14 research outputs found

    Transcriptome Analysis of Metapenaeus affinis Reveals Genes Involved in Gonadal Development

    Get PDF
    Metapenaeus affinis is a crustacean with important commercial value in the fishery of the South China Sea. Overfishing has resulted in the decline of the wild population and germplasm degradation. However, there is little background knowledge about its gonadal development, and there is a lack of research on the development of this species. To better understand the molecular regulatory mechanisms during gonadal development, here, we performed RNA-Seq on immature and mature ovaries and compared their transcriptomic signatures. 126,930,488 and 122,677,356 clean sequencing reads were obtained from the Illumina sequencing platform, respectively. 394 differentially expressed genes (DEGs) were identified, of which 136 were up-regulated, and 258 were down-regulated. Further analysis revealed rich transcriptional sequences, which have homology with genes related to reproduction and development. Expression patterns of COX, GPX, E3s, PCNA, STPK, and other genes were changed during ovarian development. Validation by qRT-PCR demonstrated the reliability of RNA-Seq. This study has made a significant contribution to the currently available sequence data of M. affinis and provided reference data for the development of genetic and breeding work

    Neuroparsin 1 (MrNP1) and Neuroparsin 2 (MrNP2) Are Involved in the Regulation of Vitellogenesis in the Shrimp Macrobrachium rosenbergii

    Get PDF
    Neuroparsins (NP) are small-size cysteine-rich neuropeptides first discovered in insects. They are known to be involved in insect reproduction. In this study, we have cloned two neuroparsin cDNAs (i.e., MrNP1 and MrNP2) from the freshwater shrimp Macrobrachium rosenbergii. The two neuroparsins consist of 12 cysteines, which is characteristic of the neuroparsin family. These cysteines are arranged in identical relative positions that form 6-disulfide bonds. MrNP1 and MrNP2 are most similar to the corresponding neuroparsin counterparts of the shrimp Macrobrachium nipponense. Phylogenetic study results suggested that MrNP1 and MrNP2 are closely related to MnNP1 and MnNP3, respectively. Also, an additional MrNP gene similar to MnNP2 is expected to exist in M. rosenbergii. The MrNP1 expression level is the highest in the ovary, and MrNP2 expression is higher in the brain and heart of the females. In addition, during the ovary maturation cycle, MrNP1 expression in the hepatopancreas is highest in stage V; in the ovary it is variable. MrNP2 expression in the hepatopancreas and ovary is the highest in stage II and stage I, respectively. In vivo and in vitro bioassay experiment results indicate that MrNP1 and MrNP2 recombinant proteins can stimulate the expression of the MrVg gene. In contrast, silencing of MrNP1 and MrNP2 genes would suppress MrVg, VgR, and CyclinB gene expressions. The results indicate that the products of both genes can stimulate vitellogenesis by up-regulating the MrVg gene expression. Results from their difference in expression patterns indicate that they might have different regulatory roles in vitellogenin synthesis. Since gene silencing of either MrNP1 or MrNP2 affected the expression of the other NP, we have hypothesized that coordinated regulatory action between MrNP1 and MrNP2 may be necessary for the normal vitellogenesis in M. rosenbergii

    The vitellogenesis-inhibiting hormone (SpVIH) of the crab Scylla paramamosain is not likely to have a vitellogenesis-inhibiting function in the ovary

    Get PDF
    The function and molecular characterization of the previously reported crab (Scylla paramamosain) vitellogenesis inhibiting hormone SpVIH is still unclear. In this experiment, SpVIH’s bioinformatic and functional characterizations were analyzed. Sequence analysis showed SpVIH was clustered with several type II MIH/GIHs of the Brachyura. The expression of SpVIH in the eyestalk was low in the early ovarian developmental stage I and increased with the advancement of ovary maturation. Interestingly, the SpVIH transcript was still at a high level in the ovary of females at the middle to late maturation stage. SpVIH’s function was studied according to the expression of vitellogenin (Vg) and vitellogenin receptor (VgR) in the ovary and hepatopancreas after incubation with SpVIH-dsRNA and recombinant SpVIH protein (rSpVIH). In vitro assay results indicated that rSpVIH has a significant stimulative effect on the expression of Vg in the hepatopancreas in the middle stage of vitellogenesis and in the ovary of each vitellogenesis stage. However, SpVIH-dsRNA has no significant effect on the expression of Vg when SpVIH-dsRNA is introduced to the ovary and hepatopancreas at the early, middle, and late stages of vitellogenesis. Furthermore, the co-incubation experiment result indicated that the eyestalk consists of factor(s) that can significantly up-regulate the expression of Vg in the ovary, with a further combination of dsVIH and eyestalk under incubation, the significant increase of Vg-mRNA expression levels were also found in the ovary of four ovarian development stages of S. paramamosain. Contrary to the previous reports, SpVIH does not possess a vitellogenesis-inhibiting function but can promote vitellogenesis in the ovary and hepatopancreas

    Transcriptome analysis of flathead grey mullet (Mugil cephalus) ovarian development induced by recombinant gonadotropin hormones

    Get PDF
    Background: Treatment with recombinant gonadotropin hormones (rGths), follicle-stimulating hormone (rFsh) and luteinizing hormone (rLh), was shown to induce and complete vitellogenesis to finally obtain viable eggs and larvae in the flathead grey mullet (Mugil cephalus), a teleost arrested at early stages of gametogenesis in intensive captivity conditions. This study aimed to investigate the transcriptomic changes that occur in the ovary of females during the rGths-induced vitellogenesis. Methods: Ovarian samples were collected through biopsies from the same five females at four stages of ovarian development. RNASeq libraries were constructed for all stages studied, sequenced on an Illumina HiSeq4000, and a de novo transcriptome was constructed. Differentially expressed genes (DEGs) were identified between stages and the functional properties of DEGs were characterized by comparison with the gene ontology and Kyoto Encyclopedia. An enrichment analysis of molecular pathways was performed. Results: The de novo transcriptome comprised 287,089 transcripts after filtering. As vitellogenesis progressed, more genes were significantly upregulated than downregulated. The rFsh application induced ovarian development from previtellogenesis to early-to-mid-vitellogenesis with associated pathways enriched from upregulated DEGs related to ovarian steroidogenesis and reproductive development, cholesterol metabolism, ovarian growth and differentiation, lipid accumulation, and cell-to-cell adhesion pathways. The application of rFsh and rLh at early-to-mid-vitellogenesis induced the growth of oocytes to late-vitellogenesis and, with it, the enrichment of pathways from upregulated DEGs related to the production of energy, such as the lysosomes activity. The application of rLh at late-vitellogenesis induced the completion of vitellogenesis with the enrichment of pathways linked with the switch from vitellogenesis to oocyte maturation. Conclusion: The DEGs and enriched molecular pathways described during the induced vitellogenesis of flathead grey mullet with rGths were typical of natural oogenesis reported for other fish species. Present results add new knowledge to the rGths action to further raise the possibility of using rGths in species that present similar reproductive disorders in aquaculture, the aquarium industry as well as the conservation of endangered species.info:eu-repo/semantics/publishedVersio

    Multi-tissue transcriptome analysis identifies key sexual development-related genes of the ornate spiny lobster (Panulirus ornatus)

    Get PDF
    Sexual development involves the successive and overlapping processes of sex determination, sexual differentiation, and ultimately sexual maturation, enabling animals to reproduce. This provides a mechanism for enriched genetic variation which enables populations to withstand ever-changing environments, selecting for adapted individuals and driving speciation. The molecular mechanisms of sexual development display a bewildering diversity, even in closely related taxa. Many sex determination mechanisms across animals include the key family of “doublesex- and male abnormal3-related transcription factors” (Dmrts). In a few exceptional species, a single Dmrt residing on a sex chromosome acts as the master sex regulator. In this study, we provide compelling evidence for this model of sex determination in the ornate spiny lobster Panulius ornatus, concurrent with recent reports in the eastern spiny lobster Sagmariasus verreauxi. Using a multi-tissue transcriptomic database established for P. ornatus, we screened for the key factors associated with sexual development (by homology search and using previous knowledge of these factors from related species), providing an in-depth understanding of sexual development in decapods. Further research has the potential to close significant gaps in our understanding of reproductive development in this ecologically and commercially significant order

    RNA interference in Artemia franciscana applied to sex determining genes

    Get PDF

    An investigation into the molecular determinants of salmon louse (Lepeophtheirus salmonis (Krøyer, 1837)) susceptibility to the antiparasitic drug emamectin benzoate.

    Get PDF
    Caligid copepods, also called sea lice, are ectoparasites of marine fish, with Lepeophtheirus salmonis (Krøyer, 1837) emerging as a problem for mariculture of Atlantic salmon (Salmo salar Linnaeus, 1758) in the northern hemisphere. Annual costs of sea lice to global salmon farming was estimated to be in excess of €300 million in 2006, with the majority of this accounted for through expenses accrued from chemical treatments. Only a limited range of anti-sea louse drugs are available and licensed for the treatment of fish, and the continued use of only a few compounds creates a situation potentially favouring the development of drug resistance. Emamectin benzoate (EMB) is currently used as a salmon delousing agent, being employed as a 0.2 % in-feed pre-mix (SLICE®). Atlantic salmon farmers have reported increased incidence of reduced L. salmonis sensitivity to SLICE®, which has highlighted the requirement for further research into the molecular mechanisms controlling salmon louse resistance to EMB. Genomic and transcriptomic research concerning L. salmonis drug resistance mechanisms has not often been reported, with previous transcriptomic studies using candidate gene approaches and genetic studies focussing on population genetics. Drug resistance in ecdysozoan invertebrates is associated with a variety of molecular mechanisms including target site mutations and changes in the expression of components in drug detoxification pathways. The research reported in this thesis was aimed at the exploration of mechanisms employed by L. salmonis to reduce the toxicity of EMB exposure, following a transcriptomic approach that utilised custom oligonucleotide (oligo) microarrays and a genetic approach that utilised Restriction-site associated DNA sequencing (RAD-seq) to identify Single Nucleotide Polymorphism (SNP) markers. An EMB-resistant (PT) and drug-susceptible (S) L. salmonis laboratory-maintained strain were to be used as a model for this research, as these two strains differ in EMB susceptibility (~ 7-fold) and show stable susceptibility profiles through multiple generations, suggesting that this drug resistance phenotype may be a heritable trait. Sequence resources available for salmon lice are limited as an annotated L. salmonis genome is currently under construction. Therefore, a significant amount of this study involved creating new resources to facilitate the analysis of EMB susceptibility. Suppression subtractive hybridisation (SSH) was used to enrich for transcripts that were differentially expressed between strains PT and S, which provided sufficient target sequence for the development of 15K oligo microarrays when combined with sequences assembled from existing L. salmonis ESTs. Additionally, transcripts were generated through sequencing a pooled sample representing key developmental stages of the L. salmonis life cycle, which were later used in the construction of a 44K oligo microarray. The toxicity of EMB and other avermectins (AVMs) against ecdysozoan invertebrates is reported to be based mainly on their interaction with ligand-gated ion channels (LGIC), specifically glutamate-gated chloride channels (GluCl). However, -aminobutyric acid (GABA)-gated chloride channels (GABA-Cls) are also believed to be targeted by AVMs and neuronal acetylcholine receptors (nAChRs) can be allosterically modulated by the AVM compound ivermectin. Transcriptional responses in PT and S salmon lice were investigated using custom 15K L. salmonis oligo microarrays. In the absence of EMB exposure, 359 targets differed in transcript abundance between the two strains. GABA-Cl and nAChR subunits showed significantly lower transcript levels in PT compared to S lice, which was estimated at ~1.4-fold for GABA-Cl and ~2.8-fold for nAChR using RT-qPCR, suggesting their involvement in AVM toxicity in caligids. Although, salmon lice from the PT strain showed few transcriptional responses following acute exposure (1 or 3 h) to 200 µg L-1 of EMB, a drug concentration tolerated by PT lice, but toxic for S lice. RAD-seq analysis of both genders from L. salmonis strains S and PT identified 15 RAD-markers that show complete association with salmon louse strain, although these preliminary results will need further analysis to confirm marker association with reduced EMB susceptibility. Additionally, RAD marker Lsa101901 showed complete association with sex for all individuals analysed, being heterozygous in females and homozygous in males. Using an allele-specific PCR assay, this SNP association pattern was further confirmed for three unrelated salmon louse strains. Marker Lsa101901 was located in the coding region of the prohibitin-2 gene, which showed a sex-dependent differential expression, with mRNA levels determined by RT-qPCR about 1.8-fold higher in adult female than adult male salmon lice. In conclusion, the identification of decreased transcript abundances for LGIC subunits in EMB-resistant salmon lice, and polymorphic SNP markers showing complete association with L. salmonis strains S or PT, provides suitable candidates for further investigation into their association with reduced EMB susceptibility. Further analysis will also be required to confirm whether EMB-induced mechanisms are not associated with reduced EMB susceptibility in L. salmonis. Additionally, the identification of sex-linked SNP Lsa101901 suggests that sex determination in the salmon louse is genetic and follows a female heterozygous system, with marker Lsa101901 providing a tool to determine the genetic sex of salmon lice. Improved knowledge of L. salmonis biology and the mechanisms potentially involved in EMB resistance, obtained during this study, may provide molecular markers that contribute to successful monitoring and management of this commercially important parasite of Atlantic salmon

    The molecular basis of circadian and seasonal rhythms in the blue mussel Mytilus edulis

    Get PDF
    Exposure to regular environmental oscillations such as day/night have allowed organisms to evolve biological mechanisms to adaptively anticipate and prepare for rhythmic environmental change. A network of gene-protein interactions between clock genes and their proteins comprise the molecular clock mechanism at the heart of regulating biological rhythms. Though this is an endogenous and self-regulating system, elements of this network can be entrained by exogenous biotic and abiotic factors. This synchronisation process between environmental cycles and endogenous rhythms is facilitated by cues like light and temperature, which influence clock gene expression patterns.Marine bivalves often inhabit intertidal habitats under the influence of numerous oscillating environmental conditions, though little is known about how they regulate their biological timekeeping. In this thesis, we investigate the molecular regulation of biological rhythms in the ecologically and commercially important blue mussel, M. edulis, over different timeframes. For the first time in this species, we isolate and characterise a number of clock genes (Clk, Cry1, ROR/HR3, Per and Rev-erb) and clock-associated genes (ARNT, Timeout-like and aaNAT). Rhythmic clock gene expression is demonstrated in the absence of light cues, indicative of endogenous clock control. Differential expression of Cry1 expression between males and females under the same conditions indicates sex-specific regulation and/or function. In addition, diurnal temperature cycles modulated the otherwise rhythmic expression of Rev-erb to constant levels demonstrating an interaction of temperature with clock function. Instances of seasonal clock mRNA expression differences were found, in addition to a number of other putative seasonal genes, indicating a possible mechanism by which seasonal cues can inform rhythmic biological processes.Understanding the influence of environmental cues on the molecular clock is essential in predicting the outcomes of future environmental change on fundamental rhythmic processes, in particular the impacts of decoupled environmental cues on the already highly dynamic and stressful intertidal zone

    Endocrine disruption in the terrestrial isopod porcellio scaber

    Get PDF
    Doutoramento em BiologiaNas últimas décadas tem-se assistido a uma preocupação crescente relativamente às possíveis consequências da exposição a compostosxenóbioticos capazes de modular ou causar disrupção do sistema endócrino, os denominados Compostos Disruptores Endócrinos (CDEs). A maioria dos estudos efectuados tem-se centrado principalmente nos efeitos dos CDEs em vertebrados, enquanto que os seus efeitos em invertebrados têmsido negligenciados, embora este grupo represente mais de 95% de todas asespécies animais. Isópodes como o Porcellio scaber, combinam características associadas às mudas e aos processos reprodutivos mediados por mecanismos endócrinosconhecidos com um modo de vida terrestre, tornando-os potenciais espécies sentinela para estudos de disrupção endócrina (DE) em ambientes terrestres. Neste estudo, isópodes machos adultos, machos e fêmeas juvenis e casais foram expostos a concentrações crescentes dedois CDEs, vinclozolina (Vz) e bisfenol A (BPA). Testou-se a hipótese nula que a Vz e o BPA não interferem com o desenvolvimento e reprodução deste isópode terrestre. Foi investigadaa possível ligação entre os efeitos causados pelos compostos propostos e DE assim como a ligação a outros potenciais mecanismos de toxicidade.Parâmetros como concentração de 20-hidroxiecdisona (20E), muda, crescimento, rácios sexuais ediversos parâmetros reprodutivos foram estudados. Adicionalmente, de modo a estudar os alvos moleculares destes tóxicos, analisou-se a expressão proteica do intestino, hepatopâncreas e testículos do isópode após exposição aos químicos. Os resultados demonstram que a Vz e o BPA estimulam o aumento dos níveis de 20E de um modo dependente da dose. Excepção feita para a concentração mais baixa de BPA testada (10 mg/kg solo), para a qual concentrações significativamente mais altas de 20E foram determinadas, sugerindo a ocorrência dos “efeitos de baixas doses típicos de DE” já demonstrados por outros autores. O BPA também distorceu o rácio sexual favorecendo asfêmeas na concentração mais baixa. A mortalidade devido à ecdise incompleta foi relacionada com o hiper-ecdisonismo nas concentrações mais elevadas de Vz. Mais ainda, a Vz tende a atrasar a muda e o BPA a induzi-la. Não obstante, ambos os compostos provocam toxicidade no desenvolvimento,uma vez que foi encontrada uma diminuição generalizada nos parâmetros de crescimento. Os juvenis mostraram ser mais sensíveis à exposição aos tóxicos que os adultos. Estes compostos provocaram ainda toxicidade reprodutiva, com um decréscimo generalizado do “output” reprodutivo. A toxicidadecausada pelos ecdisteróides e o seu papel na síntese de vitelogenina são alguns dos factores chave que poderão influenciar negativamente a reprodução.A Vz e o BPA afectaram a expressão de proteínas envolvidas nometabolismo energético e induziram várias respostas de stress. Interferiram ainda com proteínas intimamente ligadas com o sucesso reprodutivo. Conclui-se assim,que ambos os CDEs propostos provocam toxicidade nodesenvolvimento e na reprodução de P. scaber, tendo sido evidenciada umaligação a DE. Alvos moleculares de natureza não-endócrina foram também revelados, através da expressão diferencial de algumas proteínaspreviamente descritas para invertebrados aquáticos e mesmo alguns vertebrados.In the past few decades there has been a growing concern about the possibleconsequences of exposure to xenobiotic compounds that are able to modulate or cause a disruption in the endocrine system, the Endocrine DisruptingCompounds (EDCs). The majority of research done has focused mainly on theeffects of EDCs on vertebrates while the effects on invertebrates have been largely ignored, despite this group representing more than 95% of all animalspecies. Isopods like Porcellio scaber combine the features of molting and reproductive processes mediated by known endocrine mechanisms with a terrestrial modeof life, making them potential sentinel species for the study of endocrinedisruption (ED) in soil environments. In this study, male adult isopods, male and female juveniles, and couples wereexposed to increasing soil concentrations of two proposed EDCs, vinclozolin (Vz) and bisphenol A (BPA). The null hypothesis was that Vz and BPA does not disrupt the normal development nor compromise the reproduction of theterrestrial isopod P. scaber. A causal link to ED and to other potential mechanisms by which these chemicals could elicit toxicity was addressed. Several parameters such as 20-hydroxyecdysone (20E) levels, molting parameters, growth, sex ratio and several reproductive parameters wereassessed. Furthermore, to study a vast array of molecular targets, the protein expression of the isopod gut, hepatopancreas and testes was investigated. Results demonstrated that both Vz and BPA induced increased 20E levels in adose-dependent way. An exception was BPA at the lowest concentration tested(10 mg/kg soil), at which high levels of 20E were also found, suggesting thepresence of “ED typical low-dose effects”. BPA also induced a sex ratio shift favouring females at the lowest concentration. Mortality due to incompleteecdysis was related to hyperecdysonism at the highest concentrations of Vz.Also, Vz tended to postpone molt and BPA induced it. Nevertheless, both compounds elicited developmental toxicity since an overall growth decreasewas found. Juveniles were more sensitive to toxicant exposure than adults.These compounds also elicited reproductive toxicity with an overall decrease ofthe reproductive output. Ecdysteroidal toxicity and its role in vitellogeninsynthesis are some of the key factors possibly causing the reproductiveimpairment. BPA and Vz affectedproteins involved in energy metabolism, induced a variety of stress responses and interfered with proteins intimatelylinked with the isopods’ reproductive success. It is concluded from this investigation that Vz and BPA elicit reproductive anddevelopmental toxicity and a causal link to ED was provided. Non-endocrine molecular targets were also revealed for both compounds, with a differentialexpression of some proteins previously reported for some aquatic invertebrates and also some vertebrates
    corecore