Evaluation of the antibacterial synergism of two plant extracts belonging to Bignoniaceae family and development of a topical formulation

Abstract Fridericia caudigera and Cuspidaria convoluta (Bignoniaceae) species, which grow in the northwest of Argentina, have shown antibacterial effect against strains isolated from skin infections, and each one displayed synergism with commercial antibiotics. The aims of this work were to evaluate the antibacterial activity and toxicity of the combination of these two plant species, and to design a stable gel for topical use including the blend of extracts. The combination of extracts was evaluated for synergistic effects (chequerboard assay), genotoxicity (Ames test) and cytotoxicity (Artemia salina test). A gel was subsequently formulated with the combination of extracts using carboxymethylcellulose as a polymer. The following physico- chemical characteristics of the gel formulation: pH, viscosity, spreadability and total phenol content, as well as resistance to severe temperature changes, biological activity (diffusion in agar), in vitro permeation (Franz cells) and primary dermal irritation (Draize test) were analyzed. The combination of extracts showed a synergistic effect on pathogenic bacteria and was not toxic in the in vitro tests. The gel was stable and retained the antimicrobial activity of the original extracts. The formulation proposed in this work could constitute an alternative for primary skin infections since it proved to be safe for topical administration.

A note on the biogeographical origin of the brine shrimp Artemia urmiana Günther, 1899 from Urmia Lake, Iran.

The brine shrimp Artemia urmiana, an abundant inhabitant of the hypersaline Urmia Lake in northwestern Iran, has recently been described from Lake Koyashskoe, also a shallow hypersaline lake that is located on the Black Sea coast of the Crimean Peninsula (Ukraine). This discovery has questioned the endemicity of A. urmiana in Urmia Lake and has also brought into question the biogeographical origin of this species. In the present study, we combined recent genetic divergence data (mtDNA-COI) with palaeoecological evidence to address the biogeographical origin of A. urmiana. Calibration of the molecular clock of the COI region was set by assigning the age of the micro-crustacean Daphnia pulex minimally at 145 Mya. The divergence age of A. urmiana in Urmia Lake dates back to 383,000 years, whereas Ukrainian Artemia reflects a very young populations that diverged about 196,000 years ago. Palaeoecological evidence suggests that the age of the major habitat of A. urmiana i.e. Urmia Lake goes back to the Tertiary Period while the Ukranian habitats of the species are very young, by virtue of geological features of the Holocene age. We conclude that the biogeographical origin of A. urmiana is outside of Europe and the current state of knowledge strongly suggests that Urmia Lake has been the major source of its expansion into its modern habitats in Europe.

Morphological differentiation of seven parthenogenetic Artemia (Crustacea: Branchiopoda) populations from China, with special emphasis on ploidy degrees.

Parthenogenetic Artemia from seven Chinese locations with different elevations and various ploidies are characterized by phenotypic and morphometric analyses. Our findings show that the studied populations exhibit dissimilar patterns of ovisac. Four phenotypic patterns of furca are qualified and one of them is shared among di-, tetra- and pentaploid Artemia. Results of discriminant analysis based on morphometric data reveal that tetra- and pentaploid populations are grouped together, but the Aqqikkol Lake population is clearly differentiated. Previous hypothesis/conclusion that polyploid Artemia are larger than diploids is only partly supported by the present results, which show that pentaploid and tetraploid populations are larger than the mostly diploid populations in terms of the total length, but the body size of the Aibi Lake triploids has not significant difference with the sympatric diploids and the mostly diploid Aqqikkol population that inhabit in very high altitude has the largest body size among all parthenogenetic populations. The founding confirms that body size of Artemia is following with Bergmann's rule.

Estudo preliminar toxicológico, antibacteriano e fitoquímico do extrato etanólico das folhas de Jatropha mollissima (Pohl) Baill. (pinhão-bravo, Euphorbiaceae), coletada no Município de Tauá, Ceará, Nordeste Brasileiro / Toxicological, antibacterial, and phytochemical preliminary study of the ethanolic extract of Jatropha mollissima (Pohl) Baill (pinhão-bravo, Euphorbiaceae) leaves, collected in Tauá, Ceará, Northeastern Brazil

RESUMO A cada dia, cepas bacterianas estão tornando-se resistentes a diversos antibióticos, o que faz necessária a busca de novas substâncias eficazes para o tratamento de doenças. Desta forma, este trabalho reporta o estudo preliminar toxicológico, antibacteriano e fitoquímico do extrato etanólico das folhas de Jatropha mollissima (pinhão-bravo, Euphorbiaceae), coletada no Município de Tauá, Ceará, Nordeste Brasileiro. Inicialmente, realizou-se o teste de toxicidade do extrato contra Artemia salina. Na sequencia, foi realizado o ensaio antibacteriano contra quatro cepas bacterianas Gram-negativas (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Hafnia alvei ATCC 51873, Klebsiella pneumoniae ATCC 13883) e uma cepa Gram-positiva (Enterococcus faecalis ATCC 29212). Finalmente, fez-se a análise fitoquímica preliminar do extrato ativo para detecção das principais classes de metabólitos especiais. Como resultado, o extrato etanólico das folhas de J. mollissima se mostrou tóxico para Artemia salina, pois apresentou CL50 igual a 406,02 μg/mL. Quanto à ação antibacteriana, o extrato se mostrou ativo contra a bactéria Gram-positiva Enterococcus faecalis ATCC 29212, apresentando moderada atividade antibacteriana (halo de inibição igual a 7,03 mm). Evidenciou-se no extrato bioativo a presença de cumarinas, fenóis, taninos, flavonoides (flavonóis e flavanonas), alcaloides e esteroides, ambas as classes reportadas como antimicrobianos. Portanto, esse extrato tem potencial para ser usado na produção de fármacos contra infecções causadas por bactérias Gram-positivas. No entanto, as informações direcionam estudos futuros para o isolamento e identificação dos compostos bioativos, monitorados sob a ação antibacteriana mais expressiva.

ABSTRACT Each day, bacterial strains are becoming more resistant to various antibiotics, which requires the search for new effective substances for the treatment of diseases. Thus, this study reports the toxicological, antibacterial, and phytochemical preliminary study of the ethanolic extracts of Jatropha mollissima (pinhão-bravo, Euphorbiaceae) leaves, collected in Tauá, Ceará, Northeast of Brazil. Initially, we performed the toxicity testing of the extract against Artemia salina. Then, we conducted the antibacterial assay against four Gram-negative bacterial strains (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Hafnia alvei ATCC 51873, Klebsiella pneumoniae ATCC 13883), and one Gram-positive strain (Enterococcus faecalis ATCC 29212). Finally, we carried out the preliminary phytochemical analysis of the active extract to detect the main classes of special metabolites. As a result, the ethanolic extract of J. mollissima leaves was toxic to Artemia salina, because it presented LC50 equal to 406.02 µg/mL. Regarding antibacterial action, the extract was active against the Gram-positive bacteria Enterococcus faecalis ATCC 29212, with moderate antibacterial activity (inhibition zone equal to 7.03 mm). The bioactive extract had the presence of coumarins, phenols, tannins, flavonoids (flavanols and flavonones), alkaloids and steroids, both classes reported as antimicrobials. Therefore, this extract has the potential to be used in the production of drugs against infections caused by Gram-positive bacteria. However, these information require further studies for the isolation and identification of bioactive compounds, monitored under the more expressive antibacterial action.

Composição química e toxicidade frente Aedes aegypti L. e Artemia salina Leach do óleo essencial das folhas de Myrcia sylvatica (G. Mey. ) DC. / Chemical composition and toxicity against Aedes aegypti L. and Artemia salina Leach of the essential oil from Myrcia sylvatica (G. Mey. ) DC. leaves.

RESUMO A dengue está entre as doenças virais de propagação vetorial mais importante no mundo, causando sérios impactos de morbidade e mortalidade. Desta forma, o presente trabalho teve como objetivo analisar a composição química e a toxicidade do óleo essencial de Myrcia sylvatica (G. Mey) D.C. frente Aedes aegypti e Artemia salina. Folhas de M. sylvatica foram coletadas no Parque Nacional da Chapada das Mesas, no município de Carolina (MA) no mês de fevereiro de 2012. O óleo foi obtido por hidrodestilação e sua composição química foi determinada por cromatografia gasosa acoplada à espectrometria de massa (CG/EM). O bioensaio frente Artemia salina e às larvas de 3° estádio de Aedes aegypti foram realizados em diferentes concentrações. Os dados de mortalidade foram avaliados por regressão linear para determinar os valores de CL50. Obteve-se 0,5% de rendimento, sendo o (E)-cariofileno o constituinte majoritário. O óleo essencial apresentou uma CL50 = 79,44 µg/mL frente A. salina, sendo considerado altamente tóxico. No entanto, este óleo não demonstrou efeito sobre as larvas de A. aegypti. Considerando que o teste de Artemia salina tem correlação com atividades biológicas de grande interesse terapêutico como antitumoral, o óleo essencial das folhas de M. sylvatica demonstrou potencial para desenvolvimento de produtos farmacêuticos.

ABSTRACT Dengue is among the most important viral diseases of vector spread in the world, causing serious impacts of morbidity and mortality. Thus, this study aimed to analyze the chemical composition and toxicity of the Myrcia sylvatica (G. Mey.) DC essential oil against Aedes aegypti and Artemia salina Leach. Leaves of M. sylvatica. were collected in the Tables Chapada National Park, in the municipality of Carolina (MA) in february 2012. The oil was obtained by hydrodistillation and their chemical composition was determined by GC / MS. The bioassay front and Artemia salina larvae of the 3° stage of Aedes aegypti were performed at different concentrations. Mortality data were evaluated by linear regression to determine the LC50. Was obtained 0.5% yield values, and (E) -caryophyllene the major constituent. The essential oil showed a LC50 = 79.44 mg / mL front A. saline and is considered highly toxic. However, this oil showed no effect on larvae of A. aegypti. Whereas the brine shrimp test has regard to several biological activities, as antitumoral property, the essential oil from the leaves of M. sylvatica demonstrated potential for development of medicines.

Antitumor compounds from Streptomyces sp. KML-2, isolated from Khewra salt mines, Pakistan.

BACKGROUND: Actinomycetes are gram positive bacteria with high G + C content in their DNA and are capable of producing variety of secondary metabolites. Many of these metabolites possess different biological activities and have the potential to be developed as therapeutic agents. The aim of the present study was to screen actinomycetes inhabiting halophilic environment such as Khewra salt mines present in Pakistan for cytotoxic and antitumor compounds. RESULTS: An actiomycetes strain designated as Streptomyces sp. KML-2 was isolated from a saline soil of Khewra salt mines, Pakistan. The strain Streptomyces sp. KML-2 showed 84 % cytotoxic activity against larvae of Artemia salina. In the screening phase, the strain exhibited significant antitumor activity with IC50 values of 12, 48 and 56 µg/ml against Hela, MDBK and Vero cell lines, respectively. After that extract from 20 l fermentation was used to purify secondary metabolites by several chromatographic techniques. Structure elucidation of isolated compounds revealed that it is highly stable producer of Chromomycin SA (1) and 1-(1H-indol-3-yl)-propane-1,2,3-triol (2). Both of the isolated compounds showed significant antitumor activity against Hela and MCF-7 cancer cell lines (IC50 values 8.9 and 7.8 µg/ml against Hela; 12.6 and 0.97 µg/ml against MCF-7, respectively). The 16S rRNA gene sequence (1437 bp) of the strain confirm its identity (99 %) with Streptomyces griseus. CONCLUSIONS: From this research work we were successful in isolating two potent antitumor compounds, Chromomycin SA and 1-(1H-indol-3-yl)-propane-1,2,3-triol from Streptomyces KML-2 strain, isolated from Khewra salt mine. As such this is the second report which confirms that S. griseus can produce Chromomycin SA without introducing any mutagenesis in its biosynthesizing gene cluster and isolated indole derivative is being reported first time from any member of actinomycetes group with having novel antitumor activity against Hela and MCF-7 cells. Nucleotide sequences: Nucleotide sequence data reported are available in the GenBank database under the accession number: GenBank KJ009562.

Comparación de las propiedades antioxidantes y contenido de polifenoles de extractos acuosos de las algas marinas Bryothamnion triquetrum y Halimeda opuntia / Comparation of antioxidants properties and polyphenols content of aqueous extract from seaweeds Bryothamnion triquetrum and Halimeda opuntia

Objetivos. Evaluar y comparar las propiedades antioxidantes mediante ensayos in vitro de extractos acuosos de las algas roja Bryothamnion triquetrum y verde Halimeda opuntia y su relación con el contenido de polifenoles. Material y Métodos. Se utilizaron las técnicas in vitro: DPPH, Capacidad reductora, Inhibición de la peroxidación lipídica e inhibición de la hemólisis inducida por AAPH. Resultados. B. triquetrum: DPPH; CI50=1,15 ± 0,06, capacidad reductora a concentración; 128 mg/mL, DO=2,798, inhibición de la peroxidación lipídica; CI50=5,09± 0,25 e inhibición de la hemólisis con 12 mg/ mL; 35 %. H. opuntia: DPPH; CI50=12,34 ± 0,30 mg/mL, capacidad reductora; DO=0,800, inhibición de la peroxidación lipídica; CI50=1,25± 0,31 mg/mL e inhibición de la hemólisis; 82%. Conclusiones. Los resultados evidencian que B triquetrum resulto mucho más eficiente en los ensayos de DPPH y Capacidad reductora mientras H opuntia resulta más eficiente en Inhibición de la peroxidación lipídica e inhibición de la hemólisis. Se discuten algunos aspectos acerca de sus posibles mecanismos de acción

Objective. To evaluate and compare the antioxidant activity displayed by seaweed H. opuntia and B. triquetrum using different experimental in vitro antioxidant assessment models. Material and Methods. The following techniques are utilized: DPPH, Reducing power, inhibition of lipid peroxidation and inhibition of haemolysis. Results. B. triquetrum: DPPH; IC =1.15 ± 0.06, Reducing power (concentration 128 mg/mL), OD=2.798, inhibition of lipid peroxidation; IC 50 =5.09± 0.25 and inhibition of haemolysis; with 12 mg/mL; 35 %. H. opuntia: DPPH; IC 50 50 =12.34 ± 0.30 mg/mL, reducing power; OD=0.800, inhibition of lipid peroxidation; IC 50 =1.25± 0.31 mg/mL, inhibition of lipid peroxidation; C =1.25± 0.31 mg/mL and inhibition of haemolysis; 82%. 50 Conclusion. It was demonstrated that B.triquetrum extract was more effective than H. opuntia in reducing power and DPPH assays while H.opuntia was more effective in inhibition of lipid peroxidation in rat brain homogenates and the inhibition of red blood cell (RBC) haemolysis induced by AAPH. We discuss some aspects about their possible mechanisms of action

Análise fitoquímica, potencial antioxidante e toxicidade do extrato bruto etanólico e das frações da espécie Senecio westermaniiDusén frente à Artemia salina / Phytochemical analysis, antioxidant potential and toxicity of crude ethanol extract and fractions of the species Senecio westermanii Dusén against Artemia salina

RESUMO A espécie Senecio westermanii Dusén pertencente à família Asteraceae é planta endêmica e nativa do Brasil encontrada na região da Floresta Atlântica, nos estados do Paraná e São Paulo. O objetivo deste trabalho foi avaliar a composição fitoquímica do extrato bruto etanólico e frações das partes aéreas (folha e caule) de S. westermanii utilizando-se a análise fitoquímica qualitativa e cromatografia líquida de alta eficiência com detector de arranjo de diodos (CLAE-DAD), avaliar in vitro a toxicidade preliminar utilizando Artemia salina e o potencial antioxidante. O estudo fitoquímico qualitativo revelou a presença de alcaloides, flavonoides, iridoides, esteroides/triterpenos, heterosídeos saponínicos e aminogrupos. Através da análise realizada por CLAE-DAD obteve-se o fingerprint característico de cada amostra. No ensaio frente à A. salina houve ausência de toxicidade das amostras, o resultado da DL50 para todas as amostras foi superior a 1000 μg/mL. Todas as amostras apresentaram atividade antioxidante pela redução do complexo fosfomolibdênio, com destaque para a fração clorofórmio que apresentou atividade antioxidante de 92,51% em relação ao padrão rutina. Com relação ao ensaio de redução do radical DPPH•, a fração acetato de etila apresentou IC50 de 26,98 μg/mL. Pelo ensaio do ácido tiobarbitúrico (TBARS) a fração hexano apresentou o melhor índice antioxidante em relação ao padrão rutina. Os resultados obtidos demonstram evidências de que a espécie é fonte potencial de antioxidantes naturais, estimulando assim novos estudos que viabilizam sua utilização no tratamento de patologias associadas aos radicais livres. Além disso, a espécie não apresentou atividade tóxica preliminar, assegurando sua aplicabilidade.

ABSTRACT The species Senecio westermanii Dusén, which belongs to the Asteraceae family, is an endemic and native plant from Brazil. It is found mainly in the Atlantic Forest region, in Paraná and São Paulo states. This study aimed to evaluate the phytochemical composition using a qualitative phytochemical analysis and high performance liquid chromatography with diode array detector (HPLC-DAD), to evaluate preliminary toxicity in vitro using Artemia salina and antioxidant potential. Qualitative phytochemical analysis revealed the presence of alkaloids, flavonoids, iridoids, steroids, triterpenes, saponinic glycosides and aminogroups. The analysis by HPLC-DAD provided the characteristic fingerprint of each sample. In the A. salina assay, the results of LD50 over 1000 μg/mL for all samples did not indicate toxicity of the evaluated extracts. All samples demonstrated antioxidant activity by reducing phosphomolybdenum complex. The highest activity was detected in the chloroform fraction, which presented activity of 92.51% compared to the standard rutin. Regarding the trial to reduce the radical DPPH•, the ethyl acetate fraction showed an IC50 of 26.98 µg/mL. In the thiobarbituric acid assay (TBARS), the hexane fraction showed the highest antioxidant activity compared to the standard rutin. These findings indicate that the Senecio westermanii is a potential source of natural antioxidants, stimulating new studies that enable their use in the treatment of disorders associated with free radicals. In addition, the species did not present toxicity in its evaluation, ensuring its applicability.

Antitumor compounds from Streptomyces sp. KML-2, isolated from Khewra salt mines, Pakistan

BACKGROUND: Actinomycetes are gram positive bacteria with high G + C content in their DNA and are capable of producing variety of secondary metabolites. Many of these metabolites possess different biological activities and have the potential to be developed as therapeutic agents. The aim of the present study was to screen actinomycetes inhabiting halophilic environment such as Khewra salt mines present in Pakistan for cytotoxic and antitumor compounds. RESULTS: An actiomycetes strain designated as Streptomyces sp. KML-2 was isolated from a saline soil of Khewra salt mines, Pakistan. The strain Streptomyces sp. KML-2 showed 84 % cytotoxic activity against larvae of Artemiasalina. In the screening phase, the strain exhibited significant antitumor activity with IC50 values of 12, 48 and 56 µg/ml against Hela, MDBK and Vero cell lines, respectively. After that extract from 20 l fermentation was used to purify secondary metabolites by several chromatographic techniques. Structure elucidation of isolated compounds revealed that it is highly stable producer of Chromomycin SA (1) and 1-(1H-indol-3-yl)-propane-1,2,3-triol (2). Both of the isolated compounds showed significant antitumor activity against Hela and MCF-7 cancer cell lines (IC50 values 8.9 and 7.8 µg/ml against Hela; 12.6 and 0.97 µg/ml against MCF-7, respectively). The 16S rRNA gene sequence (1437 bp) of the strain confirm its identity (99 %) with Streptomyces griseus. CONCLUSIONS: From this research work we were successful in isolating two potent antitumor compounds, Chromomycin SA and 1-(1H-indol-3-yl)-propane-1,2,3-triol from Streptomyces KML-2 strain, isolated from Khewra salt mine. As such this is the second report which confirms that S. griseus can produce Chromomycin SA without introducing any mutagenesis in its biosynthesizing gene cluster and isolated indole derivative is being reported first time from any member of actinomycetes group with having novel antitumor activity against Hela and MCF-7 cells Nucleotide sequences: Nucleotide sequence data reported are available in the GenBank database under the accession number: GenBank KJ009562.

Avaliação do potencial antioxidante frente à oxidação lipídica e da toxicidade preliminar do extrato e frações obtidas das frondes de Dicksonia sellowiana (Presl. ) Hook / Evaluation of antioxidant potential against lipid oxidation and preliminary toxicity of extract and fractions obtained from the fronds of Dicksonia sellowiana (Presl. ) Hook

RESUMOA espécie Dicksonia sellowiana, conhecida popularmente como xaxim, é uma samambaia natural do continente americano e encontrada no Brasil na Mata Atlântica. Em 2001 foi inserida na lista do IBAMA como espécie ameaçada de extinção em decorrência da exploração para a confecção de vasos para a jardinagem. O presente trabalho descreve o potencial antioxidante lipídico (TBARS e Sistema β-caroteno/ácido linoleico) do extrato bruto e frações obtidos através de aparato de Soxhlet de frondes de Dicksonia sellowiana, além da atividade citotóxica e hemolítica in vitro. Pelo método TBARS, todas as amostras testadas apresentaram atividade, destacando a fração acetato de etila e extrato bruto cuja atividade foi comparável ao padrão ácido ascórbico. No sistema β-caroteno/ácido linoleico, a fração acetato de etila e extrato bruto apresentaram inibição da oxidação do ácido linoleico, destaque para a fração acetato de etila que não se diferenciou estatisticamente do padrão BHT. Na avaliação da toxicidade preliminar, não fora observado atividade citotóxica e hemolítica do extrato bruto e frações nos modelos testados. Os resultados demonstram o potencial antioxidante da espécie vegetal nos modelos de inibição da oxidação lipídica sem apresentar toxicidade.

ABSTRACTThe species Dicksonia sellowiana, popularly known as tree fern, are a natural fern from American continent found in the Atlantic Forest in Brazil. In 2001 it was added to the list of IBAMA as endangered species due to its exploitation for pots craft for gardening species. This paper describes the antioxidant lipid potential (TBARS and System β-carotene / linoleic acid) of the crude extract and fractions obtained by Soxhlet apparatus of Dicksonia sellowianafronds, besides the cytotoxic and hemolytic activity in vitro. By the TBARS method, all samples tested showed activity, particularly the fraction ethyl acetate and the crude extract whose activity was comparable to standard ascorbic acid. In the system β-carotene / linoleic acid, the fraction of ethyl acetate and crude extract inhibited the oxidation of linoleic acid, especially the ethyl acetate fraction which did not differ statistically from the standard BHT. In a preliminary assessment of toxicity,cytotoxic and hemolytic activity of crude extracts and fractions in the tested models were not observed. The results of this study indicate the antioxidant potential of this plant species in models of inhibiting lipid oxidation without producing toxicity.

Artemia biodiversity in Asia with the focus on the phylogeography of the introduced American species Artemia franciscana Kellogg, 1906.

Asia harbors a diverse group of sexual and asexual Artemia species, including the invasive Artemia franciscana, which is native to the Americas. The phylogeny of Asian Artemia species and the phylogeography of the introduced A. franciscana from 81 sampling localities in Eurasia, Africa and America were elucidated using mitochondrial (COI) and nuclear DNA (ITS1) sequences. According to a COI phylogeny, 6 distinctive genetic groups were recognized, with a complex phylogeographic structure among Asian Artemia. A haplotype complex which includes parthenogenetic lineages is distributed in 39 inland geographical localities in Asia, illustrating a wide distribution with a narrow genetic structure on this continent. The invasive A. franciscana was discovered in 31 geographical localities along the southern and eastern coastal regions of Asia. Three sexual species (A. sinica, A. tibetiana and A. urmiana) have a restricted distribution in certain geographical localities in Asia. In contrast to COI phylogeny reconstruction, ITS1 sequences showed inconsistency with the COI tree, indicating incomplete lineage sorting which provided the low genetic divergence in the Asian clade. Asian A. franciscana showed higher haplotype diversity as compared to the source population from the Great Salt Lake (USA), which could be attributed to multiple introductions by mass dispersal in Asia via human activities. The invasive success of A. franciscana in Asia could lead to a long-term biodiversity disturbance of the autochthonous Artemia species on the continent.

The potential role of As-sumo-1 in the embryonic diapause process and early embryo development of Artemia sinica.

During embryonic development of Artemia sinica, environmental stresses induce the embryo diapause phenomenon, required to resist apoptosis and regulate cell cycle activity. The small ubiquitin-related modifier-1 (SUMO), a reversible post-translational protein modifier, plays an important role in embryo development. SUMO regulates multiple cellular processes, including development and other biological processes. The molecular mechanism of diapause, diapause termination and the role of As-sumo-1 in this processes and in early embryo development of Artemia sinica still remains unknown. In this study, the complete cDNA sequences of the sumo-1 homolog, sumo ligase homolog, caspase-1 homolog and cyclin B homolog from Artemia sinica were cloned. The mRNA expression patterns of As-sumo-1, sumo ligase, caspase-1, cyclin B and the location of As-sumo-1 were investigated. SUMO-1, p53, Mdm2, Caspase-1, Cyclin B and Cyclin E proteins were analyzed during different developmental stages of the embryo of A. sinica. Small interfering RNA (siRNA) was used to verify the function of sumo-1 in A. sinica. The full-length cDNA of As-sumo-1 was 476 bp, encoding a 92 amino acid protein. The As-caspases-1 cDNA was 966 bp, encoding a 245 amino-acid protein. The As-sumo ligase cDNA was 1556 bp encoding, a 343 amino acid protein, and the cyclin B cDNA was 739 bp, encoding a 133 amino acid protein. The expressions of As-sumo-1, As-caspase-1 and As-cyclin B were highest at the 10 h stage of embryonic development, and As-sumo ligase showed its highest expression at 0 h. The expression of As-SUMO-1 showed no tissue or organ specificity. Western blotting showed high expression of As-SUMO-1, p53, Mdm2, Caspase-1, Cyclin B and Cyclin E at the 10 h stage. The siRNA caused abnormal development of the embryo, with increased malformation and mortality. As-SUMO-1 is a crucial regulation and modification protein resumption of embryonic diapause and early embryo development of A. sinica.

Origin and genetic diversity of diploid parthenogenetic Artemia in Eurasia.

There is wide interest in understanding how genetic diversity is generated and maintained in parthenogenetic lineages, as it will help clarify the debate of the evolution and maintenance of sexual reproduction. There are three mechanisms that can be responsible for the generation of genetic diversity of parthenogenetic lineages: contagious parthenogenesis, repeated hybridization and microorganism infections (e.g. Wolbachia). Brine shrimps of the genus Artemia (Crustacea, Branchiopoda, Anostraca) are a good model system to investigate evolutionary transitions between reproductive systems as they include sexual species and lineages of obligate parthenogenetic populations of different ploidy level, which often co-occur. Diploid parthenogenetic lineages produce occasional fully functional rare males, interspecific hybridization is known to occur, but the mechanisms of origin of asexual lineages are not completely understood. Here we sequenced and analysed fragments of one mitochondrial and two nuclear genes from an extensive set of populations of diploid parthenogenetic Artemia and sexual species from Central and East Asia to investigate the evolutionary origin of diploid parthenogenetic Artemia, and geographic origin of the parental taxa. Our results indicate that there are at least two, possibly three independent and recent maternal origins of parthenogenetic lineages, related to A. urmiana and Artemia sp. from Kazakhstan, but that the nuclear genes are very closely related in all the sexual species and parthenogegetic lineages except for A. sinica, who presumable took no part on the origin of diploid parthenogenetic strains. Our data cannot rule out either hybridization between any of the very closely related Asiatic sexual species or rare events of contagious parthenogenesis via rare males as the contributing mechanisms to the generation of genetic diversity in diploid parthenogenetic Artemia lineages.

Efeito da adição de farinha de linhaça à dieta sobre a concentração de colesterol e ácidos graxos em camarões / The effect of feed added of linseed on cholesterol and fatty acids in shrimp

Avaliou-se a expressão de ácidos graxos, macronutrientes e colesterol de camarões-da-malásia alimentados com dieta adicionada de sementes de linhaça. Quinhentos camarões foram distribuídos em dois grupos e alimentados durante 90 dias com dietas contendo: 1) lipídeo proveniente de linhaça e 2) óleo de soja (controle). A concentração de proteína foi maior (Р<0,05) para o grupo-linhaça, 15,8±0,09%, em relação ao grupo-controle, 15,2±0,04%. O peso corporal também se apresentou maior no grupo-linhaça, 1,38±0,93g, em relação ao grupo-controle, 0,93±0,48g. O grupo-linhaça apresentou teor de colesterol de 120,8±23,9mg/100g e o grupo-controle, 130,2±13,9mg/100g, porém sem diferença estatística significativa. Em relação ao perfil lipídico, a suplementação da dieta com linhaça influenciou a incorporação do ácido oleico (C18:1, ω-9) e melhorou a relação ω-6/ω-3 na carne do camarão (Р<0,05). Concluiu-se que a adição de linhaça à ração promoveu importantes modificações na qualidade nutricional dos tecidos do camarão.

The aim of the present study was to analyze the chemical composition of shrimp fed with a diet added of linseed flour. Five hundred post larvae shrimp were divided into two groups: a linseed group fed with a diet based on casein complemented with linseed and a control group, fed a diet without linseed flour. The cholesterol content for the linseed group was 120.8±23.9 mg/100g while for the control group it was 130.2±13.9 mg/100g. However, the protein content of 15.8±0.09 % and the body weight of 1.38±0.93g for the linseed group were higher than for the control group. It was concluded that the addition of linseed to the diet seems to increase the protein, weight and oleic acid (C18:1, ω-9) in the shrimp tissue. The ratio of polyunsaturated fatty acids/saturated fatty acids and ω-6/ω-3 for the linseed group was more suitable than for the control group.

Mitochondrial genome sequences of Artemia tibetiana and Artemia urmiana: assessing molecular changes for high plateau adaptation.

Brine shrimps, Artemia (Crustacea, Anostraca), inhabit hypersaline environments and have a broad geographical distribution from sea level to high plateaus. Artemia therefore possess significant genetic diversity, which gives them their outstanding adaptability. To understand this remarkable plasticity, we sequenced the mitochondrial genomes of two Artemia tibetiana isolates from the Tibetan Plateau in China and one Artemia urmiana isolate from Lake Urmia in Iran and compared them with the genome of a low-altitude Artemia, A. franciscana. We compared the ratio of the rate of nonsynonymous (Ka) and synonymous (Ks) substitutions (Ka/Ks ratio) in the mitochondrial protein-coding gene sequences and found that atp8 had the highest Ka/Ks ratios in comparisons of A. franciscana with either A. tibetiana or A. urmiana and that atp6 had the highest Ka/Ks ratio between A. tibetiana and A. urmiana. Atp6 may have experienced strong selective pressure for high-altitude adaptation because although A. tibetiana and A. urmiana are closely related they live at different altitudes. We identified two extended termination-associated sequences and three conserved sequence blocks in the D-loop region of the mitochondrial genomes. We propose that sequence variations in the D-loop region and in the subunits of the respiratory chain complexes independently or collectively contribute to the adaptation of Artemia to different altitudes.

High prevalence of cestodes in Artemia spp. throughout the annual cycle: relationship with abundance of avian final hosts.

Brine shrimp, Artemia spp., act as intermediate hosts for a range of cestode species that use waterbirds as their final hosts. These parasites can have marked influences on shrimp behavior and fecundity, generating the potential for cascading effects in hypersaline food webs. We present the first comprehensive study of the temporal dynamics of cestode parasites in natural populations of brine shrimp throughout the annual cycle. Over a 12-month period, clonal Artemia parthenogenetica were sampled in the Odiel marshes in Huelva, and the sexual Artemia salina was sampled in the Salinas de Cerrillos in Almería. Throughout the year, 4-45 % of A. parthenogenetica were infected with cestodes (mean species richness = 0.26), compared to 27-72 % of A. salina (mean species richness = 0.64). Ten cestode species were recorded. Male and female A. salina showed similar levels of parasitism. The most prevalent and abundant cestodes were those infecting the most abundant final hosts, especially the Greater Flamingo Phoenicopterus ruber. In particular, the flamingo parasite Flamingolepis liguloides had a prevalence of up to 43 % in A. parthenogenetica and 63.5 % in A. salina in a given month. Although there was strong seasonal variation in prevalence, abundance, and intensity of cestode infections, seasonal changes in bird counts were weak predictors of the dynamics of cestode infections. However, infection levels of Confluaria podicipina in A. parthenogenetica were positively correlated with the number of their black-necked grebe Podiceps nigricollis hosts. Similarly, infection levels of Anomotaenia tringae and Anomotaenia microphallos in A. salina were correlated with the number of shorebird hosts present the month before. Correlated seasonal transmission structured the cestode community, leading to more multiple infections than expected by chance.

Brine shrimp (Artemia salina Leach) bioassay of extracts from Lychnophoriopsis candelabrum and different Lychnophora species

O presente estudo teve como objetivo uma citotoxicidade sobre Artemia salina de vinhos e dois extratos de cinco espécies do gênero Lychnophora e de uma espécie de Lychnophoriopsis . Os extratos solubilizados em DMSO, preparados nas concentrações finais de 100, 250, 375, 500 e 600 µg mL -1 , foram adicionados a recipientes contendo náuplios de Artemia salina (10 unidades cada) e completo em volume para 5 mL de solução marinha. Lapachol e DMSO 5% foram como controles positivo e negativo, respectivamente. Como amostras foram mantidas sob iluminação e como larvas mortas foram contadas após 24 horas de contato. O cálculo da LC 50 foi feito com o programa Probitos. Os extratos brutos etanólicos de cinco Espécies apresentaram baixa letalidade nd Seguinte Ordem: Lychnophora trichocarpha (LC 50 = 672,38 ng mL -1 )> Lychnophora pinaster (LC 50 = 678,73 ng mL -1 )> Lychnophora ericoides (LC 50 = 738,09 µg mL -1 )> Lycellophoriopsis candelabro (LC 50 = 812,57 µg mL -1 )> Lychnophora passerina (LC 50 = 921,78 µg mL -1 ). Todos os extratos testados de Lychnophoriopsis candelabro eo extrato clorofórmico de Lychnophora staavioides mostraram leve toxicidade sobre A. salina . Os resultados indicaram que existem substâncias com potencial atividade farmacológica em todas as espécies testadas.

The present study aimed to evaluate on Artemia salina the citotoxicity of twenty-two extracts from five species of the genus Lychnophora and one species of the genus Lychnophoriopsis. The extracts solubilized in DMSO and prepared at the final concentrations of 100, 250, 375, 500 and 600 g mL-1 were added to tubes containing Artemia salina nauplii (10 units each) and filled to 5 mL total volume with artificial salt water. Lapachol and 5% DMSO were used as positive and negative controls, respectively. The samples were kept under light and dead larvae were counted after 24 hours of contact. LC50 was calculated by using Probit software. The crude ethanol extracts from five species showed low lethality in the following order: Lychnophora trichocarpha (LC50 = 672.38 g mL-1) > Lychnophora pinaster (LC50 = 678.73 g mL-1) > Lychnophora ericoides (LC50 = 738.09 g mL-1) > Lychnophoriopsis candelabrum (LC50 = 812.57 g mL-1) > Lychnophora passerina (LC50 = 921.78 g mL-1). All tested extracts from L. candelabrum and chloroform extract from L. staavioides showed light toxicity on A. salina. Results indicated that there are substances with potential pharmacological activity in all tested species

Expression pattern of a Gram-negative bacteria-binding protein in early embryonic development of Artemia sinica and after bacterial challenge.

Gram-negative bacteria-binding proteins (GNBPs) are important pattern recognition receptors (PRRs) in invertebrate innate immune systems. These immune proteins are capable of identifying and binding to specific targets on the surface of microorganisms, and thereby trigger a variety of defense reactions through the activation of protease cascades and intracellular immune signaling pathways. In this paper, a full-length cDNA of GNBP coding for a conserved ß-glucan-binding domain was isolated from Artemia sinica. Bioinformatic analysis showed that it belonged to a new member of the GNBP family. The expression of GNBP was investigated at various stages during the embryonic development of A. sinica using real-time PCR and immunohistochemistry assay. The result showed that the highest expression level was on the 10th day of the embryo. The larvae showed a remarkable down-regulation of GNBP transcript after challenged with Gram-negative bacteria. In contrast, the level of GNBP transcript in the larvae increased gradually after challenged with Gram-positive bacteria. Immunohistochemistry assay showed that during embryonic development, GNBP protein was mainly expressed in the head, thorax, and on the alimentary canal surface. This new GNBP member showed a constitutive and regional expression during the early embryonic development of A. sinica, and may therefore play an important role at the adult stage, as well as during infection.

Potential heterogeneity in crustacean zooplankton assemblages in southern chilean saline lakes.

The Chilean saline lakes are distributed mainly in the Atacama desert in northern Chile and the southern Patagonian plains. The scarce studies are restricted mainly to northern Chilean saline lakes, and these revealed that the main component in these ecosystems are the halophylic copepod Boeckella poopoensis Marsh 1906, or the brine shrimp Artemia franciscana (Kellog, 1906), and both species do not coexist. The present study consisted of field observations in zooplankton assemblages in southern Chilean saline lakes (51-53 ºS). These first observations revealed three different patterns, one saline lake only with A. persimilis (Piccinelli and Prosdocimi, 1968), a second lake only with B. poopensis, and a third lake with A. persimilis, B. poopoensis and unidentified harpacticoid copepod. These results are different in comparison with the observations in the literature that described the non-coexistence between B. poopoensis with brine shrimps. Ecological and biogeographical topics were discussed.

The New World Artemia species A. franciscana and A. persimilis are highly differentiated for chromosome size and heterochromatin content.

Chromosomal rearrangements have played a key role in the speciation of the New World sexual Artemia species (Crustacea, Anostraca) A. franciscana and A. persimilis. The species differ by a chromosome duplication (2n+2=44 in A. persimilis vs 2n=42 in A. franciscana), and a greater amount of heterochromatin (HCH) in A. franciscana. To investigate this difference in HCH, four parameters were compared for the first time in Artemia: 1) the absolute sizes of one A. persimilis and four A. franciscana karyotypes; 2) the relative lengths of all chromosome; 3) the number of heterochromatic bands and 4) the relative amounts of HCH per chromosome and its position. The two A.franciscana karyotypes with the largest HCH amount (26%), have twice (139.26 microm and 134.05 microm) the absolute size of the A.persimilis karyotype (64.91 microm; HCH: 1.97%). Interspecific and intraspecific (A. franciscana) differences in chromosome size and HCH were observed, although the two sets of information are not positively correlated. While A. persimilis shares plesiomorphic karyological traits with Old World species, A. franciscana has apomorphic features such as longer chromosomes and greater HCH content, mainly dispersed towards telomeres. The impacts of such chromosome rearrangements are discussed in relation to the wider geographic distribution, greater colonizing ability, and life history plasticity of A. franciscana. An additional, though preliminary, point of this paper is the observation that the female would be the heterogametic sex.