RESUMEN
Evidence of immune memory in invertebrates (immune priming) has accumulated in various organisms, and both cellular and humoral immune reactions are speculated to be involved in immune priming. However, there is a lack of understanding of the molecular mechanisms involved. In the present study, the protective effect of primed haemolymph was further validated by the increased survival rate of naïve crabs receiving a transfusion of primed haemolymph. By proteomic analysis, there were 474 proteins identified from the primed haemolymph, and most of them were functionally annotated in transport and metabolism classes. A total of 70 proteins were found to be differentially expressed in haemolymph at 12 hours and 7 days after priming stimulation with Aeromonas hydrophila, among which anti-lipopolysaccharide factor 1 (EsALF-1) and 3 (EsALF-3) were identified as the most significant (p < 0.05). After being challenged with A. hydrophila, EsALF-1 and EsALF-3 were highly expressed at both mRNA (in haemocytes) and protein (in haemolymph) levels compared with blank crabs, and the mRNA expressions of components in the EsTLR1-EsMyd88-EsPelle-EsALF pathway also increased significantly (p < 0.05). The EsALF-3 and EsMyd88 were even significantly higher expressed in response to the second A. hydrophila challenge, but their expressions all decreased (p < 0.05) when EsTLR1 was knocked down by RNAi. After the naïve crabs received an injection with the recombinant protein of EsALF-1 (rEsALF-1) or EsALF-3 (rEsALF-3), their survival rate increased significantly (p < 0.05) upon A. hydrophila stimulation. In contrast, the survival rate of the primed crabs reduced significantly (p < 0.05) after they received an injection with the antibody of EsALF-1 or EsALF-3. The enhanced expressions of EsALF-1 and EsALF-3 after A. hydrophilap riming stimulation could sustain for four weeks. All the results suggested that the EsTLR1-mediated productions of EsALF-1 and EsALF-3 in haemolymph played an indispensable role in the month-long humoral immune protection induced by A. hydrophila, which provides solid evidence of immune priming in crabs and a valuable reference for further understanding immune memory in invertebrates.
Asunto(s)
Aeromonas hydrophila/inmunología , Péptidos Antimicrobianos/biosíntesis , Proteínas de Artrópodos/biosíntesis , Braquiuros/inmunología , Lipopolisacáridos/toxicidad , Anciano , Animales , Especificidad de Anticuerpos , Péptidos Antimicrobianos/genética , Péptidos Antimicrobianos/inmunología , Acuicultura , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/inmunología , Braquiuros/genética , Braquiuros/microbiología , Clonación Molecular , Femenino , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Hemocitos/metabolismo , Hemolinfa/inmunología , Humanos , Inmunidad Humoral , Ratones , Proteómica , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Receptores Toll-Like/fisiologíaRESUMEN
Spider silk, as one of the hardest natural and biocompatible substances with extraordinary strength and flexibility, have become an ideal option in various areas of science and have made their path onto the biomedical industry. Despite its growing popularity, the difficulties in the extraction of silks from spiders and farming them have made it unaffordable and almost impossible for industrial scale. Biotechnology helped production of spider silks recombinantly in different hosts and obtaining diverse morphologies out of them based on different processing and assembly procedures. Herein, the characteristics of these morphologies and their advantages and disadvantages are summarized. A detailed view about applications of recombinant silks in skin regeneration and cartilage, tendon, bone, teeth, cardiovascular, and neural tissues engineering are brought out, where there is a need for strong scaffolds to support cell growth. Likewise, spider silk proteins have applications as conduit constructs, medical sutures, and 3D printer bioinks. Other characteristics of spider silks, such as low immunogenicity, hydrophobicity, homogeneity, and adjustability, have attracted much attention in drug and gene delivery. Finally, the challenges and obstacles ahead for industrializing the production of spider silk proteins in sufficient quantities in biomedicine, along with solutions to overcome these barriers, are discussed.
Asunto(s)
Proteínas de Artrópodos/química , Bioingeniería , Biotecnología , Seda/química , Arañas/química , Animales , Proteínas de Artrópodos/biosíntesis , Proteínas de Artrópodos/genética , Bioingeniería/métodos , Biotecnología/métodos , Fenómenos Químicos , Sistemas de Liberación de Medicamentos , Fenómenos Mecánicos , Impresión Tridimensional , Proteínas Recombinantes , Medicina Regenerativa , Ingeniería de TejidosRESUMEN
Spider silk, which has remarkable mechanical properties, is a natural protein fiber produced by spiders. Spiders cannot be farmed because of their cannibalistic and territorial nature. Hence, large amounts of spider silk cannot be produced from spiders. Genetic engineering is an alternative approach to produce large quantities of spider silk. Our group has produced synthetic spider silk proteins in E. coli to study structure/function and to produce biomaterials comparable to the silks produced by orb-weaving spiders. Here we give a detailed description of our cloning, expression, and purification methods of synthetic spider silk proteins ranging from ~30 to ~200 kDa. We have cloned the relevant genes of the spider Nephila clavipes and introduced them into bacteria to produce synthetic spider silk proteins using small and large-scale bioreactors. We have optimized the fermentation process, and we have developed protein purification methods as well. The purified proteins are spun into fibers and are used to make alternative materials like films and adhesives with various possible commercial applications.
Asunto(s)
Proteínas de Artrópodos , Escherichia coli , Expresión Génica , Seda , Arañas/genética , Animales , Proteínas de Artrópodos/biosíntesis , Proteínas de Artrópodos/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Seda/biosíntesis , Seda/genéticaRESUMEN
Zooplankton organisms face a variable food supply in their habitat. Metabolic adjustments during periods of starvation were analysed from changes in metabolite level to gene expression in the microcrustacean Daphnia pulex during starvation. The animals exploited their carbohydrate stores first, but their lipid and protein reserves were also degraded, albeit more slowly. Glycogenolysis and probably gluconeogenesis led to hyperglycaemia after 16 h of starvation. The concentration of α-ketoglutarate and the rate of oxygen consumption also reached maxima during this period. Nuclear HIF-1α levels and α-ketoglutarate concentration showed inverse correlation. Effects of this 2-oxoacid on prolyl hydroxylase activity, HIF-1α stability and the role of this transcription factor in the changes of the expression level of several putatively HIF-1-mediated metabolic genes are discussed. Transcriptome profiling via RNA-Seq revealed a downregulation of genes for protein biosynthesis and an upregulation of genes for carbohydrate metabolism during starvation. Thus, the adjustments of energy metabolism in response to food deprivation were quantified from the level of metabolites, signal transduction and gene expression, and possible connections of the respective dynamics of observed changes were analysed.
Asunto(s)
Proteínas de Artrópodos/biosíntesis , Daphnia/metabolismo , Metabolismo Energético , Regulación de la Expresión Génica , Gluconeogénesis , Glucogenólisis , Animales , Inanición/metabolismoRESUMEN
In Japan, there are two species of scorpions, Madara scorpion (Isometrus maculatus) and Yaeyama scorpion (Liocheles australasiae), and both of them are living in Yaeyama island. It has been shown that Liocheles australasiae has venom including ß-toxin acting on K+-channels (ß-KTx) (Juichi et al., 2018) [1]. Interestingly, LaIT2, one of the toxins found in the venom of Liocheles australasiae, displays the virulence for insects but almost not for mammals. Until now, molecular mechanism of the functional specificity of LaIT2 is unknown. To clear this issue, we tried to establish the overexpression system of LaIT2 in Rosetta-gami B (DE3) pLysS, which have trxB/gor mutations to induce the disulfide bond formation. In this study, we have succeeded to overexpress the recombinant LaIT2 (rLaIT2) as a thioredoxin (Trx)-tagged protein, and established the purification protocol with Ni2+-NTA column chromatography, enterokinase digestion, and HPLC. We succeeded to obtain approximately 0.5 mg of rLaIT2 from the E. coli cells cultured in 1 L of M9 culture medium. Intramolecular disulfide bonding pattern of rLaIT2 was identified by endopeptidase fragmentation and mass spectrometry. rLaIT2 showed insecticidal activity and antimicrobial activity, and these are almost identical to those of natural LaIT2. 1H-15N HSQC spectrum of 15N-labeled rLaIT2 indicated that the rLaIT2 has a stable conformation.
Asunto(s)
Proteínas de Artrópodos , Biosíntesis de Péptidos , Péptidos , Venenos de Escorpión , Escorpiones , Animales , Proteínas de Artrópodos/biosíntesis , Proteínas de Artrópodos/química , Proteínas de Artrópodos/aislamiento & purificación , Escherichia coli/genética , Escherichia coli/metabolismo , Péptidos/química , Péptidos/genética , Péptidos/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Venenos de Escorpión/química , Venenos de Escorpión/genética , Escorpiones/química , Escorpiones/genéticaRESUMEN
Venoms have evolved over a hundred times in animals. Venom toxins are thought to evolve mostly by recruitment of endogenous proteins with physiological functions. Here we report phylogenetic analyses of venom proteome-annotated venom gland transcriptome data, assisted by genomic analyses, to show that centipede venoms have recruited at least five gene families from bacterial and fungal donors, involving at least eight horizontal gene transfer events. These results establish centipedes as currently the only known animals with venoms used in predation and defence that contain multiple gene families derived from horizontal gene transfer. The results also provide the first evidence for the implication of horizontal gene transfer in the evolutionary origin of venom in an animal lineage. Three of the bacterial gene families encode virulence factors, suggesting that horizontal gene transfer can provide a fast track channel for the evolution of novelty by the exaptation of bacterial weapons into animal venoms.
Asunto(s)
Proteínas de Artrópodos/genética , Venenos de Artrópodos/genética , Quilópodos/genética , Transferencia de Gen Horizontal , Genes Bacterianos , Genes Fúngicos , Animales , Proteínas de Artrópodos/biosíntesis , Proteínas de Artrópodos/clasificación , Venenos de Artrópodos/biosíntesis , Venenos de Artrópodos/clasificación , Quilópodos/clasificación , Quilópodos/microbiología , Quilópodos/patogenicidad , Expresión Génica , Filogenia , Proteómica/métodos , TranscriptomaRESUMEN
In crustaceans, the regulation of sex differentiation is mediated by insulin-like androgenic hormone (IAG) and crustacean female sex hormone (CFSH). CFSH is reported to inhibit IAG gene (Sp-IAG) expression in the mud crab Scylla paramamosain, but the regulatory mechanism is not well understood. A 2674 bp 5' flanking Sp-IAG contains many potential transcription factor binding sites. In this study, analysis of serially deleted 5' flanking Sp-IAG and site-directed mutation (SDM) of transcription factor binding sites of the same gene showed that the promoter activity of reporter vectors with Sox-5-binding site, signal transducers and activators of transcription (STAT)-binding site and activator protein 1 (AP-1)-binding site were significantly higher than that of vectors without these regions, suggesting that they were involved in transcriptional regulation of Sp-IAG expression. The expression analysis of these transcription factor showed that there was no difference in the level of mRNA in Sox-5 and AP-1 in androgenic gland treated with recombinant CFSH, but expression of Sp-STAT was significantly reduced, suggesting that CFSH regulates the expression of Sp-STAT, inhibiting its function to regulate Sp-IAG. Further experiment revealed that RNAi mediated Sp-STAT gene knockdown reduced the expression of Sp-IAG. These results suggested that Sp-CFSH regulates Sp-IAG by inhibiting STAT. This is a pioneering finding on the transcriptional mechanism of IAG gene in crustaceans.
Asunto(s)
Proteínas de Artrópodos/biosíntesis , Braquiuros/metabolismo , Regulación de la Expresión Génica/fisiología , Hormonas de Invertebrados/metabolismo , Diferenciación Sexual/fisiología , Transcripción Genética/fisiología , Animales , Proteínas de Artrópodos/genética , Braquiuros/genética , Femenino , Hormonas de Invertebrados/genéticaRESUMEN
The Northern spot shrimp, Pandalus platyceros, a protandric hermaphrodite of commercial importance in North America, is the primary target species for shrimp fisheries within Southeast Alaska. Fishery data obtained from the Alaska Department of Fish and Game indicate that spot shrimp populations have been declining significantly over the past 25 years. We collected spot shrimps in Southeast Alaska and measured reproductive-related morphological, gonadal and molecular changes during the entire life history. The appendix masculina, a major sexual morphological indicator, is indicative of the reproductive phase of the animal, lengthening during maturation from juvenile to the male phase and then gradually shortening throughout the transitional stages until its complete disappearance upon transformation to a female. This morphological change occurs in parallel with the degeneration of testicular tissue in the ovotestis and enhanced ovarian vitellogenesis. Moreover, we obtained the entire mRNA sequence of the yolk protein precursor, vitellogenin, and monitored its transcript levels throughout the entire shrimp life-cycle. Vitellogenin transcript levels in the hepatopancreas increased in the early transitional stage until reaching a peak prior to extruding eggs. Such transcriptomic analyses, coupled with a comprehensive description of the gonad, external sex characters and timing of the reproductive life history of spot shrimps contribute to a better understanding of the hermaphroditic reproduction process in the cold Southeast Alaskan waters. This knowledge can contribute to a revision of current conservation efforts to maintain wild populations sustainable for both commercial and ecological considerations.
Asunto(s)
Proteínas de Artrópodos , Explotaciones Pesqueras , Pandalidae , ARN Mensajero , Análisis de Secuencia de ARN , Transcriptoma , Alaska , Animales , Proteínas de Artrópodos/biosíntesis , Proteínas de Artrópodos/genética , Conservación de los Recursos Energéticos , Pandalidae/genética , Pandalidae/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genéticaRESUMEN
Crustins are cysteine-rich antimicrobial peptides (AMPs) widely distributed across crustaceans. From the four described crustin Types (I to IV), crustins from the subtype IIa are the most abundant and diverse members found in penaeid shrimp. Despite the critical role of Type IIa crustins in shrimp antimicrobial defenses, there is still limited information about their synthesis and antimicrobial properties. Here, we report the subcellular localization and the antibacterial spectrum of crusFpau, a Type IIa crustin from the pink shrimp Farfantepenaeus paulensis. The recombinantly expressed crusFpau showed antimicrobial activity against both Gram-positive and Gram-negative bacteria at low concentrations. Results from immunofluorescence using anti-rcrusFpau antiserum revealed that crusFpau is synthetized and stored by both granular and semigranular hemocytes, but not by hyaline cells. Interestingly, not all granular and semigranular hemocytes stained for crusFpau, revealing that this crustin is produced by specific granule-containing hemocyte subpopulations. Finally, we showed that the granule-stored peptides are not constitutively secreted into the plasma of healthy animals.
Asunto(s)
Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/biosíntesis , Proteínas de Artrópodos/biosíntesis , Hemocitos/metabolismo , Penaeidae/inmunología , Animales , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Penaeidae/metabolismo , Penaeidae/microbiologíaRESUMEN
Rhythmic changes in reactive oxygen species (ROS) levels with time have been previously reported, but circadian rhythmic changes in ROS levels in Daphnia pulex remain unexplored. Here, in light-dark (L/D 16:8, L/D 8:16), continuous light (LL), and continuous dark (DD) conditions were set up to analyze the accumulation of ROS and activity of antioxidant enzymes in D. pulex at different time points. We observed that ROS levels significantly varied from day to night. By fluorescence probe labeling, we noted that ROS majorly accumulated in the thoracic limbs, brood chamber, and rectum in D. pulex. In addition, in the L/D 16:8 and 8:16 groups, ROS level was negatively correlated to the gene expression level of catalase, glutathione S-transferase, and glutathione peroxidase. In the LL and DD groups, food supply emerged as a zeitgeber, driving D. pulex to maintain a specific biological rhythm. The ability to scavenge hydroxyl radicals and activity of antioxidant enzymes were also affected by L/D cycles. To summarize, we indicate that different L/D cycles can induce different oxidation-antioxidant system responses in D. pulex and that Daphnia can adapt to the living environment by circadian rhythm regulation of the oxidation-antioxidant balance.
Asunto(s)
Proteínas de Artrópodos , Ritmo Circadiano/fisiología , Daphnia , Especies Reactivas de Oxígeno/metabolismo , Animales , Proteínas de Artrópodos/biosíntesis , Proteínas de Artrópodos/genética , Daphnia/genética , Daphnia/metabolismo , Oxidación-ReducciónRESUMEN
Cladocera are small freshwater crustaceans that have attracted considerable attention in recent years. They are commonly used for studying senescence. In this study, we used LC-MS/MS with eight-plex iTRAQ to perform a comparative proteomic analysis of senescence in Daphnia pulex. Of 3076 primordial proteins, 2325 were credible (the remaining were low-confidence proteins) and 247 significantly differentially expressed proteins (DEPs). Of the latter, 87, 91, and 69 DEPs were identified in the Day 15 vs. Day 5, Day 20 vs. Day 5, and Day 25 vs. Day 5 groups, respectively. Gene ontology enrichment analysis showed that oxidative damage may be the main cause of senescence in D. pulex. Using Kyoto Encyclopedia of Genes and Genomes pathway analysis, we found that the peroxisome pathway played an important role in aging. Our results suggest that D. pulex alleviates excessive oxidative damage by altering key enzymes involved in carbohydrate and protein metabolism.
Asunto(s)
Envejecimiento/metabolismo , Proteínas de Artrópodos/biosíntesis , Daphnia/metabolismo , Proteómica , AnimalesRESUMEN
Acute hepatopancreatic necrosis disease (AHPND), caused by marine bacteria Vibrio Parahaemolyticus, is a huge problem in shrimp farms. The V. parahaemolyticus infecting material is contained in a plasmid which encodes for the lethal toxins PirABVp, whose primary target tissue is the hepatopancreas, causing sloughing of epithelial cells, necrosis, and massive hemocyte infiltration. To get a better understanding of the hepatopancreas response during AHPND, juvenile shrimp Litopenaeus vannamei were infected by immersion with V. parahaemolyticus. We performed transcriptomic mRNA sequencing of infected shrimp hepatopancreas, at 24 hours post-infection, to identify novel differentially expressed genes a total of 174,098 transcripts were examined of which 915 transcripts were found differentially expressed after comparative transcriptomic analysis: 442 up-regulated and 473 down-regulated transcripts. Gene Ontology term enrichment analysis for up-regulated transcripts includes metabolic process, regulation of programmed cell death, carbohydrate metabolic process, and biological adhesion, whereas for down-regulated transcripts include, microtubule-based process, cell activation, and chitin metabolic process. The analysis of protein- protein network between up and down-regulated genes indicates that the first gene interactions are connected to oxidation-processes and sarcomere organization. Additionally, protein-protein networks analysis identified 20-top highly connected hub nodes. Based on their immunological or metabolic function, ten candidate transcripts were selected to measure their mRNA relative expression levels in AHPND infected shrimp hepatopancreas by RT-qPCR. Our results indicate a close connection between the immune and metabolism systems during AHPND infection. Our RNA-Seq and RT-qPCR data provide the possible immunological and physiological scenario as well as the molecular pathways that take place in the shrimp hepatopancreas in response to an infectious disease.
Asunto(s)
Proteínas de Artrópodos/biosíntesis , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Hepatopáncreas , Penaeidae , Vibriosis , Vibrio parahaemolyticus , Animales , Hepatopáncreas/metabolismo , Hepatopáncreas/microbiología , Necrosis , Penaeidae/metabolismo , Penaeidae/microbiología , Vibriosis/metabolismo , Vibriosis/microbiologíaRESUMEN
Agonistic behaviour is common in an encounter between two crustaceans. It often causes limb disability and consumes a lot of energy, which is harmful for the growth and survival of commercially important crustaceans. In the present study, we mainly focused on the agonistic behaviour of the Chinese mitten crab, Eriocheir sinensis, which is an important species of the aquaculture industry in China. We recorded agnostic behaviour with a high-definition camera and preliminarily evaluated the role of serotonin (5-HT) or dopamine (DA)-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway and eyestalk in the behaviour. The results showed that agonistic behaviour in E. sinensis consisted of three stages: approach, contact and fight. We found that the number of fights and cumulative time of fight were significantly higher in the male vs. male group than in the female vs. female and female vs. male groups (Pâ¯<â¯0.05). After 1â¯h of agonistic behaviour, 5-HT concentration showed a significant increase and DA concentration showed a significant decrease when compared with the control group (no encounter; Pâ¯<â¯0.05). 5-HT1B and 5-HT2B mRNA levels showed a significant increase in the eyestalk (Pâ¯<â¯0.05). 5-HT7 mRNA levels showed significant downregulation in the thoracic ganglia and DA1A mRNA levels showed upregulation in the intestine (Pâ¯<â¯0.05). DA2 mRNA levels showed a significant decrease in the eyestalk (Pâ¯<â¯0.05). These changes were accompanied by a significant increase in cAMP level and significant decrease in PKA level in the haemolymph (Pâ¯<â¯0.05). In addition, a significant decrease in glucose levels was detected after the agonistic behaviour. Crustacean hyperglycemic hormone (CHH) mRNA levels showed significant upregulation in the eyestalk and significant downregulation in the intestine (Pâ¯<â¯0.05). The number of fights and cumulative time of fight in the left eyestalk ablation (L-X vs. L-X) group were more and longer than those in the intact eyestalk (C vs. C), right eyestalk ablation (R-X vs. R-X) and bilateral eyestalk ablation (D-X vs. D-X) groups. In short, E. sinensis shows special agonistic behaviour modulated by 5-HT or DA-cAMP-PKA pathway and eyestalk, especially the left eyestalk.
Asunto(s)
Conducta Agonística/fisiología , Braquiuros/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , AMP Cíclico/fisiología , Dopamina/fisiología , Serotonina/fisiología , Transducción de Señal/fisiología , Animales , Proteínas de Artrópodos/biosíntesis , Proteínas de Artrópodos/metabolismo , Femenino , Glucosa/metabolismo , Hemolinfa/metabolismo , Hormonas de Invertebrados/biosíntesis , Hormonas de Invertebrados/metabolismo , Masculino , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/metabolismo , Receptores Dopaminérgicos/biosíntesis , Receptores Dopaminérgicos/metabolismo , Receptores de SerotoninaRESUMEN
Scorpions, a characteristic group of arthropods, are among the earliest diverging arachnids, dating back almost 440 million years. One of the many interesting aspects of scorpions is that they have venom arsenals for capturing prey and defending against predators, which may play a critical role in their evolutionary success. Unfortunately, however, scorpion envenomation represents a serious health problem in several countries, including Iran. Iran is acknowledged as an area with a high richness of scorpion species and families. The diversity of the scorpion fauna in Iran is the subject of this review, in which we report a total of 78 species and subspecies in 19 genera and four families. We also list some of the toxins or genes studied from five species, including Androctonus crassicauda, Hottentotta zagrosensis, Mesobuthus phillipsi, Odontobuthus doriae, and Hemiscorpius lepturus, in the Buthidae and Hemiscorpiidae families. Lastly, we review the diverse functions of typical toxins from the Iranian scorpion species, including their medical applications.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Antineoplásicos/química , Proteínas de Artrópodos/química , Venenos de Escorpión/química , Escorpiones/química , Animales , Péptidos Catiónicos Antimicrobianos/biosíntesis , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/uso terapéutico , Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Proteínas de Artrópodos/biosíntesis , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/uso terapéutico , Descubrimiento de Drogas/métodos , Expresión Génica , Humanos , Canales Iónicos/agonistas , Canales Iónicos/antagonistas & inhibidores , Canales Iónicos/metabolismo , Irán , Metaloproteasas/biosíntesis , Metaloproteasas/aislamiento & purificación , Metaloproteasas/toxicidad , Fosfolipasas A2/biosíntesis , Fosfolipasas A2/aislamiento & purificación , Fosfolipasas A2/toxicidad , Filogenia , Picaduras de Escorpión/fisiopatología , Venenos de Escorpión/biosíntesis , Venenos de Escorpión/aislamiento & purificación , Escorpiones/clasificación , Escorpiones/patogenicidad , Escorpiones/fisiología , Inhibidores de Serina Proteinasa/biosíntesis , Inhibidores de Serina Proteinasa/aislamiento & purificación , Inhibidores de Serina Proteinasa/toxicidad , Especificidad de la EspecieRESUMEN
To cope with harsh environments, the Artemia shrimp produces gastrula embryos in diapause, a state of obligate dormancy, having cellular quiescence and suppressed metabolism. The mechanism behind these cellular events remains largely unknown. Here, we study the regulation of cell quiescence using diapause embryos of Artemia We found that Artemia DEK (Ar-DEK), a nuclear factor protein, was down-regulated in the quiescent cells of diapause embryos and enriched in the activated cells of post-diapause embryos. Knockdown of Ar-DEK induced the production of diapause embryos whereas the control Artemia released free-swimming nuaplii. Our results indicate that Ar-DEK correlated with the termination of cellular quiescence via the increase in euchromatin and decrease in heterochromatin. The phenomena of quiescence have many implications beyond shrimp ecology. In cancer cells, for example, knockdown of DEK also induced a short period of cellular quiescence and increased resistance to environmental stress in MCF-7 and MKN45 cancer cell lines. Analysis of RNA sequences in Artemia and in MCF-7 revealed that the Wnt and AURKA signaling pathways were all down-regulated and the p53 signaling pathway was up-regulated upon inhibition of DEK expression. Our results provide insight into the functions of Ar-DEK in the activation of cellular quiescence during diapause formation in Artemia.
Asunto(s)
Artemia/embriología , Proteínas de Artrópodos/biosíntesis , Diapausa/fisiología , Embrión no Mamífero/enzimología , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Receptores de la Familia Eph/biosíntesis , Vía de Señalización Wnt/fisiología , Animales , Artemia/genética , Proteínas de Artrópodos/genética , Aurora Quinasa A/genética , Aurora Quinasa A/metabolismo , Humanos , Células MCF-7 , Receptores de la Familia Eph/genéticaRESUMEN
Cherax quadricarinatus, as one of the world's most valuable freshwater shrimp species, has received extensive attention in recent years. As males grow larger and faster than females, development of the sex control breeding techniques is of great interest, but knowledge on sex determination and differentiation in C. quadricarinatus remains poorly unknown. Sxl (Sex-lethal) is an important gene in the sexual differentiation regulatory hierarchy. It reflects the ratio of sex chromosomes to autosomes into molecule changes and directs sex-specific splicing forms of precursor mRNA. In the present study, the full-length cDNA sequences of four Sxl splice variants were identified from C. quadricarinatus, designated as CqSxl1, CqSxl2, CqSxl3 and CqSxl4, respectively. Sequence analysis determined different splicing sites near the translation termination region of four Sxl transcript isoforms. Two highly conserved classical RRM domains were found according to predicted secondary structures of Sxl proteins. mRNA expression of CqSxl in different tissues, developmental stage of embryos and testes were investigated by real-time quantitative PCR. Among four isoforms, CqSxl3 showed tissue specificity with higher expression levels in testis than in ovary. CqSxl1 and CqSxl4 were found widely expressed in various tissues and CqSxl2 was almost undetectable. In early developmental stages, the expression levels of CqSxl1/3/4 gradually increased along with embyonic development. In addition, CqSxl genes presented the higher transcript levels in the early stage of testis development. Furthermore, CqSxl3 silencing induced a significant decrease of the transcript of Cq-IAG, an androgenic hormone-encoding gene responsible for masculine development. These data indicate that CqSxl3 might be involved in male sex determination in C. quadricarinatus. Our study will contribute to understanding the mechanism of sex determination in C. quadricarinatus, and also can provide theoretical guidance for establishing a sex control technology.
Asunto(s)
Proteínas de Artrópodos , Decápodos , Regulación del Desarrollo de la Expresión Génica/fisiología , Genes Letales , Empalme del ARN/fisiología , ARN Mensajero , Diferenciación Sexual/fisiología , Animales , Proteínas de Artrópodos/biosíntesis , Proteínas de Artrópodos/genética , Decápodos/embriología , Decápodos/genética , Femenino , Perfilación de la Expresión Génica , Masculino , ARN Mensajero/biosíntesis , ARN Mensajero/genéticaRESUMEN
Methyl farnesoate (MF), a sesquiterpenoid synthesized in the mandibular organ, regulates many physiological processes in crustaceans including growth and reproduction. In the present study, farnesoic acid O-methyltransferase (FAMeT), the key enzyme responsible for final step conversion of farnesoic acid (FA) to methyl farnesoate (MF), was cloned and characterized from the nervous tissues of Penaeus indicus. Multiple sequence alignment, prediction of conserved domain regions, phosphorylation sites identification and phylogenetic analysis indicated that putative FAMeT fragment from P. indicus (PiFAMeT), shares a high degree of sequence identity to FAMeT proteins isolated from other crustaceans species. Quantitative real-time PCR analysis revealed ubiquitous expression of PiFAMeT in all the tissues examined, with comparative higher mRNA levels in nervous tissue and ovary. Additionally, the levels of PiFAMeT also showed gradual increase of expression correlating with the advancement in ovarian maturation. Further to support their role in promoting ovarian development, serotonin treatment (5HT, 50⯵g/g body weight) was given to eyestalk intact and unilaterally eyestalk ablated females which resulted in significant increase in PiFAMeT transcript levels at day 7 and day 14. The relatively higher levels of PiFAMeT, reflecting higher levels of MF, suggest a role during secondary vitellogenesis thereby regulating ovarian development in P. indicus. Further research is required to understand the synergistic interaction of MF pathways with serotonergic and other regulatory pathways in regulating ovarian maturation in penaeid shrimps.
Asunto(s)
Proteínas de Artrópodos , Regulación Enzimológica de la Expresión Génica/fisiología , Metiltransferasas , Ovario/enzimología , Penaeidae , Vitelogénesis/fisiología , Animales , Proteínas de Artrópodos/biosíntesis , Proteínas de Artrópodos/genética , Clonación Molecular , Femenino , Metiltransferasas/biosíntesis , Metiltransferasas/genética , Ovario/citología , Penaeidae/enzimología , Penaeidae/genéticaRESUMEN
The capacity of crustaceans to biosynthesise long-chain polyunsaturated fatty acids has yet to be fully defined, due to the lack of evidence on the functional activities of enzymes involved in desaturation or elongation of fatty acid substrates. We report here the cloning and in vitro functional analysis of an elongase from the orange mud crab, Scylla olivacea. Sequence and phylogenetic analysis placed the elovl close to the vertebrate Elovl1 and Elovl7 clade, which is distinct from the other remaining five Elovl families. The elongase was also clustered together with several elongases from crustaceans and insects. This elongase showed activities towards 16:1n-7, and at lower rate, linoleic acid (18:2n-6) and linolenic acid (18:3n-3). To our knowledge this is the first description of a functional enzyme involved in biosynthesis of long-chained polyunsaturated fatty acids in a crustacean species. Expression of the S. olivacea elovl7-like mRNA was prominent in stomach, intestine and gill tissues, due to the need to regulate the permeability of epithelial tissue through modification of fatty acid compositions. The implication of our findings, in terms of ability of Crustacea phylum to biosynthesise polyunsaturated fatty acids is discussed.
Asunto(s)
Acetiltransferasas , Proteínas de Artrópodos , Braquiuros , Ácidos Grasos Insaturados , Regulación Enzimológica de la Expresión Génica/fisiología , Filogenia , Acetiltransferasas/biosíntesis , Acetiltransferasas/química , Acetiltransferasas/genética , Animales , Proteínas de Artrópodos/biosíntesis , Proteínas de Artrópodos/química , Proteínas de Artrópodos/genética , Braquiuros/enzimología , Braquiuros/genética , Clonación Molecular , Elongasas de Ácidos Grasos , Ácidos Grasos Insaturados/biosíntesis , Ácidos Grasos Insaturados/genética , Especificidad de Órganos/fisiologíaRESUMEN
Mitochondrial uncoupling proteins (UCP) transport protons from the intermembrane space to the mitochondrial matrix uncoupling oxidative phosphorylation. In mammals, these proteins have been implicated in several cellular functions ranging from thermoregulation to antioxidant defense. In contrast, their invertebrate homologs have been much less studied despite the great diversity of species. In this study, two transcripts encoding mitochondrial uncoupling proteins were, for the first time, characterized in crustaceans. The white shrimp Litopenaeus vannamei transcript LvUCP4 is expressed in all tested shrimp tissues/organs, and its cDNA includes a coding region of 954 bp long which encodes a deduced protein 318 residues long and a predicted molecular weight of 35.3 kDa. The coding region of LvUCP5 transcript is 906 bp long, encodes a protein of 302 residues with a calculated molecular weight of 33.17 kDa. Both proteins share homology with insect UCPs, their predicted structures show the conserved motifs of the mitochondrial carrier proteins and were confirmed to be located in the mitochondria through a Western blot analysis. The genic expression of LvUCP4 and LvUCP5 was evaluated in shrimp at oxidative stress conditions and results were compared to some antioxidant enzymes to infer about their antioxidant role. LvUCP4 and LvUCP5 genes expression did not change during hypoxia/re-oxygenation, and no coordinated responses were detected with antioxidant enzymes at the transcriptional level. Results confirmed UCPs as the first uncoupling mechanism reported in this species, but their role in the oxidative stress response remains to be confirmed.
Asunto(s)
Proteínas de Artrópodos/biosíntesis , Regulación de la Expresión Génica/fisiología , Mitocondrias/metabolismo , Proteínas Desacopladoras Mitocondriales/biosíntesis , Penaeidae/metabolismo , Animales , Proteínas de Artrópodos/genética , Mitocondrias/genética , Proteínas Desacopladoras Mitocondriales/genética , Especificidad de Órganos/fisiología , Penaeidae/genéticaRESUMEN
In addition to the typical electron transport system (ETS) in animal mitochondria responsible for oxidative phosphorylation, in some species there exists an alternative oxidase (AOX) pathway capable of catalyzing the oxidation of ubiquinol and the reduction of oxygen to water. The discovery of AOX in animals is recent and further investigations into its expression, regulation, and physiological role have been hampered by the lack of a tractable experimental model organism. Our recent DNA database searches using bioinformatics revealed an AOX sequence in several marine copepods including Tigriopus californicus. This species lives in tidepools along the west coast of North America and is subject to a wide variety of daily environmental stresses. Here we verify the presence of the AOX gene in T. californicus and the expression of AOX mRNA and AOX protein in various life stages of the animal. We demonstrate that levels of the AOX protein increase in T. californicus in response to cold and heat stress compared to normal rearing temperature. We predict that a functional AOX pathway is present in T. californicus, propose that this species will be a useful model organism for the study of AOX in animals, and discuss future directions for animal AOX research.