RESUMO
The Pacific white shrimp Penaeus vannamei is the most cultured shrimp species around the world. Because females grow larger than males, the culture of 'only females' is of great interest, but knowledge on sex determination and differentiation is required for producing only females. In an effort to obtain information associated with reproduction in P. vannamei, transcriptomic data from female gonads was generated, and partial sequences of a transcript were identified as Sex-lethal (Sxl). Its characterization indicated that, differently from other penaeids in which this gene has been isolated, there are six isoforms of the Sxl transcript in P. vannamei (PvanSxl 1-6). These isoforms result from alternative splicing at three splice sites (SS1, SS2, SS3). The first splice-site is unique to P. vannamei, as it has not been reported for other Arthropod species; the second splice-site (SS2) is common among crustaceans, and the third splice-site (SS3) is also unique to P. vannamei and when spliced-out, it is always together with SS2. All isoforms are expressed during embryogenesis as well as gametogenesis of both genders. The two shorter isoforms, PvanSxl-5 and PvanSxl-6, which result from the splicing of SS2 and SS3, were found mostly expressed in adult testis, but PvanSxl-6 was also expressed in oocytes during gametogenesis. During oogenesis, the second largest isoform, PvanSxl-2, which splices-out only SS1, and PvanSxl-4 that splices-out SS1 and SS2 were highly expressed. These two isoforms were also highly expressed during embryonic development. In situ hybridization allowed pinpointing more specifically the cells where the PvanSxl transcripts were expressed. During embryogenesis, hybridization was observed from the one-cell stage embryo to late gastrula. In the female gonad in previtellogenesis, hybridization occurred in the nucleus of oocytes, whereas in secondary vitellogenesis the transcript also hybridized cytoplasmic granules and cortical crypts. Finally, in situ hybridization corroborated the expression of PvanSxl also in the male gonad during spermatogenesis, mostly occurring in the cytoplasm from spermatogonia and spermatocytes.
Assuntos
Proteínas de Artrópodes/genética , Penaeidae/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/classificação , Proteínas de Artrópodes/metabolismo , Desenvolvimento Embrionário/genética , Feminino , Gametogênese/genética , Gônadas/metabolismo , Masculino , Especificidade de Órgãos , Penaeidae/embriologia , Penaeidae/genética , Penaeidae/crescimento & desenvolvimento , Filogenia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Alinhamento de SequênciaRESUMO
Ploidy manipulation has been rarely used in the genetic improvement of cultured marine shrimps. Although polyploid induction has been proven to be successful in Penaeids, including the species Litopenaeus vannamei, the methodology still requires some improvements. In the present work, different thermal shock treatments on ploidy manipulation were tested and a protocol for detecting polyploid individuals was also established. Fertilized eggs were treated by cold (10°C) and heat (38°C) thermal shocks for 8, 12, 15, 18, 20, and 22 min to induce polyploidy. Nuclear measurements within distinct treatments revealed a significant deviation in relation to the mean diameter of nuclei in the control individuals. Triploid and tetraploid metaphases were observed within treated individuals, confirming the increase of interphasic nuclear diameter. The cold thermal shock was more efficient than the hot ones, besides leading to a higher and more homogeneous hatchery rate. A mean number of three nucleoli per nucleus were observed in diploid individuals, while treated samples usually presented up to five nucleoli per nucleus. The standardization of protocols to obtain and detect polyploid products allows further utilization of such methods on a commercial scale in order to evaluate the performance of polyploid individuals in the genetic improvement of L. vannamei.
Assuntos
Penaeidae/genética , Poliploidia , Adaptação Fisiológica/genética , Animais , Aquicultura/métodos , Temperatura Baixa , Análise Citogenética/métodos , Temperatura Alta , Penaeidae/embriologia , Ploidias , Estresse FisiológicoRESUMO
A genetic transformation system for penaeid shrimp could provide a powerful technique for the improvement of different production traits of importance for a sustainable aquaculture. The development of a successful transformation system depends on the ability to efficiently introduce exogenous DNA into the target species. The ability of the nuclear localization signal (NLS) peptide of the SV40 T antigen to facilitate nuclear import and transient gene expression is known from vertebrate systems and for the first time, is shown here to be efficient in a crustacean species, i.e. the shrimp Litopenaeus schmitti. Electroporation was used to introduce the pCMV-lacZ plasmid that contains the human cytomegalovirus promoter/enhancer (CMV) fused to the beta-galactosidase (lacZ) coding region, into L. schmitti zygotes. Supercoiled DNA was used at 50 or 500 ng/microl naked or bound to NLS peptide. The hatching rate of electroporated zygotes was around 60% for all groups, except from the pCMV-lacZ:NLS group at 500 ng/microl (43%). Based on Southern blot analyses of polymerase chain reaction (PCR) products the gene transfer frequency was 2-fold higher using DNA:NLS complexes than with naked DNA (23.8% vs. 11.5%, with 50 ng/microl of plasmid DNA, 44.3% vs. 28.8% with 500 ng/microl). The beta-galactosidase activity assay indicated that nuclear uptake is faster for the DNA:NLS complexes than for naked DNA. The beta-galactosidase activity was always higher in the DNA:NLS groups than in the naked DNA groups. To our knowledge, this is the first report on the use of an NLS peptide to improve gene transfer and nuclear uptake in crustaceans.
Assuntos
Antígenos Transformantes de Poliomavirus/genética , Núcleo Celular/metabolismo , Embrião não Mamífero/metabolismo , Sinais de Localização Nuclear/genética , Penaeidae/genética , Transporte Ativo do Núcleo Celular , Animais , Southern Blotting , DNA/administração & dosagem , DNA/genética , DNA/metabolismo , DNA Recombinante/genética , DNA Recombinante/metabolismo , Eletroporação , Embrião não Mamífero/efeitos dos fármacos , Desenvolvimento Embrionário , Feminino , Expressão Gênica , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Óperon Lac/genética , Óvulo/efeitos dos fármacos , Óvulo/crescimento & desenvolvimento , Óvulo/metabolismo , Penaeidae/embriologia , Reação em Cadeia da Polimerase , Fatores de Tempo , Transfecção/métodos , beta-Galactosidase/metabolismoRESUMO
The quantification of oxygen consumption and ammonia-N excretion rates is essential in determining energy requirements for development of larval invertebrates. In larval energetics, there is a need for accurate and uncomplicated techniques to quantify metabolic rates. A method for simultaneous measurements of oxygen and ammonia-N concentrations is presented. It employs sealed respirometric chambers (ca. 30 ml) in which embryos and larvae are incubated. Analysis is carried out in end-point samples by Winkler's titration and indophenol-blue for oxygen and ammonia-N, respectively. Water is sampled into volume-calibrated glass syringes and oxygen consumption and ammonia-N excretion rates were determined by the difference between experimental and control (no animals) units. The method was successfully used to measure metabolic rates in embryo and larval stages of the shrimp Farfantepenaeus paulensis and in veliger of the mussel Perna perna. The accuracy denoted by the coefficient of variation is comparable to previous results on larval metabolic rates. A biomass: volume (microg ml(-1)) is proposed to extend its application to further species of marine invertebrates. The method is simple to operate, involves non-expensive material and is portable enough for field work. A substantial number of replicates can be analyzed at the same time and O:N ratio, an indicator of the catabolized substrate, can be calculated.
Assuntos
Amônia/urina , Bivalves/embriologia , Bivalves/crescimento & desenvolvimento , Nitrogênio/urina , Consumo de Oxigênio , Penaeidae/embriologia , Penaeidae/crescimento & desenvolvimento , Animais , Embrião não Mamífero/metabolismo , Feminino , Larva/metabolismo , Fisiologia/instrumentação , Fisiologia/métodosRESUMO
Energy metabolism in early life stages of the shrimp Farfantepenaeus paulensis subjected to temperature reduction (26 and 20 degrees C) was determined using the activities of citrate synthase (CS) and pyruvate kinase (PK). At both temperatures, weight-specific activity of CS decreased throughout the ontogenetic development from protozoea II (PZ II) to postlarva XII-XIV (PL XII-XIV). PK activity reached a pronounced peak in PL V-VI, followed by a further decrease in PL XII-XIV. Temperature reduction produced variation in oxygen consumption rates (QO(2)), ammonia-N excretion and in enzyme activities. Ammonia-N excretion was higher at 20 degrees C in mysis III (M III), PL V-VI and PL XII-XIV, resulting in substantially lower O:N ratios in these stages. QO(2) was increased in protozoea II (PZ II) and mysis I (M I) at 26 degrees C, while no difference in QO(2) was detected in the subsequent stages at either temperature. This fact coincided with higher CS and PK activities in M III, PL V-VI and PL XII-XIV at 20 degrees C compared with 26 degrees C. Regressions between individual enzyme activities and dry weight exhibited slope values of 0.85-0.92 for CS and 1.1-1.2 for PK and temperature reduction was reflected by higher slope values at 20 than at 26 degrees C for both enzymes. Weight-specific CS activity was positively correlated with QO(2) at 20 and 26 degrees C, and may thus be used as an indicator of aerobic metabolic rate throughout the early stages of F. paulensis. The variation in enzyme activities is discussed in relation to possible metabolic adaptations during specific ontogenetic events of the F. paulensis life cycle. Here, the catalytic efficiency of energy-metabolism enzymes was reflected in ontogenetic shifts in behaviour such as larval settlement and the adoption of a benthic existence in early postlarvae. In most cases, enhanced enzyme activities appeared to counteract negative effects of reduced temperature.