Identification of putative crustacean neuropeptides using in silico analyses of publicly accessible expressed sequence tags

The development of expressed sequence tags (ESTs) for crustacean cDNA libraries and their deposition in publicly accessible databases has generated a rich resource for peptide discovery in this commercially and ecologically important arthropod subphylum. Here, we have conducted in silico searches of these databases for unannotated ESTs encoding putative neuropeptide precursors using the BLAST program tblastn, and have predicted the mature forms of the peptides encoded by them. The primary strategy used was to query the database with known decapod prepro-hormone sequences or, in some instances, insect precursor protein sequences. For neuropeptides for which no prepro-hormones are known, the peptides themselves were used as queries. For those peptides expected to originate from a common precursor, the individual sequences were combined, with each peptide flanked by a dibasic processing site and, if amidated, a glycine residue. Using these approaches, 13 unannotated ESTs encoding putative neuropeptide precursors were found. For example, using the first strategy, putative Marsupenaeus japonicus prepro-hormones encoding B-type allatostatins, neuropeptide F (NPF), and orcokinins were identified. Similarly, several Homarus americanus ESTs encoding putative orcokinin precursors were found. In addition to the decapod prepro-hormones, ESTs putatively encoding a NPF isoform and a red pigment concentrating hormone-like peptide were identified from the cladoceran Daphnia magna, as was one EST putatively encoding multiple tachykinin-related peptides from the isopod Eurydice pulchra. Using the second strategy, we identified a Carcinus maenas EST encoding HIGSLYRamide, a peptide recently discovered via mass spectrometry from Cancer productus. Using mass spectral methods we confirmed that this peptide is also present in Carcinus maenas. Collectively over 50 novel crustacean peptides were predicted from the identified ESTs, providing a strong foundation for future investigations of the evolution, regulation and function of these and related molecules in this arthropod taxon.     

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.     

Neuropeptide evolution: neurohormones and neuropeptides predicted from the genomes of Capitella teleta and Helobdella robusta

Genes encoding neurohormones and neuropeptide precursors were identified in the genomes of two annelids, the leech Helobdella robusta and the polychaete worm Capitella teleta. Although no neuropeptides have been identified from these two species and relatively few neuropeptides from annelids in general, 43 and 35 such genes were found in Capitella and Helobdella, respectively. The predicted peptidomes of these two species are similar to one another and also similar to those of mollusks, particular in the case of Capitella. Helobdella seems to have less neuropeptide genes than Capitella and it lacks the glycoprotein hormones bursicon and GPA2/GPB5; in both cases the genes coding the two subunits as well as the genes coding their receptors are absent from its genome. In Helobdella several neuropeptide genes are duplicated, thus it has five NPY genes, including one pseudogene, as well as four genes coding Wwamides (allatostatin B). Genes coding achatin, allatotropin, allatostatin C, conopressin, FFamide, FLamide, FMRFamide, GGRFamide, GnRH, myomodulin, NPY, pedal peptides, RGWamide (a likely APGWamide homolog), RXDLamide, VR(F/I)amide, WWamide were found in both species, while genes coding cerebrin, elevenin, GGNG, LFRWamide, LRFYamide, luqin, lymnokinin and tachykinin were only found in Capitella.     

Neurohormones and neuropeptides encoded by the genome of Lottia gigantea, with reference to other mollusks and insects

The Lottia gigantea genome was prospected for the presence of genes coding neuropeptides and neurohormones. Four genes code insulin-related peptides: two genes code molluscan insulin-like growth hormones, one gene an insulin very similar to vertebrate insulin, and the fourth a peptide related to drosophila insulin-like peptide 7. Four other genes encode the cysteine-knot proteins GPA2/GPB5 and bursicon/parabursicon. Another 37 genes code for precursors of the following neuropeptides: achatin, APGWamide, allatostatin C, allatotropin, buccalin (perhaps an allatostatin A homolog), cerebrin, CCAP, conopressin, elevenin (the predicted neuropeptide made by abdominal neuron 11 in Aplysia), egg laying hormone (two genes), enterin, feeding circuit activating neuropeptide (FCAP), FFamide, FMRFamide, GGNG, a GnRH-like peptide, the newly discovered LASGLVamide, LFRFamide, LFRYamide, LRNFVamide, luqin, lymnokinin, myomodulin (two genes), the newly discovered NKY, NPY, pedal peptide (three genes), PKYMDT, pleurin, PXFVamide, small cardioactive peptides, tachykinins (two genes) and WWamide (an allatostatin B homolog). One gene was found to encode FWISamide, while about 20 closely related genes were found to encode WWFamide. These small neuropeptides appear homologous to the NdWFamide, which contains d-Trp; these genes are similar to the Aplysia gene encoding NWFamide. Some of these peptides had not been previously identified from mollusks, such as the predicted hormones similar to Drosophila and vertebrate insulins, bursicon, the putative proctolin homolog PKYMDT and allatostatin C. Together with neuropeptides which are likely homologs of other insect neuropeptides, such as cerebrin and WWamide, this shows that despite significant differences the molluscan and arthropod neuropeptidomes are more similar than generally recognized.     

Adipokinetic hormone signaling through the gonadotropin-releasing hormone receptor modulates egg-laying in Caenorhabditis elegans

In mammals, hypothalamic gonadotropin-releasing hormone (GnRH) is a neuropeptide that stimulates the release of gonadotropins from the anterior pituitary. The existence of a putative functional equivalent of this reproduction axis in protostomian invertebrates has been a matter of debate. In this study, the ligand for the GnRH receptor in the nematode Caenorhabditis elegans (Ce-GnRHR) was found using a bioinformatics approach. The peptide and its precursor are reminiscent of both insect adipokinetic hormones and GnRH-preprohormone precursors from tunicates and higher vertebrates. We cloned the AKH-GnRH-like preprohormone and the Ce-GnRHR and expressed the GPCR in HEK293T cells. The GnRHR was activated by the C. elegans AKH-GnRH-like peptide (EC₅₀ = 150 nM) and by Drosophila AKH and other nematode AKH-GnRHs that we found in EST databases. Analogous to both insect AKH receptor and vertebrate GnRH receptor signaling, Ce-AKH-GnRH activated its receptor through a Gα_q protein with Ca²⁺ as a second messenger. Gene silencing of Ce-GnRHR, Ce-AKH-GnRH, or both resulted in a delay in the egg-laying process, comparable to a delay in puberty in mammals lacking a normal dose of GnRH peptide or with a mutated GnRH precursor or receptor gene. The present data support the view that the AKH-GnRH signaling system probably arose very early in metazoan evolution and that its role in reproduction might have been developed before the divergence of protostomians and deuterostomians.     

Growth hormone and prolactin in Andrias davidianus: cDNA cloning, tissue distribution and phylogenetic analysis

The Chinese giant salamander (Andrias davidianus) is one of the largest and ‘living fossil’ species of amphibian. To obtain genetic information for this species, the cDNAs encoding growth hormone (adGH) and prolactin (adPRL) were cloned from a pituitary cDNA library. The isolated adGH cDNA consisted of 864 bp and encoded a propeptide of 215 amino acids, while the cDNA of adPRL was 1106 bp in length and encoded a putative peptide of 229 amino acids. Expression of the GH and PRL mRNA was only detected in the pituitary. Phylogenetic analyses were performed based on the isolated pituitary hormone sequences using maximum parsimony and neighbor-joining algorithms. The clustering results are similar to that based on the morphological characteristics or the rRNA genes, which indicate that the two orders (Anura and Caudata) of amphibian were monophyletic, and that A. davidianus was diverged early in the Caudate clade. These results indicated that both the GH and PRL sequence might be useful to study the phylogenies of relatively moderate evolved groups.     

Incrimination of Eratyrus cuspidatus (Stal) in the transmission of Chagas’ disease by molecular epidemiology analysis of Trypanosoma cruzi isolates from a geographically restricted area in the north of Colombia

Following the report of two cases of acute Chagas’ disease and the appearance of several triatomine species in human dwellings in an area considered non-endemic for domestic transmission of Trypanosoma cruzi; a epidemiological, entomological and T. cruzi molecular epidemiology analysis was performed in order to establish the transmission dynamic of the parasite in the studied area. 2 T. cruzi isolates from human patients, 5 from Eratyrus cuspidatus, 4 from Rhodnius pallescens, 4 from Panstrongylus geniculatus and 7 reference stocks were analyzed by mini-exon gene, random amplified polymorphic DNA (RAPD) and multilocus enzyme electrophoresis (MLEE).All isolates from vectors and human resulted T. cruzi group I by mini-exon, RAPD and MLEE. While mini-exon and MLEE did not showed any differences between the studied isolates, RAPD analysis identified a common T. cruzi genotype for the E. cuspidatus isolates and human isolates and distinguished different strains from R. pallescens and P. geniculatus isolates. The presence of the same T. cruzi genotype in isolates from patients and E. cuspidatus suggests that this species can be responsible for the transmission of Chagas’ disease in the study area. RAPD analysis showed better resolution and discrimination of T. cruzi strains than mini-exon and MLEE and can be considered a useful tool for molecular epidemiology studies. Incrimination of sylvatic triatomine species in the transmission of Chagas’ disease indicates that more knowledge about the ecology of these vectors is necessary to improve control strategies.     

Bombyx prothoracicostatic peptides activate the sex peptide receptor to regulate ecdysteroid biosynthesis

Insect molting and metamorphosis are induced by steroid hormones named ecdysteroids, whose production is regulated by various neuropeptides. We cloned the gene and analyzed the expression of the prothoracicostatic peptide, a unique neuropeptide shown to suppress the production of ecdysteroids in the prothoracic gland of the silkworm, Bombyx mori. We also characterized a Bombyx G protein-coupled receptor, which has previously been identified as an ortholog of the Drosophila sex peptide receptor, as a functional prothoracicostatic peptide receptor. This receptor responded specifically to the prothoracicostatic peptides when examined using a heterologous expression system. The receptor was highly expressed in the prothoracic gland on the day before each larval and pupal ecdysis, when prothoracicostatic peptides are synthesized at a high level in the epiproctodeal glands. These results suggest that the sex peptide receptor functions as a prothoracicostatic peptide receptor in Bombyx and that the peripheral neurosecretory cells as well as the central neuroendocrine system play stage-specific roles in regulating ecdysteroidogenesis.     

Genetic analyses of Trypanosoma cruzi isolates from naturally infected triatomines and humans in northeastern Brazil

Trypanosoma cruzi genetic diversity was investigated in 25 isolates (vectors and humans) from the semiarid zone of the State of Rio Grande do Norte, Brazil. Molecular markers (3′ region of the 24Sα rRNA; mitochondrial cytochrome oxidase subunit 2 (COII) gene; spliced leader intergenic region (SL-IR) gene; allelic size microsatellite polymorphism) identified 56% TcIII (100% Panstrongyluslutzi; 50% Triatomabrasiliensis); 40% TcII (91.7% humans; 50% T. brasiliensis) and 4% TcI (human). Microsatellite analysis revealed monoclonal and heterozygous patterns on one or more microsatellite loci in 64% of T. cruzi isolates (92.3% triatomines; 33.3% humans) and 36% putative polyclonal populations (66.7% humans; 7.7% triatomines) by loci SCLE10, SCLE11, TcTAT20, TcAAAT6, all belonging to TcII. Identical T. cruzi polyclonal profiles (88.9%) were detected, mostly from humans. The adaptative natural plasticity of TcII and TcIII and their potential for maintaining human infection in T. brasiliensis were confirmed. Intraspecific and phylogenetic T. cruzi diversity in the sylvatic and domestic transmission cycles in this specific region will provide exclusive control strategies.