Distribution of pigment dispersing hormone- and tachykinin-related peptides in the central nervous system of the copepod crustacean Calanus finmarchicus

Peptides represent the largest class of signaling molecules used by nervous systems, functioning as locally-released paracrines and circulating hormones in both invertebrates and vertebrates. While many studies have focused on elucidating peptidergic systems in higher crustaceans, little is known about neuropeptides in the more primitive crustacean taxa. Here, we have begun an investigation of the peptides present in the central nervous system (CNS) of the copepod crustacean Calanus finmarchicus, presenting immunohistochemical data on the presence and distribution of pigment dispersing hormone (PDH) and tachykinin-related peptide (TRP). In this species, strong PDH-like immunoreactivity was restricted to one pair of somata in the protocerebrum (PC) and the axonal projections emanating from them. TRP-like immunopositive structures were present in the PC, deutocerebrum (DC), tritocerebrum (TC), and ventral nerve cord (VNC). In the PC, a single soma in the left hemisphere was labeled. This neuron appears to be the source of a centrally located, bilaterally symmetric plexus present within the PC. In the DC, two pairs of intensely immunopositive somata were labeled, each projecting axons toward the posterior and producing an extensive collection of putative release terminals that spans the DC, TC, and anterior portion of the VNC. Several other more weakly labeled somata were also present in the DC. Double-labeling studies indicated that no co-localization of PDH- and TRP-like peptides is present in the C. finmarchicus CNS. As preadsorption controls completely abolished each label, we feel these data represent accurate distributions of PDH- and TRP-like peptides within the C. finmarchicus CNS, thus providing a framework for future studies of the functional roles members of these peptide families play in this copepod species.     

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.     

SIFamide peptides in clawed lobsters and freshwater crayfish (Crustacea, Decapoda, Astacidea): a combined molecular, mass spectrometric and electrophysiological investigation

Recently, we identified the peptide VYRKPPFNGSIFamide (Val(1)-SIFamide) in the stomatogastric nervous system (STNS) of the American lobster Homarus americanus using matrix-assisted laser desorption/ionization-Fourier transform mass spectrometry (MALDI-FTMS). Given that H. americanus is the only species thus far shown to possess this peptide, and that a second SIFamide isoform, Gly(1)-SIFamide, is broadly conserved in other decapods, including another astacidean, the crayfish Procambarus clarkii, we became interested both in confirming our identification of Val(1)-SIFamide via molecular methods and in determining the extent to which this isoform is conserved within other members of the infraorder Astacidea. Here, we present the identification and characterization of an H. americanus prepro-SIFamide cDNA that encodes the Val(1) isoform. Moreover, we demonstrate via MALDI-FTMS the presence of Val(1)-SIFamide in a second Homarus species, Homarus gammarus. In contrast, only the Gly(1) isoform was detected in the other astacideans investigated, including the lobster Nephrops norvegicus, a member of the same family as Homarus, and the crayfish Cherax quadricarinatus, P. clarkii and Pacifastacus leniusculus, which represent members of each of the extant families of freshwater astacideans. These results suggest that Val(1)-SIFamide may be a genus (Homarus)-specific isoform. Interestingly, both Val(1)- and Gly(1)-SIFamide possess an internal dibasic site, Arg(3)-Lys(4), raising the possibility of the ubiquitously conserved isoform PPFNGSIFamide. However, this octapeptide was not detected via MALDI-FTMS in any of the investigated species, and when applied to the isolated STNS of H. americanus possessed little bioactivity relative to the full-length Val(1) isoform. Thus, it appears that the dodeca-variants Val(1)- and Gly(1)-SIFamide are the sole bioactive isoforms of this peptide family in clawed lobsters and freshwater crayfish.     

Finding of Parastrongylus cantonensis (Chen, 1935) in Rattus rattus in Tenerife, Canary Islands (Spain)

Parastrongylus cantonensis is a parasite of murid rodents that can infect humans and cause health problems as eosinophilic meningitis. Although it is endemic in south Asia, the Pacific islands, Australia, USA, and a few Caribbean islands, it has been extended to new geographical regions. In the Canary Islands (Spain) a survey of helminths of Rattus rattus, Rattus norvegicus and Mus musculus domesticus was carried out. Furthermore, five species of molluscs were examined for nematode larvae to determine whether they are potential intermediate hosts of P. cantonensis. Nematodes were found in the lungs of 15% of 67 R. rattus examined in Tenerife, one of the four studied islands, with a prevalence of 20% in the highest focus of infection. Based on morphological and molecular analysis, with the complete internal transcribed spacer 2 (ITS-2) and a fragment of the small subunit ribosomal RNA (SSU rRNA) nucleotide sequences, nematodes were identified as P. cantonensis. Larval nematodes found from snails and slugs were identified as third-stage (L₃) Metastrongyloidea, but the molecular study showed that they did not belong to P. cantonensis. This is the first finding of angiostrongyliasis in rats in the Canary Islands (Spain). New molecular data for this species and Parastrongylus dujardini are reported. The presence of P. cantonensis in Tenerife could be of importance from the public health point of view. Further studies are required in order to look for other potential foci of infections in the Canary Islands.     

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.     

Effects of angiotensin II and natriuretic peptides of the eel on prolactin and growth hormone release in the tilapia,Oreochromis mossambicus

The effects of angiotensin II (ANG II) and natriuretic peptides (NPs) of the eel (ANP, atrial natriuretic peptide; CNP, C-type natriuretic peptide; and VNP, ventricular natriuretic peptide) on prolactin (PRL(188) and PRL(177)) and growth hormone (GH) release from the organ-cultured tilapia pituitary were examined. Eel ANG II at concentrations greater than 1 nM stimulated the release of PRL(188) and PRL(177) in a dose-related manner during the first hour of incubation. Significant stimulation by 100 nM ANG II on PRL(177) release was observed until 4h of incubation, and on PRL(188) release until 12 h. No effect of ANG II was seen on GH release. None of the NPs altered the release of PRLs at any time point. On the other hand, eel VNP at concentrations greater than 1 nM stimulated GH release in a dose-related manner after 4 h, and significant stimulation was observed until 48 h. Eel CNP was less effective than eel VNP; significant stimulation of GH release was observed at 1 and 10 nM during 24-48 h of incubation. No significant effect of eel ANP on GH release was seen at any concentration. ANG II had no effect on GH release at any time point. There was no change in mRNA levels of PRLs or GH in the pituitaries incubated with ANG II for 8 h or those incubated with the NPs for 48 h. These results indicate rapid and short-lasting stimulation by ANG II on PRL release and slow and long-lasting stimulation by VNP and CNP on GH release from the tilapia pituitary.     

Identification of A-type allatostatins possessing -YXFGI/Vamide carboxy-termini from the nervous system of the copepod crustacean Calanus finmarchicus

The copepod crustacean Calanus finmarchicus plays a critical role in the ecology of the Gulf of Maine and other regions of the North Atlantic. To increase our understanding of the physiology of this species, a normalized, whole organism cDNA library was constructed, and expressed sequence tags (ESTs) of the clones were generated. Among these ESTs was one with homology to known cDNAs encoding prepro-A-type allatostatins (A-type ASTs), a well-known family of arthropod peptides that regulate juvenile hormone production in insects. Sequence analysis of the clone from which the EST was generated, with subsequent translation of its open reading frame, showed it to encode five novel A-type ASTs, whose mature structures were predicted to be APYGFGIamide, pE/EPYGFGIamide, ALYGFGIamide, pE/EPYNFGIamide, and pQ/QPYNFGVamide. Each of the peptides is present as a single copy within the prepro-hormone with the exception of APYGFGIamide, which is present in three copies. Surprisingly, the organization of the Calanus prepro-A-type AST, specifically the number of encoded A-type peptides, is more similar to those of insects than it is to the known decapod crustacean prepro-hormones. Moreover, the Calanus A-type ASTs possess isoleucine or valine residues at their carboxy (C)-termini rather than leucine, which is present in most other family members. Wholemount immunohistochemistry suggests that six pairs of somata produce the native Calanus A-type ASTs: five in the protocerebrum and one in the suboesophageal region. To the best of our knowledge, our report is the first characterization of a neuropeptidergic system in a copepod, the first identification of A-type ASTs from a non-decapod crustacean, the first report of crustacean A-type ASTs possessing isoleucine C-terminal residues, and the first report from any species of an A-type peptide possessing a valine C-terminal residue.     

Thyroid-stimulating hormone (TSH): measurement of intracellular, secreted, and circulating hormone in Xenopus laevis and Xenopus tropicalis

Thyroid-stimulating hormone (TSH) is an important regulator of the hypothalamic-pituitary-thyroid (HPT) axis in Xenopus laevis. To evaluate the role of this hormone on developing tadpoles, immunologically-based Western blots and sandwich ELISAs were developed for measuring intracellular (within pituitaries), secreted (ex vivo pituitary culture), and circulating (serum) amounts. Despite the small size of the tadpoles, these methods were able to easily measure intracellular and secreted TSH, and circulating TSH was measurable in situations where high levels were induced. The method was validated after obtaining a highly purified and enriched TSH sample using anti-TSH-β antibodies conjugated to magnetic beads. Subsequent mass-spectrometric analysis of the bands from SDS-PAGE and Western procedures identified the presence of amino acid sequences corresponding to TSH subunits. The purified sample was also used to prepare standard curves for quantitative analysis. The Western and ELISA methods had limits of detection in the low nanogram range. While the majority of the developmental work for these methods was done with X. laevis, the methods also detected TSH in Xenopus tropicalis. To our knowledge this is the first report of a specific detection method for TSH in these species, and the first to measure circulating TSH in amphibians. Examples of the utility of the methods include measuring a gradual increase in pituitary TSH at key stages of development, peaking at stages 58-62; the suppression of TSH secretion from cultured pituitaries in the presence of thyroid hormone (T4); and increases in serum TSH following thyroidectomy.     

Expression profiles of Dax1, Dmrt1, and Sox9 during temperature sex determination in gonads of the sea turtle Lepidochelys olivacea

Sex determination is controlled either by genetic or environmental factors. In mammals Sry initiates determination but no homologue of this gene exists in non-mammalian species. Other genes of the mammalian sex-determining pathway have been identified in gonads of different vertebrates. Sox9, Dax1, and Dmrt1 are expressed at the onset of gonadal development in birds and reptiles. In the sea turtle Lepidochelys olivacea, a species with temperature sex determination (TSD), Sox9 is expressed in undifferentiated gonads at male- (MPT) or female-promoting temperatures (FPT). At MPT, Sox9 remains expressed in male gonads, but at FPT it is downregulated coinciding with the onset of the ovarian morphologic differentiation and female sex determination. At MPT however, male sex is determined early than at FPT in still undifferentiated gonads suggesting that other genes maintain Sox9 expression in testis. Here we used RT-PCR to study the expression profiles of Dax1, Dmrt1, and Sox9 in gonads of embryos of L. olivacea incubated at MPT or at FPT. The profiles were correlated with sex determination during and after the temperature-sensitive period (TSP). Dax1 maintained similar levels at both temperatures during the TSP. The Dax1 expression level increased significantly in ovaries compared to testes at stage 27, once they were morphologically distinct. The expression levels of Dmrt1 were higher at MPT than at FPT at all stages, in contrast with Sox9 levels which were similar at both temperatures at stages 23-25. Together, current results suggest that, whereas Dax1 is not involved in TSD in L. olivacea, upregulation of Dmrt1 and downregulation of Sox9 may play a role in male and female sex determination, respectively.     

Roles of ecdysteroid and juvenile hormone in vitellogenesis in an endoparasitic wasp, Pteromalus puparum (Hymenoptera: Pteromalidae)

To elucidate the endocrine regulation of vitellogenesis in an endoparastic wasp (Pteromalus puparum), the titers of ecdysteroid and juvenile hormone (JH) from the whole bodies are measured using the method of radioimmunoassay and GC-MS, and compared with the levels of vitellogenin (Vg) mRNA in the fat bodies, hemolymph Vg and ovarian vitellin (Vt), respectively. The results show that the ecdysteroid titer and fat body Vg mRNA level have a similar dynamics tendency, and the peak titer is at adult eclosion. The titer of JH III and ovarian Vt also have a similar dynamics tendency, and the peak titer is at 48 h after eclosion. The profiles of hemolymph Vg, Vg mRNA in fat bodies and ovarian Vt, are also measured in the wasps after treated with different amounts of 20-hydroxyecdysone (20HE) or JH III in female pupa and adults. The results show that 20HE stimulates Vg synthesis in the fat bodies and its release into the hemolymph, and that JH III only accelerates Vg sequestration in the oocytes. Decapitation, which is believed to terminate synthesis of JH in insects, can not inhibit vitellogenesis and oocyte maturation in P. puparum. Furthermore, Vg gene is expressed with a lower titer of JH and depressed by a higher titer of JH III. These studies suggest that ecdysteroids play a role in Vg synthesis and believed to be the dominant hormones in regulation of vitellogenesis in P. puparum, and JHs are not the essential factors to female reproduction in this wasp.