Three new species of Liparis s.l. (Orchidaceae: Malaxideae) from Southwest China based on morphological characters and phylogenetic evidence

Liparis aureolabella and L. mengziensis, two new species from the karst region of southwestern China, and L. bingzhongluoensis, a new species from montane region in Yunnan, are described and illustrated. L. aureolabella is easily distinguished from its relatives by having abaxially purple leave with purple reticulate veins prominent adaxially, a lip auriculate at base, and falcate-lanceolate pollinia. Liparis mengziensis is closely related to L. petiolata and L. auriculata, but differs from them by having an ovate to broadly ovate leaf, purple lip and apex connate along the margins. Liparis bingzhongluoensis is similar to Liparis nanlingensis, but the new species is characterized by having a lip with two transparent ridges on its disc, longitudinally concave basal callus and triangular column wings. Phylogenetic analyses based on nuclear ribosomal ITS and plastid matK sequences showed that L. aureolabella and L. mengziensis are nested with L. petiolata or L. auriculata in a monophyletic clade. L. bingzhongluoensis is sister to a clade formed by L. nanlingensis, L. tsii, L. sasakii and L. krameri. Moreover, morphological comparisons strongly support that the three species as separated species newly to science.     

Morphological evidence of species differentiation within Lepus capensis Linnaeus, 1758 (Leporidae,Lagomorpha) in Cape Province,South Africa

A morphological study was carried out of the hares (Lepus) from Cape Province previously assigned to the subspecies L. capensis capensis, L. c. centralis, and L. c. grantii. The purpose of the study was to characterize the species L. capensis Linnaeus, 1758. In doing so, it was possible to distinguish two populations which we consider different species, as each shows homogeneous cranial, dental and pelage features. One of them, defined as L. capensis, is distributed near Cape Town not far from the coast, between Lambert's Bay and Cape Agulhas. The other species, defined as L. centralis, which includes L. grantii as a synonym, is distributed in central and western Cape Province. L. capensis and L. centralis have a parapatric distribution, with a small area of sympatry in a contact zone in Compagnies Drift area, near Lambert's Bay. With respect to cranial differences between the two species, L. capensis has a stronger maxilla and more robust dental series, while L. centralis has larger tympanic bullae. Among dental characters, L. centralis usually has a deeper groove and more abundant cement than L. capensis in the first upper incisor, and its internal lobe is squared, while in L. capensis is rounded. As for pelage color and pattern, L. capensis shows a more extended white ventral area than L. centralis. Our results are of interest for further research on taxonomic problems regarding Old World hare populations in which L. capensis is concerned.     

Role of Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenase in Visceral Organ Infection by Leishmania donovani

The initial 7 steps of the glycolytic pathway from glucose to 3-phosphoglycerate are localized in the glycosomes in Leishmania, including step 6, catalyzed by the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In L. donovani and L. mexicana, there exists a second GAPDH enzyme present in the cytosol that is absent in L. braziliensis and that has become a pseudogene in L. major. To investigate the role of the cytosolic GAPDH (cGAPDH), an L. donovani cGAPDH-null mutant was generated, and conversely, the functional L. donovani cGAPDH was introduced into L. major and the resulting engineered parasites were characterized. The L. donovani cGAPDH-null mutant was able to proliferate at the same rate as the wild-type parasite in glucose-deficient medium. However, in the presence of glucose, the L. donovani cGAPDH-null mutant consumed less glucose and proliferated more slowly than the wild-type parasite and displayed reduced infectivity in visceral organs of experimentally infected mice. This demonstrates that cGAPDH is functional in L. donovani and is required for survival in visceral organs. Restoration of cGAPDH activity in L. major, in contrast, had an adverse effect on L. major proliferation in glucose-containing medium, providing a possible explanation of why it has evolved into a pseudogene in L. major. This study indicates that there is a difference in glucose metabolism between L. donovani and L. major, and this may represent an important factor in the ability of L. donovani to cause visceral disease.     

Speciation and paraphyly in western mediterranean hares (Lepus castroviejoi,L. europaeus,L. granatensis,andL. capensis) revealed by mitochondrial DNA phylogeny

Mitochondrial DNA (mtDNA) variation among specimens of the northwestern African hare (Lepus capensis schlumbergeri) and three European hares sampled in Spain (L. castroviejoi andL. granatensis, which are endemic to the Iberian Peninsula, andL. europaeus) was analyzed using seven restriction endonucleases. Fourteen haplotypes were found among the 34 animals examined. Restriction site maps were constructed and the phylogeny of the haplotypes was inferred. mtDNA ofL. capensis was the most divergent, which is consistent with its allopatric African distribution and with an African origin of European hares. We estimated that mtDNA in hares diverges at a rate of 1.5–1.8% per MY assuming that the European and African populations separated 5–6 MYBP. Maximum intraspecies nucleotide divergences were 1.3% inL. capensis, 2.7% inL. castroviejoi, and 2.3% inL. granatensis but 13.0% inL. europaeus. The latter species contained two main mtDNA lineages, one on the branch leading toL. castroviejoi and the other on that leading toL. granatensis. The separation of these two lineages from theL. castroviejoi orL. granatensis lineages appears to be much older than the first paleontological record ofL. europaeus in the Iberian peninsula. This suggests that the apparent polyphyly ofL. europaeus is due not to secondary introgression, but to the retention of ancestral polymorphism inL. europaeus. The results suggest thatL. europaeus either has evolved as a very large population for a long time or has been fractionated. Such a pattern of persistence of very divergent lineages has also been reported in other species of highly mobile terrestrial mammals. As far as mtDNA is concerned,L. europaeus appears to be the common phylogenetic trunk which has diversified during dispersion over the European continent and from whichL. castroviejoi andL. granatensis speciated separately in southwest Europe.     

A New Oligonucleotide Microarray for Detection of Pathogenic and Non-Pathogenic Legionella spp.

Legionella pneumophila has been recognized as the major cause of legionellosis since the discovery of the deadly disease. Legionella spp. other than L. pneumophila were later found to be responsible to many non-pneumophila infections. The non-L. pneumophila infections are likely under-detected because of a lack of effective diagnosis. In this report, we have sequenced the 16S-23S rRNA gene internal transcribed spacer (ITS) of 10 Legionella species and subspecies, including L. anisa, L. bozemanii, L. dumoffii, L. fairfieldensis, L. gormanii, L. jordanis, L. maceachernii, L. micdadei, L. pneumophila subspp. fraseri and L. pneumophila subspp. pasculleii, and developed a rapid oligonucleotide microarray detection technique accordingly to identify 12 most common Legionella spp., which consist of 11 pathogenic species of L. anisa, L. bozemanii, L. dumoffii, L. gormanii, L. jordanis, L. longbeachae, L. maceachernii, L. micdadei, and L. pneumophila (including subspp. pneumophila, subspp. fraseri, and subspp. pasculleii) and one non-pathogenic species, L. fairfieldensis. Twenty-nine probes that reproducibly detected multiple Legionella species with high specificity were included in the array. A total of 52 strains, including 30 target pathogens and 22 non-target bacteria, were used to verify the oligonucleotide microarray assay. The sensitivity of the detection was at 1.0 ng with genomic DNA or 13 CFU/100 mL with Legionella cultures. The microarray detected seven samples of air conditioner-condensed water with 100% accuracy, validating the technique as a promising method for applications in basic microbiology, clinical diagnosis, food safety, and epidemiological surveillance. The phylogenetic study based on the ITS has also revealed that the non-pathogenic L. fairfieldensis is the closest to L. pneumophila than the nine other pathogenic Legionella spp.     

Plasmids in Listeria

Thirty-two Listeria strains, including L. monocytogenes of the different Seeliger serovars, L. grayi and L. murrayi, have been analyzed for the presence of plasmids. A cryptic plasmid of 38.5 Md was found in seven strains: in four L. monocytogenes (serovars $\text{1}\text{2}\text{c}$, 4b, 4d, and Ivanov 1), and in both the L. grayi and the single L. murrayi strains. Restriction enzyme cleavage analysis with two restriction enzymes suggests that the plasmid is a single molecular species, pRYC16. Conjugation experiments between Streptococcus agalactiae BM6101 containing the resistance plasmid pIP501 and L. monocytogenes, L. grayi, or L. Murrayi demonstrated that pIP501 is transferred to Listeria at a mean frequency of 10^?6. pIP501 is fully expressed in Listeria and promotes its own transfer between strains of Listeria and from Listeria back to Streptococcus. It is suggested that a circulation of plasmids can take place among members of the genera Listeria and Streptococcus.     

Evolution of a Novel Appendage Ground Plan in Water Striders Is Driven by Changes in the Hox Gene Ultrabithorax

Water striders, a group of semi-aquatic bugs adapted to life on the water surface, have evolved mid-legs (L2) that are long relative to their hind-legs (L3). This novel appendage ground plan is a derived feature among insects, where L2 function as oars and L3 as rudders. The Hox gene Ultrabithorax (Ubx) is known to increase appendage size in a variety of insects. Using gene expression and RNAi analysis, we discovered that Ubx is expressed in both L2 and L3, but Ubx functions to elongate L2 and to shorten L3 in the water strider Gerris buenoi. Therefore, within hemimetabolous insects, Ubx has evolved a new expression domain but maintained its ancestral elongating function in L2, whereas Ubx has maintained its ancestral expression domain but evolved a new shortening function in L3. These changes in Ubx expression and function may have been a key event in the evolution of the distinct appendage ground plan in water striders.     

Lycoperdon rupicola and L. subumbrinum: two new puffballs from Europe

Lycoperdon rupicola and L. subumbrinum are here described as new species. The former is a morphologically characteristic species forming small subglobose to pyriforme fruiting bodies with a prominent, protruding and stellately lobed stoma. In the phylogenetic analyses the six sequences of L. rupicola form a clade with 100% BS and 1.0 BPP, clustering with L. dermoxanthum. The proposed species grows among mosses on siliceous boulders and flat rocks as well as on open, sandy soil in temperate and hemiboreal regions. The records to date indicate its presence in Fennoscandia and Spain (Catalunya). L. subumbrinum morphologically resembles L. umbrinum but differs in having coarser ornamentation of its spores. In the phylogenetic analyses the five sequences of L. subumbrinum form a clade with 100% BS and 1.0 BPP, clustering with L. muscorum. The records to date, originating from southernmost Sweden and NW Slovakia, indicate that L. subumbrinum is a species of rich deciduous forests. L. muscorum is recorded for the first time from the Iberian peninsula (Spain).     

A taxonomic revision of Limnobaris Bedel in the strict sense (Coleoptera,Curculionidae, Baridinae), with particular emphasis on the species found in China

The genus name Limnobaris Bedel is applied in a restricted sense to baridine weevils with a covered pygidium and non-prominent, decussate mandibles which occur on sedges in the Palaearctic Region and immediately adjacent parts of tropical Southeast Asia. Calyptopygus Marshall and Pertorcus Voss are syn. n. of Limnobaris. Some species from Africa and the Americas are maintained provisionally in Limnobaris in the widest sense but will need to be transferred to other genera in future studies. A total of eleven species is recognized in Asia, two of which are widespread and occur also in the Western Palaearctic Region. Limnobaris martensi Korotyaev sp. n. is described from Nepal. Pertorcus tibialis basalis Voss is raised to species rank, as L. basalis (stat. prom.). New or reestablished synonyms are L. dolorosa (Goeze) (= L. jucunda Reitter, = L. koltzei Reitter), L. tibialis (Voss) (= Pertorcus tibialis pilifer Voss) and L. t-album (Linnaeus) (= L. bedeli Reitter, = Baridius crocopelmus Gyllenhal, = L. sahlbergi Reitter, = L. scutellaris Reitter, = Baris t-album sculpturata Faust). Calandra uniseriata Dufour is considered a junior synonym of Sitophilus oryzae (L.) (syn. n.). A key for identification and a distribution map are provided.     

Taxonomic and morphological survey of the Lygephila lusoria (Linnaeus, 1758) species-group with description of a new species (Lepidoptera,Erebidae, Toxocampinae)

The taxa of the Lygephila lusoria (Linnaeus, 1758) species-group are revised. The genital features of all known taxa are described and illustrated with special reference to the structure of vesica. The male genitalia of L. pallida pallida (Bang-Haas, 1907) are described and illustrated for the first time. L. pallida subpicata (Wiltshire, 1971) is treated here as a species, L. pallida subpicata (Wiltshire, 1971), stat. n., distinct from L. pallida. A new species, L. minima, sp. n.,from South Russia is described. Illustrations of the holotype and its genitalia are provided; a diagnostic comparison with L. pallida is given. L. alaica Remm, 1983 is included in the L. lusoria species-group for the first time.     

Leptographium wushanense sp. nov., associated with Tomicus armandii on Pinus armandii in Southwestern China

Leptographium wushanense sp. nov., which is associated with Tomicus armandii, is described based on molecular sequence data and morphological characters. The phylogenetic analyses based on ITS2-partial LSU rDNA region, β-tubulin and elongation factor 1-α genes showed that L. wushanense formed a well-supported clade and was closely related to L. conjunctum and L. yunnanense, and then nested within L. lundbergii complex. The species is characterized that its conidiophores were longer than those of L. yunnanense and shorter than those of L. conjunctum, while its conidia were larger than those of L. conjunctum and smaller than those of L. yunnanense. Furthermore, the colonial growth of L. wushanense is faster than those of L. conjunctum and L. yunnanense at 25 °C on 2% MEA. Both molecular data and morphological comparison confirmed the placement of the new species in genus of Leptographium.     

Interspecific differences in prey items in relation to morphological characteristics among four lutjanid species (Lutjanus decussatus, L. fulviflamma, L. fulvus and L. gibbus)

Clarifying interspecific differences in prey items in relation to morphological characteristics is a fundamental aspect to understand the mechanism enabling the diversity of feeding ecology of fishes. The aim of the present study was to clarify the relationship between prey items and body shape variation, teeth and mandible characteristics for four lutjanid species: Lutjanus decussatus, L. fulviflamma, L. fulvus and L. gibbus. Stomach contents analysis revealed that the main prey items of L. decussatus were fishes, L. fulviflamma were crabs and fishes, and L. fulvus and L. gibbus was crabs. Body shape analysis revealed that L. decussatus and L. fulviflamma had a shallower body depth whereas L. fulvus and L. gibbus had a deeper body depth. The two species with a shallower body had long teeth whereas the other two species with a deeper body depth had shorter teeth. The jaw-lever mechanics were compared and L. decussatus and L. fulviflamma have a faster mouth opening–closing mechanism. Canonical correspondence analysis revealed that fishes was the major prey item for the species having a shallower body depth, higher teeth length and lower ratios of in-lever to out-lever of mandibles, whereas crabs was the major prey item for the species having a deeper body depth, lower teeth length and higher ratios of in-lever to out-lever of mandibles. It is suggested that the interspecific differences in main prey items among the four species are directly related to behavioral differences based on body shape, teeth characteristics and jaw-lever mechanics.     

Field assessment of hybridization between Laricobius nigrinus and L. rubidus,predators of Adelgidae

Two adelgid predators, Laricobius nigrinus Fender and Laricobius rubidus LeConte, were recently discovered to produce hybrid progeny in the eastern United States. L. rubidus is native to eastern North America where it feeds on pine bark adelgid (Pineus strobi Hartig) and L. nigrinus is native to western North America and was introduced to the eastern United States in 2003 for biological control of hemlock woolly adelgid (Adelges tsugae Annand). Currently, L. nigrinus and L. rubidus form a mosaic of hybrid zones throughout the eastern United States. It is not known whether these zones will be maintained over time and whether hybridization will impact the efficacy of biological control or result in displacement of L. rubidus. Sampling from 2007 to 2012 on eastern hemlock (Tsuga canadensis Carrière) showed a clear increase in L. nigrinus, a decrease in L. rubidus, and a steady proportion of hybrids. Sampling from 2010 to 2012 on both eastern hemlock and white pine (Pinus strobus L.) at ten sites showed that L. nigrinus was more dominant on hemlock and L. rubidus was more dominant on white pine, which may demonstrate habitat preference and promote segregation between the two species. Site factors were tested for a relationship with the proportion of hybrids. The number of years L. nigrinus had been present at the site was the only factor that showed a relationship. The results suggest that L. nigrinus may displace L. rubidus on hemlock, but not on white pine at sites where they are both present.     

Production and Targeting of the Brucella abortus Antigen L7/L12 in Lactococcus lactis: a First Step towards Food-Grade Live Vaccines against Brucellosis

Brucella abortus is a facultative intracellular gram-negative bacterial pathogen that infects humans and animals by entry mainly through the digestive tract. B. abortus causes abortion in pregnant cattle and undulant fever in humans. The immunogenic B. abortus ribosomal protein L7/L12 is a promising candidate antigen for the development of oral live vaccines against brucellosis, using food-grade lactic acid bacteria (LAB) as a carrier. The L7/L12 gene was expressed in Lactococcus lactis, the model LAB, under the nisin-inducible promoter. Using different signals, L7/L12 was produced in cytoplasmic, cell-wall-anchored, and secreted forms. Cytoplasmic production of L7/L12 gave a low yield, estimated at 0.5 mg/liter. Interestingly, a secretable form of this normally cytoplasmic protein via fusion with a signal peptide resulted in increased yield of L7/L12 to 3 mg/liter; secretion efficiency (SE) was 35%. A fusion between the mature moiety of the staphylococcal nuclease (Nuc) and L7/L12 further increased yield to 8 mg/liter. Fusion with a synthetic propeptide (LEISSTCDA) previously described as an enhancer for heterologous protein secretion in L. lactis (Y. Le Loir, A. Gruss, S. D. Ehrlich, and P. Langella, J. Bacteriol. 180:1895-1903, 1998) raised the yield to 8 mg/liter and SE to 50%. A surface-anchored L7/L12 form in L. lactis was obtained by fusing the cell wall anchor of Streptococcus pyogenes M6 protein to the C-terminal end of L7/L12. The fusions described allow the production and targeting of L7/L12 in three different locations in L. lactis. This is the first example of a B. abortus antigen produced in a food-grade bacterium and opens new perspectives for alternative vaccine strategies against brucellosis.     

Further notes on European taxa of the lichen genus Leptogium,with emphasis on the small species

Abstract:Leptogium imbricatum P.M. Jørg. sp. nov. and L. intermedium (Arnold) Arnold are established as the correct names for two fairly widespread, small species: L. imbricatum has an arctic-alpine distribution. Leptogium intermedium was previously mostly named L. minutissimum (Flörke) Fr., the type of which is L. subtile (Schrader) Torss., a distinct species usually growing on rotting wood or debris. Leptogium magnussonii Degel. & P.M. Jørg, mainly from maritime rock outcrops in Norway and Sweden, is described as new. The species complexes of L. plicatile (Ach.) Leighton and L. schraderi (Bernh.) Nyl. are discussed. The differences between L. byssinum (Hoffm.) Nyl. and L. biatorinum (Nyl.) Leighton are given, and L. aquale (Arnold) P.M. Jørg. comb. nov., a taxon related to the latter, is considered to be a distinct species growing in mountain brooks in Central Europe. Leptogium rivale Tuck. is recorded as new to Europe, from Romania. Leptogium bellopratense Hy is shown to be a synonym of L. brebissonii Mont. New records for L. coralloideum (Meyen & Flotow) Vainio, L. massiliense Nyl and L. rivulare (Ach.) Mont. are given. A preliminary key to the small European species (north of the Alps) is appended. The following names have been lecto- or neotypified: Collema byssinum Hoffm., Collema pulvinatum Hoffm., Leptogium bellopratense Hy., Leptogium crenulatum Watson, Leptogium massiliense Nyl., Leptogium minutissimum var. intermedium Arnold, Leptogium minutissimum f. plumbeum Zwackh ex Arnold, Leptogium pusillum var. aquale Arnold, Leptogium rivulare var. crenatulum Nyl., Leptogium scotinum var. crenatum Nyl., Lichen byssinus Hoffm., Lichen minutissimus Flörke, Lichen schraderi Bemh., Lichen subtilis Schrader and Parmelia scotina var. lophaea Ach.     

Rhythmic Conidiation in Constant Light in Vivid Mutants of Neurospora crassa

In Neurospora crassa, a circadian rhythm of conidiation (asexual spore formation) can be seen on the surface of agar media. This rhythm has a period of 22 hr in constant darkness (D/D). Under constant illumination (L/L), no rhythm is visible and cultures show constant conidiation. However, here we report that strains with a mutation in the vivid (vvd) gene, previously shown to code for the photoreceptor involved in photo-adaptation, exhibit conidiation rhythms in L/L as well as in D/D. The period of the rhythm of vvd strains ranges between 6 and 21 hr in L/L, depending upon the intensity of the light, the carbon source, and the presence of other mutations. Temperature compensation of the period also depends on light intensity. Dark pulses given in L/L shift the phase of the rhythm. Shifts from L/L to D/D show unexpected after effects; i.e., the short period of a vvd strain in L/L gradually lengthens over 2–3 days in D/D. The rhythm in L/L requires the white collar (wc-1) gene, but not the frequency (frq) gene. FRQ protein shows no rhythm in L/L in a vvd strain. The conidiation rhythm in L/L in vvd is therefore driven by a FRQ-less oscillator (FLO).     

Compared chemical properties of dermonecrotic and lethal toxins from spiders of the genusLoxosceles (Araneae)

Loxosceles spider venom usually causes a typical dermonecrotic lesion in bitten patients, but it may also cause systemic effects that may be lethal. Gel filtration on Sephadex G-100 ofLoxosceles gaucho, L. laeta, orL. intermedia spider venoms resulted in three fractions (A, containing higher molecular mass components, B containing intermediate molecular mass components, and C with lower molecular mass components). The dermonecrotic and lethal activities were detected exclusively in fraction A of all three species. Analysis by SDS-PAGE showed that the major protein contained in fraction A has molecular weight approximately 35 kDa inL. gaucho andL. intermedia, but 32 kDa inL. laeta venom. These toxins were isolated from venoms ofL. gaucho, L. laeta, andL. intermedia by SDS-PAGE followed by blotting to PVDF membrane and sequencing. A database search showed a high level of identity between each toxin and a fragment of theL. reclusa (North American spider) toxin. A multiple sequence alignment of theLoxosceles toxins showed many common identical residues in their N-terminal sequences. Identities ranged from 50.0% (L. gaucho andL. reclusa) to 61.1% (L. intermedia andL. reclusa). The purified toxins were also submitted to capillary electrophoresis peptide mapping afterin situ partial hydrolysis of the blotted samples. The results obtained suggest thatL. intermedia protein is more similar toL. laeta toxin thanL. gaucho toxin and revealed a smaller homology betweenL. intermedia andL gaucho. Altogether these findings suggest that the toxins responsible for most important activities of venoms ofLoxosceles species have a molecular mass of 32–35 kDa and are probably homologous proteins.     

Two Different Tetracycline Resistance Mechanisms, Plasmid-Carried tet(L) and Chromosomally Located Transposon-Associated tet(M), Coexist in Lactobacillus sakei Rits 9

Lactobacillus sakei is extensively used as functional starter culture in fermented meat products. One of the safety criteria of a starter culture is the absence of potentially transferable antibiotic resistance determinants. However, tetracycline-resistant L. sakei strains have already been observed. In this paper, we show that tetracycline resistance in L. sakei Rits 9, a strain isolated from Italian Sola cheese made from raw milk, is mediated by a transposon-associated tet(M) gene coding for a ribosomal protection protein and a plasmid-carried tet(L) gene coding for a tetracycline efflux pump. pLS55, the 5-kb plasmid carrying the tet(L) gene, is highly similar to the pMA67 plasmid recently described for Paenibacillus larvae, a species pathogenic to honeybees. pLS55 could be transferred by electroporation into the laboratory strain L. sakei 23K. While the L. sakei 23K transformant containing pLS55 displayed an intermediate tetracycline resistance level (MIC, <32 μg/ml), L. sakei Rits 9, containing both tetracycline-resistant determinants, had a MIC of <256 μg/ml, suggesting that Tet L and Tet M confer different levels of resistance in L. sakei. Remarkably, in the absence of tetracycline, a basal expression of both genes was detected for L. sakei Rits 9. In addition, subinhibitory concentrations of tetracycline affected the expression patterns of tet(M) and tet(L) in different ways: the expression of tet(M) was induced only at high tetracycline concentrations, whereas the expression of tet(L) was up-regulated at lower concentrations. This is the first time that two different mechanisms conferring resistance to tetracycline are characterized for the same strain of a lactic acid bacterium.