Cloning of two SOS1 transporters from the seagrass <Emphasis Type="Italic">Cymodocea nodosa</Emphasis>. SOS1 transporters from <Emphasis Type="Italic">Cymodocea</Emphasis> and <Emphasis Type="Italic">Arabidopsis</Emphasis> mediate potassium uptake in bacteria

Two cDNAs isolated from Cymodocea nodosa, CnSOS1A, and CnSOS1B encode proteins with high-sequence similarities to SOS1 plant transporters. CnSOS1A expressed in a yeast Na⁺-efflux mutant under the control of a constitutive expression promoter mimicked AtSOS1 from Arabidopsis; the wild type cDNA did not improve the growth of the recipient strain in the presence of Na⁺, but a cDNA mutant that expresses a truncated protein suppressed the defect of the yeast mutant. In similar experiments, CnSOS1B was not effective. Conditional expression, under the control of an arabinose responsive promoter, of the CnSOS1A and CnSOS1B cDNAs in an Escherichia coli mutant defective in Na⁺ efflux was toxic, and functional analyses were inconclusive. The same constructs transformed into an E. coli K⁺-uptake mutant revealed that CnSOS1A was also toxic, but that it slightly suppressed defective growth at low K⁺. Truncation in the C-terminal hydrophilic tail of CnSOS1A relieved the toxicity and proved that CnSOS1A was an excellent low-affinity K⁺ and Rb⁺ transporter. CnSOS1B mediated a transient, extremely rapid K⁺ or Rb⁺ influx. Similar tests with AtSOS1 revealed that it was not toxic and that the whole protein exhibited excellent K⁺ and Rb⁺ uptake characteristics in bacteria.     

Metapopulation structure in the lagoon cockle <Emphasis Type="Italic">Cerastoderma lamarcki</Emphasis> in the northern Wadden Sea

Benthic invertebrates in discontinuous inshore habitats and with short or no pelagic larval dispersal are likely to exhibit regional metapopulation dynamics with partially isolated local populations. Near the island of Sylt, the bivalve Cerastoderma (Cardium) lamarcki (Reeve, 1844, syn. in part with C. glaucum Bruguiére, 1789) was widespread in intertidal seagrass beds, coexisting with the sibling species C. edule (Linné, 1758). However, the last C. lamarcki in this habitat was found in 1980. At present the lagoon cockle is restricted to disjunct ditches, creeks and ponds within island salt marshes. There it differs in year-class structure between localities. Successful recruitment events did not coincide. At one locality, a period with regular recruitment was followed by 5 years of recruitment failure, resulting in an overaged population probably at the rim of extinction. In a nearby brackish pond, extinction was followed by recolonization 3 years later. Other lagoonal habitats which seem to be suitable are without cockles. It is speculated that small and isolated habitats occasionally receive colonizers by eggs and juveniles adhering to avian vectors. Electronic Publication     

Seasonal dynamics of <Emphasis Type="Italic">Zostera noltii</Emphasis> Hornem. in two Mediterranean lagoons

The density, biomass and shoot morphology of two populations of Zostera noltii were monitored from January 1998 to July 1999 at two shallow Mediterranean lagoons of Biguglia and Urbino, which differ in hydro-morphological conditions and nutrient loading. Monitoring included the principal biological and foliar parameters (shoot density, aboveground and belowground biomass, length, width and number of leaves, LAI and coefficient A: percentage of leaves having lost their apex), the organic matter contents of the sediment and the environmental conditions (salinity, turbidity, temperature, nutrient concentrations and dissolved oxygen levels). The two populations of Z. noltii displayed seasonal changes in density (1600–19600 m²), aboveground biomass (11–153 g. DW. m⁻²), leaf length (33–255 mm), and leaf width (0.9–1.8 mm). Temperature and turbidity were significant environmental factors influencing the temporal changes observed in the Z. noltii meadows studied. Conversely, the belowground biomass, the number of leaves per shoot and the LAI did not undergo any seasonal changes. In the Biguglia lagoon, the functioning dynamics of the Z. noltii seagrass beds are determined by the catchment area and the inputs of nutrients derived from it, whereas in the Urbino lagoon the dynamics of the Z. noltiibeds depend on low levels of water turbidity.     

Antibacterial activity of three South Indian seagrasses, <Emphasis Type="Italic">Cymodocea serrulata</Emphasis>, <Emphasis Type="Italic">Halophila ovalis</Emphasis> and <Emphasis Type="Italic">Zostera capensis</Emphasis>

The antibacterial properties of the three seagrasses namely Cymodocea serrulata, Halophila ovalis and Zostera capensis were tested against the human pathogens Staphylococcus aureus, Bacillus cereus, B. subtilis, Escherichia coli, Salmonella paratyphi, Salmonella typhimurium and Micrococcus luteus, using six different solvents namely, petroleum ether, chloroform, ethyl acetate, acetone, methanol and water. Ethyl acetate and methanol extracts showed maximum activity against most of the pathogens when compared to other solvents. Experiments are underway to isolate active compound(s) implicated in controlling the growth of the pathogens in vitro.     

Dynamics of Bacterial Communities in Cockles (<Emphasis Type="Italic">Cerastoderma edule</Emphasis>) with Respect to Trematode Parasite (<Emphasis Type="Italic">Bucephalus minimus</Emphasis>) Infestation

The bacterial communities associated with the cockle (Cerastoderma edule) were investigated at the individual level through a 10-month monitoring programme. Temporal changes and those changes associated with a common parasite of the cockle, Bucephalus minimus, were investigated by monthly sampling of individuals, selected based on their shell length (cohort monitoring). Cockle bacterial community abundance (CBCA) and diversity (CBCD) were estimated by epifluorescence microscopy counts and automated ribosomal intergenic spacer analysis, respectively. CBCA showed a temporal pattern peaking at 30 × 10⁶ cells per gram of cockle flesh and intervalval liquid in October and a significant 1.8-fold increase linked with B. minimus occurrence. CBCD was characterized by 112 ± 26 intergenic transcribed spacer (ITS) per individual and showed a relative homology between individuals (52 ± 6%, Jaccard similarity) in spite of more than 30% of rare ITS. Consistent with an undisturbed evolution of the condition index of the studied cohort individuals as an estimate of their physiological state, neither temporal nor parasite-induced change in CBCA has been related to marked changes in CBCD.     

Comparative Feeding Ecology of Two Sympatric Greenling Species, <Emphasis Type="Italic">Hexagrammos otakii</Emphasis> and <Emphasis Type="Italic">Hexagrammos agrammus</Emphasis> in Eelgrass <Emphasis Type="Italic">Zostera marina</Emphasis> Beds

Synopsis Feeding ecology was compared between sympatric greenling species, Hexagrammos otakii and H. agrammus in the eelgrass beds in Jindong Bay, Korea, from January to December. These two species had similar diets composed of crustaceans, polychaetes, gastropods and fishes; both species consumed primarily crustaceans throughout study periods. H. otakii, however, fed a greater proportion of polychaetes and fishes than H. agrammus. H. agrammus had a greater proportion of gastropods in their diets. The diet of both species underwent size-related changes; smaller individuals of H. otakii and H. agrammus consumed amphipods (gammarid amphipods and caprellid amphipods), while larger individuals of H. otakii ate polychaetes and fishes and those of H. agrammus fed mainly on gastropods and crabs. The diet of H. otakii underwent seasonal changes; H. otakii consumed mainly polychaetes and fishes during January and February 2002 but amphipods during March and May 2002. H. agrammus, however, ate mainly gastropods and crabs all seasons. H. otakii underwent also a significant diel changes that could be related to difference of prey availability. Thus the nocturnal emergence of gammarid amphipods, polychaetes and fishes explained their greater consumption by H. otakii. Dietary breadth of both species was lower in the smallest individuals (<5 cm SL) and in March and April 2002. This was due to the disproportionate dry mass attributable to the consumption of amphipods by both H. otakii and H. agrammus. Dietary overlap of both species was relatively moderate to high, in particular in <9.9 cm SL (0.62 – 0.71) from May to July 2002 (0.63 – 0.71). This is coincident with higher abundances of crabs, caridean shrimps and polychaetes in the study area, and it was assumed that these prey species were not limited resources. Higher dietary overlap was correlated with an abundance of a shared resource and did not indicate the interspecific competition between H. otakii and H. agrammus.     

Bio-resolution of glycidyl (<Emphasis Type="Italic">o</Emphasis>, <Emphasis Type="Italic">m</Emphasis>, <Emphasis Type="Italic">p</Emphasis>)-methylphenyl ethers by <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

A newly isolated Bacillus megaterium with epoxide hydrolase activity resolved racemic glycidyl (o, m, p)-methylphenyl ethers to give enantiopure epoxides in 84–99% enantiomeric excess and with 21–73 enantiomeric ratios. The (S)-enantiomer was obtained from rac-glycidyl (o or m)-methylphenyl ether while the (R)-epoxides was obtained from glycidyl p-methylphenyl ether. The observations are explained at the level by enzyme-substrate docking studies.     

<Emphasis Type="Italic">STAYGREEN</Emphasis> (<Emphasis Type="Italic">CsSGR</Emphasis>) is a candidate for the anthracnose (<Emphasis Type="Italic">Colletotrichum orbiculare</Emphasis>) resistance locus <Emphasis Type="Italic">cla</Emphasis> in Gy14 cucumber

Key message

Map-based cloning identified a candidate gene for resistance to the anthracnose fungal pathogen Colletotrichum orbiculare in cucumber, which reveals a novel function for the highly conserved STAYGREEN family genes for host disease resistance in plants.

Abstract

Colletotrichum orbiculare is a hemibiotrophic fungal pathogen that causes anthracnose disease in cucumber and other cucurbit crops. No host resistance genes against the anthracnose pathogens have been cloned in crop plants. Here, we reported fine mapping and cloning of a resistance gene to the race 1 anthracnose pathogen in cucumber inbred lines Gy14 and WI 2757. Phenotypic and QTL analysis in multiple populations revealed that a single recessive gene, cla, was underlying anthracnose resistance in both lines, but WI2757 carried an additional minor-effect QTL. Fine mapping using 150 Gy14?×?9930 recombinant inbred lines and 1043 F₂ individuals delimited the cla locus into a 32 kb region in cucumber Chromosome 5 with three predicted genes. Multiple lines of evidence suggested that the cucumber STAYGREEN (CsSGR) gene is a candidate for the anthracnose resistance locus. A single nucleotide mutation in the third exon of CsSGR resulted in the substitution of Glutamine in 9930 to Arginine in Gy14 in CsSGR protein which seems responsible for the differential anthracnose inoculation responses between Gy14 and 9930. Quantitative real-time PCR analysis indicated that CsSGR was significantly upregulated upon anthracnose pathogen inoculation in the susceptible 9930, while its expression was much lower in the resistant Gy14. Investigation of allelic diversities in natural cucumber populations revealed that the resistance allele in almost all improved cultivars or breeding lines of the U.S. origin was derived from PI 197087. This work reveals an unknown function for the highly conserved STAYGREEN (SGR) family genes for host disease resistance in plants.

     

Actinomycetemcomitin: a new bacteriocin produced by <Emphasis Type="Italic">Aggregatibacter</Emphasis> (<Emphasis Type="Italic">Actinobacillus</Emphasis>) <Emphasis Type="Italic">actinomycetemcomitans</Emphasis>

Aggregatibacter (Actinobacillus) actinomycetemcomitans P_7–20 strain isolated from a periodontally diseased patient has produced a bacteriocin (named as actinomycetemcomitin) that is active against Peptostreptococcus anaerobius ATCC 27337. Actinomycetemcomitin was produced during exponential and stationary growth phases, and its amount decreased until it disappeared during the decline growth phase. It was purified by ammonium sulphate precipitation (30–60% saturation), and further by FPLC (mono-Q ionic exchange and Phenyl Superose hydrophobic interaction) and HPLC (C-18 reversed-phase). This bacteriocin loses its activity after incubation at a pH below 7.0 or above 8.0, following heating for 30 min at 45°C, and after treatment with proteolytic enzymes such as trypsin, α-chymotrypsin, and papain. Actinomycetemcomitin has a molecular mass of 20.3 KDa and it represents a new bacteriocin from A. actinomycetemcomitans.     

Multiple paternity in <Emphasis Type="Italic">Rhipicephalus</Emphasis> (<Emphasis Type="Italic">Boophilus</Emphasis>) <Emphasis Type="Italic">microplus</Emphasis> confirmed by microsatellite analysis

The aim of this study was to determine if individual ticks among the progeny of a single female Rhipicephalus (Boophilus) microplus tick removed from cattle under natural conditions are the result of mating with one or several males. To this end, simulations were run using an existing dataset of genotypes from 8 microsatellite loci to predict the number of samples required and the best locus. Subsequently, 14–22 progeny from each of 15 engorged female ticks removed from three cows, and the engorged females themselves, were genotyped for the BmM1 locus and the minimum number of potential male parents was determined for each progeny group. Of the 15 progeny groups, 10 must have been sired by more than one male, as indicated by the presence of five unique alleles among the progeny or three unique alleles that could not have been contributed by the female. This finding demonstrates multiple paternity in R. microplus.     

Evaluation of the toxicity of <Emphasis Type="Italic">Arthrospira</Emphasis> (<Emphasis Type="Italic">Spirulina</Emphasis>) <Emphasis Type="Italic">platensis</Emphasis> extract

In this study, the methanol extract of Arthrospira (Spirulina) platensis was examined for acute and subchronic toxicities. The extract did not produce any sign of toxicity within 7 days after feeding it at a single high dose of 6 g kg⁻¹ body weight to female and male Swiss mice. For the subchronic toxicity test, the extract at doses of 6, 12, and 24 mg kg⁻¹ body weight was orally administered to six male and six female Wistar rats daily for 12 weeks. Throughout the study period, we did not observe any abnormalities on behavior, food and water intakes, and health status among the treated animals. The hematology and clinical chemistry parameters of treated groups did not significantly differ from those of the controls in both sexes. Postmortem examination of the test groups also showed no abnormalities in both gross and histological findings. These results thus suggest that the methanol extract of A. platensis did not cause acute or subchronic toxicity in our experimental animals.     

First record of the tick <Emphasis Type="Italic">Ixodes</Emphasis> (<Emphasis Type="Italic">Pholeoixodes</Emphasis>) <Emphasis Type="Italic">kaiseri</Emphasis> in Turkey

Nymphs and larvae belonging to Ixodes spp. were collected from a red fox in Turkey. The ticks were identified morphologically and molecularly (16S rDNA PCR and phylogenetic analysis) as I. kaiseri. Sequence and phylogenetic analyses show that our I. kaiseri isolate is very similar to I. kaiseri isolates collected from Germany, Serbia, Romania, and Hungary. Therefore, the existence of I. kaiseri has been demonstrated for the first time in Turkey. More studies relating to the regional distribution and vectorial competence of I. kaiseri are needed.     

Swimming restricted foraging behavior of two zooplanktivorous fishes <Emphasis Type="Italic">Pseudorasbora parva</Emphasis> and <Emphasis Type="Italic">Rasbora daniconius</Emphasis> (Cyprinidae) in a simulated structured environment

In littoral zones of aquatic systems, submerged macrophytes have marked structural variation that can modify the foraging activity of planktivores. Swimming and feeding behavior of Pseudorasbora parva and Rasbora daniconius (Cyprinidae) on their prey Daphnia pulex and Artemia salina, respectively, was studied in a series of laboratory experiments with varying stem densities. A range of stem densities was tested for each of the two species to compare the effect of simulated macrophytes on prey attack rates and swimming speed, average stem distance (D) was measured in fish body lengths for each of the two fish species. We found that, with reducing average stem distance, the attack rate decreased in the similar trend and this trend was similar for both fish species. However, the species differed in the degree to which swimming activity was hindered at increased stem densities, and this was due to species-specific differences in the distance moved with one tail beat. Therefore, we conclude that the reductions in swimming speed with reduced average stem distance are due to the differences in fish movement per tail beat.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

<Emphasis Type="Italic">Sex combs reduced</Emphasis> (<Emphasis Type="Italic">Scr</Emphasis>) regulatory region of <Emphasis Type="Italic">Drosophila</Emphasis> revisited

The Hox gene Sex combs reduced (Scr) is responsible for the differentiation of the labial and prothoracic segments in Drosophila. Scr is expressed in several specific tissues throughout embryonic development, following a complex path that must be coordinated by an equally complex regulatory region. Although some cis-regulatory modules (CRMs) have been identified in the Scr regulatory region (~75 kb), there has been no detailed and systematic study of the distinct regulatory elements present within this region. In this study, the Scr regulatory region was revisited with the aim of filling this gap. We focused on the identification of Initiator elements (IEs) that bind segmentation factors, Polycomb response elements (PREs) that are recognized by the Polycomb and Trithorax complexes, as well as insulators and tethering elements. To this end, we summarized all currently available information, mainly obtained from high throughput ChIP data projects. In addition, a bioinformatic analysis based on the evolutionary conservation of regulatory sequences using the software MOTEVO was performed to identify IE and PRE candidates in the Scr region. The results obtained by this combined strategy are largely consistent with the CRMs previously identified in the Scr region and help to: (i) delimit them more accurately, (ii) subdivide two of them into different independent elements, (iii) identify a new CRM, (iv) identify the composition of their binding sites and (v) better define some of their characteristics. These positive results indicate that an approach that integrates functional and bioinformatic data might be useful to characterize other regulatory regions.     

<Emphasis Type="Italic">In silico</Emphasis> and <Emphasis Type="Italic">in situ</Emphasis> characterization of the zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) <Emphasis Type="Italic">gnrh3</Emphasis> (sGnRH) gene

Background

Gonadotropin releasing hormone (GnRH) is responsible for stimulation of gonadotropic hormone (GtH) in the hypothalamus-pituitary-gonadal axis (HPG). The regulatory mechanisms responsible for brain specificity make the promoter attractive for in silico analysis and reporter gene studies in zebrafish (Danio rerio).

Results

We have characterized a zebrafish [Trp⁷, Leu⁸] or salmon (s) GnRH variant, gnrh 3. The gene includes a 1.6 Kb upstream regulatory region and displays the conserved structure of 4 exons and 3 introns, as seen in other species. An in silico defined enhancer at -976 in the zebrafish promoter, containing adjacent binding sites for Oct-1, CREB and Sp1, was predicted in 2 mammalian and 5 teleost GnRH promoters. Reporter gene studies confirmed the importance of this enhancer for cell specific expression in zebrafish. Interestingly the promoter of human GnRH-I, known as mammalian GnRH (mGnRH), was shown capable of driving cell specific reporter gene expression in transgenic zebrafish.

Conclusions

The characterized zebrafish Gnrh3 decapeptide exhibits complete homology to the Atlantic salmon (Salmo salar) GnRH-III variant. In silico analysis of mammalian and teleost GnRH promoters revealed a conserved enhancer possessing binding sites for Oct-1, CREB and Sp1. Transgenic and transient reporter gene expression in zebrafish larvae, confirmed the importance of the in silico defined zebrafish enhancer at -976. The capability of the human GnRH-I promoter of directing cell specific reporter gene expression in zebrafish supports orthology between GnRH-I and GnRH-III.

     

<Emphasis Type="Italic">Myrmica</Emphasis> host-ants limit the density of the ant-predatory large blue <Emphasis Type="Italic">Maculinea nausithous</Emphasis>

Butterflies of the highly endangered genus Maculinea are parasites of red Myrmica ants. Prior to the adoption by Myrmica worker ants Maculinea caterpillars feed on a specific host plant. This field study aims to answer the question whether the density and distribution of the host plant Sanguisorba officinalis or the density of the host ant M. rubra limit the density of M. nausithous egg, larval and adult stage. We found that the density of M. nausithous egg stage and adult stage increased with the density of the host ant. The density of M. nausithous caterpillars was not associated with ant density or plant density. This study suggests that the density of M. nausithous is limited by the density of the host ant M. rubra. We conclude that habitat management for M. nausithous should focus on the maintenance of habitats that hold both resources, but that enable high densities of M. rubra. In addition, it is discussed why high densities of host ants might be more important in predatory than in cuckoo-feeding Maculinea.     

Photoregulation of morphological structure and its physiological relevance in the cyanobacterium <Emphasis Type="Italic">Arthrospira</Emphasis> (<Emphasis Type="Italic">Spirulina</Emphasis>) <Emphasis Type="Italic">platensis</Emphasis>

The spiral structure of the cyanobacterium Arthrospira (Spirulina) platensis (Nordst.) Gomont was previously found to be altered by solar ultraviolet radiation (UVR, 280–400 nm). However, how photosynthetic active radiation (PAR, 400–700 nm) and UVR interact in regulating this morphological change remains unknown. Here, we show that the spiral structure of A. platensis (D-0083) was compressed under PAR alone at 30°C, but that at 20°C, the spirals compressed only when exposed to PAR with added UVR, and that UVR alone (the PAR was filtered out) did not tighten the spiral structure, although its presence accelerated morphological regulation by PAR. Their helix pitch decreased linearly as the cells received increased PAR doses, and was reversible when they were transferred back to low PAR levels. SDS-PAGE analysis showed that a 52.0 kDa periplasmic protein was more abundant in tighter filaments, which may have been responsible for the spiral compression. This spiral change together with the increased abundance of the protein made the cells more resistant to high PAR as well as UVR, resulting in a higher photochemical yield.