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

Center for Marine Functional Genomics, Mount Desert Island Biological Laboratory, Salisbury Cove, ME 04672, USA.
General and Comparative Endocrinology (Impact Factor: 2.47). 02/2009; 160(3):271-87. DOI: 10.1016/j.ygcen.2008.12.014
Source: PubMed


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.

1 Follower
24 Reads
    • "To identify Tigriopus transcripts encoding putative circadian protein homologs, known circadian sequences from C. finmarchicus (Christie et al., 2013a) were used as the input queries for BLAST searches of the publicly accessible T. californicus TSA data; this method is a wellvetted strategy that has been used previously for the identification of transcripts/genes encoding a variety of proteins, both from this and other crustacean species (Christie et al., 2008; Gard et al., 2009; Ma et al., 2009; Christie et al., 2010; Ma et al., 2010; Christie et al., 2011; McCoole et al., 2011; Tilden et al., 2011; McCoole et al., 2012a,b; Christie et al., 2013a,b,c; Christie, 2014a, 2014b, 2014c, 2014d, 2014e, 2014f; Christie et al., 2014a,b; Lenz et al., 2014). Specifically, the database of the online program tblastn (National Center for Biotechnology Information, Bethesda, MD; was set to " Transcriptome Shotgun Assembly (TSA) " and restricted to sequence data from " Tigriopus californicus (taxid:6832) " . "
    [Show abstract] [Hide abstract]
    ABSTRACT: Copepods of the genus Tigriopus have been proposed as marine models for investigations of environmental perturbation. One rapidly increasing anthropogenic stressor for intertidal organisms is light pollution. Given the sensitivity of circadian rhythms to exogenous light, the genes/proteins of a Tigriopus circadian pacemaker represent a potential system for investigating the influences of artificial light sources on circadian behavior in an intertidal species. Here, the molecular components of a putative Tigriopus californicus circadian clock were identified using publicly accessible transcriptome data; the recently deduced circadian proteins of the copepod Calanus finmarchicus were used as a reference. Transcripts encoding homologs of all commonly recognized ancestral arthropod core clock proteins were identified (i.e. CLOCK, CRYPTOCHROME 2, CYCLE, PERIOD and TIMELESS), as were ones encoding proteins likely to modulate the core clock (i.e. CASEIN KINASE II, CLOCKWORK ORANGE, DOUBLETIME, PROTEIN PHOSPHATASE 1, PROTEIN PHOSPHATASE 2A, SHAGGY, SUPERNUMERARY LIMBS and VRILLE) or to act as inputs to it (i.e. CRYPTOCHROME 1). PAR DOMAIN PROTEIN 1 was the only circadian-associated protein not identified in Tigriopus; it appears absent in Calanus too. These data represent just the third full set of molecular components for a crustacean circadian pacemaker (Daphnia pulex and C. finmarchicus previously), and only the second obtained from transcribed sequences (C. finmarchicus previously). Given Tigriopus’ proposed status as a model for investigating the influences of anthropogenic stressors in the marine environment, these data provide the first suite of gene/protein targets for understanding how light pollution may influence circadian physiology and behavior in an intertidal organism.
    No preview · Article · Sep 2014 · Comparative Biochemistry and Physiology Part D Genomics and Proteomics
  • Source
    • "The SIFamides represent a family of neuropeptides, which are highly conserved within the arthropods [89]. Since their first characterisation in the flesh fly Neobelleria bullata (as a myotropic agent) [90], an increasing number of these peptides have also been identified in the Hexapoda [91]–[94], in the Crustacea [7], [19], [34], [74], [88], [95]–[97] and in the Chelicerates [24]. They are highly abundant peptides and therefore relatively easy to isolate using techniques such as HPLC, MALDI and QTOF, but are also present in the molecular databases of genome sequences, ESTs and transcriptomes. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The Ice krill, Euphausia crystallorophias is one of the species at the base of the Southern Ocean food chain. Given their significant contribution to the biomass of the Southern Ocean, it is vitally important to gain a better understanding of their physiology and, in particular, anticipate their responses to climate change effects in the warming seas around Antarctica. Illumina sequencing was used to produce a transcriptome of the ice krill. Analysis of the assembled contigs via two different methods, produced 36 new pre-pro-peptides, coding for 61 neuropeptides or peptide hormones belonging to the following families: Allatostatins (A, B et C), Bursicon (α and β), Crustacean Hyperglycemic Hormones (CHH and MIH/VIHs), Crustacean Cardioactive Peptide (CCAP), Corazonin, Diuretic Hormones (DH), the Eclosion Hormone (EH), Neuroparsin, Neuropeptide F (NPF), small Neuropeptide F (sNPF), Pigment Dispersing Hormone (PDH), Red Pigment Concentrating Hormone (RPCH) and finally Tachykinin. LC/MS/MS proteomics was also carried out on eyestalk extracts, which are the major site of neuropeptide synthesis in decapod crustaceans. Results confirmed the presence of six neuropeptides and six precursor-related peptides previously identified in the transcriptome analyses. This study represents the first comprehensive analysis of neuropeptide hormones in a Eucarida non-decapod Malacostraca, several of which are described for the first time in a non-decapod crustacean. Additionally, there is a potential expansion of PDH and Neuropeptide F family members, which may reflect certain life history traits such as circadian rhythms associated with diurnal migrations and also the confirmation via mass spectrometry of several novel pre-pro-peptides, of unknown function. Knowledge of these essential hormones provides a vital framework for understanding the physiological response of this key Southern Ocean species to climate change and provides a valuable resource for studies into the molecular phylogeny of these organisms and the evolution of neuropeptide hormones.
    Full-text · Article · Aug 2013 · PLoS ONE
  • Source
    • "Also in Crustacea, MIPs have been found and with five exons, the AstB gene is rather complex in Daphnia pulex (Dircksen et al., 2011). However, most of the crustacean MIPs have been identified in decapod species, such as Cancer borealis, Homarus americanus and Marsupenaeus japonicus (Fu et al., 2005, 2007; Gard et al., 2009; Ma et al., 2008, 2009, 2010). A MIP has also been found in the mollusc, Aplysia californica (Moroz et al., 2006). "
    [Show abstract] [Hide abstract]
    ABSTRACT: In many animal species, copulation elicits a number of physiological and behavioral changes in the female partner. In Drosophila melanogaster, the main molecular effector of these physiological responses has been identified as sex peptide (SP). The sex peptide receptor (SPR) has been characterized and recently, its activation by Drosophila myoinhibiting peptides (MIPs) - in addition to SP - has been demonstrated. The myoinhibiting peptides are members of a conserved peptide family, also known as B-type allatostatins, which generally feature the C-terminal motif -WX6Wamide.
    Full-text · Article · Feb 2013 · General and Comparative Endocrinology
Show more