Diversity of the Bacterial Communities Associated with the Azooxanthellate Deep Water Octocorals <Emphasis Type="Italic">Leptogorgia minimata</Emphasis>, <Emphasis Type="Italic">Iciligorgia schrammi</Emphasis>, and <Emphasis Type="Italic">Swiftia exertia</Emphasis>

This study examined the microbiota associated with the marine azooxanthellate octocorals Leptogorgia minimata, Swiftia exertia, and Iciligorgia schrammi collected from moderate depths (45 m). Traditional aerobic plate culture, fluorescence in situ hybridization (FISH), and molecular identification of the 16S rDNA region were used for this purpose. In general, cultures were found to be selective for Gammaproteobacteria, Alphaproteobacteria, and Firmicutes. Interestingly, FISH counts for Firmicutes in the whole coral (holobiont) were near the detection limit of this assay, representing less than 6% of the total detectable microbiota in all counts. Proteobacteria, especially Alpha- and Gammaproteobacteria, made up the majority of the total microbiota in the holobionts. In addition, the absence of zooxanthellae in these three corals was confirmed by the use of polymerase chain reaction (PCR) and dinoflagellate-specific primers, and spectrophotometric chlorophyll pigment measurements. No evidence of zooxanthellae could be found in any of the corals by either of these techniques. This is the first study examining the microbiota marine octocorals, which grow at moderate depth (40 to 100 m) in the absence of direct sunlight. Thomas B. Brück and Wolfram M. Brück have made equal contributions to this publication.     

Characterization of <Emphasis Type="Italic">Francisella</Emphasis> sp., GM2212, the first <Emphasis Type="Italic">Francisella</Emphasis> isolate from marine fish,Atlantic cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>)

A Francisella sp., isolate GM2212^T, previously isolated from diseased farmed Atlantic cod Gadus morhua in Norway is characterized. The complete 16S rDNA, 16S–23S intergenic spacer, 23S rDNA, 23S–5S intergenic spacer, 5S rDNA, FopA, lipoprotein TUL4 (LpnA), malate dehydrogenase and a hypothetical lipoprotein (LpnB) is sequenced and compared with Francisella tularensis and Francisella philomiragia. All these sequences support a close relationship between GM2212^T and F. philomiragia. The bacterium grows at 10–25°C with an optimum at about 20°C, a temperature range clearly different from F. tularensis and F. philomiragia. GM2212^T is catalase-positive, indole positive, oxidase-negative, do not produce H₂S in Triple Sugar Iron agar, and does not hydrolyze gelatin, is resistant to erythromycin and susceptible to ceftazidime, the latter five characteristics separating it from F. philomiragia. Cysteine enhances growth. Acid is produced from d-glucose, maltose, sucrose (weak) but not from lactose or glycerol. GM2212^T grows on both MacConkey agar and in nutrient broth (6% NaCl). The bacterium is resistant to trimethoprim-sulfamethoxazole, penicillines, cefuroxime and erythromycin; but is susceptible to ceftazidime, tetracycline, gentamicin, ciprofloxacin. Based on the molecular and phenotypical characteristics, we suggest that this GM2212 isolate, may represent a new species of Francisella. Isolate GM2212^T (=CNCM I-3481^T = CNCM I-3511^T = DSM 18777^T).     

Bacterial Communities Associated with <Emphasis Type="Italic">Chenopodium album</Emphasis> and <Emphasis Type="Italic">Stellaria media</Emphasis> Seeds from Arable Soils

The bacterial community compositions in Chenopodium album and Stellaria media seeds recovered from soil (soil weed seedbank), from bulk soil, and from seeds harvested from plants grown in the same soils were compared. It was hypothesized that bacterial communities in soil weed seedbanks are distinct from the ones present in bulk soils. For that purpose, bacterial polymerase chain reaction denaturing gradient gel electrophoresis (PCR–DGGE) fingerprints, made from DNA extracts of different soils and seed fractions, were analyzed by principal component analysis. Bacterial fingerprints from C. album and S. media seeds differed from each other and from soil. Further, it revealed that bacterial fingerprints from soil-recovered and plant-harvested seeds from the same species clustered together. Hence, it was concluded that microbial communities associated with seeds in soil mostly originated from the mother plant and not from soil. In addition, the results indicated that the presence of a weed seedbank in arable soils can increase soil microbial diversity. Thus, a change in species composition or size of the soil weed seedbank, for instance, as a result of a change in crop management, could affect soil microbial diversity. The consequence of increased diversity is yet unknown, but by virtue of identification of dominant bands in PCR–DGGE fingerprints as Lysobacter oryzae (among four other species), it became clear that bacteria potentially antagonizing phytopathogens dominate in C. album seeds in soil. The role of these potential antagonists on weed and crop plant growth was discussed.     

Population variation in thermal growth responses of juvenile Atlantic cod (<Emphasis Type="Italic">Gadus morhua</Emphasis> L.)

Controlled environment experiments were carried out to investigate thermal influences and population differences on growth of wild-caught juvenile Atlantic cod Gadus morhua L. from two regions of differing thermal regime off Scotland; the Clyde Sea on the west coast and St Andrews Bay on the east coast. Cod from the Clyde demonstrated significantly higher growth rates than cod from St Andrews. In both populations the growth rate was greater at 12°C than at 8°C. These population and temperature effects act to reinforce one another and it could therefore be predicted that the growth differences between the two areas in the wild should be even more pronounced. The results are consistent with the suggestion that cod may be locally adapted to their thermal environment.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Preliminary data on the variability of three microsatellite loci in Pacific <Emphasis Type="Italic">Gadus macrocephalus</Emphasis> and Atlantic <Emphasis Type="Italic">G. morhua</Emphasis> cod (Gadidae)

Variability of microsatellite DNA loci Gmo3, Gmo34, and Gmo35 is studied in samples of Pacific cod Gadus macrocephalus and Atlantic cod G. morhua. The results show high values of identity of the samples within the North Pacific basin (0.9766–0.9924) and within the Northeast Atlantic basin (0.9580). Based on the pairwise assessment of genetic differentiation, the F _ST values are significantly different in all variants between the samples of Pacific and Atlantic cod (F _ST = 0.5235–0.6719, p < 0.001). Within the basins, the significant differences in the frequencies of main alleles are revealed in the loci Gmo3 and Gmo34 for the samples from the Pacific and Atlantic oceans, respectively.     

Occurrence of the tuna nematode <Emphasis Type="Italic">Hysterothylacium cornutum</Emphasis> (Stossich, 1904) in farmed Atlantic cod <Emphasis Type="Italic">Gadus morhua</Emphasis> L. in North Norway

Adult and fourth-stage larval nematodes found in the stomachs of farmed cod in North Norway in October 2006 were identified as Hysterothylacium cornutum (Stossich, 1904), a nematode considered to be specific at the adult stage to tunas of the genus Thunnus. As far as we are aware, this is the first report of an adult form of this nematode from any host other than members of the genus Thunnus, and is also the first report from a polar region. The two infected cod, one with one large adult worm and another with six larvae, were in a sample of 17 that had been captured from the wild about 1 year before sampling and held in floating sea cages in Øksfjord, Finnmark County, North Norway, where the examinations took place. No further infections were found in any other samples of wild and farmed cod, totalling 261 fish, examined by us during the period 2006 and 2007 from five locations distributed along the coast of Norway from Øksfjord in the north to Ålesund in the south. Possible sources of this unusual infection are discussed, but no firm conclusion could be reached.     

Diversity and recombination in <Emphasis Type="Italic">Wolbachia</Emphasis> and <Emphasis Type="Italic">Cardinium</Emphasis> from <Emphasis Type="Italic">Bryobia</Emphasis>spider mites

BACKGROUND: Wolbachia and Cardinium are endosymbiotic bacteria infecting many arthropods and manipulating host reproduction. Although these bacteria are maternally transmitted, incongruencies between phylogenies of host and parasite suggest an additional role for occasional horizontal transmission. Consistent with this view is the strong evidence for recombination in Wolbachia, although it is less clear to what extent recombination drives diversification within single host species and genera. Furthermore, little is known concerning the population structures of other insect endosymbionts which co-infect with Wolbachia, such as Cardinium. Here, we explore Wolbachia and Cardinium strain diversity within nine spider mite species (Tetranychidae) from 38 populations, and quantify the contribution of recombination compared to point mutation in generating Wolbachia diversity. RESULTS: We found a high level of genetic diversity for Wolbachia, with 36 unique strains detected (64 investigated mite individuals). Sequence data from four Wolbachia genes suggest that new alleles are 7.5 to 11 times more likely to be generated by recombination than point mutation. Consistent with previous reports on more diverse host samples, our data did not reveal evidence for co-evolution of Wolbachia with its host. Cardinium was less frequently found in the mites, but also showed a high level of diversity, with eight unique strains detected in 15 individuals on the basis of only two genes. A lack of congruence among host and Cardinium phylogenies was observed. CONCLUSIONS: We found a high rate of recombination for Wolbachia strains obtained from host species of the spider mite family Tetranychidae, comparable to rates found for horizontally transmitted bacteria. This suggests frequent horizontal transmission of Wolbachia and/or frequent horizontal transfer of single genes. Our findings strengthens earlier reports of recombination for Wolbachia, and shows that high recombination rates are also present on strains from a restrictive host range. Cardinium was found co-infecting several spider mite species, and phylogenetic comparisons suggest also horizontal transmission of Cardinium among hosts.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<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">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

The effect of food odor background on gustatory preferences and gustatory behavior of carp <Emphasis Type="Italic">Cyprinus carpio</Emphasis> and cod <Emphasis Type="Italic">Gadus morhua</Emphasis>

It was shown that stimulation by food odor (aquatic extract of food organisms, 10^?2 and 10^?3 g/l) does not cause shifts in gustatory preferences in carp Cyprinus carpio and cod Gadus morhua but modifies gustatory behavior. The level of consumption by carp of control granules and granules with attractive, by taste, L-proline (0.1 M) or deterrent L-lysine (0.1 M) (item by item presentation of granules) and by cod of control granules and granules with indifferent, to it, L-asparagine (0.1 M) (presentation of 10 granules simultaneously) is similar prior to and during olfactory stimulation. In the presence of food odor, the duration of taste testing for most types of granules, as well as the number of repeated graspings of granules with an attractive taste do not change in fish. At the same time, granules with indifferent or repulsive gustatory properties are rejected and repeatedly grasped by fish against the background of food odor more frequently than in water without odor. Olfactory stimulation leads to a considerable increase in the average number of graspings per one grasped granule with an indifferent or repulsive taste. Such behavior manifested by fish in the presence of food odor in response to granules with unattractive gustatory properties is apparently caused by the contradiction between the information coming via different chemosensory canals—olfactory and gustatory. The obtained results indicate that food stimulation caused by food odor in nature can lead to an increase in the actual consumption of only those accessible food items that have an attractive taste for fish.     

Domestication causes rapid changes in heart and brain morphology in Atlantic cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>)

Brain and heart development is very plastic in teleost fishes, and receptive to changes in social and environmental conditions. Domestication in salmonids has been reported to result in pronounced changes in both heart and brain morphology. In particular, a high prevalence of heart deformities has been reported in farmed salmonids, which has been linked to increased stress responsiveness that can impair survival of both farmed and escaped fish. Here we report for the first time that significant changes in heart and brain morphology occur following domestication of Atlantic cod (Gadus morhua), an emerging aquaculture species. Juvenile farmed cod developed significantly larger hearts and smaller brains, by weight, compared to their wild conspecifics. These differences occurred within the first captive generation, suggesting that they were driven largely by the strong contrast in environmental and social conditions experienced within their respective rearing environments. Changes in brain and heart morphology, as a consequence of domestication could affect the well-being and survival of Atlantic cod raised under intensive aquaculture conditions.     

New species in the genus <Emphasis Type="Italic">Francisella</Emphasis> (Gammaproteobacteria; Francisellaceae); <Emphasis Type="Italic">Francisella piscicida</Emphasis> sp. nov. isolated from cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>)

A Francisella strain, GM2212, previously isolated from moribund farmed Atlantic cod (Gadus morhua) in Norway, is closely related to Francisella philomiragia among Francisella spp. according to its complete 16S rDNA, 16S-23S intergenic spacer, 23S rDNA, 23S–5S intergenic spacer, 5S rDNA, FopA, lipoprotein TUL4 (LpnA), malate dehydrogenase and hypothetical lipoprotein (LpnB) sequences. A comparison between GM2212 and the type strain of Francisella philomiragia were performed by DNA–DNA hybridization and fatty acid analysis. The DNA–DNA hybridization showed a 70% similarity. The fatty acid analysis showed only minor differences between the Francisella isolates. Due to the inconclusive result from the DNA–DNA hybridisation, major emphasis concerning the status of this isolate is made on previously published molecular, phenotypic and biochemical characters. All characteristics taken together support the establishment of GM2212 as a novel species, for which the name Francisella piscicida sp. nov. is proposed (=CNCM I-3511^T = DSM 18777^T = LMG registration number not yet available).     

Ectomycorrhizae of <Emphasis Type="Italic">Tuber huidongense</Emphasis> and <Emphasis Type="Italic">T. liyuanum</Emphasis> with <Emphasis Type="Italic">Castanea mollissima</Emphasis> and <Emphasis Type="Italic">Pinus armandii</Emphasis>

Tuber huidongense and T. liyuanum are common commercial white truffles in China that belong to the Rufum and Puberulum groups of the genus Tuber, respectively. Their mycorrhizae were successfully synthesized with two native trees—Castanea mollissima and Pinus armandii—under greenhouse conditions. The identities of the mycorrhizae were confirmed through internal transcribed spacer (ITS) sequence analyses, and their morphological characteristics were described. All of the obtained mycorrhizae have an interlocking pseudoparenchymatous mantle, which is a typical feature of truffle mycorrhizae. The mycorrhizae of T. huidongense on the two trees have hyaline branched emanating hyphae, similar to the documented mycorrhizae of the Rufum group. The unramified, spiky, and hyaline cystidia on the mycorrhizae of T. liyuanum with both C. mollissima and P. armandii further confirmed that this characteristic is constant for the mycorrhizae of the Puberulum group. The successful mycorrhizal syntheses on the two nut-producing trees will be of economic importance in the cultivation of the two truffles.     

The tyrosine <Emphasis Type="Italic">O</Emphasis>-prenyltransferase SirD catalyzes <Emphasis Type="Italic">O</Emphasis>-, <Emphasis Type="Italic">N</Emphasis>-, and <Emphasis Type="Italic">C</Emphasis>-prenylations

Recently, the prenyltransferase SirD was found to be responsible for the O-prenylation of tyrosine in the biosynthesis of sirodesmin PL in Leptosphaeria maculans. In this study, the behavior of SirD towards phenylalanine/tyrosine and tryptophan derivatives was investigated. Product formation has been observed with 12 of 19 phenylalanine/tyrosine derivatives. It was shown that the alanine structure attached to the benzene ring and an electron donor, e.g., OH or NH₂, at its para-position are essential for the enzyme activity. Modifications were possible both at the side chain and the benzene ring. Enzyme products from seven phenylalanine/tyrosine derivatives were isolated and characterized by MS and NMR analyses including HSQC and HMBC and proven to be O- or N-prenylated derivatives at position C4 of the benzene rings. K _M values of six selected derivatives were found in the range of 0.10–0.68 mM. Catalytic efficiencies (K _cat/K _M ) were determined in the range of 430–1,110 s⁻¹·M⁻¹ with l-tyrosine as the best substrate. In addition, 7 of 14 tested tryptophan analogs were also accepted by SirD and converted to C7-prenylated derivatives, which was confirmed by comparison with products obtained from enzyme assays using a 7-dimethylallyltryptophan synthase 7-DMATS from Aspergillus fumigatus.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.