Axenic culture of <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> using antibiotics

The rotifer Brachionus plicatilis culture is composed of complex microcosms including bacteria, protozoans, algae, and fungi. Previous studies reported methods to establish axenic rotifer cultures, but further refinement of these techniques is needed, for molecular biological research which requires pure culture to isolate nucleic acids from rotifers only. In order to render rotifer culture axenic, we tested five antibiotics: ampicillin (Amp), chloramphenicol (Cp), kanamycin (Km), nalidixic acid (Na), and streptomycin (Sm) at 30–100 μg/ml. Except for Cp, which reduces rotifer reproduction, all other antibiotics at the tested concentrations did not affect rotifer reproduction or show any toxic effects. A rotifer disinfection method was finally established by treating the resting eggs with 0.25% (w/v) sodium hypochlorite (NaOCl) for 3 min, washing with sterilized sea water, and then exposing the neonates to an Amp, Km, Na, and Sm mixture. Using four nutrient media, we confirmed that this protocol renders the rotifer culture bacterial and fungus free. The axenic rotifer culture generated here is useful not only for genetic analysis of Brachionus plicatilis, but for studying the rotifer life cycle without bacterial influence.     

Inheritance of mitochondrial DNA in the rotifer <Emphasis Type="Italic">Brachionus plicatilis</Emphasis>

By crossing Brachionus plicatilis s.s. NH1L strain and German strain, we obtained two types of hybrids, NH1L female × German male designated as NXG and German female × NH1L male designated as GXN. To confirm the crossing of the two hybrid strains at the genetic level, random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis using 10 kinds of primers (10 and 12 mers) was carried out. Some amplified DNA fragments from RAPD of the hybrid strain showed mixed patterns of both parental strains, thus confirming that both hybrids were crossbreeds of the NH1L and German strains. Using these hybrids, we investigated the mode of mitochondrial inheritance in B. plicatilis. Full-length mtDNA of the four strains was amplified by PCR, and digested with restriction enzymes to obtain restriction fragment length polymorphism (RFLP) patterns. Both hybrid strains had the same RFLP patterns as their female parents. This result shows that mitochondrial inheritance in rotifers is maternal. Guest editors: S. S. S. Sarma, R. D. Gulati, R. L. Wallace, S. Nandini, H. J. Dumont and R. Rico-Martínez Advances in Rotifer Research     

Factors Affecting Swimming Speed in the Rotifer <Emphasis Type="Italic">Brachionus plicatilis</Emphasis>

This study examines the swimming speed in amictic females of Brachionus plicatilis in laboratory cultures. Five different stages were examined: recently hatched females, juveniles, adult non-ovigerous females, ovigerous females with 1 attached egg and ovigerous females with 2 attached eggs. We tested the speed at two temperatures, 15 °C and 25 °C, and two feeding conditions, presence and absence of microalgal cells. An automated motion analysis system was used to measure speed which was then video recorded. Swimming speed (μm s⁻¹) increased with increasing body size. There was a slight decrease in the speed of adult females as the number of attached eggs increased. Swimming activity was higher at 25 °C than at 15 °C and in the absence of food than if microalgae were present. Average values under the different experimental conditions ranged between 500 μm s⁻¹ for the recently hatched and fed females and 1500 μm s⁻¹ for the adult non-ovigerous females in the absence of microalgae. Mass-specific swimming speed decreased with body mass increase.     

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.     

Individual- and population-level effects of antibiotics on the rotifers, <Emphasis Type="Italic">Brachionus calyciflorus</Emphasis> and <Emphasis Type="Italic">B. plicatilis</Emphasis>

The toxicity of three common antibiotics (streptomycin sulfate, tetracycline hydrochloride, and tylosin tartrate) to the freshwater rotifer Brachionus calyciflorus and brackish-water rotifer B. plicatilis was investigated using full-lifespan exposure durations. Effects of each antibiotic on lifespan, lifetime reproduction, and Malthusian parameter were assessed at seven nominal concentrations (ranging from 5.6 mg l⁻¹ to 2,000 mg l⁻¹) and a negative control. Lowest Observed Effect Concentrations (LOECs) were determined for reproduction and lifespan, while 1%, 10%, 25%, and 50% Inhibitory Concentrations (IC₁, IC₁₀, IC₂₅, IC₅₀) and 95% confidence intervals were estimated for all three endpoints. LOECs ranged from 5.6 mg l⁻¹ to 90 mg l⁻¹, with all LOECs less than 90 mg l⁻¹ occurring in B. calyciflorus. The lowest IC1 concentrations were 3.91 mg l⁻¹ for the effect of tetracycline on lifetime reproduction in B. calyciflorus and 4.06 mg l⁻¹ for the effect of tylosin on lifetime reproduction in B. plicatilis. Overall, lifetime reproduction was the most sensitive endpoint and the Malthusian parameter was the least sensitive. IC₁ values for lifetime reproduction were roughly one to two orders of magnitude lower than the corresponding IC₅₀ values. Guest editors: S. S. S. Sarma, R. D. Gulati, R. L. Wallace, S. Nandini, H. J. Dumont and R. Rico-Martínez Advances in Rotifer Research     

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     

<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.     

Isolation of mixis-related genes from the rotifer <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> using subtractive hybridization

The monogonont rotifer Brachionus plicatilis produces resting eggs through sexual reproduction (mixis), which is affected by external and internal factors. We collected resting eggs from rotifers cultured at 15 and 25°C and hatched them with and without 14-day dormancy in the dark. Stem females hatched from both conditions were further cultured at 15, 20 and 25°C. We obtained two phenotypes, one with high mixis (more than 50%), which was hatched from resting eggs formed at 15°C without dormancy, and another in which sexual reproduction occurred at 25°C and resting eggs were formed at 15°C with a 14-day dormancy. In the latter phenotype, mictic females appeared at 15°C, but not at 25°C. Using subtractive hybridization, we isolated one gene from the latter phenotype of females that had no significant similarity to known genes in BLAST searches. We propose that this gene is unique to rotifer mictic reproduction. Ongoing characterization of this gene attempts understand its role in mixis.     

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 <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.     

Swimming behaviour of <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> in relation to food concentration and feeding rates

We examined feeding rates and swimming speed in amictic females of Brachionus plicatilis over algal cell concentrations ranging from 15 × 10³ to 30 × 10⁶ cell ml⁻¹, to determine to what extent filtration rate is a consequence of a real modulation of swimming speed in response to food availability. Swimming rates were measured using an automated motion analysis system via video recording. The results showed that swimming speed changed as a function of food density. Swimming speed increased from the lowest tested concentration of algae to reach a maximum at 6 × 10⁶ cell ml⁻¹. Above this density, swimming speed declined slightly and then remained constant at a mean speed of 0.45 mm s⁻¹. Filtration and ingestion rates changed as cell concentration increased, following patterns consistent with those generally described for suspension feeders. However, the observed swimming pattern did not explain the recorded changes in clearance rate. These results suggest that filtration, and therefore ingestion, is mainly regulated by modifying particle retention efficiency. Guest editors: S. S. S. Sarma, R. D. Gulati, R. L. Wallace, S. Nandini, H. J. Dumont & R. Rico-Martínez Advances in Rotifer Research     

Effect of <Emphasis Type="Italic">cra</Emphasis> gene knockout together with <Emphasis Type="Italic">edd</Emphasis> and <Emphasis Type="Italic">iclR</Emphasis> genes knockout on the metabolism in <Emphasis Type="Italic">Escherichia coli</Emphasis>

To elucidate the physiological adaptation of Escherichia coli due to cra gene knockout, a total of 3,911 gene expressions were investigated by DNA microarray for continuous culture. About 50 genes were differentially regulated for the cra mutant. TCA cycle and glyoxylate shunt were down-regulated, while pentose phosphate (PP) pathway and Entner Doudoroff (ED) pathway were up-regulated in the cra mutant. The glucose uptake rate and the acetate production rate were increased with less acetate consumption for the cra mutant. To identify the genes controlled by Cra protein, the Cra recognition weight matrix from foot-printing data was developed and used to scan the whole genome. Several new Cra-binding sites were found, and some of the result was consistent with the DNA microarray data. The ED pathway was active in the cra mutant; we constructed cra.edd double genes knockout mutant to block this pathway, where the acetate overflowed due to the down-regulation of aceA,B and icd gene expressions. Then we further constructed cra.edd.iclR triple genes knockout mutant to direct the carbon flow through the glyoxylate pathway. The cra.edd.iclR mutant showed the least acetate production, resulting in the highest cell yield together with the activation of the glycolysis pathway, but the glucose consumption rate could not be improved. Dayanidhi Sarkar and Khandaker Al Zaid Siddiquee have contributed equally.     

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.     

Effect of <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis> on the proliferation of <Emphasis Type="Italic">Propionibacterium acnes</Emphasis> and <Emphasis Type="Italic">Staphylococcus epidermidis</Emphasis>

While it is generally accepted that Propionibacterium acnes is involved in the development of acne, other bacteria including Staphylococcus epidermidis have also been isolated from the acne lesion. The interaction between Lactobacillus reuteri, a probiotic bacterium, and acnegenic bacteria is unclear. This study examined the effects of L. reuteri on the proliferation of P. acnes and S. epidermidis. Human-derived L. reuteri strains (KCTC 3594 and KCTC 3678) and rat-derived L. reuteri KCTC 3679 were used. All strains exhibited significant inhibitory effects on the growth of P. acnes and S. epidermidis. The proliferation of P. acnes was decreased by 2-log scales after incubation with L. reuteri for 24 h. In addition, the proliferation of S. epidermidis was decreased by 3-log scales after incubation with L. reuteri for 24 h, whereas the growth of L. reuteri was unaffected by P. acnes or S. epidermidis. Among the L. reuteri strains examined, L. reuteri KCTC 3679 had the strongest inhibitory effect on the growth of P. acnes and S. epidermidis, followed by L. reuteri KCTC 3594 and L. reuteri KCTC 3678. Interestingly, reuterin, an antimicrobial factor, was produced only by L. reuteri KCTC 3594. The most pronounced the antibacterial activities of L. reuteri were attributed to the production of organic acids. Overall, these results suggest that L. reuteri may be a useful probiotic agent to control the growth of bacteria involved in acne inflammation and prevent acne.     

Disentangling the morphological stasis in two rotifer species of the <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> species complex

The taxonomic uncertainty surrounding cryptic species complexes has traditionally been resolved using lengthy experimental approaches, while, since the advent of PCR based techniques the number of cryptic species described in a variety of taxa is increasing steadily. Here we formally describe a new rotifer species of the Brachionus plicatilis complex: Brachionus manjavacas n.sp., disentangling what was known as a morphological stasis. Detailed morphological analyses demonstrated significant differences in body shape and size between B. manjavacas and B. plicatilis s.s., analysed by geometric morphometrics; unfortunately these statistical differences are not taxonomically reliable because of wide overlaps. Size and asymmetry of masticatory apparatus, named trophi, observed by SEM, gave similar results, with taxonomic ambiguity. Only the shape of small pieces of the trophi, named satellites, were consistently different between the species. On a strictly classical taxonomical basis it is absolutely useful to name new species on morphological bases, as we did, and to assess their status as distinct entities. Nevertheless, the two species are broadly similar; therefore, we do not suggest using the small differences in shape of satellites of trophi to identify the species for further ecological studies, but to continue discriminating them on genetic marker bases. Handling editor: K. Martens     

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.