Partial cDNA Sequence Research Articles

An anti-apoptotic B-cell lymphoma-2 (BCL-2) from Channa striatus: Sequence analysis and delayed and advanced gene expression in response to fungal, bacterial and poly I:C induction

B-cell lymphoma-2 (BCL-2) is a suppressor of apoptosis and inhibits the caspase dependent apoptosis pathway. In this study, we report molecular characterization of a cDNA sequence encoded of BCL-2 from striped murrel, Channa striatus. A partial cDNA sequence of CsBCL-2 was identified from the striped murrel cDNA library during annotation. Subsequently, the full length CsBCL-2 cDNA sequence was obtained by an internal sequencing method using a forward primer. The sequence contains 699 nucleotide base pairs which encode 232 amino acid residues. The domain and motif analysis revealed that the CsBCL-2 polypeptide consists of BCL-2 homologous domain BH4 at the N-terminal region between 4 and 21 and the BCL-2 homologous domains BH1, BH2 and BH3 between 87 and 187. The CsBCL-2 polypeptide sequence does not have a signal peptide region, but it consists of two novel transmembrane regions at 134–152 and 209–226. The sequence analysis showed that the CsBCL-2 has highest sequence identity (70%) with BCL-2 like protein 1 (BCL-2 L1) from pufferfish Takifugu rubripes. The phylogenetic analysis showed that the CsBCL-2 was situated in the BCL-2 L1 fish clade. The secondary analysis showed that the CsBCL-2 protein consists of 132 amino acid residues in the α-helical region and 100 amino acid residues in the random coil region. The validated 3D structure of CsBCL-2 showed the active residues Gly135 and Arg136 in the 7th α-helical position, whereas Trp178 is in the 9th α-helical region. CsBCL-2 mRNA transcription is predominately present in spleen and is upregulated upon being induced with fungus Aphanomyces invadans, bacteria Aeromonas hydrophila, Escherichia coli LPS, Laminaria digitata beta-1,3-glucan and poly I:C. Overall, the CsBCL-2 mRNA transcription results indicate the potential involvement of CsBCL-2 in immune system of C. striatus. However, further research at proteomic level is necessary to examine these predictions.

Vitellogenin gene characterization and expression of Asian paddle crabs (Charybdis japonica) following endocrine disrupting chemicals

Vitellogenin (VTG), the yolk-precursor lipoprotein, has been widely recognized as a biomarker for the detection of estrogenic activity in water-borne chemical pollutants. The Asian paddle crab, Charybdis japonica, is a potential bio-indicator for monitoring marine environments. The aim of this study was to identify the possibility of using C. japonica VTG as biomarkers of stress caused by endocrine disrupting chemicals (EDCs). We characterized a partial sequence of the VTG cDNA in the C. japonica crab and evaluated the crab’s mRNA expression profiles following exposure to different concentrations of bisphenol A (BPA) and 4-nonylphenol (NP) for 24 or 96 h. The sequence homology of C. japonica VTG is over 93% in nucleotide and over 98% in amino acid with the corresponding gene of other crabs. Phylogenetic analysis revealed that the C. japonica VTG is an ortholog of other species of lobster and shrimp. Tissue distribution analysis of the C. japonica VTG mRNA revealed that the expression of VTG mRNA was highest in the ovary of females and hepatopancreas. The expression of the C. japonica VTG gene in various BPA or NP concentrations during shorter and longer times was assessed. The expression of VTG transcripts was significantly increased in the C. japonica crab exposed to BPA and NP at different concentrations for 24 h. The mRNA expression of the VTG gene was significantly induced in concentration- and time-dependent manners after BPA or NP exposures for 96 h. These results indicate that crab C. japonica VTG could be used as a potential biomarker of EDCs in marine environment monitoring.

Molecular cloning, characterization and expression of a gene encoding phosphoketolase from Termitomyces clypeatus

A phosphoketolase (pk) gene from the fungus Termitomyces clypeatus (TC) was cloned and partially characterized. Oligonucleotide primers specific for the phosphoketolase gene (pk) were designed from the regions of homologies found in the primary structure of the enzyme from other fungal sources related to TC, using multiple sequence alignment technique. The cDNA of partial lengths were amplified, cloned and sequenced in three parts by 3′ and 5′ RACE and RT-PCR using these oligonucleotide primers. The full length ds cDNA was constructed next by joining these three partial cDNA sequences having appropriate overlapping regions using Overlap Extension PCR technique. The constructed full length cDNA exhibited an open reading frame of 2487 bases and 5′ and 3′ UTRs. The deduced amino acid sequence, which is of 828 amino acids, when analyzed with NCBI BLAST, showed high similarities with the phosphoketolase enzyme (Pk) superfamily with expected domains. The part of the TC genomic DNA comprising of the pk gene was also amplified, cloned and sequenced and was found to contain two introns of 68 and 74 bases that interrupt the pk reading frame. The coding region of pk cDNA was subcloned in pKM260 expression vector in correct frame and the protein was expressed in Escherichia coli BL21 (DE3) transformed with this recombinant expression plasmid. The recombinant protein purified by His-tag affinity chromatography indicated the presence of a protein of the expected size. In vivo expression studies of the gene in presence of different carbon sources indicated synthesis of Pk specific mRNA, as expected. Phylogenetic studies revealed a common ancestry of the fungal and bacterial Pk. The TC is known to secrete several industrially important enzymes involved in carbohydrate metabolism. However, the presence of Pk, a key enzyme in pentose metabolism, has not been demonstrated conclusively in this organism. Cloning, sequencing and expression study of this gene establishes the functioning of this gene in T. clypeatus. The Pk from TC is a new source for commercial exploitation.

Sexually dimorphic transcription of estrogen receptors in cod gonads throughout a reproductive cycle

The role of sex steroid regulation in gonadal maturation is a very complex process that is far from being fully understood. Hence, we have investigated seasonal changes in gonadal expression of estrogen receptors (ERs) in Atlantic cod (Gadus morhua L.), a batch spawner, throughout the annual reproductive cycle. Three nuclear ER partial cDNA sequences (esr1, esr2a, and esr2b) were cloned and all esr transcripts were detected mainly in liver and gonads of fish of both sexes. In situ hybridization of esrs along with germ cell (vasa) and gonadal somatic cell markers (gonadal soma-derived factor (gsdf), 3β-hydroxysteroid dehydrogenase (3βhsd), and anti-Müllerian hormone (amh) for testicular, or gsdf for ovarian somatic cells) showed that all three esrs were preferentially localized within interstitial fibroblasts composed of immature and mature Leydig cells in testis, whereas they were differentially expressed in both follicular cells and oocytes in ovary. Quantitative real-time PCR analysis revealed a sexually dimorphic expression pattern of the three esr paralogs in testis and ovary. A significant increase in esr2a expression was identified in testis and of esr2b in ovary, whereas esr1 transcripts were elevated in both testis and ovary in February and March before the spawning period. The localization and sexually dimorphic expression of esr genes in gonads indicate a direct function of estrogen via ERs in gonadal somatic cell growth and differentiation for Leydig cell in testis and follicular cells in ovary throughout the annual reproductive cycle in Atlantic cod.

First evidence of the P-glycoprotein gene expression and multixenobiotic resistance modulation in earthworm

Multixenobiotic resistance (MXR) is an important mechanism of cellular efflux mediated by ATP binding cassette (ABC) transporters that bind and actively remove toxic substrates from the cell. This study was the first to identify ABC transporter P-glycoprotein (P-gp/ABCB1) as a representative of the MXR phenotype in earthworm (Eisenia fetida). The identified partial cDNA sequence of ABCB1 overlapped with ABCB1 homologues of other organisms from 58.5 % to 72.5 %. We also studied the effect of five modulators (verapamil, cyclosporine A, MK571, probenecid, and orthovanadate) on the earthworm's MXR activity by measuring the accumulation of model substrates rhodamine B and rhodamine 123 in whole body tissue of the adult earthworm. MK571, orthovanadate, and verapamil significantly inhibited MXR activity, and rhodamine 123 turned out to better reflect MXR activity in that species than rhodamine B. Our results show that E. fetida can serve well as a test organism for environmental pollutants that inhibit MXR activity.

Effects of 17α-ethinyl estradiol exposure on estrogen receptors α and β and vitellogenins A, B and C mRNA expression in the liver of sand goby (Pomatoschistus minutus)

This study aims to characterize the estrogen receptor (er) in sand goby (Pomatoschistus minutus) and determine the temporal effects of 17α-ethinyl estradiol (EE2) on erα and vitellogenin (vtg) gene expression in males. Two partial cDNA sequences (erα and erβ1) are presented showing conserved structural features with ers of other species. Transcript levels for both ers were low in control fish but EE2 exposure (11 ng/L, for 29 days) increased both to a pattern similar to vitellogenic females. The relative expression of three vtg genes (vtga, vtgb and vtgc) along with erα was determined in control and male fish exposed to EE2 (11 ng/L) at multiple time-points over 29 days. All four transcripts were significantly induced due to exposure and expression rose during the time course with distinct temporal patterns and vtga reached a substantially higher level at the end of the time course coinciding with rapid elevation in erα expression.

中缅树鼩PRDM16、BMP7、PPARα、COX II及PGC-1α基因的扩增研究

本研究对中缅树鼩PRDM16、BMP7、PPARα、COX II及PGC-1α基因的部分序列进行扩增和分析，获得中缅树鼩PRDM16、BMP7、PPARα、COX II及PGC-1α片段分别为184 bp、84 bp、358 bp、212 bp、271 bp，推测其分别编码61、27、119、70、87个氨基酸。将PRDM16、BMP7、PPARα、COX II及PGC-1α基因编码氨基酸序列分别与多种哺乳动物对应的氨基酸序列进行同源性比较分析发现氨基酸的同源性较高。以获得的PRDM16、BMP7、PPARα、COX II及PGC-1α基因序列分别构建系统进化树，结果显示中缅树鼩与灵长类动物的亲缘关系较近。In the present study, partial cDNA sequence of PRDM16, BMP7, PPARα, COX II and PGC-1α genes in Tupaia belangeri were amplificated and analyzed. The obtained partial sequences of PRDM16, BMP7, PPARα, COX II and PGC-1α in T. belangeri were 184 bp, 84 bp, 358 bp, 212 bp and 271 bp, respectively. The coding protein of these sequences included 61 amino acids in PRDM16; 27 amino acids in BMP7; 119 amino acids in PPARα, 70 amino acids in COX II and 87 amino acids in PGC-1α. Comparison of the amino acids of PRDM16, BMP7, PPARα, COX II and PGC-1α with the amino acids in many mammals found that the homology of amino acids was higher. The PRDM16, BMP7, PPARα, COX II and PGC-1α genes were used to reconstruct phylogenetic trees which showed that T. belangeri had the closest relationship relative to primates.

Isolation and characterisation of four partial cDNA sequences encoding multidrug resistance-associated proteins (MRPs) in the salmon louse Lepeophtheirus salmonis (Krøyer, 1837)

The salmon louse Lepeophtheirus salmonis (Krøyer, 1837) is an ectoparasite of salmonid fish severely affecting cultured salmon production in the North Atlantic. Salmon louse control on farms currently relies in part upon the use of veterinary drugs; however, as only a limited number of salmon delousing agents are available, there are concerns about development of drug resistance in salmon lice. The common anti-salmon louse drug SLICE® (Merck Animal Health) contains the avermectin emamectin benzoate (EMB). Members of the large gene superfamily of ABC (ATP-binding cassette) transporters have been identified as potential avermectin resistance factors in parasitic nematodes. In salmon lice, only three ABC transporters have been cloned and studied to date. We report here upon the isolation of four novel L. salmonis ABC transporters, and employ an inhibitor-based approach to assess roles of L. salmonis ABC transporters in the toxicology of EMB. To isolate salmon louse ABC transporters, publicly available L. salmonis expressed sequence tags (ESTs) were subjected to homology searches, and the retrieved ESTs assembled into contiguous sequences and annotated. Potential ABC drug transporters isolated by this approach included four multidrug resistance-associated proteins (MRPs). In addition, five ABC proteins likely having roles unrelated to drug resistance were obtained. Quantitative real time PCR (RT-qPCR) was used to analyse mRNA levels of the MRPs in L. salmonis strains differing in EMB susceptibility. In the absence of EMB, all studied MRPs showed similar transcript levels in the drug-susceptible strain S and the moderately EMB-resistant strain R. Moreover, mRNA expression of the four studied MRPs remained unaffected by exposure to EMB. Taken together, the results of RT-qPCR analyses did not provide evidence for roles of the studied MRPs as factors affecting EMB susceptibility. Further experiments used an inhibitor-based approach to investigate the roles of ABC transporters in EMB toxicity. In immotility bioassays, salmon lice of the two strains were exposed to EMB, provided alone or in combination with cyclosporin A or verapamil, which are known inhibitors of P-glycoprotein and MRPs. Cyclosporin A increased EMB toxicity to a similar degree with both strains, suggesting the biochemical factors affected by this inhibitor show similar levels in both strains. In contrast, verapamil increased EMB effects only in the R strain. This result could be indicative of an enhanced expression of hitherto unknown ABC transporters in the moderately EMB-resistant R strain. More research is required to identify the target of verapamil in salmon lice.

Use of heat shock protein mRNA expressions as biomarkers in wild crucian carp for monitoring water quality

In organisms, the production of heat shock proteins (HSPs) can be induced after exposure to various stressors. In the present study, partial cDNA sequences encoding of HSP30, HSP60, HSP70, and HSP90 genes were isolated and sequenced in the crucian carp (Carassius carassius). Next, quantitative real-time reverse transcription PCR (qRT-PCR) assays normalized to beta-actin were developed to quantify HSP mRNA expression. The same methods were used to detect HSP mRNA expression in the wild crucian carp that were collected from the Hun River. In fish located downstream of the river, we found that the levels of HSP70 and HSP30 in the liver and kidney were higher than fish located upstream, and these differences coincided with changes of the water quality. These results suggest that kidney HSP30 and liver HSP70 expression can serve as sensitive biomarkers for the presence of field environmental stressors in wild crucian carp.

Wood chemistry analysis and expression profiling of a poplar clone expressing a tyrosine-rich peptide

Our study has identified pathways and gene candidates that may be associated with the greater flexibility and digestibility of the poplar cell walls. With the goal of facilitating lignin removal during the utilization of woody biomass as a biofuel feedstock, we previously transformed a hybrid poplar clone with a partial cDNA sequence encoding a tyrosine- and hydroxyproline-rich glycoprotein from parsley. A number of the transgenic lines released more polysaccharides following protease digestion and were more flexible than wild-type plants, but otherwise normal in phenotype. Here, we report that overexpression of the tyrosine-rich peptide encoding sequence in these transgenic poplar plants did not significantly alter total lignin quantity or quality (S/G lignin ratio), five- and six-carbon sugar contents, growth rate, or susceptibility to a major poplar fungal pathogen, Septoria musiva. Whole-genome microarray analysis revealed a total of 411 differentially expressed transcripts in transgenic lines, all with decreased transcript abundance relative to wild-type plants. Their corresponding genes were overrepresented in functional categories such as secondary metabolism, amino acid metabolism, and energy metabolism. Transcript abundance was decreased primarily for five types of genes encoding proteins involved in cell-wall organization and in lignin biosynthesis. The expression of a subset of 19 of the differentially regulated genes by qRT-PCR validated the microarray results. Our study has identified pathways and gene candidates that may be the underlying cause for the enhanced flexibility and digestibility of the stems of poplar plants expressing the TYR transgene.

An abiotic stress-responsive WRKY gene is transiently induced in response to cold and drought stresses in Poncirus trifoliata

A partial cDNA sequence sharing significant homology with WRKY transcription factors (TFs) was previously amplified from Poncirus trifoliata by reverse transcription polymerase chain reaction (RT-PCR) using degenerate primers. In this study, the full-length cDNA sequence of this cDNA, PtrWRKY3, was obtained by RACE method from P. trifoliata. The length of PtrWRKY3 cDNA was 1908 bp and contained open reading frame coding for a 512-amino-acid polypeptide. The ortholog of this gene, CpWRKY3, was also cloned and sequenced from Citrus paradisi (grapefruit). The gene consists of 1550 bp cDNA encoding a protein of 516 amino acids. Both genes have double WRKY domains with Cys2His2 signature motif and showed 98% amino acid sequence identity with each other. They were tightly clustered together and showed a close phylogenetic relation with putative WRKY TFs from different woody perennial plants. The expression of the WRKY3 gene was analyzed quantitatively by northern blot and real-time RT-PCR in response to cold and drought stresses in both cold-hardy Poncirus and one of the cold-sensitive Citrus species, C. paradisi. While the expression of the WRKY3 gene was initially induced transiently in response to cold in cold-hardy Poncirus, no significant changes in the expression of this gene were observed in cold-sensitive grapefruit plants. On the other hand, the expression of the WRKY3 gene was transiently induced by drought stress in both Poncirus and grapefruit plants. These results indicated that the WRKY3 gene from Poncirus and Citrus is conserved and its response to drought stress was similar in both species. However, its expression in response to cold was different in cold-hardy and cold-sensitive species indicating that it is transiently induced early in cold acclimation and may be involved in cold tolerance in Poncirus.

Liver glucose-6-phosphatase proteins in suckling and weaned grey seal pups: structural similarities to other mammals and relationship to nutrition, insulin signalling and metabolite levels

Phocid seals have been proposed as models for diabetes because they exhibit limited insulin response to glucose, high blood glucose and increasing insulin resistance when fasting. Liver glucose-6-phosphatase (G6Pase) catalyses the final step in glucose production and is central to glucose regulation in other animals. G6Pase comprises a translocase (SLC37A4) and a catalytic subunit (G6PC). G6PC and SLC37A4 expression and activity are normally regulated by nutritional state and glucostatic hormones, particularly insulin, and are elevated in diabetes. We tested the hypotheses that (1) grey seal G6PC and SLC37A4 cDNA and predicted protein sequences differ from other species' at functional sites, (2) relative G6Pase protein abundances are lower during feeding than fasting and (3) relative G6Pase protein abundances are related to insulin, insulin receptor phosphorylation and key metabolite levels. We show that G6PC and partial SLC37A4 cDNA sequences encode proteins sharing 82-95% identity with other mammals. Seal G6PC contained no differences in sites responsible for activity, stability or subcellular location. Several substitutions in seal SLC37A4 were predicted to be tolerated with low probability, which could affect glucose production. Suckling pups had higher relative abundance of both subunits than healthy, postweaned fasting pups. Furthermore, relative G6PC abundance was negatively related to glucose levels. These findings contrast markedly with the response of relative hepatic G6Pase abundance to feeding, fasting, insulin, insulin sensitivity and key metabolites in other animals, and highlight the need to understand the regulation of enzymes involved in glucose control in phocids if these animals are to be informative models of diabetes.

Expression of amino acid transporter genes in developmental stages and adult tissues of Antarctic echinoderms

Epithelial cells in the body wall of adult and developmental stages of marine invertebrates absorb dissolved organic material directly from seawater. Despite over a century of study, little is known about the molecular biological mechanisms responsible for this transport process. Previous studies on embryonic and larval Antarctic echinoderms show that amino acid uptake could provide an important supplement of metabolic substrates. In the present study, partial cDNA sequences of 11 putative amino acid transporter genes were isolated from six species of Antarctic echinoderms including the Antarctic sea stars Acodontaster hodgsoni, Diplasterias brucei, Odontaster meridionalis, Odontaster validus, and Perknaster fuscus, and the Antarctic sea urchin Sterechinus neumayeri. Conserved domains of cDNA-deduced amino acid sequences characterized these genes as being members of a family of amino acid transporters (solute carrier family 6). Expression of these genes was detected throughout embryonic and larval development of two species that have contrasting developmental modes (A. hodgsoni: lecithotrophic; O. meridionalis: planktotrophic). In all six species studied, the expression of amino acid transporter genes was detected in tube feet and digestive organs of adult animals, demonstrating that members of a single amino acid transporter gene family are expressed during the entire life history of a marine invertebrate. The identification of these genes is an important step toward developing a mechanistic understanding of amino acid transport capacities in Antarctic marine invertebrates.

Quantification and in situ localisation of abcb1 and abcc9genes in toxicant-exposed sea urchin embryos

A multixenobiotic resistance (MXR) mechanism mediated by ABC binding cassette (ABC) transport proteins is an efficient chemical defence mechanism in sea urchin embryos. The aim of our work was to evidence whether exposure to sub-lethal doses of specific contaminants (oxybenzone (OXI), mercuric chloride (HgCl2) and trybutiltin (TBT)) would induce MXR transporter activity during embryonic development (from zygote to blastula stage) in purple sea urchin (Paracentrotus lividus) embryos. Further, we present data on molecular identification, transport function, expression levels and gene localisation of two ABC efflux transporters-P-glycoprotein (ABCB1/P-gp) and sulfonylurea-receptor-like protein (ABCC9/SUR-like). Partial cDNA sequences of abcb1 and abcc9 were identified and quantitative PCR (qPCR) evidenced an increase in mRNA transcript levels of both ABC transporters during the two-cell, as well as an overall decrease during the blastulae stage. Calcein-AM efflux activity assay indicated the activation of multidrug resistance-associated protein/ABCC-like transport in the presence of HgCl2 and TBT in exposed blastulae. The in situ hybridisation of the two-cell and blastula stages showed ubiquitous localisation of both transcripts within cells, supporting qPCR data. In conclusion, ABCB1 and ABCC9 are constitutive, as are HgCl2, TBT and OXI-inducible ABC membrane transporters, coexpressed in the zygote, two-cell and blastula stages of the P. lividus. Their ubiquitous cell localisation further fortifies their protective role in early embryonic development.

Molecular characterization of a p38 MAPK from Litopenaeus vannamei and its expression during the molt cycle and following pathogen infection

The p38 mitogen-activated protein kinase (MAPK), a serine/threonine-specific protein kinase, has been reported to be involved in innate immunity, development and muscle differentiation. To explore the function of p38 in shrimp, partial cDNA sequence of p38 in Litopenaeus vannamei (designated as Lv-p38) was characterized and the expression of Lv-p38 in hepatopancreas of the shrimp after being infected with Vibrio parahaemolyticus and in muscle of the shrimp at different molt stages was detected by quantitative RT-PCR in this study. The results showed that the open reading frame of Lv-p38 was 1098bp and encoded a protein of 365 amino acids. The protein of Lv-p38 which showed close phylogenetic relationship to Marsupenaeus japonicus p38 had a conserved TGY motif and serine/threonine protein kinase (S_TKc) domain. The expression of Lv-p38 was detected in all tested tissues, especially in the hepatopancreas and muscle. The expression of Lv-p38 in the hepatopancreas was different from that of the control at the 24th hour after the injection of V. parahaemolyticus and in the muscle significantly increased at stage C but decreased at other stages during molt, illustrating that Lv-p38 could be involved in pathogen infection and the molt cycle of shrimp. In conclusion, we identified Lv-p38 and studied its role in pathogen infection and molting, which might facilitate our understanding of the function of p38 in innate immunity and growth during molt of shrimp.

Ontogeny expression of ghrelin, neuropeptide Y and cholecystokinin in blunt snout bream, Megalobrama amblycephala

Ghrelin, neuropeptide Y (NPY) and cholecystokinin (CCK) all have important roles in the regulation of feeding in fish and mammals. To better understand the role of the three peptides in appetite regulation in the early developmental stages of blunt snout bream (Megalobrama amblycephala), partial cDNA sequences of ghrelin, NPY and CCK genes were cloned. And then, real-time quantitative PCR and RT-PCR were used to detect and quantify the mRNA expressions of these genes from zygotes to larvae of 50 days after hatching (DAH). Ghrelin, NPY and CCK were all expressed throughout the embryonic and larval development stages, and the expression levels were higher in larval stages than in embryonic stages. Ghrelin and NPY mRNA expressions were upregulated at 1, 3, 5 DAH, while CCK mRNA expression was reduced significantly at 3 DAH. The mRNA expression levels of three genes in larvae varied significantly until 30 DAH. In adult fish, all three peptides were detected to be expressed in brain and several peripheral tissues. Ghrelin mRNA was mainly expressed in the intestine, whereas NPY and CCK mRNAs were mainly expressed in the brain. Taken together, these results indicate that ghrelin, NPY and CCK may have roles in early development and participate in the regulation of feeding of larvae in blunt snout bream and will be helpful for further investigation into feed intake regulation in adults of this species.

Light and feeding entrainment of the molecular circadian clock in a marine teleost (Sparus aurata)

Daily light and feeding cycles act as powerful synchronizers of circadian rhythmicity. Ultimately, these external cues entrain the expression of clock genes, which generate daily rhythmic behavioral and physiological responses in vertebrates. In the present study, we investigated clock genes in a marine teleost (gilthead sea bream). Partial cDNA sequences of key elements from both positive (Bmal1, Clock) and negative (Per2, Cry1) regulatory loops were cloned before studying how feeding time affects the daily rhythms of locomotor activity and clock gene expression in the central (brain) and peripheral (liver) oscillators. To this end, all fish were kept under a light-dark (LD) cycle and were divided into three experimental groups, depending on the time of their daily meal: mid-light (ML), mid-darkness (MD), or at random (RD) times. Finally, the existence of circadian control on gene expression was investigated in the absence of external cues (DD + RD). The behavioral results showed that seabream fed at ML or RD displayed a diurnal activity pattern (>91% of activity during the day), whereas fish fed at MD were nocturnal (89% of activity during the night). Moreover, seabream subjected to regular feeding cycles (ML and MD groups) showed food-anticipatory activity (FAA). Regardless of the mealtime, the daily rhythm of clock gene expression in the brain peaked close to the light-dark transition in the case of Bmal1 and Clock, and at the beginning of the light phase in the case of Per2 and Cry1, showing the existence of phase delay between the positive and negative elements of the molecular clock. In the liver, however, the acrophases of the daily rhythms differed depending on the feeding regime: the maximum expression of Bmal1 and Clock in the ML and RD groups was in antiphase to the expression pattern observed in the fish fed at MD. Under constant conditions (DD + RD), Per2 and Cry1 showed circadian rhythmicity in the brain, whereas Bmal1, Clock, and Per2 did in the liver. Our results indicate that the seabream clock gene expression is endogenously controlled and in liver it is strongly entrained by food signals, rather than by the LD cycle, and that scheduled feeding can shift the phase of the daily rhythm of clock gene expression in a peripheral organ (liver) without changing the phase of these rhythms in a central oscillator (brain), suggesting uncoupling of the light-entrainable oscillator (LEO) from the food-entrainable oscillator (FEO). These findings provide the basis and new tools for improving our knowledge of the circadian system and entraining pathways of this fish species, which is of great interest for the Mediterranean aquaculture. (Author correspondence: javisan@um.es).

First evidence of a Toll signaling pathway involved in innate immune response in Lophotrochozoa

Antimicrobial peptides (AMPs) are certainly one of the major effectors of the antiinfectious innate immunity. The complete molecular cascade of signal transduction from pathogen recognition to AMP activity is only known in Drosophila (Ecdysozoa), although not totally elucidated. Only some of the molecular intermediates have been reported from Lophotrochozoa, mainly as partial cDNA sequences. In mussels, biochemical analysis completed by molecular approaches, reveal a huge diversity of AMPs, including within the same animal, suggesting a complex system of molecular effectors. Expression regulations and specificities have been investigated following injections with bacteria, fungi, and physical stresses. No strict correlation has been yet established between structure and function of AMPs in mussels. Recent scientific advances in term of high-speed sequencing have allowed to obtain a large mussel EST database, completed with Illumina reads assemblies. The scanning of these libraries, based on protein's conserved domains (by analogy with vertebrates and Drosophila) confirmed the presence of molecules involved in recognition-signal transduction and revealed new ones. First discovery was the existence in the mussel, Mytilus galloprovincialis, of 23 Tolllike receptors (TLRs) and 3 myeloid differentiation factors 88 (MyD88s). These TLRs possess the characteristic canonical structure with the presence of extracellular leucine-reach repeat (LRR) domains in variable number, followed by a transmembrane region and an intracellular part composed by a Toll-interleukine 1 receptor (TIR) domain. Study of LRR structure revealed that only 3 TLR present multiple cysteine clusters (mccTLR), found in the Toll receptor of Ecdysozoa (Drosophile Toll-1). The similarities observed in up regulation of gene expression of only one mccTLR and one MyD88 in response to microorganism injection gave some light on the existence of the cascade in mussel. We recently identified most of the other members of the NF-kB cascade. Their structures and gene expressions will be presented and discussed. In conclusion, Lophotrochozoa, as well as Ecdysozoa, appeared to rely on the NF-kB pathway for signal transduction. However, the functionality of such complex system in the course of real infection is still to be fully understood. This work was partially funded by the EU program BIVALIFE (KBBE-2010-266157).

PAPP-A in Indian Water Buffalo (Bubalus bubalis) Ovary: Phylogeny, Expression, Hormonal Regulation, and Sequence Characterization

The insulin-like growth factor binding proteins and pregnancy-associated plasma protein-A (PAPP-A) within the future dominant follicle is a critical part of selection of one follicle. This study was aimed to find out the expression and hormonal regulation of PAPP-A mRNAs in the Indian water buffalo ovary. The results showed stage-specific expression of PAPP-A in the whole follicles and postovulatory structures, corpus luteum and corpus albicans. Expression of PAPP-A mRNA was found to be up regulated in corpus luteum. The 341 nucleotide partial cDNA sequence of buffalo PAPP-A was determined. Sequence analyses have revealed that bubaline PAPP-A belongs to Zinc-metzincin superfamily (zinc-dependent metalloprotease) and pappalysin-like subfamily. It also confirmed the stimulatory effect of follicle-stimulating hormone on PAPP-A mRNA expression. The PAPP-A sequences showed more than 90 % homology with reported sequences of cattle, sheep, pig, and human. The study was an attempt in understanding local regulatory mechanism of follicular selection and development in buffalo ovary.

Proteomic profiling of cereal aphid saliva reveals both ubiquitous and adaptive secreted proteins.

The secreted salivary proteins from two cereal aphid species, Sitobion avenae and Metopolophium dirhodum, were collected from artificial diets and analysed by tandem mass spectrometry. Protein identification was performed by searching MS data against the official protein set from the current pea aphid (Acyrthosiphon pisum) genome assembly and revealed 12 and 7 proteins in the saliva of S. avenae and M. dirhodum, respectively. When combined with a comparable dataset from A. pisum, only three individual proteins were common to all the aphid species; two paralogues of the GMC oxidoreductase family (glucose dehydrogenase; GLD) and ACYPI009881, an aphid specific protein previously identified as a putative component of the salivary sheath. Antibodies were designed from translated protein sequences obtained from partial cDNA sequences for ACYPI009881 and both saliva associated GLDs. The antibodies detected all parent proteins in secreted saliva from the three aphid species, but could only detect ACYPI009881, and not saliva associated GLDs, in protein extractions from the salivary glands. This result was confirmed by immunohistochemistry using whole and sectioned salivary glands, and in addition, localised ACYPI009881 to specific cell types within the principal salivary gland. The implications of these findings for the origin of salivary components and the putative role of the proteins identified are discussed in the context of our limited understanding of the functional relationship between aphid saliva and the plants they feed on. The mass spectrometry data have been deposited to the ProteomeXchange and can be accessed under the identifier PXD000113.