RESUMEN

Aquatic environments are subject to ultraviolet B (UVB) radiation incidence, and its effects on organisms are dose-dependent. Besides DNA, mitochondria are an important target of this radiation that causes structural damage and impairs its functional dynamics. Here, we hypothesize that mitophagy acts as an organelle quality control mechanism to mitigate UVB impacts in embryonic cells. Then, freshwater prawn Macrobrachium olfersii embryos was used as a model to investigate the effects of UVB on genes (Tomm20, Opa1, Pink, Prkn, Sqstm1, and Map1lc3) and proteins (TOM20, PINK1, p62 and LC3B) involved in mitophagy modulation. The choice of genes and proteins was based on the identification of mitochondrial membrane (Tomm20, Opa1 and TOM20), mediation of mitophagy (Pink1, Prkn and PINK1), and recognition of mitochondria by the autophagosome membrane (Sqstm1, Map1lc3, p62 and LC3B). First, the phylogeny of all genes presented bootstrap values >80 and conserved domains among crustacean species. Gene expression was inherently modulated during development, with transcripts (Tomm20, Opa1, Pink, Prkn, Sqstm1, and Map1lc3) overexpressed in the initial and final stages of development. Moreover, UVB radiation induced upregulation of Tomm20, Opa1, Pink, Prkn, Sqstm1, and Map1lc3 genes at 6 h after exposure. Interestingly, after 12 h, the protein content of PINK1, p62, and LC3B increased, while TOM20 was not responsive. Despite UVB radiation's harmful effects on embryonic cells, the chronology of gene expression and protein content indicates rapid activation of mitophagy, serving as an organelle quality control mechanism, given the analyzed cells' integrity.

Asunto(s)

Mitofagia , Palaemonidae , Rayos Ultravioleta , Animales , Rayos Ultravioleta/efectos adversos , Mitofagia/efectos de la radiación , Palaemonidae/efectos de la radiación , Palaemonidae/embriología , Palaemonidae/genética , Mitocondrias/metabolismo , Mitocondrias/efectos de la radiación , Embrión no Mamífero/efectos de la radiación , Embrión no Mamífero/metabolismo , Proteínas de Artrópodos/metabolismo , Proteínas de Artrópodos/genética , Filogenia , Orgánulos/metabolismo , Orgánulos/efectos de la radiación

RESUMEN

The Macrobrachium amazonicum complex is composed of at least the Macrobrachium amazonicum and Macrobrachium pantanalense species, with the latter described from specimens originally identified as part of an endemic M. amazonicum population in the Brazilian Pantanal region. While there may be a reproductive barrier between these two Macrobrachium species, both are phylogenetically close, with small genetic distance. However, there is currently no available biochemical information of Macrobrachium pantanalense (Na+, K+)-ATPase. Here, we report the kinetic characteristics of the gill (Na+, K+)-ATPase in two populations of M. pantanalense from Baiazinha Lagoon (Miranda, MS, Brazil) and Araguari River (Uberlândia, MG, Brazil), and compare them with Macrobrachium amazonicum populations from the Paraná-Paraguay River Basin. (Na+, K+)-ATPase activities were 67.9 ± 3.4 and 93.3 ± 4.1 nmol Pi min-1 mg-1 protein for the Baiazinha Lagoon and Araguari River populations, respectively. Two ATP hydrolyzing sites were observed for the Araguari River population while a single ATP site was observed for the Baiazinha Lagoon shrimps. Compared to the Araguari River population, a 3-fold greater apparent affinity for Mg2+ and Na+ was estimated for the Baiazinha Lagoon population, but no difference in K+ affinity and ouabain inhibition was seen. The kinetic differences observed in the gill (Na+, K+)-ATPase between the two populations of M. pantanalense, compared with those of various M. amazonicum populations, highlight interspecific divergence within the Macrobrachium genus, now examined from a biochemical perspective.

Asunto(s)

Branquias , Palaemonidae , ATPasa Intercambiadora de Sodio-Potasio , Animales , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/genética , Palaemonidae/genética , Palaemonidae/enzimología , Branquias/metabolismo , Branquias/enzimología , Brasil , Ríos , Cinética

RESUMEN

BACKGROUND: The river prawn, Macrobrachium americanum (M. americanum), is one of the largest prawns of the genus in Latin America and is an amphidromous species distributed along the Pacific coast of America. This prawn has commercial value due to its size and taste, making it a good option for aquaculture production. Its culture has been attempted in ponds and concrete tanks, but no successful technique can still support commercial production. Understanding the mechanisms that regulate reproduction at the molecular level is very important. This knowledge can provide tools for manipulating transcripts, which could increase the number or size of animals in the culture. Our understanding of the mechanism that regulates the reproduction of M. americanum at the molecular level is limited. AIM: Perform and analyze the transcriptome assembly of the testes, vas deferens, and terminal ampulla of M. americanum. to provide new molecular information about its reproduction. METHODS AND RESULTS: The cDNA library was constructed and sequenced for each tissue to identify novel transcripts. A combined transcriptome with the three tissues was assembled using Trinity software. Unigenes were annotated using BLASTx and BLAST2GO. The transcriptome assembly generated 1,059,447 unigenes, of which 7222 genes had significant hits (e-value < 1 × 10-5) when compared against the Swiss-Prot database. Around 75 genes were related to sex determination, testis development, spermatogenesis, spermiogenesis, fertilization, maturation of testicular cells, neuropeptides, hormones, hormone receptors, and/or embryogenesis. CONCLUSIONS: These results provide new molecular information about M. americanum reproduction, representing a reference point for further genetic studies of this species.

Asunto(s)

Decápodos , Palaemonidae , Penaeidae , Animales , Masculino , Palaemonidae/genética , Perfilación de la Expresión Génica/métodos , Transcriptoma/genética , Decápodos/genética , Biblioteca de Genes , Penaeidae/genética

RESUMEN

BACKGROUND: Macrobrachium amazonicum is a freshwater prawn widely distributed in South America that is undergoing speciation, so the denomination "M. amazonicum complex" is used for it. The mitochondrial cytochrome c oxidase subunit I (COI) gene has been used to elucidate this speciation, but heteroplasmies and pseudogenes have been recorded, making separation difficult. Obtaining genes from cDNA (RNA) rather than genomic DNA is an effective tool to mitigate those two types of occurrences. The aim of this study was to assemble in silico the mitochondrial DNA (mtDNA) of the Amazonian coastal population of M. amazonicum inhabiting the state of Pará. RESULTS: Sequences were obtained from the prawn's transcriptome using the de novo approach. Six libraries of cDNA from the androgen gland, hepatopancreas, and muscle tissue were used. The mtDNA of M. amazonicum was 14,960 bp in length. It contained 13 protein-coding genes, 21 complete transfer RNAs, and the 12S and 16S subunits of ribosomal RNA. All regions were found on the light strand except tRNAGln, which was on the heavy strand. The control region (D-loop) was not recovered, making for a gap of 793 bp. The cladogram showed the formation of the well-defined Macrobrachium clade, with high support value in the established branches (91-100). The three-dimensional spatial conformation of the mtDNA-encoded proteins showed that most of them were mainly composed of major α-helices that typically shows in those proteins inserted in the membrane (mitochondrial). CONCLUSIONS: It was possible to assemble a large part of the mitochondrial genome of M. amazonicum in silico using data from other genomes deposited in GenBank and to validate it through the similarities between its COI and 16S genes and those from animals of the same region deposited in GenBank. Depositing the M. amazonicum mtDNA sequences in GenBank may help solve the taxonomic problems recorded for the species, in addition to providing complete sequences of candidate coding genes for use as biomarkers in ecological studies.

Asunto(s)

Genoma Mitocondrial , Palaemonidae , Animales , ADN Mitocondrial/genética , Palaemonidae/genética , ADN Complementario , Transcriptoma , ARN de Transferencia/genética , Filogenia

RESUMEN

The giant river prawn, Macrobrachium rosenbergii (de Man, 1879), is a species of great commercial importance. It is farmed under different conditions that translate to a great range of light environments, which impact their behavior and productivity. We present the first study employing both visual modeling and beha vioral data to evaluate the ontogenetic changes in color preferences of juveniles and adults of M. rosenbergii. We offered ten shelters of different colors to juveniles and adults and registered their preferences. Our results show that shelter preference changed with ontogeny: juveniles chose shelters based on chromaticity (preference for blue), while adults based their decisions on brightness (preference for dark grey). This preference adults show for dark colors is probably associated with light avoidance behavior. We recommend providing blue shelters for juveniles and dark shelters for adults.(AU)

Asunto(s)

Animales , Conducta Animal , Acuicultura/métodos , Palaemonidae/genética , Estimulación Luminosa , Bienestar del Animal

RESUMEN

Species of the decapod family Palaemonidae are common components of tropical coastal waters and coral reefs. The majority of these species are symbionts of various invertebrate phyla. Despite a long history of research on their species diversity in the Dutch Caribbean, recent field expeditions have yielded much new information. Combined with examinations of specimens housed in Naturalis Biodiversity Center and information from literature, a comprehensive list of Dutch Carribean palaemonids is provided. Newly collected material was primarily identified via morphological analyses. Additional molecular phylogenetic analyses based on mitochondrial COI and 16S and nuclear Histone 3 (H3) genes were conducted in search of cryptic species on the one hand and to check conspecifity in species that were found on multiple host species on the other hand. In total, 46 species are here listed for the Dutch Caribbean of which 24 are here recorded for the first time for one of the islands. One species new to science was discovered and is herein described. Sixty new host associations are recorded. In light of biodiversity loss and increasing anthropogenic pressure on declining coral reefs, documenting the diversity of palaemonids and other coral reef species to provide baseline data takes on a new urgency.

Asunto(s)

Decápodos , Palaemonidae , Animales , Palaemonidae/genética , Filogenia , Decápodos/genética , Indias Occidentales , Arrecifes de Coral , Región del Caribe , Biodiversidad

RESUMEN

Hox genes encode transcription factors that specify the body segment identity during development, including crustaceans, such as amphipods and decapods, that possess a remarkable diversity of segments and specialized appendages. In amphipods, alterations of specialized appendages have been obtained using knockout experiment of Hox genes, which suggests that these genes are involved in the evolution of morphology within crustaceans. However, studies of Hox genes in crustaceans have been limited to a few species. Here, we identified the homeodomain of nine Hox genes: labial (lab), proboscipedia (pb), Deformed (Dfd), Sex combs reduced (Scr), fushi tarazu (ftz), Antennapedia (Antp), Ultrabithorax (Ubx), abdominal-A (abdA), and Abdominal-B (AbdB), and evaluated their expression by RT-qPCR and RT-PCR in the ovary, during embryonic development, and at the first larval stage (Zoea I) of the decapod Macrobrachium olfersii. The transcript levels of lab, Dfd, and ftz decreased and transcripts of pb, Scr, Antp, Ubx, abdA, and AbdB increased during embryonic development. Hox genes were expressed in mature ovaries and Zoea I larval stages, except Scr and ftz, respectively. In addition, isoforms of Dfd, Scr, Ubx, and abdA, which have been scarcely reported in crustaceans, were described. New partial sequences of 87 Hox genes from other crustaceans were identified from the GenBank database. Our results are interesting for future studies to determine the specific function of Hox genes and their isoforms in the freshwater prawn M. olfersii and to contribute to the understanding of the diversity and evolution of body plans and appendages in Crustaceans.

Asunto(s)

Proteínas de Drosophila , Palaemonidae , Animales , Proteínas de Drosophila/genética , Desarrollo Embrionario , Femenino , Regulación del Desarrollo de la Expresión Génica , Genes Homeobox , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Palaemonidae/genética , Palaemonidae/metabolismo

RESUMEN

Crustaceans are major constituents of aquatic ecosystems and, as such, changes in their behavior and the structure and function of their bodies can serve as indicators of alterations in their immediate environment, such as those associated with climate change and anthropogenic contamination. We have used bioinformatics and a de novo transcriptome assembly approach to identify potential targets for developing specific antibodies to serve as nervous system function markers for freshwater prawns of the Macrobrachium spp. Total RNA was extracted from brain ganglia of Macrobrachium carcinus freshwater prawns and Illumina Next Generation Sequencing was performed using an Eel Pond mRNA Seq Protocol to construct a de novo transcriptome. Sequencing yielded 97,202,662 sequences: 47,630,546 paired and 1,941,570 singletons. Assembly with Trinity resulted in 197,898 assembled contigs from which 30,576 were annotated: 9,600 by orthology, 17,197 by homology, and 3,779 by transcript families. We looked for glutamate receptors contigs, due to their main role in crustacean excitatory neurotransmission, and found 138 contigs related to ionotropic receptors, 32 related to metabotropic receptors, and 18 to unidentified receptors. After performing multiple sequence alignments within different biological organisms and antigenicity analysis, we were able to develop antibodies for prawn AMPA ionotropic glutamate receptor 1, metabotropic glutamate receptor 1 and 4, and ionotropic NMDA glutamate receptor subunit 2B, with the expectation that the availability of these antibodies will help broaden knowledge regarding the underlying structural and functional mechanisms involved in prawn behavioral responses to environmental impacts. The Macrobrachium carcinus brain transcriptome can be an important tool for examining changes in many other nervous system molecules as a function of developmental stages, or in response to particular conditions or treatments.

Asunto(s)

Anticuerpos/inmunología , Encéfalo/metabolismo , Ecosistema , Anotación de Secuencia Molecular/métodos , Palaemonidae/genética , Receptores de Glutamato/genética , Animales , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Palaemonidae/metabolismo , Receptores de Glutamato/inmunología , Receptores de Glutamato/metabolismo , Transcriptoma

RESUMEN

Palaemonid shrimps inhabit osmotic niches from marine to continental waters. They hyper-regulate hemolymph osmolality and ionic concentrations in dilute media, hypo-regulating in concentrated media. Their gill epithelia express ion transporters like the Na+-K+-2Cl- symporter (NKCC) thought to play a role in salt secretion. To examine Cl- hypo-regulatory capability and phylogenetic correlations between gill NKCC mRNA levels and protein expression, we used palaemonids ranging from marine tide pools through estuaries (Palaemon) to coastal and continental fresh waters (Macrobrachium). We established the species' upper critical salinity limits (UL50) and short- (24 h) and long-term (120h) hypo-regulatory abilities at salinities of 80% of their UL50's (80%UL50). The Palaemon species exhibited the highest UL50's and greatest hypo-regulatory capabilities; among the Macrobrachium species, UL50's were higher in the diadromous than in the hololimnetic species. While basal transcript levels of gill NKCC mRNA were highest in P. pandaliformis, levels were unaffected by salinity or exposure time in all species. However, gill NKCC protein abundance increased after 120-h exposure at the 80%UL50 in all Macrobrachium species, except M. potiuna. Unexpectedly, hemolymph hyper-osmoregulatory capability in acclimatization media correlated with gill NKCC protein synthesis, while gill NKCC mRNA expression correlated with hemolymph hyper-Cl- regulation in Macrobrachium. These findings, together with the evolutionary history of osmoregulation in this shrimp clade, suggest a role for the gill NKCC symporter in both salt uptake and secretion. The evolution of NKCC protein expression responsiveness, unlike hemolymph hypo-regulation and NKCC mRNA expression, may have been driven by environmental salinity during niche radiation. SUMMARY STATEMENT: While mRNA expression of the gill Na+-K+-2Cl- symporter is unchanged during acclimation of palaemonid shrimps to saline media, protein expression is up regulated, revealing a role in chloride secretion.

Asunto(s)

Branquias/fisiología , Palaemonidae/genética , Palaemonidae/fisiología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Aclimatación , Animales , Evolución Biológica , Ecosistema , Femenino , Agua Dulce , Hemolinfa/metabolismo , Iones , Cinética , Masculino , Concentración Osmolar , Osmorregulación , Ósmosis , Filogenia , ARN Mensajero/metabolismo , Salinidad , Sodio/metabolismo , Especificidad de la Especie , Simportadores/genética , Simportadores/metabolismo , Resultado del Tratamiento , Equilibrio Hidroelectrolítico/fisiología

RESUMEN

The mitochondrial cytochrome oxidase c subunit 1 (COI) gene has been widely used in phylogenetic studies of crustaceans and analyses in population genetics. As COI studies have become more popular, there has been an increase in the number of reports of the presence of nuclear insertions of mitochondrial DNA (Numts) and mitochondrial heteroplasmy. Here, we provide evidence of both types of event in the COI sequences of Macrobrachium amazonicum, an economically important freshwater prawn, which is widespread in South America. Heteroplasmy and Numts were confirmed by different methods of DNA extraction (genomic, mitochondrial, and nuclear-enriched DNA), cloning, and sequencing, and were observed in 11 of the 14 populations sampled, primarily in the Amazon region. We discuss how the occurrence of these events affects the interpretation of the genetic relationships among the M. amazonicum populations, and we recommend caution when using COI for genetic inferences in prawns of the genus Macrobrachium, and in particular that any analysis should include nuclear markers.

Asunto(s)

Núcleo Celular/genética , Código de Barras del ADN Taxonómico/métodos , Complejo IV de Transporte de Electrones/genética , Mitocondrias/genética , Palaemonidae/clasificación , Animales , Proteínas de Artrópodos/genética , Brasil , Clonación Molecular , ADN Mitocondrial/genética , Genética de Población , Heteroplasmia , Palaemonidae/genética , Paraguay , Filogenia , Filogeografía , Análisis de Secuencia de ADN

RESUMEN

Freshwater prawns of the genus Macrobrachium are one of the important components of circumtropical marine, estuarine, and freshwater environments. They have been extensively exploited for human consumption for many years. More than 250 species reflect the evolutionary success of this highly diversified group, with a complex and challenging taxonomy due to morphological variations and vast geographical distribution. Although genetic approaches have been used to clarify phylogenetic and taxonomic aspects of Macrobrachium species, cytogenetic information is still very scarce and mostly focused on chromosome number and morphology. Here, we present chromosome data for three species from the Neotropical region, M. carcinus, M. acanthurus, and M. amazonicum, and one species from the Oriental region, M. rosenbergii. Using conventional cytogenetic approaches and chromosome mapping of repetitive DNAs by fluorescence in situ hybridization (FISH), we identified numerical diversification of the diploid set, within and between both zoogeographic regions. These included M. acanthurus and M. amazonicum sharing diploid chromosomes of 98, while M. carcinus has 94, and M. rosenbergii has 118 chromosomes. Argentophilic sites are also variable in number, but they occur in a much higher number than 18S rDNA, representing two to 10 sites within the study species. Microsatellites repeat motifs are also abundant in the chromosomes, with a co-localization and uniform distribution along the chromosome arms, but completely absent in the AT-rich centromeric regions. As a whole, our study suggests that the 2n divergence was followed by a considerable rDNA diversification. The abundance of the exceptional amount of microsatellite sequences in the chromosomes also suggests that they are essential components of the Macrobrachium genome and, therefore, maintained as a shared feature by the species, the reason for which is yet unknown.

Asunto(s)

Análisis Citogenético , Palaemonidae/genética , Animales , ADN Ribosómico/genética , Humanos , Hibridación Fluorescente in Situ , Cariotipo , Repeticiones de Microsatélite , Palaemonidae/clasificación

RESUMEN

The extracellular matrix (ECM) is a non-cellular and three-dimensional structure, constituted by a macromolecular dynamic network that involves the cells in all animal tissues, including embryonic ones. Several studies with vertebrates and cell cultures have reported deleterious effects of ultraviolet-B (UVB) radiation on the components associated with the ECM. However, studies focusing on the UVB radiation effects on ECM components of crustaceans during embryonic development are very scarce. Thus, the aim of this study was to identify the coding sequences of components associated with the ECM and to evaluate the effect of UVB radiation on embryos of the ecologically-important decapod Macrobrachium olfersii. To evaluate the modulation of these ECM components during embryonic development, the transcript levels of Col4α1, Itgß, Lamα, Mmp1 and Timp in M. olfersii embryos were analyzed at early developmental stages (E1, E3 and E4), intermediate developmental stage (E7) and late developmental stages (E10 and E14). In addition, embryos at E7, which correspond to a landmark of crustacean development, were analyzed after 12 h of UVB exposure to verify UVB effects on the ECM components. The ECM component sequences were similar to other decapods, suggesting conservation of these genes among crustaceans. The results showed modulations of the ECM components of M. olfersii embryos that reflect the need for each component in the cellular mechanisms, necessary for normal embryonic development. After UVB exposure, embryos showed opacity of embryonic tissues and it was found the overexpression of Col4α1, Itgß, Mmp1 and Timp transcript levels (1.82-, 1.52-, 2.34- and 6.27-fold, respectively). These impairments can compromise important events for normal embryonic development, such as growth of optic lobes, caudal papilla, ramification of appendages and differentiation of organic systems. The results presented here, together with the effects on morphology, cell proliferation, differentiation, and apoptosis demonstrated previously, strengthen the knowledge of the complex impacts of UVB radiation on freshwater embryos. Nevertheless, our results encourage further investigations focusing on the assessment of UVB effects on different organisms in order to better understand the myriad of UVB effects on ECM components.

Asunto(s)

Embrión no Mamífero/efectos de la radiación , Desarrollo Embrionario/efectos de la radiación , Matriz Extracelular/efectos de la radiación , Palaemonidae/efectos de la radiación , Transcripción Genética/efectos de la radiación , Rayos Ultravioleta , Animales , Apoptosis/efectos de la radiación , Diferenciación Celular/efectos de la radiación , Proliferación Celular/efectos de la radiación , Embrión no Mamífero/metabolismo , Embrión no Mamífero/patología , Desarrollo Embrionario/genética , Matriz Extracelular/genética , Agua Dulce/química , Palaemonidae/genética , Palaemonidae/crecimiento & desarrollo

RESUMEN

Glyphosate-based herbicides (GBH) are the most used herbicides worldwide and are considered as endocrine-disrupting compounds (EDC) for non-target organisms. However, effects of GBH on their endocrine systems remain poorly understood. Thus, the aim of this study was to assess the effects of low concentrations of Roundup WG® on growth and reproduction process molecules in both males and females of the decapod crustacean Macrobrachium potiuna, by the relative transcript expression levels of the ecdysteroid receptor (EcR), the molt-inhibiting hormone (MIH), and the vitellogenin (Vg) genes. Prawns were exposed to three concentrations of GBH (0.0065, 0.065, and 0.28 mg L-1) for 7 and 14 days. The results revealed that only in males the three genes transcript levels were influenced by the GBH concentration, time of exposure, and the interaction between the concentrations and time of exposure, suggesting that males were more sensitive to GBH than females. For males, after 7 days of exposure at 0.065 mg L-1, EcR and MIH were over-expressed, while the Vg expression was only over-expressed after 14 days. The present study highlighted that GBH impacted endocrine systems of M. potiuna. Moreover, EcR and MIH gene expressions could be promising EDC biomarkers of exposure in crustaceans. These results also indicate that GBH concentrations, considered secure by regulatory agencies, should be reviewed to minimize the effects on non-target organisms. Potential effects of glyphosate-based herbicides on the endocrine system of decapods Macrobrachium sp.

Asunto(s)

Disruptores Endocrinos/toxicidad , Glicina/análogos & derivados , Herbicidas/toxicidad , Palaemonidae/fisiología , Animales , Sistema Endocrino , Femenino , Glicina/toxicidad , Hormonas de Invertebrados , Masculino , Palaemonidae/genética , Receptores de Esteroides/genética , Glifosato

RESUMEN

Palaemonetes argentinus, an abundant freshwater prawn species in the northern and central region of Argentina, has been used as a bioindicator of environmental pollutants as it displays a very high sensitivity to pollutants exposure. Despite their extraordinary ecological relevance, a lack of genomic information has hindered a more thorough understanding of the molecular mechanisms potentially involved in detoxification processes of this species. Thus, transcriptomic profiling studies represent a promising approach to overcome the limitations imposed by the lack of extensive genomic resources for P. argentinus, and may improve the understanding of its physiological and molecular response triggered by pollutants. This work represents the first comprehensive transcriptome-based characterization of the non-model species P. argentinus to generate functional genomic annotations and provides valuable resources for future genetic studies. Trinity de novo assembly consisted of 24,738 transcripts with high representation of detoxification (phase I and II), anti-oxidation, osmoregulation pathways and DNA replication and bioenergetics. This crustacean transcriptome provides valuable molecular information about detoxification and biochemical processes that could be applied as biomarkers in further ecotoxicology studies.

Asunto(s)

Fase II de la Desintoxicación Metabólica/genética , Fase I de la Desintoxicación Metabólica/genética , Palaemonidae/genética , Palaemonidae/metabolismo , Transcriptoma , Animales , Argentina , Biomarcadores/análisis

RESUMEN

The Malaysian giant prawn is among the most commonly cultured species of the genus Macrobrachium. Stocks of giant prawns from four rivers in Peninsular Malaysia have been used for aquaculture over the past 25 years, which has led to repeated harvesting, restocking, and transplantation between rivers. Consequently, a stock improvement program is now important to avoid the depletion of wild stocks and the loss of genetic diversity. However, the success of such an improvement program depends on our knowledge of the genetic variation of these base populations. The aim of the current study was to estimate genetic variation and differentiation of these riverine sources using novel expressed sequence tag-microsatellite (EST-SSR) markers, which not only are informative on genetic diversity but also provide information on immune and metabolic traits. Our findings indicated that the tested stocks have inbreeding depression due to a significant deficiency in heterozygotes, and FIS was estimated as 0.15538 to 0.31938. An F-statistics analysis suggested that the stocks are composed of one large panmictic population. Among the four locations, stocks from Johor, in the southern region of the peninsular, showed higher allelic and genetic diversity than the other stocks. To overcome inbreeding problems, the Johor population could be used as a base population in a stock improvement program by crossing to the other populations. The study demonstrated that EST-SSR markers can be incorporated in future marker assisted breeding to aid the proper management of the stocks by breeders and stakeholders in Malaysia.

Asunto(s)

Etiquetas de Secuencia Expresada , Repeticiones de Microsatélite , Palaemonidae/genética , Polimorfismo Genético , Animales , Malasia , Selección Artificial

RESUMEN

RT-qPCR is a sensitive and highly efficient technique that is widely used in gene expression analysis and to provide insight into the molecular mechanisms underlying embryonic development. The freshwater prawn, Macrobrachium olfersii is an emerging model organism, but, the stable reference genes of this species need to be identified and validated for RT-qPCR analysis. Thus, the aim of this study was to evaluate the expression stability of six genes (ß-act, GAPDH, EF-1α, RpL8, RpS6, AK) in embryos and in adult tissues (cerebral ganglia, muscle and hepatopancreas) of M. olfersii. The expression stabilities of these genes were evaluated using geNorm, NormFinder, BestKeeper, ΔCt method and integrated tool RefFinder. In the general ranking, RpL8 and RpS6 were the most stable genes in embryos, while RpS6 and RpL8 were the most stable in a combined adult tissue analysis. Analysis of the adult tissues revealed that ß-act and AK were the most stable genes in cerebral ganglia, RpL8 and AK in muscle, and RpS6 and ß-act in hepatopancreas. EF-1α and GAPDH were the least stable genes and as normalizer genes in RT-qPCR affected expression of the Distal-less gene during M. olfersii development. This study provides suitable reference genes for RT-qPCR analysis and allows future studies of the gene expression in M. olfersii for understanding the molecular mechanisms of their development. To our knowledge, this is the first published study that identifies and evaluates reference genes for RT-qPCR analysis in M. olfersii and could be useful as basis for evaluations of reference genes in other prawns.

Asunto(s)

Palaemonidae/embriología , Palaemonidae/genética , Animales , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Modelos Genéticos , Palaemonidae/crecimiento & desarrollo , Reacción en Cadena en Tiempo Real de la Polimerasa , Distribución Tisular/genética

RESUMEN

Imidazole derivative KK-42 is a well-known regulator of insect growth. KK-42 pretreatment has been shown to promote the survival of Macrobrachium nipponense infected with Aeromonas hydrophila, possibly via activation of superoxide dismutase (SOD). In this study, the cytMnSOD gene was cloned from the hepatopancreas of M. nipponense using the rapid amplification of cDNA ends technique. The full-length cDNA of cytMnSOD was 1233 bp long, and the open reading frame was 858 bp long, encoding a 286-aa protein with a 60-aa leader sequence. The calculated molecular mass of the translated cytMnSOD protein was 31.33 kDa, with an estimated isoelectric point of 5.62. cytMnSOD contained two N-glycosylation sites, four conserved amino acids responsible for binding manganese, and a manganese SOD domain (DVWEHAYY). Real-time RT-PCR analysis showed that cytMnSOD was expressed in all tissues examined with the highest expression observed in the hepatopancreas. Levels of the cytMnSOD transcript in the hepatopancreas were highest in stage C of the molting cycle. Real-time PCR analysis revealed that cytMnSOD expression increased significantly 3, 6, and 12 h after KK-42 treatment, with simultaneous increases in SOD activity from 6 to 12 h. Our results demonstrate that cytMnSOD expression and SOD activity may be induced by KK-42, which may represent one of the molecular mechanisms through which KK-42 promotes increased survival of prawns infected with A. hydrophila.

Asunto(s)

Hepatopáncreas/efectos de los fármacos , Imidazoles/farmacología , Hormonas Juveniles/farmacología , Palaemonidae/efectos de los fármacos , ARN Mensajero/genética , Superóxido Dismutasa/genética , Aeromonas hydrophila/crecimiento & desarrollo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Citosol/efectos de los fármacos , Citosol/enzimología , Citosol/inmunología , Citosol/microbiología , Regulación del Desarrollo de la Expresión Génica , Hepatopáncreas/enzimología , Hepatopáncreas/inmunología , Hepatopáncreas/microbiología , Interacciones Huésped-Patógeno , Peso Molecular , Sistemas de Lectura Abierta , Palaemonidae/genética , Palaemonidae/inmunología , Palaemonidae/microbiología , Dominios Proteicos , Señales de Clasificación de Proteína , ARN Mensajero/inmunología , Superóxido Dismutasa/inmunología , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

Nitric oxide synthase (NOS) produces nitric oxide (NO) by catalyzing the conversion of l-arginine to l-citrulline, with the concomitant oxidation of nicotinamide adenine dinucleotide phosphate. Recently, various studies have verified the importance of NOS invertebrates and invertebrates. However, the NOS gene family in the oriental river prawn Macrobrachium nipponense is poorly understood. In this study, we cloned the full-length NOS complementary DNA from M. nipponense (MnNOS) and characterized its expression pattern in different tissues and at different developmental stages. Real-time quantitative polymerase chain reaction (RT-qPCR) showed the MnNOS gene to be expressed in all investigated tissues, with the highest levels observed in the androgenic gland (P < 0.05). Our results revealed that the MnNOS gene may play a key role in M. nipponense male sexual differentiation. Moreover, RT-qPCR revealed that MnNOS mRNA expression was significantly increased in post-larvae 10 days after metamorphosis (P < 0.05). The expression of this gene in various tissues indicates that it may perform versatile biological functions in M. nipponense.

Asunto(s)

Proteínas de Artrópodos/genética , Regulación del Desarrollo de la Expresión Génica , Óxido Nítrico Sintasa/genética , Palaemonidae/genética , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos/metabolismo , Secuencia de Bases , China , Clonación Molecular , Conservación de los Recursos Naturales , ADN Complementario/genética , ADN Complementario/metabolismo , Embrión no Mamífero , Femenino , Larva/genética , Larva/crecimiento & desarrollo , Masculino , Óxido Nítrico Sintasa/metabolismo , Técnicas de Amplificación de Ácido Nucleico , Especificidad de Órganos , Palaemonidae/clasificación , Palaemonidae/crecimiento & desarrollo , Filogenia , Ríos , Alineación de Secuencia , Homología de Secuencia de Aminoácido

RESUMEN

Feminization-1 homolog b (Fem1b) is one of the genes essential for male development and play central roles in sex determination of Caenorhabditis elegans. In this study, we cloned and characterized the full-length Fem1b cDNA from the freshwater prawn Macrobrachium nipponense (MnFem1b) in different tissues and at different developmental stages. Real-time quantitative reverse polymerase chain reaction (RT-qPCR) showed that the MnFem1b gene was expressed in all investigated tissues, with the highest expression level found in the testes. The results revealed that the MnFem1b gene might play roles in aspects of development of the male prawn phenotype. The RT-qPCR also revealed that MnFem1b mRNA expression was significantly increased at 10 days after metamorphosis. The expression levels in all investigated tissues showed a certain degree of sexually dimorphism, the expression levels in males were significantly higher than those in females (P < 0.05). Notably, the highest expression of MnFem1b was found in the testes. The expression of MnFem1b in different tissues indicates that it plays multiple biological functions in M. nipponense.

Asunto(s)

Síndrome de Resistencia Androgénica/genética , Proteínas de Artrópodos/genética , Metamorfosis Biológica/genética , Palaemonidae/genética , Procesos de Determinación del Sexo , Secuencia de Aminoácidos/genética , Animales , Caenorhabditis elegans/genética , Clonación Molecular , ADN Complementario/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Masculino , Ovario/crecimiento & desarrollo , Ovario/metabolismo , Palaemonidae/crecimiento & desarrollo , Filogenia , ARN Mensajero/biosíntesis , ARN Mensajero/genética

RESUMEN

Actin is a highly conserved protein that is found in all eukaryotic cells, and has been widely used as an internal control gene in gene expression studies. In this study, we cloned an actin gene (named Ecß-actin) from Exopalaemon carinicauda and determined its expression levels. The full-length cDNA of Ecß-actin was 1335 bp long, comprising a 1131-bp ORF encoding 376 amino acids, a 65-bp 5'-UTR, and a 139-bp 3'-UTR with a poly(A) tail. The A + T content was approximately 79% in the 3'-UTR of the Ecß-actin mRNA. The 3'-UTR contained two repeats of the AUUUA motif. The putative protein Ecß-actin showed high identity (97-99%) with other actins from various species. Phylogenetic analysis revealed that Ecß-actin belongs to Crustacea, although it formed a singleton sub-branch that was located a short distance from crabs and other shrimp species. Ecß- actin expression was detected in the hepatopancreas, ovary, muscle, gill, stomach, and hemocytes, and was strongly expressed in the hemocytes and ovary of E. carinicauda. Ecß-actin mRNA expression varied during ovarian development, with high levels observed at stages I and V. Therefore, caution should be taken when using the Ecß-actin gene as an endogenous control gene.

Asunto(s)

Actinas/biosíntesis , Palaemonidae/genética , Filogenia , Actinas/genética , Animales , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Hepatopáncreas/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética