Evidence of Symbiosis Between the Soil Yeast <Emphasis Type="Italic">Cryptococcus laurentii</Emphasis> and a Sclerophyllous Medicinal Shrub, <Emphasis Type="Italic">Agathosma betulina</Emphasis> (Berg.) Pillans

The interaction between a common soil yeast, Cryptococcus laurentii, and a slow-growing medicinal plant adapted to low-nutrient soils, Agathosma betulina (Berg.) Pillans, was studied. C. laurentii CAB 578 was isolated from the rhizosphere of wild A. betulina, and liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis revealed that the yeast was capable of producing polyamines, such as cadaverine and spermine, while growing in vitro in a chemically defined medium. Since the exogenous application of polyamines are known to impact on root growth, these findings supported the results obtained when axenic cultures of A. betulina seedlings were inoculated with C. laurentii CAB 578 and cultivated for 5 months under glasshouse conditions. The presence of the yeast increased root growth by 51%. Using soil dilution plates, it was demonstrated that yeast numbers were greater in the vicinity of the roots than in the bulk soil. In addition, fluoromicroscopy, in combination with the fluorescent probes Fungolight and Calcofluor white, revealed the presence of metabolic active yeast colonies on the rhizoplane 5 months after initiation of the experimentation. The study provided evidence for a symbiosis between C. laurentii and A. betulina. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Smilodon populator et Protocyon troglodytes, deux superprédateurs du Pléistocène supérieur de la Serra da Capivara (Piauí) au Nordeste du Brésil

Among the fossiliferous sites of the Serra da Capivara national park region (South Eastern Piauí), two have yielded interesting remains of the two large Upper Pleistocene Carnivora, Protocyon troglodytes (Lund, 1838) and Smilodon populator Lund, 1842, the Toca da Cima dos Pilão and the Toca das Moendas, both in the municipality of Coronel José Dias. In these two caverns, numerous remains of preys were found, namely, for the Toca da Cima dos Pilão, the two peccaries Dicotyles tajacu and Tayassu pecari, the medium sized ground sloth Catonyx cuvieri and the giant armadillo Pampatherium; for the Toca das Moendas Palaeolama, Equids and Cervids add to that list. The two sites were used as dens by the two Carnivora species. In the Toca das Moendas, a near complete adult skull and a hemimandible of the large Canid P. troglodytes were found. The skull is the third ever known for the species. Its dimensions are very similar to those of a complete skull from the Toca da Boa Vista (Campo Formoso, Bahia, Brazil) described by Cartelle et Langguth (1999), and to those of an incomplete skull from the banks of the Bermejo River (Formosa Province, Argentina), described by Prevosti et al. (2005). It fits perfectly, like the teeth, with the anatomical characteristics of the species P. troglodytes: wide muzzle, short palate, very reduced and forwardly offset protocone of the P4/, etc. The dimensions of the cheek teeth correspond well with those of numerous teeth from Lagoa Santa (Minas Gerais, Brazil) of the Lund Collection preserved in the Zoological Museum of Copenhagen. The same site of Toca das Moendas yielded two left hemimandibles of the Machairodontine S. populator Lund, 1842, an adult and a very young ones. We study them together with other remarkable remains from the Toca da Cima dos Pilão, namely an adult upper canine and an adult hemimandible. The upper canine is one of the longest ever described for S. populator. The P/4 are somewhat longer than those of the Californian Smilodon fatalis (Leidy, 1868), and the M/1 are about of the same average length but somewhat narrower. The segment P/4-M/1 is relatively short. The dimensions of the upper canine and of the lower cheek teeth fit well with those of some teeth from Lagoa Santa (Minas Gerais, Brazil) and from Argentina preserved in the Zoological Museum of Copenhagen. We describe particularly the decidual lower dentition, badly known in that species. The rather weak milk canine has a piriform outline. The D/3 is constituted by a strong protoconid flanked with two small cuspids, an anterior and a posterior ones. The D/4 (deciduous carnassial) shows a strong paraconid followed by a higher protoconid and a small posterior cuspid. These milk teeth, without any abrasion mark, suggest that our young specimen was less than three month old.     

Enhancing the enzymatic activity of the endochitinase by the directed evolution and its enzymatic property evaluation

Endochitinase has an important application in the biological treatment of chitin, the second most abundant and renewable resource in nature. In order to enhance its activity, a double mutant strain MECH-Y185/S226 was obtained by the directed evolution using the error-prone PCR with the mature endochitinase cDNA from Trichoderma viride as the template. Compared to those of the primitive strain MECH, endochitinase activities of the mutant one were 1.8-fold higher towards 4-nitrophenyl-N-acetyl-β-d-glucosaminide and 3.5-fold higher towards the colloidal chitin. Sequence alignments indicated that 9 nucleotides and 2 amino acids (Y185F and S226P) were mutant. The SDS–PAGE analysis showed that a single band with an estimated molecular weight of 46 kDa was obtained when the Ech42-Y185/S226 was purified sequentially by ammonium sulfate precipitation, DE52 anion-exchanging column chromatography and Sephadex G-100 column chromatography. Kinetic parameters K_m and V_max of the Ech42-Y185/S226 were 0.25 ± 0.02 mmol/l and 4.59 ± 0.32 μmol/l min, respectively. The analysis of enzymatic properties showed that the Ech42-Y185/S226 had a higher thermal stability at higher temperatures and a higher pH stability within a wider pH range than the Ech42. Observed activities of the Ech42-Y185/S226 are the highest in the presence of Mg²⁺ and the lowest in the presence of Zn²⁺.     

<Emphasis Type="Italic">Fomitopsis betulina (</Emphasis>formerly <Emphasis Type="Italic">Piptoporus betulinus)</Emphasis>: the Iceman’s polypore fungus with modern biotechnological potential

Higher Basidiomycota have been used in natural medicine throughout the world for centuries. One of such fungi is Fomitopsis betulina (formerly Piptoporus betulinus), which causes brown rot of birch wood. Annual white to brownish fruiting bodies of the species can be found on trees in the northern hemisphere but F. betulina can also be cultured as a mycelium and fruiting body. The fungus has a long tradition of being applied in folk medicine as an antimicrobial, anticancer, and anti-inflammatory agent. Probably due to the curative properties, pieces of its fruiting body were carried by Ötzi the Iceman. Modern research confirms the health-promoting benefits of F. betulina. Pharmacological studies have provided evidence supporting the antibacterial, anti-parasitic, antiviral, anti-inflammatory, anticancer, neuroprotective, and immunomodulating activities of F. betulina preparations. Biologically active compounds such as triterpenoids have been isolated. The mushroom is also a reservoir of valuable enzymes and other substances such as cell wall (1→3)-α-d-glucan which can be used for induction of microbial enzymes degrading cariogenic dental biofilm. In conclusion, F. betulina can be considered as a promising source for the development of new products for healthcare and other biotechnological uses.     

Phylogeography using mitogenomes: A rare Dipodidae,Sicista betulina,in North‐western Europe

Repeated climatic and vegetation changes during the Pleistocene have shaped biodiversity in Northern Europe including Denmark. The Northern Birch Mouse (Sicista betulina) was one of the first small rodent species to colonize Denmark after the Late Glacial Maximum. This study analyses complete mitochondrial genomes and two nuclear genes of the Northern Birch Mouse to investigate the phylogeographical pattern in North‐western Europe and test whether the species colonized Denmark through several colonization events. The latter was prompt by (i) the present‐day distinct northern and southern Danish distribution and (ii) the subfossil record of Northern Birch Mouse, supporting early Weichselian colonization. Samples from Denmark, Norway, Sweden, Russia, Latvia, Estonia, and Slovakia were included. Mitogenomes were obtained from 54 individuals, all representing unique mitogenomes supporting high genetic variation. Bayesian phylogenetic analysis identified two distinct evolutionary linages in Northern Europe diverging within the Elster glaciation period. The results of the two nuclear genomes showed lower genetic differentiation but supported the same evolutionary history. This suggests an allopatric origin of the clades followed by secondary contact. Individuals from southern Denmark were only found in one clade, while individuals from other areas, including northern Denmark, were represented in both clades. Nevertheless, we found no evidence for repeated colonization''s explaining the observed fragmented distribution of the species today. The results indicated that the mitogenome pattern of the Northern Birch Mouse population in southern Denmark was either (i) due to the population being founded from northern Denmark, (ii) a result of climatic and anthropogenic effects reducing population size increasing genetic drift or (iii) caused by sampling bias.     

Connection of Gnomonia intermedia to Discula betulina and its relationship to other taxa in Gnomoniaceae

Discula betulina is a foliar pathogen on birch (Betula) and Gnomonia intermedia is found on overwintered birch leaves. Perithecia of G. intermedia developed in vitro on colonies of D. betulina isolated from birch tissues in late summer, and single ascospores of G. intermedia consistently developed into colonies similar to D. betulina, producing typical D. betulina conidia. Isolates of D. betulina could be grouped into two mating types, which produced fertile perithecia of G. intermedia when mated with each other. Mycelia from single-ascospore and single-conidial isolates were inoculated onto shoots of downy birch, causing lesions and die-back from which D. betulina was consistently isolated. ITS region ribosomal DNA sequence analysis confirmed the results of the morphological and mating studies, and found that the closest known relatives of G. intermedia/D. betulina are Gnomoniella nana and Sirococcus clavigignenti-juglandacearum. The conclusion from these studies is that D. betulina is the anamorph of G. intermedia.     

Dientes de Theropoda (Dinosauria: Saurischia) de la Formación Bajo Barreal, Cretácico Superior, Provincia del Chubut, Argentina

Theropod teeth from the Bajo Barreal Formation (Upper Cretaceous: Cenomanian-Turonian) at the “estancia Ocho Hermanos”, Chubut Province are described. Most of them show features of Abelisauroidea, a clade already represented in the Bajo Barreal Formation by Xenotarsosaurus bonapartei and other isolated remains including a left maxilla. Dromaeosauridae and Carcharodontosauridae represent the first record of these taxa for the Bajo Barreal Formation. Besides this, at least three theropod clades (Abelisauroidea, Carcharodontosauridae, and Dromaeosauridae) coexisted in central Patagonia during the Cenomanian-Turonian along with other theropods as Aniksosaurus darwini and Megaraptor sp.     

Rongeurs et lagomorphes du Miocène inférieur de Béon 2 (MN4, Montréal-du-Gers,SW France)

About 200 micromammal isolated teeth (rodents, lagomorphs) originating from the middle Orleanian locality of Béon 2 (Montréal-du-Gers, SW France) are described. The rodent fauna is dominated by myomorphs (7 species), including the cricetid Democricetodon aff. hispanicus Fahlbusch, the melissiodontid Melissiodon sp., the glirids Peridyromys murinus (Pomel), Pseudodryomys aff. ibericus, Pseudodryomys aff. simplicidens, and Glirudinus modestus (Dehm), and the eomyid Ligerimys aff. florancei. Sciuromorphs are represented only by Heteroxerus rubricati Crusafont, Villalta and Truyols. Within lagomorphs, 2 dental morphs referred to the ochotonid Prolagus Pomel are identified in Béon 2. They correspond to Prolagus oeningensis (König) and P. aff. vasconiensis. On a biostratigraphical point of view, this study confirms the location of Béon 2 at the early part of the MN4; this locality is older than other regional sites such as Pellecahus, Béon 1, La Romieu, and Bézian (MN4b), and it is coeval to Artenay, in the Loire basin. In particular, glirids and lagomorphs from Béon 2 testify to close relationships with micromammal localities referred to the MN3 biozone.     

Fungal Keratitis Secondary to <Emphasis Type="Italic">Trametes betulina</Emphasis>: A Case Report and Review of Literature

Purpose

We report the first case of human infection and keratitis secondary to Trametes betulina, a rare filamentous fungus.

Methods

Clinical examination including external and slit-lamp examination and corneal scrapings with microbiologic evaluation were performed on a patient with chronic allergic conjunctivitis, entropion and a long-standing corneal ulcer resistant to treatment.

Results

The culture from the corneal scraping revealed a basidiomycetous fungus which was submitted for identification. DNA extraction with sequencing and analysis of the ITS and D1/D2 regions were performed on the isolate and demonstrated 100% similarity to Lenzites betulina/Trametes betulina. Susceptibility testing demonstrated potent in vitro activity of voriconazole (MIC < 0.03 μg/ml). The patient was treated with voriconazole, and the corneal ulcer and infiltrate resolved. The infection resulted in corneal thinning and a dense central corneal scar. Penetrating keratoplasty was performed 5 months after diagnosis and treatment and revealed stromal scarring without fungal elements.

Conclusion

This is the first reported case of keratitis caused by Trametes betulina. This organism should be considered in the differential diagnosis for rare filamentous fungal keratitis and its treatment with voriconazole also noted.

     

Application of vibrational spectroscopy in the quality assessment of Buchu oil obtained from two commercially important Agathosma species (Rutaceae)

Quality assessment of natural raw materials and derived consumer products is often done using conventional analytical techniques such as liquid and gas chromatography which are expensive and time consuming. This paper reports on the use of vibrational spectroscopy techniques as possible alternatives for the rapid and inexpensive assessment of the quality of ‘buchu oil’ obtained from two South African species; Agathosma betulina and Agathosma crenulata belonging to the Rutaceae family. Samples of A. betulina (55) and A. crenulata (16) were collected from different natural localities and cultivation sites in South Africa. The essential oil was obtained by hydrodistillation and scanned on Near infrared (NIR), mid infrared (MIR) and Raman spectrometers. The spectral data obtained was processed using chemometric techniques and orthogonal partial least squares discriminant analysis (OPLS-DA) was used to clearly differentiate between A. betulina and A. crenulata. The OPLS-DA technique also proved to be a useful tool to identify wave regions that contain biomarkers (peaks) that contributed to the separation of the two species. The three spectroscopy techniques were also evaluated for their ability to accurately predict the percentage composition of seven major compounds that occur in A. betulina ‘buchu’ oil. Using GC–MS reference data, calibration models were developed for the MIR, NIR and Raman spectral data to predict/profile the major compounds in ‘buchu oil’. A comparison of the three spectroscopy techniques showed that MIR together with PLS algorithms produced the best model (R²X = 0.96; R²Y = 0.88 and Q²Ycum = 0.85) for the quantification of six of the seven major oil constituents. The MIR model showed high predictive power for pseudo-diosphenol (R² = 0.97), isomenthone (R² = 0.97), menthone (R² = 0.90), limonene (R² = 0.91), pulegone (R² = 0.96) and diosphenol (R² = 0.85). These results illustrate the potential of MIR spectroscopy as a rapid and inexpensive alternative to predict the major compounds in buchu oil.     

Molecular characterization and estimation of cellulolytic potential of gut bacteria isolated from four white grub species native to Indian Himalayas

An investigation was carried out to isolate, identify and molecularly characterize the cellulose-degrading bacterial isolates from the guts of four white grub species (Anomala bengalensis, Brahmina coriacea, Holotrichia longipennis and Holotrichia setticollis) native to Uttarakhand, Himalayas through 16S rRNA sequencing. A total of 178 bacterial strains were isolated from different gut compartments of selected white grub species, of which 95 bacterial isolates showed cellulose metabolizing activities in the CMC assay. Maximum degraders i.e., 38 were isolated from A. bengalensis, of which 18 were isolated from the fermentation chamber. The value of cellulolytic index ranged between 0.05 and 16 showing a variable cellulolytic activity by degraders. A total of 25 potent strains of cellulose-degrading bacteria recording cellulolytic activity > 1 were isolated and sequenced for 16S rRNA gene. Bacillus stratosphericus strain CBG4MG1 (10.78 ± 4.18), Bacillus cereus strain CBG2FC1 (10.33 ± 3.53), Bacillus sp. strain CBG3MG2 (7.28 ± 0.16) and Paenibacillus ginsengagri strain CBG1FC2 (5.66 ± 2.67) were the most potent cellulose-degrading bacteria isolated from the gut of B. coriacea, H. longipennis, H. setticollis and A. bengalensis, respectively. Thus, the cellulolytic bacteria isolated from the gut of selected white grub species may be good sources for profiling novel isolates for industrial use besides identifying eco-friendly solutions for agro-waste management.     

AFLP markers in the detection of Scirpus × mariqueter (CYPERACEAE) hybrid in China

Scirpus × mariqueter Tang & F.T. Wang (CYPERACEAE) is a pioneer plant of the tidal zone of estuaries of East Asia. Amplified fragment length polymorphisms (AFLP) were used to infer the relationship between S. × mariqueter and its putative progenitors, Scirpus planiculmis F. Schmidt and Scirpus triqueter L. The genetic distance between S. × mariqueter and S. triqueter was much higher than that between S. × mariqueter and S. planiculmis, and samples of S. × mariqueter and those of S. planiculmis did not form a monophyletic group, while the S. triqueter group formed a distinct monophyletic group. However, we also detected an apparent clustering of individuals of S. × mariqueter and S. planiculmis separately. Our results disprove the hybrid origin of S. × mariqueter and rather suggest that it is more closely related to S. planiculmis.     

Potential effect of Allium sativum bulb for the treatment of biofilm forming clinical pathogens recovered from periodontal and dental caries

Biofilm producing clinical bacterial isolates were isolated from periodontal and dental caries samples and identified as, Lactobacillus acidophilus, Streptococcus sanguis, S. salivarius, S. mutansand Staphylococcus aureus. Among the identified bacterial species, S. aureus and S. mutansshowed strong biofilm producing capacity. The other isolated bacteria, Streptococcus sanguis, S. salivarius showed moderate biofilm formation. These pathogens were subjected for the production of extracellular polysaccharides (EPS) in nutrient broth medium and the strain S. aureus synthesized more amounts of EPS (610 ± 11.2 µg/ml) than S. sanguis (480 ± 5.8 µg/ml).EPS production was found to be less in S. salivarius (52 ± 3.8 µg/ml).The solvent extract of A. sativum bulb showed the phytochemicals such as, carbohydrate, total protein, alkaloids, saponins, flavonoids, tannins and sterioids. The solvent extract of A. sativum bulb showed wide ranges of activity against the selected dental pathogens. The difference in antibacterial activity of the solvent extract revealed differences in solubility of phytochemicals in organic solvents. Ethanol extract was highly active againstS. aureus (25 ± 2 mm). The Minimum Inhibitory Concentration (MIC) of crude garlic bulb varied widely and this clearly showed that bacteria exhibits different level of susceptibility to secondary metabolites. MIC value ranged between 20 ± 2 mg/ml and 120 ± 6 mg/ml and Minimum Bactericidal Concentration (MBC) value ranged from 60 ± 5 mg/l to 215 ± 7 mg/ml. To conclude, A. sativum bulb can be effectively used to treat periodontal and dental caries infections.     

Contributions to the tooth morphology in early embryos of three species of hammerhead sharks (Elasmobranchii: Sphyrnidae) and their evolutionary implications

The tooth types in the embryos of the hammerhead sharks Sphyrna tiburo, Sphyrna tudes and Eusphyra blochii are here described in labial and lingual views, and, in some cases, in additional views. The presence of cusplets was observed in the anterior teeth of S. tiburo and S. tudes, which is secondarily lost after early embryonic stages. Many aligned root foramina were detected in the sphyrnids, which, as the cusplets, are shared by many phylogenetic-related carcharhinids. Other anatomic features, related to the root and central cusp, are presented for the first time. Such characters represent the first step to compare the teeth of extant and fossil species.     

Interactions between root and shoot competition among four plant species in an alpine meadow on the Tibetan Plateau

At low altitudes, the interactions between root and shoot competition on plant growth have been extensively studied, and such interaction effects were found to be positive, negative, or neutral. However, little is known about such effects at high altitudes where the environmental conditions are harsher than those at low altitudes. We carried out a field experiment in an alpine meadow in the northeast Tibetan Plateau to test the hypothesis that a negative interaction between root and shoot competition exists for alpine plant species. Root and shoot competition were experimentally manipulated in the four grass species (Kobresia humilis, Saussurea superba, Stipa aliena and Elymus nutans). We found that K. humilis and S. aliena grew better without competition, whereas S. superba grew better with shoot competition and E. nutans grew better with root competition. The interactions between root and shoot competition were negative in K. humilis, positive in S. superba, but neutral in S. aliena and E. nutans, suggesting that the interaction effects are species-specific. This study also suggested that alpine plants may trade off both plant–plant interactions and competition shift between root and shoot to adapt to stressful environments.     

MALDI-TOF-MS and 16S rRNA characterization of lead tolerant metallophile bacteria isolated from saffron soils of Kashmir for their sequestration potential

Toxic metal contamination in soils due industrialization is nowadays a concern to the scientists worldwide. The current study deals with the evaluation of response and tolerance by isolated metallophilic bacteria in different lead concentrations (100 ppm to 1000 ppm). By taking optical densities of the isolates, the minimum inhibitory concentration (MIC) of Pb²⁺ were determined.16S rRNA and MALDI-TOF MS were used for the identification of the bacteria. Total of 37 isolates were observed, among them 04 (Staphylococcus equorum, Staphylococcus warneri, Bacillus safensis and Bacillus thuringiensis), isolated were detected having efficacy of Pb²⁺tolerance and sequestration at varying MIC. Furthermore, B. thuringiensis was observed to have highest (900 ppm) tolerance for lead and lowest (500 ppm) for Staphylococcus warneri. Moreover, the highest (65.3%) sequestration potential has been observed for B. thuringiensis and least (52.8%) for S. warneri. The tolerance and sequestration potential properties of these isolated species can be utilised to exterminate heavy metals and reduce their toxicity from the contaminated environment.     

Strangler unmasked: Parasitism of Cystoderma amianthinum by Squamanita paradoxa and S. pearsonii

The mushroom-forming genus Squamanita comprises 10 described species, all parasitic on basidiomes of other members of the order Agaricales, including members of the genera Cystoderma, Galerina, Inocybe and Hebeloma. Here we report an anatomical investigation of the stipitate “mycocecidium’ (=fungus gall) formed on the basidiome of Cystoderma amianthinum (“powdercap”) by S. paradoxa (“powdercap strangler”), alongside the development of taxon-specific-PCR primer to localise the presence of S. paradoxa/C. amianthinum mycelia within mycocecidia, in associated plant tissues and apparently healthy host basidiomes. Dissection of fungarium samples also confirmed these findings, whilst ITS barcode sequencing of all available samples held at the RBG Kew and Edinburgh fungaria did not reveal any variation in ITS sequences within UK populations of S. paradoxa or the closely related S. pearsonii. The absence of any ¹³C or ¹⁵N isotopic differences between C. amianthinum and S. paradoxa suggests that S. paradoxa is nutritionally dependent on its host. The status of C. amianthinum as host of S. pearsonii is also confirmed.     

Les Cervidae (Mammalia, Artiodactyla) du Pléistocène supérieur-Holocène ancien de la région du Parc National Serra da Capivara (Piauí, Brésil)

The Upper Pleistocene/Lower Holocene fossil-bearing sites of the Serra da Capivara National Park Region have yielded three cervid species: Mazama gouazoubira, M. americana and Blastocerus dichotomus, all currently living in South America, the two first in the region. A grand total of more than one hundred remains demonstrates the presence of Mazama gouazoubira in seven sites, mainly the Toca das Moendas, the Toca do Serrote do Artur, the Toca da Cima dos Pilão. This small species shows, since the Upper Pleistocene, a conspicuous tendency to reduce the average dimensions of its teeth and long bones. From the taller M. americana, only a dozen of remains were found in four sites, mainly the Sitio do Meio. In all of these it is sympatric with M. gouazoubira. It differs from this last one by its cheek teeth and its limb bones size and proportions. The oldest site where the species is known is Tarija (Bolivia, Middle Pleistocene) and it does not show any significant changes in size and proportions between recent and fossil samples. Sixteen remains of the large B. dichotomus were found in five sites, mainly the Toca das Moendas and the Toca da Barra do Antonião. The species is a rare fossil, but is frequently figured in the rock art painting of the region, where it is presently unknown.     

In vitro leukocyte response of three-spined sticklebacks (Gasterosteus aculeatus) to helminth parasite antigens

Helminth parasites of teleost fish have evolved strategies to evade and manipulate the immune responses of their hosts. Responsiveness of fish host immunity to helminth antigens may therefore vary depending on the degree of host-parasite counter-adaptation. Generalist parasites, infective for a number of host species, might be unable to adapt optimally to the immune system of a certain host species, while specialist parasites might display high levels of adaptation to a particular host species. The degree of adaptations may further differ between sympatric and allopatric host-parasite combinations. Here, we test these hypotheses by in vitro exposure of head kidney leukocytes from three-spined sticklebacks (Gasterosteus aculeatus) to antigens from parasites with a broad fish host range (Diplostomum pseudospathaceum, Triaenophorus nodulosus), a specific fish parasite of cyprinids (Ligula intestinalis) and parasites highly specific only to a single fish species as second intermediate host (Schistocephalus pungitii, which does not infect G. aculeatus, and Schistocephalus solidus, infecting G. aculeatus). In vitro responses of stickleback leukocytes to S. solidus antigens from six European populations, with S. solidus prevalence from <1% to 66% were tested in a fully crossed experimental design. Leukocyte cultures were analysed by means of flow cytometry and a chemiluminescence assay to quantify respiratory burst activity. We detected decreasing magnitudes of in vitro responses to antigens from generalist to specialist parasites and among specialists, from parasites that do not infect G. aculeatus to a G. aculeatus-infecting species. Generalist parasites seem to maintain their ability to infect different host species at the costs of relatively higher immunogenicity compared to specialist parasites. In a comparison of sympatric and allopatric combinations of stickleback leukocytes and antigens from S. solidus, magnitudes of in vitro responses were dependent on the prevalence of the parasite in the population of origin, rather than on sympatry. Antigens from Norwegian (prevalence 30–50%) and Spanish (40–66%) S. solidus induced generally higher in vitro responses compared to S. solidus from two German (<1%) populations. Likewise, leukocytes from stickleback populations with a high S. solidus prevalence showed higher in vitro responses to S. solidus antigens compared to populations with low S. solidus prevalence. This suggests a rather low degree of local adaptation in S. solidus populations, which might be due to high gene flow among populations because of their extremely mobile final hosts, fish-eating birds.     

In vitro and in vivo evaluation of the wound healing properties and safety assessment of two seaweeds (Sargassum ilicifolium and Ulva lactuca)

Seaweeds have been regarded as a reservoir of biologically active molecules that are important in the pharmaceutical industry. The aim of the present study was to explore the wound healing properties and to assess the safety of the seaweed Sargassum ilicifolium and Ulva lactuca. Enhanced cell proliferation and cell migration activities were observed in L929 cells treated with S. ilicifolium extract compared to U. lactuca extract treated cells and the control group. In-vivo experiments were conducted using five groups (10 in each) of Albino mice (BALB/c). Mice in group I and group II were treated (Orally, 100 mg/kg BW/day) with aqueous extracts of S. ilicifolium and U. lactuca, respectively for 14 days. Treatment group III received a topical application of the aqueous extract of S. ilicifolium (25% w/w) and ointment base (75% w/w) (2 g/kg BW/day, for 14 days). Group IV (Control) received an equal amount of distilled water, orally and mice in group V kept without wounds. The extract from S. ilicifolium showed stronger wound healing properties than the one from Ulva lactuca. Histopathological findings also revealed that the healing process was significantly enhanced in the mice group treated orally with S. ilicifolium aqueous extract. These findings show that S. ilicifolium species possess promising wound healing properties in-vitro and in-vivo.