The Suppressor of AAC2 Lethality SAL1 Modulates Sensitivity of Heterologously Expressed Artemia ADP/ATP Carrier to Bongkrekate in Yeast

The ADP/ATP carrier protein (AAC) expressed in Artemia franciscana is refractory to bongkrekate. We generated two strains of Saccharomyces cerevisiae where AAC1 and AAC3 were inactivated and the AAC2 isoform was replaced with Artemia AAC containing a hemagglutinin tag (ArAAC-HA). In one of the strains the suppressor of ΔAAC2 lethality, SAL1, was also inactivated but a plasmid coding for yeast AAC2 was included, because the ArAACΔsal1Δ strain was lethal. In both strains ArAAC-HA was expressed and correctly localized to the mitochondria. Peptide sequencing of ArAAC expressed in Artemia and that expressed in the modified yeasts revealed identical amino acid sequences. The isolated mitochondria from both modified strains developed 85% of the membrane potential attained by mitochondria of control strains, and addition of ADP yielded bongkrekate-sensitive depolarizations implying acquired sensitivity of ArAAC-mediated adenine nucleotide exchange to this poison, independent from SAL1. However, growth of ArAAC-expressing yeasts in glycerol-containing media was arrested by bongkrekate only in the presence of SAL1. We conclude that the mitochondrial environment of yeasts relying on respiratory growth conferred sensitivity of ArAAC to bongkrekate in a SAL1-dependent manner. © 2013 Wysocka-Kapcinska et al

Recombinant DnaK orally administered protects axenic European sea bass against vibriosis

Vibrio anguillarum causes high mortality in European sea bass (Dicentrarchus labrax) larviculture and is a hindering factor for successful sustainable aquaculture of this commercially valuable species. Priming of the innate immune system through administration of immunostimulants has become an important approach to control disease outbreaks in marine fish larviculture. This study was conducted to evaluate immunostimulation by Escherichia coli HSP70 (DnaK) in axenic European sea bass larvae in order to protect the larvae against vibriosis. DnaK stimulates the immune response in crustaceans and juvenile fish against bacterial infections. The use of axenic fish larvae allows to study immunostimulation in the absence of an interfering microbial community. At 7 days post-hatching, larvae received a single dose of alginate encapsulated recombinant DnaK. Two non-treated control groups in which animals either received empty alginate microparticles (C1) or no alginante microparticles (C2 and C3) were included in the study. Eighteen hours later, all larvae, except the ones from group C3 (non-infected control) were challenged with V. anguillarum (10(5) CFU, bath infection). Mortality was daily recorded until 120 h post infection and at 18, 24, and 36 h post infection, larvae were sampled for expression of immune related genes. Results showed that V. anguillarum induced an immune response in axenic sea bass larvae but that the innate immune response was incapable to protect the larvae against deadly septicaemic disease. In addition, we showed that administration of alginate encapsulated DnaK to axenic European sea bass larvae at DAH7 resulted in a significant, DnaK dose dependent, upreglation of immune sensor, regulatory and effector genes. Significant upregulation of cxcr4, cas1 and especially of hep and dic was correlated with significant higher survival rates in V. anguillarum infected larvae. In the future recombinant DnaK might perhaps be used as a novel immunostimulant in sea bass larviculture

Colonization and dispersal patterns of the invasive American brine shrimp Artemia franciscana (Branchiopoda: Anostraca) in the Mediterranean region

Cysts of the brine shrimp Artemia franciscana are harvested from the Great Salt Lake (GSL) and San Francisco Bay (SFB) saltworks in the USA, and marketed worldwide to provide live food for aquaculture. This species has become invasive across several countries. We investigated (1) if the introduced populations in the Mediterranean region could have originated from these USA populations, (2) how the genetic diversity of Mediterranean compares to that at GSL and SFB, and (3) if genetic patterns in the Mediterranean can shed light on colonization routes. We sequenced a fragment of the cytochrome c oxidase subunit I and screened microsatellites loci from Mediterranean populations and the two putative USA sources. Haplotypes from Mediterranean populations were identical or closely related to those from SFB and GSL, and not related to other available American populations. Microsatellite analyses showed a reduced population diversity for most Mediterranean populations suggesting bottleneck effects, but few populations were showing similar or higher genetic diversity than native ones, which are likely to be admixed from both GSL and SFB because of multiple introductions. Results suggest natural dispersal, potentially via flamingos, between two Spanish populations. Our analyses show that all invaded populations could have originated from those commercialized USA populations. © 2013 Springer Science+Business Media Dordrecht

Identification of the alternative oxidase gene and its expression in the copepod Tigriopus californicus

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

Moisture source and diet affect development and reproduction of Orius thripoborus and Orius naivashae, two predatory anthocorids from southern Africa

The effect of moisture source and diet on the development and reproduction of the pirate bugs, Orius thripoborus (Hesse) and Orius naivashae (Poppius) (Hemiptera: Anthocoridae) was examined in the laboratory. Both species had been collected in and around sugarcane fields in South Africa. Supplementing eggs of the flour moth Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae) with a green bean pod as a moisture source yielded better nymphal survival and faster development, as compared with free water encapsulated in Parafilm, suggesting that the predators may extract extra nutrients from the bean pod. The impact of two factitious foods and moist honey bee pollen on developmental and reproductive parameters of both predators was also investigated. The overall performance of both Orius species on E. kuehniella eggs and cysts of brine shrimp, Artemia franciscana Kellogg (Crustacea: Artemiidae) was better than on pollen. Nonetheless, a pollen diet alone allowed 66 and 78% of the nymphs of O. thripoborus and O. naivashae, respectively, to reach adulthood. Overall, developmental and reproductive performance of O. thripoborus on the tested diets was superior to that of O. naivashae. The implications of these findings for the mass production of these predators and their potential role in biological control programs in southern Africa are discussed

A first AFLP-based genetic linkage map for brine shrimp Artemia franciscana and its application in mapping the sex locus

We report on the construction of sex-specific linkage maps, the identification of sex-linked markers and the genome size estimation for the brine shrimp Artemia franciscana. Overall, from the analysis of 433 AFLP markers segregating in a 112 full-sib family we identified 21 male and 22 female linkage groups (2n = 42), covering 1,041 and 1,313 cM respectively. Fifteen putatively homologous linkage groups, including the sex linkage groups, were identified between the female and male linkage map. Eight sex-linked AFLP marker alleles were inherited from the female parent, supporting the hypothesis of a WZ-ZZ sex-determining system. The haploid Artemia genome size was estimated to 0.93 Gb by flow cytometry. The produced Artemia linkage maps provide the basis for further fine mapping and exploring of the sex-determining region and are a possible marker resource for mapping genomic loci underlying phenotypic differences among Artemia species

Biometry of Artemia Franciscana From Three Different Batches

Biometry of Artemia franciscana from three different batches were characterized under laboratoryconditions. The results from diameter measurement of cyst from PR, SI and AS batches were 240 μm, 238μm and 245 μm respectively. The diameter of decapsulated cyst from the same batches were 223 μm (PR),221 μm (SI) and 220 μm (AS). The length of Instar I nauplii from PR, SI and AS batches were 476 μm, 497μm and 498 μm. Hatching efficiency after 48 hours of incubation were 2.76 x 105 nauplii/g cyst, 2.80 x 105nauplii/g cyst and 2.90 x 105 nauplii/g cyst from PR, SI and AS batches. The statistical analysis of theresults indicated that there were no significant differences (P=0.1) in diameter of cyst, length of Instar Inauplii and hatching efficiency of cyst. The hatching percentage of cyst from AS batch at 24 hours (89%),and respectively 48 hours (91%), was significantly higher (P=0.1) than those of SI (83% and 88%) and PR(74% and 80%).The hatching percentage of cyst from PR batch was significantly lower (P=0.1) than SI

Alachlor oxidation by the filamentous fungus Paecilomyces marquandii

Alachlor, a popular chloroacetanilide herbicide, can be a potential health risk factor. Soil microorganisms are primarily responsible for conversion and migration of alachlor in natural environment, but knowledge concerning alachlor biodegradation is not complete. Therefore, we studied the ability of Paecilomyces marquandii, soil fungus tolerant to heavy metals, to eliminate alachlor and proposed a new pathway of its transformation. After 7 days of incubation only 3.3% of alachlor was detected from an initial concentration 50 mg L-1 and 20.1% from a concentration 100 mg L-1. The qualitative IDA LC-MS analysis showed the presence of ten metabolites. All of them were dechlorinated mainly through oxidation, but also reductive dechlorination was observed. The main route of alachlor conversion progressed via N-acetyl oxidation resulting in the formation of mono-, di- and trihydroxylated byproducts. N-acetyl oxidation as a dominant route of alachlor metabolism by fungi has not been described so far. The toxicity of alachlor tested with Artemia franciscana did not increase after treatment with P. marquandii cultures. Paecilomyces marquandii strain seems to be an interesting model for the research on alachlor conversion by soil microscopic fungi, due to its dechlorination and hydroxylation ability as well as high tolerance to heavy metals.Grant of the National Centre for Science in Cracow, Poland, No UMO-2011/01/B/NZ9/0289