LFRFamides: a novel family of parasitation-induced -RFamide neuropeptides that inhibit the activity of neuroendocrine cells in Lymnaea stagnalis

We report the characterization of a cDNA encoding a novel -RFamide neuropeptide precursor that is up-regulated during parasitation in the snail Lymnaea stagnalis. Processing of this precursor yields five structurally related neuropeptides, all but one ending with the C-terminal sequence -LFRFamide, as was confirmed by direct mass spectrometry of brain tissue. The LFRFamide gene is expressed in a small cluster of neurons in each buccal ganglion, three small clusters in each cerebral ganglion, and one cluster in each lateral lobe of the cerebral ganglia. Application of two of the LFRFamide peptides to neuroendocrine cells that control either growth and metabolism or reproduction induced similar hyperpolarizing K+-currents, and inhibited electrical activity. We conclude that up-regulation of inhibitory LFRFamide neuropeptides during parasitation probably reflects an evolutionary adaptation that allows endoparasites to suppress host metabolism and reproduction in order to fully exploit host energy recourses.     

Characterization of Insulin-like Peptide (ILP) and Its Potential Role in Ovarian Development of the Cuttlefish Sepiella japonica

The insulin-like peptide (ILP) family is well known for regulating reproduction in invertebrates, while its role in mollusks remains largely unknown. In this study, we first isolated and characterized the ILP gene in the cuttlefish Sepiella japonica. The full-length SjILP cDNA obtained was 926 bp and encoded a precursor protein of 161 amino acids. The precursor protein consisted of a signal peptide, a B chain, a C-peptide, and an A chain. It possessed the typical features of ILP proteins, including two cleavage sites (KR) and eight conserved cysteines. To define the function of SjILP, the expression of SjILP in different tissues and ovarian development stages were analyzed using qRT-PCR. SjILP was mainly expressed in the ovary, and its gene expression correlated with ovarian development. Furthermore, silencing SjILP using RNA interference (RNAi) dramatically decreased the expression levels of four ovarian-development-related genes (vitellogenin1, vitellogenin2, cathepsin L1-like, and follistatin). These data suggest the critical role of SjILP in the regulation of ovarian development in S. japonica.     

Molecular analysis of a novel FMRFamide-related peptide gene (SOFaRP(2)) and its expression pattern in the brain of the European cuttlefish Sepia officinalis

FMRFamide-related peptides (FaRPs) are among several neurotransmitters known to regulate the chromatophore function in the European cuttlefish Sepia officinalis. Here we report the cloning and sequencing of a novel S. officinalis FaRP gene (SOFaRP(2)). The complete 835-base pair cDNA sequence of the SOFaRP(2) gene contains an open reading frame of 567 base pairs encoding 188 amino acids and four putative FaRPs, NSLFRFamide, GNLFRFamide, TIFRFamide and PHTPFRFamide. All except TIFRFamide cause chromatophore expansion when assayed in an in vitro chromatophore bioassay. To investigate the expression pattern of SOFaRP(2) gene in the cuttlefish brain, in situ hybridization was performed using a full length RNA probe. The SOFaRP(2) gene was expressed primarily in the posterior chromatophore, anterior chromatophore, lateral basal and optic lobes among other brain locations. The SOFaRP(2) gene appears to be expressed in all brain regions involved in chromatophore regulation. These data suggests that some or all of the four FaRPs encoded by SOFaRP(2) might be involved in controlling chromatophore activity in cuttlefish.     

Genetic diversity and population structure of Sepiella japonica (Mollusca: Cephalopoda: Decapoda) inferred by mitochondrial DNA (COI) variations

In order to describe the genetic diversity of five geographical populations of cuttlefish (Sepiella japonica) along with Chinese coast and determined their phylogenetic relationship, partial mitochondrial COI gene (681bp in length) was amplified from 96 individuals collected from these populations and sequenced. The 5 populations of cuttlefish inhabit Yellow Sea, East China Sea and South China Sea. Out of 22 polymorphic nucleotides identified, 8 were represented by a single sequence, 12 were parsimony informative, which defined 22 haplotypes. Haplotype and nucleotide diversity were low among populations. Of 22 haplotypes, 15 appeared only in a single population, 6 appeared in 2 or 3 populations and 1 was shared by all populations. The COI gene was monomorphic in Qingdao population. The haplotypes identified clustered into 2 clades, each covered individuals from 5 populations each. Pairwise F_ST were not proportional to the geographical distances. Among the 5 populations, relatively high level of genetic diversity was found in Ningde population which was recommended to be the best choice of germplasm resources for artificial releasing project.     

The actions of rfamide neuroactive peptides on the isolated heart of the giant african snail,Achatina fulica

1. The effects of a range of RFamide peptides were examined on the frequency, amplitude and tone of the isolated heart of Achatina fulica.2. FMRFamide, FLRFamide and SDPNFLRFamide were potent excitants while LPLRFamide, KHEYLRFamide and GSFFRFamide were weak excitants.3. DNFLRFamide and SSLFRFamide were potent inhibitory peptides while LFRFamide, GSLFRFamide and KNEFIRFamide were weak inhibitory peptides.4. MRFamide, LRFamide and RFamide were inactive.5. It is concluded that a tetrapeptide sequence is the minimal requirement for activity on the Achatina heart. With the exceptions of DNFLRFamide and in most preparations GSFFRFamide, LRFamide peptides are excitatory while FRFamide peptides are inhibitory.6. Due to its inhibitory effect and high potency, the sequence DNFLRFamide warrants further investigation.     

曼氏无针乌贼神经肽sCAP基因克隆与组织表达定位

为研究曼氏无针乌贼(Sepiella japonica)小心激肽(small cardioactive peptides, sCAP)生理功能, 通过RACE技术克隆曼氏无针乌贼sCAP基因(简称SjsCAP, GenBank登录号:MG779491), 得到总长度为696 bp的cDNA序列, 包括111 bp的5′非编码区(UTR)和324 bp的3′UTR, 预测的开放阅读框(ORF)共261 bp, 编码86个氨基酸, 相对分子量(MW)为9.331 kD, 等电点(pI)为8.52。信号肽以及跨膜区预测结果表明, sCAP中含有明显的信号肽序列和跨膜区结构。因此, 推测该蛋白可能由细胞内分泌到细胞外发挥作用。亲水性分析显示该蛋白为亲水性蛋白。基于sCAP氨基酸序列进行的系统进化分析表明曼氏无针乌贼与商乌贼(Sepia officinalis)亲缘关系最近, 相似性达到90%。通过荧光定量PCR (Quantitative real-time PCR, qRT-PCR)技术对SjsCAP基因在成熟雄性和雌性曼氏无针乌贼不同组织中的表达量进行分析, 结果显示sCAP主要在视叶中显著表达, 在脑中也有较高的表达量, 其中雄性个体表达量显著高于雌性个体。原位杂交实验结果显示sCAP基因在曼氏无针乌贼的脑组织的视叶、食道上神经团的垂直叶、亚垂直、脑脚叶、背外侧叶和视腺均可以观察到明显的阳性杂交信号。sCAP基因的成功克隆以及组织表达定位分析为sCAP的亚细胞定位以及生物学功能研究奠定一定的基础, 同时为曼氏无针乌贼的种质资源保护与开发提供一定的理论支持。     

Nucleotide Sequence of Polymerase Chain Reaction Product Amplified from Rickettsia japonica DNA Using Rickettsia rickettsii 190-Kilodalton Surface Antigen Gene Primers

The PCR product amplified from Rickettsia japonica with the primer pair Rr 190.70p and Rr 190.602n of R. rickettsii 190-kDa antigen gene was cloned into M13mp19 RF DNA at the EcoRI site and sequenced by chemiluminescent DNA sequencing. The sequence revealed a molecular size of 533 base pairs (bp). The primer-flanking region of 491 bp, an open reading frame, was compared with the corresponding region of R. rickettsii, demonstrating 35 nucleotide substitutions in R. japonica. The sequence of primer portions in R. japonica DNA was also analyzed, revealing one nucleotide substitution in the Rr 190.70p and two in the Rr 190.602n portion. The homology in the overall sequence of PCR-amplified regions between R. japonica and R. rickettsii was 93% in nucleotide and 85% in putative amino acid structure. The sequence contains no cleavage site for the restriction endonuclease AfaI but two PstI sites giving three fragments of 121, 159, and 253 bp, which differentiated R. japonica from other spotted fever group rickettsiae in addition to R. rickettsii. The cleavage sites for endonucleases AluI, HinfI, and MunI that disappeared or appeared in the sequence by nucleotide substitution differentiated R. japonica from others, as did PstI. The estimation of molecular size of DNA fragments on polyacrylamide gel electrophoresis is discussed.     

Molecular cloning and characterization of prohormone convertase 1 gene in abalone (Haliotis diversicolor supertexta)

Prohormone convertases (PCs) are calcium-dependent serine endoproteases of the subtilisin family that play a key role in the posttranslational processing of precursors for bioactive peptides. In this study, the cDNA of PC1 from abalone (Haliotis diversicolor supertexta) was cloned and sequenced. The PC1 cDNA consisted of 2216 bp with an open reading frame of 2010 bp encoding a 670 amino acid peptide. Comparative structural analysis revealed that abalone PC1 shared high similarity and identity with most PC counterparts. The profile of deduced peptide of PC1 was composed of an N-terminal signal peptide, a prosegment domain, a catalytic domain and a P domain, which were common in many species. Sequence analysis indicated that the abalone PC1 was highly conserved in catalytic domain, including three conserved serine catalytic signatures that comprised a catalytic triad active center. Also conserved were the potential cleavage site for release of the mature peptide, a cognate integrin binding site RGD in P domain, and four cysteine residues involved in forming an intrachain disulfide bridge. To further investigate the functions of PC1 in abalone, real-time quantitative PCR was performed to determine the expression level of this gene at three different reproduction stages (i.e. pre-, during- and post-breeding). Results indicated that PC1 was expressed throughout the three stages but the expression levels varied with the timepoints and different tissues in abalone. The expression levels of PC1 in digestive gland were much higher than those of the gonad. In female abalone, the expression of PC1 was higher at pre-breeding and during-breeding stages (P < 0.05), and the expression declined at the subsequent stage. Whereas, the level of PC1 in male individual did not exhibit a significant difference in various reproduction stages. Also, the natural enzyme activity of PC1 partially exhibited a similar tendency with the mRNA expression. According to the results, it can be concluded that PC1 gene is involved in the abalone reproduction process (e.g. spawning or sperming). PC1 is a potential prohormone processing enzyme and it may play a critical role in abalone physiological processes related to reproduction.     

CYP341B14: A cytochrome P450 involved in the specific epoxidation of pheromone precursors in the fall webworm Hyphantria cunea

Two of the four sex pheromone components in the fall webworm Hyphantria cunea (Lepidoptera: Arctiidae), cis-9,10-epoxy-(3Z,6Z)-3,6-henicosadiene and cis-9,10-epoxy-(3Z,6Z)-1,3,6-henicosatriene, possess an epoxy ring within their molecules. These compounds have been suggested to be biosynthesized from dietary linolenic acid via the following enzymatic reactions; chain elongation, terminal desaturation (in the case of the latter component), decarboxylation, and epoxidation. The last step of this biosynthesis, epoxidation, is known to occur specifically in the sex pheromone gland of females. We identified the enzyme involved in the epoxidation of pheromone precursors by focusing on cytochromes P450, which are known to catalyze the oxidation of various compounds. Three P450-like sequences (Hc_epo1, Hc_epo2, and Hc_epo3) were identified in the cDNA library prepared from the sex pheromone gland of H. cunea. Among these clones, only Hc_epo1 was specifically expressed in the pheromone gland. The full-length sequence of Hc_epo1 contained an ORF of 1527 bp, which encoded a protein of 509 amino acids with a predicted molecular weight of 57.9 kDa. The deduced Hc_epo1 amino acid sequence possessed the characteristics of P450. A phylogenetic analysis of the sequence indicated that Hc_epo1 belonged to the CYP341B clade in the CYP341 family. Therefore, it was named CYP341B14. A subsequent functional assay using Sf-9 cells transiently expressing CYP341B14 demonstrated that this P450 protein was able to specifically epoxidize a (Z)-double bond at the 9th position in the pheromone precursor, (3Z,6Z,9Z)-3,6,9-henicosatriene.     

Molecular Cloning of Cathelicidin-like cDNA from <Emphasis Type="Italic">Andrias davidianus</Emphasis>

Antimicrobial peptides (AMPs) as part of host defense systems has been widely recognized in most organisms. Cathelicidin is an important family of AMPs acting as multifunctional effector molecules in innate immunity and exists in organisms with cathelicidin-like precursor. Andrias davidianus (A. davidianus) is a unique species in China and the biggest amphibians in the world. With the rapid growth of A. davidianus aquaculture, pathogens of bacteria, virus and fungus were reported, however little is known about antimicrobial peptides derived from A. davidianus. To investigate antimicrobial peptides of cathelicidin-like in A. davidianus, cathelicidin-like precursor gene cloning and bioinformatic analysis was carried out. The results showed that 1106 bp full-length cDNA of cathelicidin-like precursor was obtained, which was including a 35 bp 5' terminal UTR, a 546 bp open reading frame (ORF) and a 525 bp 5' terminal UTR. The cathelicidin-like precursor amino acid (AA) sequence of A. davidianus comprised N-terminal signal peptide (21 AA), highly conserved cathelin domain and C-terminal mature peptide. The cathelicidin-like precursor gene nucleotide sequence showed low identify with other cathelicidin-like sequences, while AA sequence displayed relatively higher similarity with cathelicidin-like isolated from other species. Phylogenetic tree indicated cathelicidinlike precursor of A. davidianus was firstly clade with Tylototrition verrucosus, which also belonged to Caudata, Amphibian. The precursor gene expression was detected by RT-qPCR. The result displayed this gene was abundant expression in A. davidianus skin. According the specificity proteases cleavage and characteristic of cathelicidin, five putative mature cathelicidin were predicted. This study confirms the presence of cathelicidin in A. davidianus. Their results not only reveal innate immune system of A. davidianus but also enlarge the AMP knowledge of urodele amphibians.     

Cloning of Wap65 in sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) and expression in sea bass tissues

The complementary DNA encoding WAP65 protein was cloned from the liver of two fish species sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata). Full-length cDNA sequences were obtained from reverse transcribed total RNA, followed by 5′ and 3′ rapid amplification of cDNA end (RACE) experiments. The full-length cDNA sequence of D. labrax is 1709 bp and the coding sequence is flanked by a 67 bp 5′-UTR and a 358 bp 3′-UTR. The full-length cDNA sequence of S. aurata is 1599 bp, and the coding sequence is flanked by a 48 bp 5′-UTR and a 273 bp 3′-UTR. The deduced amino acid putative primary sequences are composed of 427 and 425 amino acid residues for D. labrax and S. aurata, respectively. They display high homologies with previously described fish WAP65 and other hemopexin-like proteins from rabbit (Oryctolagus cuniculus). Expression of Wap65 has proved to be a natural physiological adaptive answer of teleost fish to warm temperature acclimation. In all fish species studied to date, Wap65 was found expressed mainly by the liver, although other tissues seem able to express Wap65 in response to a warm temperature acclimation, in a specie specific manner. Here, we investigate the tissue specific expression of Wap65 in D. labrax and S. aurata in response to a warm temperature acclimation, by RT-PCR analysis.     

Tissue expression and bioinformatics analysis of the vitellogenin gene of Asian arowana (Scleropages formosus)

The RACE technique was used to clone the full‐length vitellogenin (VTG) cDNA sequence of Asian arowana (Scleropages formosus). The full‐length sequence was 5,550 bp with an open reading frame of 5,238 bp, encoding 1,745 amino acids, and 5′ and 3′ UTRs (untranslated regions) of 45 bp and 267 bp, respectively. Phylogenetic analysis showed that S. formosus and silver arowana (Osteoglossum bicirrhosum) share a close evolutionary relationship (bootstrap 100%). The quantitative real‐time PCR results showed that vtg expression was significantly higher in liver and gonads of male and female fish compared with its expression in the other tissues tested (p < 0.01). The relative expression levels of vtg in liver, gland, kidney, heart, head kidney, and brain of female fish were significantly higher than in the corresponding tissues of male fish (p < 0.05).     

Ovarian jelly-peptides (OJPs), a new family of regulatory peptides identified in the cephalopod Sepia officinalis

In marine invertebrates, numerous water-borne peptides involved in reproductive behavior have been characterized. In this study, we focused on three ovarian water-borne peptides, released by full-grown oocytes (FGO) in the genital coelom and in the lumen of the oviduct in the cuttlefish Sepia officinalis. The first one (DQVKIVL), was characterized by the monitoring of HPLC purified fraction using a myotropic bioassay. Subsequently, a peptidomic approach consisting of a mass spectrometry comparative screening performed between the peptide content of FGO with that of FGO-conditioned medium, led to the identification of two additional water-borne peptides. The second peptide identified (DEVKIVL) was characterized by MS/MS and the primary structure of the third one (DEVKIVLD) was elucidated by a combination of Edman degradation, acid hydrolysis and MS/MS analysis. Sequence homology, tissue mapping and bioactivity demonstrate that these peptides belong to the same family. DQVKIVL-related-peptides strictly localized in the female genital tract modulate the whole female genital tract and the main nidamental gland contractions. Furthermore, these peptides form a jelly, when resuspended in water. This particular property could play an important role in the kinetics of peptide diffusion in the external medium. Thus, these regulatory peptides were named ovarian jelly-peptides (OJPs).     

A neuromedin-pyrokinin-like neuropeptide signaling system in Caenorhabditis elegans

Neuromedin U (NMU) in vertebrates is a structurally highly conserved neuropeptide of which highest levels are found in the pituitary and gastrointestinal tract. In Drosophila, two neuropeptide genes encoding pyrokinins (PKs), capability (capa) and hugin, are possible insect homologs of vertebrate NMU. Here, the ligand for an orphan G protein-coupled receptor in the nematode Caenorhabditis elegans (Ce-PK-R) was found using a bioinformatics approach. After cloning and expressing Ce-PK-R in HEK293T cells, we found that it was activated by a neuropeptide from the C. elegans NLP-44 precursor (EC₅₀ = 18 nM). This neuropeptide precursor is reminiscent of insect CAPA precursors since it encodes a PK-like peptide and two periviscerokinin-like peptides (PVKs). Analogous to CAPA peptides in insects and NMUs in vertebrates, whole mount immunostaining in C. elegans revealed that the CAPA precursor is expressed in the nervous system. The present data also suggest that the ancestral CAPA precursor was already present in the common ancestor of Protostomians and Deuterostomians and that it might have been duplicated into CAPA and HUGIN in insects. In vertebrates, NMU is the putative homolog of a protostomian CAPA-PK.     

Egg capsule secretion in invertebrates: a new ovarian regulatory peptide identified by mass spectrometry comparative screening in Sepia officinalis

Mass spectrometry comparative screening was used to identify ovarian regulatory peptides involved in the successive steps of egg-laying in the cuttlefish Sepia officinalis. The peptide content of full-grown oocytes (FGO) was compared with that of oocyte-conditioned medium, which resulted in the detection of peptides that were present in both samples. These peptides, which are suspected of being released by the oocyte in the genital tract, were submitted to a structural analysis. This strategy led to the characterization of a new ovarian regulatory peptide (EISLDKD) able to inhibit the contractions of the whole female genital tract and of the main nidamental glands (MNG). As EISLDKD appeared to be the first regulatory peptide directly involved, at physiological concentrations, in the secretion of the egg capsule by the main nidamental glands, it was named SepCRP for Sepia Capsule Releasing Peptide. Mass spectrometry analysis clearly demonstrated that SepCRP was expressed during vitellogenesis by the ovarian follicles and released by the FGO in the lumen of the female genital tract. In association with the ovarian 5-HT, SepCRP would be responsible for the storage of FGO to avoid the spawning of unfertilized oocytes before mating. Released by the distal oviduct in the mantle cavity, SepCRP probably in association with a cocktail of ovarian regulatory factors targets the MNG to regulate the egg capsule secretion. Thus, the ovary appeared to be one of the main sources of regulatory peptides involved in the successive steps of egg-laying in the cephalopod mollusk S. officinalis.