Petra H. Lenz

Identification of putative peptide paracrines/hormones in the water flea Daphnia pulex (Crustacea; Branchiopoda; Cladocera) using transcriptomics and immunohistochemistry

The cladoceran crustacean Daphnia pulex has emerged as a model species for many biological fields, in particular environmental toxicology and toxicogenomics. Recently, this species has been the subject of an extensive transcriptome project, resulting in the generation and public deposition of over 150,000 expressed sequence tags (ESTs). This resource makes D. pulex an excellent model for protein discovery using bioinformatics. Here, in silico searches of the D. pulex EST database were conducted to identify transcripts encoding putative peptide precursors. Moreover, the mature peptides contained within the deduced prepro-hormones were predicted using online peptide processing programs and homology to known arthropod isoforms. In total, 63 putative peptide-encoding ESTs were identified encompassing 14 distinct peptide families/subfamilies: A-type allatostatin, B-type allatostatin, C-type allatostatin, bursicon (both alpha and beta subunit peptides), crustacean cardioactive peptide (CCAP), crustacean hyperglycemic hormone (CHH)/ion transport peptide (both CHH- and moult-inhibiting hormone-like subfamilies), diuretic hormone (calcitonin-like), ecdysis-triggering hormone (ETH), FMRFamide (both neuropeptide F and short neuropeptide F subfamilies), orcokinin and pigment dispersing hormone. From these transcripts, the structures of 76 full-length/partial peptides were predicted, which included the first C-type allatostatin-like peptide identified from a crustacean, the first crustacean calcitonin-like diuretic hormone, an undescribed CCAP isoform, two hitherto unknown ETH variants, and two new orcokinins. Neuronal localization of several of the identified peptide families was confirmed using immunohistochemitry (i.e. A-type allatostatin, CCAP, FMRFamide and PDH). In addition, immunohistochemical analyses identified other putative neuropeptides for which no ESTs had been found (i.e. corazonin, insect kinin, proctolin, red pigment concentrating hormone, SIFamide, sulfakinin and tachykinin-related peptide). Collectively, the data presented here not only catalog an extensive array of putative D. pulex peptide paracrines/hormones, but also provide a strong foundation for future investigations of the effects of environmental/anthropogenic stressors on peptidergic control in this model organism.

Combining in silico transcriptome mining and biological mass spectrometry for neuropeptide discovery in the Pacific white shrimp Litopenaeus vannamei

The shrimp Litopenaeus vannamei is arguably the most important aquacultured crustacean, being the subject of a multi-billion dollar industry worldwide. To extend our knowledge of peptidergic control in this species, we conducted an investigation combining transcriptomics and mass spectrometry to identify its neuropeptides. Specifically, in silico searches of the L. vannamei EST database were conducted to identify putative prepro-hormone-encoding transcripts, with the mature peptides contained within the deduced precursors predicted via online software programs and homology to known isoforms. MALDI-FT mass spectrometry was used to screen tissue fragments and extracts via accurate mass measurements for the predicted peptides, as well as for known ones from other species. ESI-Q-TOF tandem mass spectrometry was used to de novo sequence peptides from tissue extracts. In total 120 peptides were characterized using this combined approach, including 5 identified both by transcriptomics and by mass spectrometry (e.g. pQTFQYSRGWTNamide, Arg(7)-corazonin, and pQDLDHVFLRFamide, a myosuppressin), 49 predicted via transcriptomics only (e.g. pQIRYHQCYFNPISCF and pQIRYHQCYFIPVSCF, two C-type allatostatins, and RYLPT, authentic proctolin), and 66 identified solely by mass spectrometry (e.g. the orcokinin NFDEIDRAGMGFA). While some of the characterized peptides were known L. vannamei isoforms (e.g. the pyrokinins DFAFSPRLamide and ADFAFNPRLamide), most were novel, either for this species (e.g. pEGFYSQRYamide, an RYamide) or in general (e.g. the tachykinin-related peptides APAGFLGMRamide, APSGFNGMRamide and APSGFLDMRamide). Collectively, our data not only expand greatly the number of known L. vannamei neuropeptides, but also provide a foundation for future investigations of the physiological roles played by them in this commercially important species.

The need for speed. I. Fast reactions and myelinated axons in copepods

Abstract A rapid and powerful escape response decreases predation risk in planktonic copepods. Calanoid copepods are sensitive to small and brief hydrodynamic disturbances: they respond with multiple nerve impulses to a vibrating sphere. Some species, such as Pleuromamma xiphias and Labidocera madurae, respond with very large spikes (1–4 mV), whereas maximum spike heights are an order of magnitude smaller in others, such as Undinula vulgaris and Neocalanus gracilis. A comparative study of the escape responses showed that all species reacted within 10 ms of the initiation of a hydrodynamic stimulus. However, U. vulgaris and N. gracilis had significantly shorter reaction times (minimum reaction times: 1.5 ms and 1.6 ms) than the other two, P. xiphias (6.6 ms) and L. madurae (3.1 ms). Examination of the first antenna and the central nervous system using transmission electron microscopy revealed extensive myelination of sensory and motor axons in the two species with the shorter reaction times. Axons of the other two species resembled typical crustacean unmyelinated fibers. A survey of 20 calanoids revealed that none of the species in two of the more ancient superfamilies possessed myelin, but myelination was present in the species from three more recently-evolved superfamilies.

Myelin-like sheaths in copepod axons

Copepods, the small planktonic crustaceans that are the most abundant metazoans in the oceans, are so successful partly because they have an escape response that accelerates them to 200 body lengths per second within milliseconds. We find that nerve fibres of many copepods seem to be designed for rapid signalling. They have well-developed myelin-like sheaths, like those that give a tenfold boost to the conduction speed of nerve impulses in vertebrates. By reducing a copepods reaction time to predatory attack, these sheaths may be crucial to the survival of the large copepod populations that inhabit dangerous oceanic ecosystems.

Environmental information stored in otoliths: insights from stable isotopes

The present study compares the stable oxygen-and carbon-isotope ratios (180:16O;13C:12C) in the otoliths of Atlantic cod,Gadus morhua, with those expected at equilibrium with seawater. Otoliths from juveniles reared for a 3 mo period under controlled conditions indicate that otoliths are formed in isotopic disequilibrium with seawater. This is probably due to positive metabolic fractionating of the heavier isotopes. This “vital effect” remains constant over the temperature range studied here (9 to 16°C) but may differ among other species. Our data indicate that the concentration of18O in calcium carbonate is inversely related to temperature and is described as ∂18Oa − ∂w − 3.79 − 0.200(T°C). The13C:12C ratios of otoliths and body tissues are related to the carbon ratio in the food source, although we found that the13C concentration is considerably higher in the otoliths relative te, the body tissues and the diet.

Effects of increasing salinity on anArtemia population from Mono Lake, California

SummarySalinity increased from 48 to 93 g/l total dissolved solids (TDS) in Mono Lake, California between 1941 and 1982, and is expected to fluctuate between 169 and 248 g/l at equilibrium by the middle of the next century. In order to predict the consequences of this trend on the Mono Lake ecosystem, we determined effects of salinity on survival, growth, reproduction and hatching ofArtemia monica, Mono Lakes only macrozooplankton species. Seven salainities ranging from 76 to 179 g/l were tested in a long-term experiment to determine both lethal and sublethal responses. The salt tolerance limit for subadultA. monica was between 159 and 179 g/l. Adult size, growth rates, and brood sizes decreased, and female mortality during reproduction increased, as salinity increased. Hatching of diapause eggs was delayed and total percent hatch decreased as salinity increased, and hatching failed at 159 g/l. The life-time reproductive potential of individual females decreased linearly over the seven salinities tested. Based on this study, we predict a decrease in the productivity of theA. monica population in Mono Lake and extinction of the species is probable before the lake reaches equilibrium.

Genomic analyses of the Daphnia pulex peptidome.

Genome mining has provided a valuable tool for peptide discovery in many species, yet no crustacean has undergone this analysis. Currently, the only crustacean with a sequenced genome is the cladoceran Daphnia pulex, a model organism in many fields of biology. Here, we have mined the D. pulex genome for peptide-encoding genes. For each gene identified, the encoded precursor protein was deduced, and its mature peptides predicted. Twenty-four peptide-encoding genes were identified, including ones predicted to produce members of the A-type allatostatin, B-type allatostatin, C-type allatostatin, allatotropin (ATR), bursicon α, bursicon β, calcitonin-like diuretic hormone, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone, ecdysis-triggering hormone, eclosion hormone (EH), insulin-like peptide (ILP), molt-inhibiting hormone, neuropeptide F, orcokinin (two genes), pigment-dispersing hormone, proctolin, red pigment concentrating hormone/adipokinetic hormone (RPCH/AKH), short neuropeptide F, SIFamide, sulfakinin, and tachykinin-related peptide (TRP) families/subfamilies. In total, 96 peptides were predicted from these genes. Our identification of isoforms of corazonin, EH, ILP, proctolin, RPCH/AKH, sulfakinin and TRP are the first for D. pulex, while our prediction of ATR from this species is the first from any crustacean. The number of peptides predicted in our study shows the power of genome mining for peptide discovery, and provides a model for future genomic analyses of the peptidomes of other crustaceans. In addition, the data presented in our study provide foundations for future molecular, biochemical, anatomical, and physiological investigation of peptidergic signaling in D. pulex and other cladoceran species.

Peptidergic signaling in Calanus finmarchicus (Crustacea, Copepoda): in silico identification of putative peptide hormones and their receptors using a de novo assembled transcriptome.

The copepod Calanus finmarchicus is the most abundant zooplankton species in the North Atlantic. While the life history of this crustacean is well studied, little is known about its peptidergic signaling systems despite the fact that these pathways are undoubtedly important components of its physiological/behavioral control systems. Here we have generated and used a de novo assembled transcriptome for C. finmarchicus (206,041 sequences in total) to identify peptide precursor proteins and receptors. Using known protein queries, 34 transcripts encoding peptide preprohormones and 18 encoding peptide receptors were identified. Using a combination of online software programs and homology to known arthropod isoforms, 148 mature peptides were predicted from the deduced precursors, including members of the allatostatin-A, allatostatin-B, allatostatin-C, bursicon, crustacean cardioactive peptide (CCAP), crustacean hyperglycemic hormone, diuretic hormone 31 (DH31), diuretic hormone 44 (DH44), FMRFamide-like peptide (myosuppressin, neuropeptide F [NPF] and extended FL/IRFamide subfamilies), leucokinin, neuroparsin, orcokinin, orcomyotropin, periviscerokinin, RYamide and tachykinin-related peptide (TRP) families. The identified receptors included ones for allatostatin-A, allatostatin-C, bursicon, CCAP, DH31, DH44, ecdysis-triggering hormone, NPF, short NPF, FMRFamide, insulin-like peptide, leucokinin, periviscerokinin, pigment dispersing hormone, and TRP. Developmental profiling of the identified transcripts in embryos, early nauplii, late nauplii, early copepodites, late copepodites, and adult females was also undertaken, with all showing the highest expression levels in the naupliar and copepodite stages. Collectively, these data radically expand the catalog of known C. finmarchicus peptidergic signaling proteins and provide a foundation for experiments directed at understanding the physiological roles served by them in this species.

De novo assembly of a transcriptome for Calanus finmarchicus (Crustacea, Copepoda)--the dominant zooplankter of the North Atlantic Ocean.

Assessing the impact of global warming on the food web of the North Atlantic will require difficult-to-obtain physiological data on a key copepod crustacean, Calanus finmarchicus. The de novo transcriptome presented here represents a new resource for acquiring such data. It was produced from multiplexed gene libraries using RNA collected from six developmental stages: embryo, early nauplius (NI-II), late nauplius (NV-VI), early copepodite (CI-II), late copepodite (CV) and adult (CVI) female. Over 400,000,000 paired-end reads (100 base-pairs long) were sequenced on an Illumina instrument, and assembled into 206,041 contigs using Trinity software. Coverage was estimated to be at least 65%. A reference transcriptome comprising 96,090 unique components (“comps”) was annotated using Blast2GO. 40% of the comps had significant blast hits. 11% of the comps were successfully annotated with gene ontology (GO) terms. Expression of many comps was found to be near zero in one or more developmental stages suggesting that 35 to 48% of the transcriptome is “silent” at any given life stage. Transcripts involved in lipid biosynthesis pathways, critical for the C. finmarchicus life cycle, were identified and their expression pattern during development was examined. Relative expression of three transcripts suggests wax ester biosynthesis in late copepodites, but triacylglyceride biosynthesis in adult females. Two of these transcripts may be involved in the preparatory phase of diapause. A key environmental challenge for C. finmarchicus is the seasonal exposure to the dinoflagellate Alexandrium fundyense with high concentrations of saxitoxins, neurotoxins that block voltage-gated sodium channels. Multiple contigs encoding putative voltage-gated sodium channels were identified. They appeared to be the result of both alternate splicing and gene duplication. This is the first report of multiple NaV1 genes in a protostome. These data provide new insights into the transcriptome and physiology of this environmentally important zooplankter.

The need for speed. II. Myelin in calanoid copepods

Abstract Speed of nerve impulse conduction is greatly increased by myelin, a multi-layered membranous sheath surrounding axons. Myelinated axons are ubiquitous among the vertebrates, but relatively rare among invertebrates. Electron microscopy of calanoid copepods using rapid cryofixation techniques revealed the widespread presence of myelinated axons. Myelin sheaths of up to 60 layers were found around both sensory and motor axons of the first antenna and interneurons of the ventral nerve cord. Except at nodes, individual lamellae appeared to be continuous and circular, without seams, as opposed to the spiral structure of vertebrate and annelid myelin. The highly organized myelin was characterized by the complete exclusion of cytoplasm from the intracellular spaces of the cell generating it. In regions of compaction, extracytoplasmic space was also eliminated. Focal or fenestration nodes, rather than circumferential ones, were locally common. Myelin lamellae terminated in stepwise fashion at these nodes, appearing to fuse with the axolemma or adjacent myelin lamellae. As with vertebrate myelin, copepod sheaths are designed to minimize both resistive and capacitive current flow through the internodal membrane, greatly speeding nerve impulse conduction. Copepod myelin differs from that of any other group described, while sharing features of every group.