Transfection of siRNA into <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> (Rotifera)

Lake Taihu is one of the most contaminated lakes in China. Surface sediment data show that the northern area of the Lake has the worst heavy metals pollution, and high heavy metal concentrations were attributed to discharge of untreated and partially treated industrial waste water from cities to the north of the lake. To study geochemical features and pollution history of heavy metals, total content and chemical fractionations of Cu, Fe, Mn, Ni, Pb, and Zn were analyzed for core sediments from western Lake Taihu using the speciation extraction procedure, proposed by the Commission of the European Communities Bureau of Reference (BCR), together with grain size and organic carbon measurements. Results show that sediments are composed of organic-poor clayey-fine silts for Cores MS and DLS, and have similar geochemical features shown by heavy metals. Cu, Fe, Ni, and Zn mainly are associated with the residue fraction, Mn is concentrated in the exchangeable-carbonate and residue fractions, and Pb is concentrated in the Fe–Mn oxide fraction and organic-sulfide fraction. The fractions of Ni, Pb, and Zn bound to Fe–Mn oxide show significant correlations with Mn from the Fe–Mn oxide fraction, and the organic-sulfide fractions of Cu, Mn, Ni, Pb, and Zn are correlated with TOC. The increase of Cu, Mn, Ni, Pb and Zn content and percentage of extractable fractions in the upper layers of the sediments are correlated with anthropogenic input of heavy metals due to rapid industrial development. This coincides with rapid economic development in the Taihu basin since late 1970s. Heavy metals in the surface sediments have certain potential biological toxicity as shown by the higher SEM/AVS ratio.     

Combined Effects of Algal (<Emphasis Type="Italic">Chlorella vulgaris</Emphasis>) Food Level and Temperature on the Demography of <Emphasis Type="Italic">Brachionus havanaensis</Emphasis> (Rotifera): a Life Table Study

We evaluated the combined effects of food (0.5 × 10⁶, 1.0 × 10⁶ and 2.0 × 10⁶ cells ml⁻¹ of Chlorella vulgaris) and temperature (15, 20 and 25 °C) on life history variables of B. havanaensis. Regardless of Chlorella density there was a steep fall in the survivorship of B. havanaensis at 25 °C. Both food level and temperature affected the fecundity of B. havanaensis. At any given food level, rotifers cultured at 15 °C showed extended but low offspring production. At 25 °C, offspring production was elevated, the duration of egg laying reduced and the fecundity was higher during the latter part of the reproductive period. The effect of food level was generally additive, at any given temperature, and higher densities of Chlorella resulted in higher offspring production. Average lifespan, life expectancy at birth and generation time were 2–3 times longer at 15 °C than at 25 °C. At 20 °C, these remained at intermediate levels. The shortest generation time (about 4 days) was observed at 25 °C. Gross and net reproductive rates and the rate of population increase (r) increased with increasing temperature and generally, at any given temperature, higher algal food levels contributed to higher values in these variables. The r varied from 0.11 to 0.66. The survival patterns and lower rates of reproduction at 15 °C suggest that the winter temperatures (10–15 °C) prevailing in many waterbodies in Mexico City allow this species to sustain throughout the year under natural conditions.     

Mate choice and sexual conflict in <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> (Rotifera)

Male mate choice in Brachionus plicatilis is based on information from coronal receptors and contact chemoreception of a glycoprotein signal on the body surface of females. Male mating behavior is modulated by mating signal quality and strength, which determines male mating persistence and circling intensity. We probe the sensory abilities of males by better resolving their discrimination of female age. Males preferentially initiate mating with young females, but not too young. Males circle 0.2 h old females just hatched from resting eggs only about 1/2 as frequently as 12 h old females. Males are even more discriminating of females with whom they copulate, preferring 3 h old females significantly more than 0.2 h or 6 h old females. In contrast, males cannot distinguish between virgin females and those who have already copulated. Similarly, males circled and copulated with females hatching from resting eggs with equal frequency as those hatching from amictic eggs. The counterpoint to male mate choice is female resistance to mating. When males contact females, the females respond with one of four behaviors: no response, acceleration, foot flipping, or coronal retraction. In 65% of male–female encounters, there was no initial response by females. However, when males began circling females, females accelerated 11.1 times more often than when males were absent. The second type of evasive female behavior was foot-flipping, which tends to knock off males attempting to circle the female. In the presence of circling males, females performed foot-flipping behavior three times more often than in the absence of males. Coronal retraction, where they stop swimming and withdraw their corona, was observed less frequently than acceleration or foot-flipping, and there was no difference in the presence or absence of males. These data are interpreted in the context of sexual conflict, where the behaviors that optimize male and female fitness differ. Guest editors: S. S. S. Sarma, R. D. Gulati, R. L. Wallace, S. Nandini, H. J. Dumont & R. Rico-Martínez Advances in Rotifer Research     

Combined effects of temperature,food (<Emphasis Type="Italic">Chlorella vulgaris</Emphasis>) concentration and predation (<Emphasis Type="Italic">Asplanchna girodi</Emphasis>) on the morphology of <Emphasis Type="Italic">Brachionus havanaensis</Emphasis> (Rotifera)

The combined effect of temperature, food level and the presence of an invertebrate predator on the body size of the rotifer Brachionus havanaensis were tested in this study. B. havanaensis was cultured at 15, 20, and 25°C under three different Chlorella vulgaris levels (0.5 × 10⁶, 1.0 × 10⁶ and 2.0 × 10⁶ cells ml⁻¹) in the presence and in the absence of Asplanchna girodi. For each treatment we maintained three replicates and constant (0.4 ind ml⁻¹) population density of B. havanaensis. In treatments containing A. girodi, the predator was separated from the prey by a mesh (pore size 50 μm). On the last day of the experiment, a portion of the B. havanaensis population was sampled for several morphometric measurements (adult lorica length, width, posterior spine length, body volume, and the egg volume). Size measurements were done by drawing the specimens using a calibrated camera lucida. Statistically significant impact of temperature as well as the predator’s presence was observed on the lorica length, posterior spine, and egg volume of B. havanaensis. The interactions of food × temperature, or predator′s presence × food × temperature were non-significant (P > 0.05) for lorica length, spine length, body volume, and egg volume. Regardless of the type of treatment, there was a direct positive correlation between lorica length and width. Egg volume was linearly related to the adult size. Notably long posterior spines were observed in treatments containing the presence of A. girodi. Guest editors: S. S. S. Sarma, R. D. Gulati, R. L. Wallace, S. Nandini, H. J. Dumont & R. Rico-Martínez. Advances in Rotifer Research     

Toxicity of Crude Extracellular Products of <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> on Rohu, <Emphasis Type="Italic">Labeo rohita</Emphasis> (Ham.)

In the present study the haemolytic and proteolytic activity of extracellular products (ECP) secreted from Aeromonas hydrophila (CAHH14 strain) were studied with respect to temperature and different time of incubation as well as its lethal toxicity on rohu, Labeo rohita. The strain was isolated from Catla catla (showing abdominal dropsy symptom) collected from the pond of Central Institute of Freshwater Aquaculture (CIFA), Bhubaneswar, India and was characterized on the basis of biochemical tests. The highest production of haemolysin was achieved when the bacteria was grown at 35°C for 30 h. The proteolytic activity was found to be highest when the bacterium was grown at 30°C for 36 h. The haemolytic and proteolytic toxin produced by Aeromonas hydrophila was found to be lethal to rohu (LD₅₀ 1.7 × 10⁴ cfu/ml). The lethality of ECP was decreased by heating and completely inactivated by boiling at 100°C for 10 min. This indicates that protease activity and haemolytic activity of A. hydrophila ECP was temperature dependant.     

A comparative study on the protective role of trehalose and LEA proteins against abiotic stresses in transgenic Chinese cabbage (<Emphasis Type="Italic">Brassica campestris</Emphasis>) overexpressing<Emphasis Type="Italic">CaLEA</Emphasis> or<Emphasis Type="Italic">otsA</Emphasis>

Trehalose and LEA proteins, representative low MW chemicals that are synthesized under dehydration, are known to protect plants from drought stress. To compare their effectiveness on enhancing tolerance against various abiotic stresses, we generated transgenic Chinese cabbage plants overexpressingE. ctdi trehalose-6-phosphate synthase gene (otsA) or hot pepper (Capsicum annuum) LEA protein gene(CaLEA). Both transgenic plants exhibited altered phenotype including stunted growth and aberrant root development When subjected to drought, salt or heat stress, these plants showed remarkably improved tolerance against those stresses compared with nontransformants. After dehydration treatment, leaf turgidity and fresh weight was better maintained in both transgenic plants. GaUEA-plants performed somewhat better under dehydrated condition. When treated with 250 mM NaCI, both otsA-plants and CaLEA-plants remained equally healthier than nontransformants in maintaining leaf turgidity and delaying necrosis. Furthermore, leaf Chi content and Fv/Fm was maintained considerably higher in both transgenic plants than nontransformants. After heat-treatment at 45°C, both transgenic plants appeared much less damaged in external shape and PSII function, but LEA proteins were more protective. Our results indicate that although both trehalose and LEA proteins are effective in protecting plants against various abiotic stresses, LEA proteins seem to be more promising in generating stress-tolerant transgenic plants.     

New combinations and typifications in <Emphasis Type="Italic">Bistorta</Emphasis>, <Emphasis Type="Italic">Persicaria</Emphasis>, <Emphasis Type="Italic">Polygonum,</Emphasis> and <Emphasis Type="Italic">Rumex</Emphasis> (Polygonaceae)

New combinations are proposed in anticipation of the Polygonaceae treatment in the forthcoming volume of Intermountain Flora: Polygonum kelloggii var. esotericum, P. kelloggii var. watsonii , Rumex densiflorus var. pycnanthus , R. salicifolius var. utahensis, and R. occidentalis var. tomentellus. Typifications are proposed to facilitate ongoing studies in Polygonaceae and to maintain current usage.     

<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.     

<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">Brachionus calyciflorus</Emphasis> is a Species Complex: Mating Behavior and Genetic Differentiation Among Four Geographically Isolated Strains

Four geographic strains of B. calyciflorus are investigated regarding their genetic similarity and ability to cross-mate. DNA sequence analysis of the mitochondrial cox1 gene (694 bp) and the nuclear ribosomal ITS region (735 bp) showed that the Florida and Georgia strains were very similar to each other (0.3% sequence divergence for the 1429 bp) and different from the Texas and Australia strains (~7% and 9% sequence divergence for the 1429 bp, respectively). Consistent with this genetic relatedness, cross-copulation occurred only between the Florida and Georgia strains. Thus, B. calyciflorus is a complex of cryptic species. While the Florida, Texas and Australia strains were reproductively isolated from one another, most combinations of cross-strain mating tests showed intense and prolonged male circling behavior following male–female encounters. This suggests that precopulatory male circling and copulation are two separate behaviors that may be controlled by different female chemicals and male coronal receptors. In some cross-strain mating tests, females regularly retracted their corona when circled by a male, indicating that they can recognize ‘foreign’ males and actively interfere with copulation.     

Micropropagation of <Emphasis Type="Italic">Ailanthus altissima</Emphasis> and <Emphasis Type="Italic">in vitro</Emphasis> heavy metal tolerance

Ailanthus altissima, a fast-growing and contamination-resistant species is investigated for its use in areas contaminated by heavy metals. A micropropagation protocol for A. altissima was developed, cultured shoots were tested for in vitro heavy metals tolerance. Proliferation rate and shoot length were affected by 6-benzylaminopurine (BAP) and Murashige and Skoog’s (MS) salt concentrations, best results were obtained in full strength MS medium supplemented with 1.32 or 2.64 μM BAP. Rooting percentage was strongly influenced by indole-3-butyric acid. Cultures of A. altissima exposed to heavy metals demonstrated a tolerance comparable to species already utilized in phytoremediation.     

Heart Rate in the Blue Mussel <Emphasis Type="Italic">Mytilus edulis</Emphasis> (Bivalvia) under Salinity Change

The heart rate (HR) was studied applying noninvasive monitoring in the blue mussel Mytilus edulis L. from the White Sea. It was shown that the sublittoral mussels had a higher cardiac activity than the littoral mussels. Mollusks responded to a drop in salinity from 25 to 15‰ with a decrease in HR, which was more pronounced in sublittoral individuals. Acclimated mollusks sharply increased their HR upon being returned to the initial salinity (deacclimation). The cardiac activity normalized completely within two days after the beginning of the deacclimation.     

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.     

Floral development and systematic position of <Emphasis Type="Italic">Arillastrum</Emphasis>, <Emphasis Type="Italic">Allosyncarpia</Emphasis>, <Emphasis Type="Italic">Stockwellia</Emphasis> and <Emphasis Type="Italic">Eucalyptopsis</Emphasis> (Myrtaceae)

We have investigated the floral ontogeny of Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis (of the eucalypt group, Myrtaceae) using scanning electron microscopy and light microscopy. Several critical characters for establishing relationships between these genera and to the eucalypts have been determined. The absence of compound petaline primordia in Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis excludes these taxa from the eucalypt clade. Post-anthesis circumscissile abscission of the hypanthium above the ovary in Stockwellia, Eucalyptopsis and Allosyncarpia is evidence that these three taxa form a monophyletic group; undifferentiated perianth parts and elongated fusiform buds are characters that unite Stockwellia and Eucalyptopsis as sister taxa. No floral characters clearly associate Arillastrum with either the eucalypt clade or the clade of Stockwellia, Eucalyptopsis and Allosyncarpia.We gratefully acknowledge Clyde Dunlop and Bob Harwood (Northern Territory Herbarium) for collecting specimens of Allosyncarpia, and Bruce Gray (Atherton) for collecting specimens of Stockwellia. The Australian National Herbarium (CANB) kindly lent herbarium specimens of Eucalyptopsis for examination. This research was supported by a University of Melbourne Research Development Grant to Andrew Drinnan.     

Pedogamy in <Emphasis Type="Italic">Neidium</Emphasis> (<Emphasis Type="Italic">Bacillariophyceae</Emphasis>)

Single (unpaired) vegetative cells of freshwater pennate diatom Neidium cf. ampliatum differentiated into gametangia and produced a single zygote (auxospore) via a pedogamic process. The gametic nuclei fused after auxospore expansion had begun. The auxospore expanded in parallel to the apical axis of the gametangium.     

<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.     

Sociality in <Emphasis Type="Italic">Euglossa</Emphasis> (<Emphasis Type="Italic">Euglossa</Emphasis>) <Emphasis Type="Italic">viridissima</Emphasis> Friese (Hymenoptera,Apidae, Euglossini)

Euglossa viridissima is an orchid bee that forms both solitary and multiple female nests, making it a suitable species for the study of factors leading to diverse degrees of sociality in Euglossines. We conducted observations in eight reused nests (where a first generation of bees had been produced) kept in artificial boxes from the Yucatan Peninsula, Mexico. Five nests were reused (reactivated) by a single female (SFN), two nests reused by a mother and one daughter (MFN1) and one nest reused by the mother and two daughters (MFN2). No single nest was reactivated by unrelated females. The number of foraging trips, their duration and the duration of cell provisioning was not different between SFN and MFN. The overall production of cells per female was not different either between both types of nest. However, in MFN although all females did lay eggs, there was a reproductive skew in favor of the mother (95 and 45% of the brood produced in MFN1 and MFN2 respectively). She showed reproductive control of her daughters through oophagy and displaying threatening behavior when the daughters tried to open a cell where she had laid an egg. Brood losses to parasites (Anthrax sp. (Bom-byliidae) and Hoplostelis bivittata (Megachilidae)) were only found in SFN which possibly reflects and advantage of MFN in this respect. Our results coupled with other studies in Euglossa, reveal that a wide range of social behaviors occur in this genus, from solitary and communal to primitive reproductive division of labor. Multiple factors involving different levels of pressure imposed by food availability and parasites may favor such a diverse range of nesting behaviors. Interestingly, female associations in E. viridissima seem a result of kin selection that is enforced by coercion from mother females on their daughters. More studies are needed to shed light upon the social organization of Euglossa and other Euglossines and on their phylogenetic relationships in order to trace the origins of eusociality in Apidae. Received 12 February 2008; revised 25 June 2008; accepted 17 July 2008.     

Replication timing of large <Emphasis Type="Italic">Sorex granarius</Emphasis> (<Emphasis Type="Italic">Soricidae</Emphasis>, <Emphasis Type="Italic">Eulipotyphla</Emphasis>) telomeres

Previously, we described the unique feature of telomeric regions in Iberian shrew Sorex granarius: its telomeres have two ranges of size, very small (3.8 kb of telomeric repeats on average) and very large discontinuous telomeres (213 kb) interrupted with 18S rDNA. In this study, we have demonstrated extraordinary replication pattern of S. granarius large telomeres that have not been shown before in other studied mammal. Using the ReD-FISH procedure, we observed prolonged, through S period, large telomere replication. Furthermore, revealed ReD-FISH asymmetric signals were probably caused by partial replication of telomeres within an hour of 5-bromodeoxyuridine treatment due to the large size and special organization. We also found that in contrast to the telomeric halo from primary fibroblasts of bovine, mink, and common shrew, telomere halo of S. granarius consists of multiple loops bundled together, some of which contain rDNA. Here, we suggested several replicons firing possibly stochastic in each large telomere. Finally, we performed the TIF assay to reveal DNA damage responses at the telomeres, and along with TIF in nuclei, we found large bodies of telomeric DNA and ?-H2AX in the cytoplasm and on the surface of fibroblasts. We discuss the possibility of additional origin activation together with recombination-dependent replication pathways, mainly homologous recombination including BIR for replication fork stagnation overcoming and further S. granarius large telomere replication.