimation of relative protein abundance in shotgun proteomics.
      ), and is also dependent on the sequence coverage of the transcriptome. However, we note that the transcriptomic database used for spectral searching was comprehensive enough to contain 1.33 Gb that matched over 7,600 total platypus Ensembl genes at 20 counts and above. In addition, we also used the Ensembl platypus genebuild, which contains 22,369 genes for peptide-spectra identification. Of course, a venom-gland transcriptome sequenced at higher depth will produce longer contigs that will lead to greater sensitivity in peptide-spectral matching with better resolution of less abundant proteins and alternative isoforms. The availability of a sequenced genome allows for resolution between alleles, gene duplicates, and alternative transcripts, which can be difficult to distinguish because of similarities in the sequences of venom transcripts.
      Despite being independently derived in each lineage, the venom of the platypus is most similar to those of snakes and other reptiles in that it contains amide oxidase, WAP, protease inhibitors, and CRISP. The similarities might reflect the two taxa's more recent common ancestry compared with other well-studied venomous taxa.
      A large number of immune-related genes were identified in platypus venom, including CD55, a chemokine, PGLYRP1, and GDF15. Immune genes might be suitable candidates for venom activity because of their role in coordinating fast-acting local and systemic responses. Genes that function in rapid physiological processes are likely to be co-opted to venom function (
      • Fry B.G.
      • Roelants K.
      • Champagne D.E.
      • Scheib H.
      • Tyndall J.D.A.
      • King G.F.
      • Nevalainen T.J.
      • Norman J.A.
      • Lewis R.J.
      • Norton R.S.
      • Renjifo C.
      • Rodríguez de la Vega R.C.
      The toxicogenomic multiverse: convergent recruitment of proteins into animal venoms.
      ). Their rapid rate of gene evolution might also render them favorable for co-option to novel functions (neofunctionalization) (
      • Wong E.S.W.
      • Papenfuss A.T.
      • Whittington C.M.
      • Warren W.C.
      • Belov K.
      A limited role for gene duplications in the evolution of platypus venom.
      ). It is notable that one class of antimicrobials, the β-defensins, gave rise to a major component of platypus venom, the venom defensin-like peptides (
      • Whittington C.M.
      • Papenfuss A.T.
      • Bansal P.
      • Torres A.M.
      • Wong E.S.W.
      • Deakin J.E.
      • Graves T.
      • Alsop A.
      • Schatzkamer K.
      • Kremitzki C.
      • Ponting C.P.
      • Temple-Smith P.
      • Warren W.C.
      • Kuchel P.W.
      • Belov K.
      Defensins and the convergent evolution of platypus and reptile venom genes.
      ).
      CRISP and WAP were not up-regulated during the mating season. Our results indicate that CRISP is constitutively expressed throughout the year, with WAP being up-regulated outside of the mating season. This suggests that these genes might not function as toxins and instead might play a role in up-keep and protection of the venom gland. It is also possible that some genes—particularly CRISP, given its similarity to lizard toxins—possess both toxin and nontoxin roles. It is also worth noting that all other venom genes were also expressed outside of the breeding season, albeit at much lower levels, suggesting that venom production is not completely abolished but is suppressed during nonbreeding periods. Alternatively, these genes might function in other capacities outside of the breeding season. Large numbers of leukocytes infiltrate the stroma, secretory epithelium, and gland lumena during gland regression (
      • Temple-Smith P.
      ), and this might lead to an increase of immune gene expression in the out-of-season transcriptome that is unrelated to toxin production.
      To our knowledge, the platypus is the only venomous animal that seasonally produces venom. Gene expression comparisons between venom glands obtained in and out of the breeding season offer a unique opportunity to inform us of venom gene regulation, providing insights into the putative functions of these genes. Although the large number of genes significantly up-regulated in the in-season venom gland (2,587) was likely due in part to the lack of biological replicates and subsequent underestimation of biological variation, our results suggest that venom gland gene expression changes dramatically during the breeding cycle. This corresponds with increases in gland weight and venom volumes and correlates directly with increases in the weight and activity of the testis and in the size of the androgen-producing Leydig cells in the testis (
      • Temple-Smith P.
      ). Up-regulated nonvenom genes are enriched in genes involved in cellular processes required for venom synthesis, such as the regulation of gene expression and RNA splicing (Fig. 2A). Similarly, a large number of genes (1,821) are up-regulated out of the breeding season. It is interesting to consider the activity of the crural gland at this time; we noted an abundance of genes functioning in cytoskeleton organization and protein secretion regulation (Fig. 2B). This might point to differences in the abundance of cell populations during the venom cycles. Indeed, secretory epithelial cells have been reported to be lost in the venom gland after the mating season, suggestive of seasonal changes in cell types (
      • Krause W.J.
      Morphological and histochemical observations on the crural gland-spur apparatus of the echidna (Tachyglossus aculeatus) together with comparative observations on the femoral gland-spur apparatus of the duckbilled platypus (Ornithorhyncus anatinus).
      ). Care should be taken when interpreting the result of the gene enrichment analyses because of the lack of biological replicates, and the use of human functional annotations to infer relationships. Biological replicates of venom glands would likely reduce the number of differentially expressed genes, producing more robust results. However, it is difficult to obtain samples, given the lack of a breeding colony for research purposes, and the two venom glands sequenced in this study were obtained opportunistically from animals found dead. It should also be noted that protein synthesis might not always correlate with gene expression.
      This is the first time, to our knowledge, that deep sequencing of the venom gland transcriptome has been used in combination to characterize the venom components from any venomous species. This rapid, genome-wide approach can be applied to any venomous species to identify venom peptides, even in species for which a genome assembly is not available. Given full-length transcripts, it will be possible to compare transcriptomic abundance and protein abundance using informatics strategies such as spectral counting (
      • Liu H.
      • Sadygov R.G.
      • Yates 3rd, J.R.
      A model for random sampling and estimation of relative protein abundance in shotgun proteomics.
      ).
      Future functional studies will allow us to understand the role of the identified venom proteins in the context of toxicity, which will shed light on their contribution to the observed symptoms of platypus envenomation. Animal venoms are a valuable source of novel drugs (
      • King G.F.
      Venoms as a platform for human drugs: translating toxins into therapeutics.
      ). Our findings might pave the way for future therapeutics and biomedical studies.

      Acknowledgments

      We thank the Tasmanian Department of Primary Industries and Water for their provision of opportunistically collected platypus tissue samples, and Dr. Kath Handasyde and Prof. Geoff Shaw for assistance in collecting the venom from platypuses in NSW. We thank the staff of The Genome Institute at Washington University, particularly Devin Locke, Vince Magrini, and Sean McGrath, for assistance with cDNA library preparation.

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