A rare new species of Polhillia (Genisteae,Fabaceae)

The new species Polhillia ignota Boatwr. is described. This species is known only from two collections, one between Vredenburg and Saldanha and the other close to Porterville. The new species is most similar to P. obsoleta, which is known only from a few localities around Worcester, in its narrow, sericeous leaflets and flowers of equal size, but differs in its flattened mature leaves with larger leaflets (up to ± 12 mm long), longer pseudo-peduncles (up to ± 15  mm long), denser inflorescences (with up to four flowers), shorter pedicels (1–2 mm long) and non-auriculate wing- and keel petals.     

Repellent activity of catmint, Nepeta cataria, and iridoid nepetalactone isomers against Afro-tropical mosquitoes, ixodid ticks and red poultry mites

The repellent activity of the essential oil of the catmint plant, Nepeta cataria (Lamiaceae), and the main iridoid compounds (4aS,7S,7aR) and (4aS,7S,7aS)-nepetalactone, was assessed against (i) major Afro-tropical pathogen vector mosquitoes, i.e. the malaria mosquito, Anopheles gambiae s.s. and the Southern house mosquito, Culex quinquefasciatus, using a World Health Organisation (WHO)-approved topical application bioassay (ii) the brown ear tick, Rhipicephalus appendiculatus, using a climbing repellency assay, and (iii) the red poultry mite, Dermanyssus gallinae, using field trapping experiments. Gas chromatography (GC) and coupled GC-mass spectrometry (GC-MS) analysis of two N. cataria chemotypes (A and B) used in the repellency assays showed that (4aS,7S,7aR) and (4aS,7S,7aS)-nepetalactone were present in different proportions, with one of the oils (from chemotype A) being dominated by the (4aS,7S,7aR) isomer (91.95% by GC), and the other oil (from chemotype B) containing the two (4aS,7S,7aR) and (4aS,7S,7aS) isomers in 16.98% and 69.83% (by GC), respectively. The sesquiterpene hydrocarbon (E)-(1R,9S)-caryophyllene was identified as the only other major component in the oils (8.05% and 13.19% by GC, respectively). Using the topical application bioassay, the oils showed high repellent activity (chemotype A RD₅₀ = 0.081 mg cm⁻² and chemotype B RD₅₀ = 0.091 mg cm⁻²) for An. gambiae comparable with the synthetic repellent DEET (RD₅₀ = 0.12 mg cm⁻²), whilst for Cx. quinquefasciatus, lower repellent activity was recorded (chemotype A RD₅₀ = 0.34 mg cm⁻² and chemotype B RD₅₀ = 0.074 mg cm⁻²). Further repellency testing against An. gambiae using the purified (4aS,7S,7aR) and (4aS,7S,7aS)-nepetalactone isomers revealed overall lower repellent activity, compared to the chemotype A and B oils. Testing of binary mixtures of the (4aS,7S,7aR) and (4aS,7S,7aS) isomers across a range of ratios, but all at the same overall dose (0.1 mg), revealed not only a synergistic effect between the two, but also a surprising ratio-dependent effect, with lower activity for the pure isomers and equivalent or near-equivalent mixtures, but higher activity for non-equivalent ratios. Furthermore, a binary mixture of (4aS,7S,7aR) and (4aS,7S,7aS) isomers, in a ratio equivalent to that found in chemotype B oil, was less repellent than the oil itself, when tested at two doses equivalent to 0.1 and 0.01 mg chemotype B oil. The three-component blend including (E)-(1R,9S)-caryophyllene at the level found in chemotype B oil had the same activity as chemotype B oil. In a tick climbing repellency assay using R. appendiculatus, the oils showed high repellent activity comparable with data for other repellent essential oils (chemotype A RD₅₀ = 0.005 mg and chemotype B RD₅₀ = 0.0012 mg). In field trapping assays with D. gallinae, addition of the chemotype A and B oils, and a combination of the two, to traps pre-conditioned with D. gallinae, all resulted in a significant reduction of D. gallinae trap capture. In summary, these data suggest that although the nepetalactone isomers have the potential to be used in human and livestock protection against major pathogen vectors, intact, i.e. unfractionated, Nepeta spp. oils offer potentially greater protection, due to the presence of both nepetalactone isomers and other components such as (E)-(1R,9S)-caryophyllene.     

Synthesis, crystal structure and luminescent behaviour of coordination complexes of copper with bi- and tridentate amines and phosphonic acids

The synthesis and crystal structure of four new copper(I) and copper(II) supramolecular amine, and amine phosphonate, complexes is reported. Reaction of copper(I) with 2-,9-dimethyl-1-10-phenanthroline (dmp) produced a stable 4-coordinate Cu(I) species, [Cu^(I)(dmp)₂]Cl · MeOH · 5H₂O (2), i.e., the increased steric hindrance in the ‘bite’ area of dmp did not prevent interaction with the metal and provided protection against oxidation which was not possible for the phen analogue [R. Clarke, K. Latham, C. Rix, M. Hobday, J. White, CrystEngCommun. 7(3) (2005), 28-36]. Subsequent addition of phenylphosphonic acid to (2) produced two structures from alternative synthetic routes. An ‘in situ’ process yielded red block Cu(I) crystals, [Cu^(I)(dmp)₂] · [C₆H₅PO₃H₂ · C₆H₅PO₃H] (4), whilst recrystallisation of (2) prior to addition of the acid (‘stepwise’ process) produced a green, needle-like Cu(II) complex, [Cu^(II)(dmp) · (H₂O)₂ · C₆H₅PO₂(OH)] [C₆H₅PO₂(OH)] (3). However, addition of excess dmp during the ‘stepwise’ process forced the equilibrium towards product (4) and resulted in an optimum yield (99%). The structure of (4) was similar to the phen analogue, [Cu^(II)Cl(phen)₂] · [C₆H₅PO₂(OH) · C₆H₅PO(OH)₂] (1) [R. Clarke, K. Latham, C. Rix, M. Hobday, J. White, CrystEngCommun. 7(3) (2005), 28-36], but the presence of dmp exerted some influence on global packing, whilst (3) exists as a polymeric layered material. In contrast, reaction of copper(I) with di-2-pyridyl ketone (dpk), followed by phenylphosphonic acid produced purple/blue Cu(II) species, [Cu^(II)(dpk · H₂O)₂] Cl₂ · 4H₂O (5), and [Cu^(II)(dpk · H₂O)₂] · [C₆H₅PO₂(OH)₂ · C₆H₅PO(OH)₂] (6), respectively, i.e., in both cases oxidation of copper occurred. Solid-state luminescence was observed in (2) and (4). The latter showing a 5-fold enhancement in intensity.     

Experimental evaluation of the pathogenicity of Perkinsusolseni in juvenile Manila clams Ruditapes philippinarum

We evaluated the pathogenicity of Perkinsus olseni towards the Manila clam, Ruditapes philippinarum, by an experimental challenge. For production of prezoosporangia of P. olseni, we injected uninfected Manila clams with cells of a pure strain of P. olseni and reared them for 7 d. Prezoosporangia were isolated from the soft tissue of the injected clams after culturing in Ray’s fluid thioglycollate medium. Hatchery-reared, uninfected juvenile clams (3-10 mm shell length) were challenged by immersion in one of two concentrations of a prezoosporangial suspension of P. olseni for 6 d. The challenged clams had significantly higher mortality at both the concentrations than the unchallenged clams. The mortality due to infection dose-dependently began approximately 4 weeks and 7 weeks after challenge in the higher and lower concentrations, respectively. This is the first experimental evidence that P. olseni causes direct mortality in Manila clams. The lethal level of infection was estimated at approximately 10⁷ pathogen cells/g soft tissue weight.     

Nutritional value of the cryptophyte Rhodomonas lens for Artemia sp.

Juvenile or adult Artemia sp. are often used as live prey for the rearing of early life stages of some crustacean, fish and cephalopod species. The improvements of both Artemia growth and its biochemical composition are key issues for the suitable use of Artemia biomass in these rearing processes. In this study we evaluated the growth and survival rates of Artemia fed with the cryptophyte Rhodomonas lens in comparison with different microalgal species commonly used in aquaculture: the prasinophyte Tetraselmis suecica, the prymnesiophyte Isochrysis galbana Parke, and the eustigmatophyte Nannochloropsis gaditana. Microalgae were cultured semi-continuously in nutrient saturated conditions and with a daily renewal rate of 30% of the volume of cultures, to obtain biomass of controlled and optimized composition. Considerable differences in Artemia growth were observed, as well as in the survival rate. At day 8 of rearing, Artemia fed R. lens had the highest length (4.9 ±0.6 mm, P < 0.001), followed by individuals fed T. suecica (4.2 ± 0.7 mm), I. galbana (3.6 ± 0.7 mm) and finally those fed N. gaditana (1.5 ± 0.2 mm). The survival rate of Artemia fed N. gaditana (18 ± 3%) was much lower (P < 0.001) than values found for the remaining groups (69 to 88%). The growth rate of Artemia obtained with R. lens was in general much higher than with other microalgal diets previously reported in the literature. The higher protein content of R. lens could explain the higher growth obtained with this species, but differences of Artemia growth with the different diets could not be explained solely on the basis of the gross composition of microalgae. Factors such as cell size and digestibility all seem to contribute to the results observed. Another trial was carried out to investigate differences in Artemia growth and on its biochemical composition when fed the best two diets: R. lens or T. suecica. The fatty acid (FA) and total amino acid (AA) composition of both microalgal species and the composition of Artemia were assessed as well. As found in the first experiment individuals fed R. lens (group ARHO) grew faster than those fed T. suecica (group ATET), attaining 3.6 ± 0.3 mm and 3.2 ± 0.4 mm (P < 0.001), respectively, after 5 days of rearing. The much higher AA content obtained in R. lens may be on the basis of the higher growth obtained with this species. Protein and carbohydrate levels in Artemia juveniles were very similar in both groups (64-68% of dry weight, and 8-10%, respectively). Lipid was slightly lower in ARHO (12%) than in ATET (15%, P < 0.01). Regarding the FA composition, juveniles from group ARHO contained higher levels of eicosapentaenoic acid (EPA, 6.2%) than juveniles from ATET (4.1%, P < 0.01), whereas docosahexaenoic acid (DHA) was only found in juveniles from ARHO (1.1%). Taking into account that the daily productivity of R. lens culture was higher than, or at least equal, the remaining microalgal species this cryptophyte is confirmed as an excellent diet to optimize the growth of Artemia, as well as to improve its biochemical composition.     

Virulence of Hypocreales fungi to pecan aphids (Hemiptera: Aphididae) in the laboratory

There is need for efficacious biocontrol agents for aphids in commercial orchards. As a preliminary step to this end we determined the virulence of several Hypocreales fungi to pecan aphids. In the first experiment we tested the virulence of Isaria fumosorosea (ARSEF 3581) blastospores to three pecan aphids Monellia caryella, Melanocallis caryaefoliae, and Monelliopsis pecanis under laboratory conditions. Rates of 1 × 10⁷ or 1 × 10⁸ spores per ml were applied in 2 ml via a spray tower to 90 mm Petri dishes containing 10 aphids each. Mortality and mycosis were determined after 24, 48 and 72 h. Treatment effects were observed by 48 h post-application, and by 72 h the higher application rate caused >90% mortality and mycosis in M. caryella and M. caryaefoliae, whereas <70% was observed in M. pecanis.We conducted two subsequent experiments (Experiments 2 and 3), using the same methodology, to compare the virulence of several Hypocreales species and strains against the aphid of primary economic concern to most pecan growers, M. caryaefoliae. In Experiment 2, we compared blastospores and conidia of two I. fumosorosea strains (ARSEF 3581 and ATCC 20874 [= strain 97]). The blastospores of ARSEF 3581 and conidia of ATCC 20874 showed higher virulence than other treatments and thus were included in Experiment 3, which also compared the virulence of conidia of Beauveria bassiana (GHA strain) and Metarhizium anisopliae (F52 strain). Results in Experiment 3 indicated the highest virulence in I. fumosorosea 3581 blastospores and M. anisopliae (F52) followed by I. fumosorosea (20874) conidia. The detection of pathogenicity to pecan aphids establishes the potential for commercial usage and additional study. Results reported here will narrow treatments to test in future greenhouse and field trials.     

Biochemical and pathogenic properties of the natural isolate of Shewanella algae from Peter the Great Bay, Sea of Japan

Pathogenic properties of the natural isolate of Shewanella algae from the coelomic fluid of the sea cucumber Apostichopus japonicus (Peter the Great Bay, Sea of Japan) were investigated. The isolate had oxydative metabolism, was positive for ornithine decarboxylase, cytochrome oxidase, catalase, DNase and gelatinase, hemolytically active, did not produce acid from carbohydrates, and did not hydrolyze urea and esculin. The strain was resistant to penicillin, amoxicillin, and ampicillin and susceptible to tetracycline and carbenicillin. Among cellular fatty acids, 13:0-i, 15:0-i, 16:0, 16:1(n-7), 17:0-i, and 17:0-ai dominated. These biochemical properties made it possible to attribute the isolated bacteria to the genus Shewanella and identified as S. algae. The cells of this bacterium were introduced into the coelomic cavity of another echinoderm, the sea urchin Strongylocentrotus nudus. As a result, in about 24 h the animals became slow and 3-8 days after the inoculation died. Dividing bacteria were being found during the experiment in the coelomic fluid as well as in the phagosomes of amoebocytes, i.e. cells acting as phagocytes in the coelomic fluid. The studies of the invasive properties of strain 156 showed that bacterial cells entered the subcuticular space of S. nudus and A. japonicus through the cuticle and stayed there for a long time without penetrating epithelium and exerting toxic effect upon the organisms of the laboratory animals. Pathogenic effect of S. algae can be manifested only if the cutaneous epithelium is destroyed permitting it to penetrate the lower tissue layers. The toxicity of S. algae is confirmed by in vitro experiments. The inoculation of the embryonic cells of S. nudus with samples of this bacterium caused the death of 10% of cells within an hour and 100% of cells within 12 h after inoculation. The results of the investigations demonstrate that S. algae could produce opportunistic infection in the sea cucumber A. japonicus and the sea urchin S. nudus, which may be natural reservoirs of this human pathogen.     

Attraction of four entomopathogenic nematodes to four white grub species

To better understand the differences in the efficacy of entomopathogenic nematode species against white grub species, we are studying the various steps of the infection process of entomopathogenic nematodes into different white grub species using nematode species/strains with particular promise as white grub control agents. In this study we compared the attraction of the entomopathogenic nematodes Steinernema scarabaei (AMK001 strain), Steinernema glaseri (NC1 strain), Heterorhabditis zealandica (X1 strain), and Heterorhabditis bacteriophora (GPS11 strain) to third-instars of the scarabs Popillia japonica, Anomala orientalis, Cyclocephala borealis, and Rhizotrogus majalis, and late-instar greater wax moth, Galleria mellonella, larvae. Individual larvae were confined at the bottom of 5.5 cm vertical sand columns, nematodes added to the sand surface after 24 h, and nematodes extracted after another 24 h. Nematode attraction to hosts was strongly affected by nematode species but the effect of insect species varied with nematode species. S. glaseri had a high innate dispersal rate (i.e., in absence of insects) and was strongly attracted to insects without significant differences among insect species. S. scarabaei had a very low innate dispersal rate so that even a strong relative response to insects resulted in low absolute dispersal rates toward insects. S. scarabaei tended to be most attracted to G. mellonella and least attracted to C. borealis. H. zealandica had a high innate dispersal rate but only responded weakly to insects without significant differences among species. H. bacteriophora had limited innate dispersal and only weakly responded to insects with G. mellonella tending to be the most attractive and C. borealis the least attractive insect. It has to be noted that we cannot exclude that the use of different rearing hosts (A. orientalis and P. japonica larvae for S. scarabaei, G. mellonella larvae for the other nematodes) might have had an impact on the nematodes dispersal and relative attraction behavior. This study indicates that host attractiveness and nematode dispersal rates may contribute but do not play a major role in the variability in white grub susceptibility and/or nematode virulence.     

Parental size and environmental conditions affect egg capsule production by Nassarius reticulatus (Linnaeus 1758) (Gastropoda: Nassariidae)

In shallow coastal habitats scavenging netted whelks Nassarius reticulatus attached egg capsules to the stipes of red algae Chondrus crispus and occasionally on Furcellaria lumbricalis and Plumaria plumose. In the laboratory egg capsules were laid on aquaria sides and lids by individuals ≥ 21 mm shell length. Larger size classes produced more egg capsules and spawned over a longer period and in doing so partitioned less energy into shell growth. Large netted whelks (25-28.9 mm) produced larger capsules which contained significantly more and larger eggs than those produced by smaller individuals (21-24.9 mm). Egg capsule production continued throughout the year by regularly fed N. reticulatus held at ambient seawater temperatures. Egg production increased in the spring and summer with peak production during June (15 °C), decreased between August and October and resumed again during the winter (November to February at ∼ 7 °C). During the summer (15-16 °C) egg capsules were smaller and contained smaller eggs than those deposited during the winter (7-10 °C), although the number of eggs · capsule⁻¹ was similar. Enforced food limitation reduced the number and size of the egg capsules, the number and size of eggs produced · female⁻¹ and the duration of the breeding period. Hatching success of N. reticulatus egg capsules was high (95%) even at winter seawater temperatures (11-8.5 °C) and the duration of embryonic development was fastest between 15 and 17.5 °C.     

Enhanced algae growth in both phototrophic and mixotrophic culture under blue light

Biomass productivity and fatty acid methyl esters (FAME) derived from intracellular lipid of a Nannochloropsis sp. isolated from Singapore’s coastal waters were studied under different light wavelengths and intensities. Nannochloropsis sp., was grown in both phototrophic and mixotrophic (glycerol as the carbon source) culture conditions in three primary monochromatic light wavelengths, i.e., red, green and blue LEDs, and also in white LED. The maximum specific growth rate (μ) for LEDs was blue > white > green > red. Nannochloropsis sp. achieved a μ of 0.64 and 0.66 d⁻¹ in phototrophic and mixotrophic cultures under blue lighting, respectively. The intracellular fatty acid composition of Nannochloropsis sp. varied between cultures exposed to different wavelengths, although the absolute fatty acid content did differ significantly. Maximum FAME yield from Nannochloropsis sp. was 20.45% and 15.11% of dry biomass weight equivalent under photo- and mixotrophic culture conditions respectively for cultures exposed to green LED (550 nm). However, maximum volumetric FAME yield was achieved for phototrophic and mixotrophic cultures (i.e., 55.13 and 111.96 mg/l, respectively) upon cell exposure to blue LED (470 nm) due to highest biomass productivity. It was calculated that incremental exposure of light intensity over the cell growth cycle saves almost 20% of the energy input relative to continuous illumination for a given light intensity.     

Flavonol tetraglycosides from fruits of Styphnolobium japonicum (Leguminosae) and the authentication of Fructus Sophorae and Flos Sophorae

The dried fruits and seeds of Styphnolobium japonicum (L.) Schott (syn. Sophora japonica L.) are used in traditional Chinese medicine and known as Fructus Sophorae or Huai Jiao. The major flavonoids in these fruits and seeds were studied by LC-MS and other spectroscopic techniques to aid the chemical authentication of Fructus Sophorae. Among the flavonoids were two previously unreported kaempferol glycosides: kaempferol 3-O-β-glucopyranosyl(1 → 2)-β-galactopyranoside-7-O-α-rhamnopyranoside and kaempferol 3-O-β-xylopyranosyl(1 → 3)-α-rhamnopyranosyl(1 → 6)[β-glucopyranosyl(1 → 2)]-β-glucopyranoside, the structures of which were determined by NMR. Two further tetraglycosides were identified for the first time in S. japonicum as kaempferol 3-O-β-glucopyranosyl(1 → 2)[α-rhamnopyranosyl(1 → 6)]-β-glucopyranoside-7-O-α-rhamnopyranoside and kaempferol 3-O-β-glucopyranosyl(1 → 2)[α-rhamnopyranosyl(1 → 6)]-β-galactopyranoside-7-O-α-rhamnopyranoside; the latter was the main flavonoid in mature seeds. The chromatographic profiles of 27 recorded flavonoids were relatively consistent among fruits of similar ages collected from five trees of S. japonicum, and those of maturing unripe and ripe fruits were similar to a market sample of Fructus Sophorae, and thus provide useful markers for authentication of this herbal ingredient. The flower buds (Huai Mi) and flowers (Huai Hua) of S. japonicum (collectively Flos Sophorae) contained rutin as the main flavonoid and lacked the flavone glycosides that were present in flower buds and flowers of Sophora flavescens Ait., reported to be occasional substitutes for Flos Sophorae. The single major flavonoid in fruits of S. flavescens was determined as 3′-hydroxydaidzein.     

Resistance of the Manila clam (Venerupis philippinarum) to infection with Mikrocytos mackini

Manila clams (Venerupis philippinarum) challenged in laboratory trials via bath exposure proved to be resistant to infections with Mikrocytos mackini (protistan parasite of unknown taxonomic affiliation), while Pacific oysters (Crassostrea gigas) challenged simultaneously using identical conditions developed infections. Although M. mackini was detected by a nucleic acid pathogen specific (PCR) assay in 10-30% of the challenged V. philippinarum that were sampled soon after exposure (0-48 h, n = 40), all of the subsequent V. philippinarum (n = 62) sampled 9-17 weeks post-exposure tested negative for M. mackini by PCR assay. Prevalence of infection for the exposed C. gigas (n = 100) during this same period ranged from 50% to 100% by PCR assay. Infection was confirmed in the oysters (58%, n = 60) by a digoxigenin-labelled DNA probe designed to detect M. mackini by in situ hybridization, but M. mackini was not found in any of the exposed Manila clams (n = 63) using this technique.     

Huernia humpatana (Apocynaceae), a new species from southern Angola

A new species, Huernia humpatana Bruyns (Apocynaceae–Ceropegieae), closely related to H. similis N.E.Br., is described from the Chela Mountains of Huila Province in southern Angola. The two species are distinguished by the 5-angled and erect stems with more prominent tubercles up to 6 mm long joined into clear angles and separated by V-shaped grooves in H. humpatana as opposed to very obtusely 4-angled stems with tubercles only 2 mm long and only indistinct grooves between the angles in H. similis. Furthermore, in H. similis the nodding corolla is ± 9 mm in diameter with sepals ± 2 mm long, while in H. humpatana the horizontally facing corolla is 18–20 mm in diameter with sepals 4–6 mm long.     

Inhibitory effect of essential oils of Allium sativum and Piper longum on spontaneous muscular activity of liver fluke, Fasciola gigantica

Effects of essential oil of Allium sativum (garlic) and Piper longum (Indian long pepper) were evaluated on muscular activity of whole Fasciola gigantica and its strip preparation. The whole flukes and longitudinal strip preparations of the flukes were isometrically mounted to record the spontaneous muscular activity (SMA) and to evaluate effects of cumulative doses (0.1, 0.3, 1.0 and 3.0 mg/ml) of the plant essential oils. Whole flukes and the strip preparations exhibited continuous SMA without any significant difference in its baseline tension, frequency and amplitude for 2 h. Essential oil of A. sativum produced significant reduction in the frequency and the amplitude of the SMA of whole fluke at 1 and 3 mg/ml concentrations. It caused complete paralysis of the fluke after 15 min of administration of 3 mg/ml concentration. Similar to whole fluke, essential oil of A. sativum (3 mg/ml) also produced flaccid paralysis in the strip preparations of the flukes. Essential oil of P. longum firstly induced marked excitatory effect and then there was flaccid paralysis of the whole fluke following 15 min exposure at 3 mg/ml concentration. Complete flaccid paralysis of the strip preparation was also ensued after 15 min of administration of 3 mg/ml concentration of P. longum. In both the essential oils, the whole fluke and strip preparations did not recover from paralysis following 2-3 washes. In conclusion, the observations demonstrated irreversible paralytic effect of essential oils of A. sativum and P. longum on F. giganticain vitro which might possibly help to developing herbal-based anthelmintic.     

Effects of Echinaforce treatment on ex vivo-stimulated blood cells

The herb Echinacea purpurea, also called purple coneflower, is regarded as an immune modulator. This study examined changes in cytokine production in blood samples from 30 volunteers before and during 8-day oral administration with an ethanolic extract of fresh Echinacea purpurea (Echinaforce^®). Daily blood samples were ex vivo stimulated by LPS/SEB or Zymosan and analysed for a series of cytokines and haematological and metabolic parameters. Treatment reduced the proinflammatory mediators TNF-α and IL-1β by up to 24% (p < 0.05) and increased anti-inflammatory IL-10 levels by 13% (p < 0.05) in comparison to baseline. This demonstrated a substantial overall anti-inflammatory effect of Echinaforce^® for the whole group (n = 28). Chemokines MCP-1 and IL-8 were upregulated by 15% in samples from subjects treated with Echinaforce^® (p < 0.05). An analysis of a subgroup of volunteers who showed low pre-treatment levels of the cytokines MCP-1, IL-8, IL-10 or IFN-γ (n = 8) showed significant stimulation of these factors upon Echinaforce^® treatment (30-49% increases; p < 0.05), whereas the levels in subjects with higher pre-treatment levels remained unaffected. We chose the term “adapted immune-modulation” to describe this observation. Volunteers who reported high stress levels (n = 7) and more than 2 colds per year experienced a significant transient increase in IFN-γ upon Echinaforce^® treatment (>50%). Subjects with low cortisol levels (n = 11) showed significant down-regulation of the acute-phase proteins IL1-β, IL-6, IL-12 and TNF-α by Echinaforce^® (range, 13-25%), while subjects with higher cortisol levels showed no such down-regulation. This is the first ex vivo study to demonstrate adapted immune-modulation by an Echinacea preparation. While Echinaforce^® did not affect leukocyte counts, we speculate that the underlying therapeutic mechanism is based on differential multi-level modulation of the responses of the different types of leukocytes. Echinaforce^® thus regulates the production of chemokines and cytokines according to current immune status, such as responsiveness to exogenous stimuli, susceptibility to viral infection and exposure to stress.     

Acylated flavonol tetraglycosides from Delphinium gracile

An ethanol extract of the aerial parts of Delphinium gracile DC. yielded five flavonol glycosides quercetin-3-O-{[β-d-xylopyranosyl (1 → 3)-4-O-(E-p-caffeoyl)-α-l-rhamnopyranosyl (1 → 6)][β-d-glucopyranosyl (1 → 2)]}-β-d-glucopyranoside (1), quercetin-3-O-{[β-d-xylopyranosyl (1 → 3)-4-O-(E-p-coumaroyl)-α-l-rhamnopyranosyl (1 → 6)][β-d-glucopyranosyl (1 → 2)]}-β-d-glucopyranoside (2), quercetin-3-O-{[β-d-xylopyranosyl (1 → 3)-4-O-(Z-p-coumaroyl)-α-l-rhamnopyranosyl (1 → 6)][β-d-glucopyranosyl (1 → 2)]}-β-d-glucopyranoside (3), kaempferol-3-O-{[β-d-glucopyranosyl (1 → 3)-4-O-(E-p-coumaroyl)-α-l-rhamnopyranosyl (1 → 6)][β-d-glucopyranoside-7-O-(4-O-acetyl)-α-l-rhamnopyranoside (4) kaempferol-3-O-{[β-d-glucopyranosyl (1 → 3)-4-O-(E-p-coumaroyl)-α-l-rhamnopyranosyl (1 → 6)][β-d-glucopyranoside-7-O-(4-O-acetyl)-α-l-rhamnopyranoside (5) in addition to 4-(β-d-glucopyranosyloxy)-6-methyl-2H-pyran-2-one (6) and rutin. Structures were elucidated by spectroscopic methods.     

The combined influence of sub-optimal temperature and salinity on the in vitro viability of Perkinsus marinus, a protistan parasite of the eastern oyster Crassostrea virginica

Perkinsus marinus is a major cause of mortality in eastern oysters along the Gulf of Mexico and Atlantic coasts. It is also well documented that temperature and salinity are the primary environmental factors affecting P. marinus viability and proliferation. However, little is known about the effects of combined sub-optimal temperatures and salinities on P. marinus viability. This in vitro study examined those effects by acclimating P. marinus at three salinities (7, 15, 25 ppt) to 10 °C to represent the lowest temperatures generally reached in the Gulf of Mexico, and to 2 °C to represent the lowest temperatures reached along the mid-Atlantic coasts and by measuring changes in cell viability and density on days 1, 30, 60 and 90 following acclimation. Cell viability and density were also measured in 7 ppt cultures acclimated to each temperature and then transferred to 3.5 ppt. The largest decreases in cell viability occurred only with combined low temperature and salinity, indicating that there is clearly a synergistic effect. The largest decreases in cell viability occurred only with both low temperature and salinity after 30 days (3.5 ppt, 2 °C: 0% viability), 60 days (3.5 ppt, 10 °C: 0% viability) and 90 days (7 ppt, 2 °C: 0.6 ± 0.7%; 7 ppt, 10 °C: 0.2 ± 0.2%).     

Inducible defence and its modulation by environmental stress in the red alga Chondrus yendoi (Yamada and Mikami in Mikami, 1965) from Honshu Island, Japan

Among the numerous anti-herbivore defences developed by macroalgae, chemical and morphological traits are best documented and understood. Plant defence theory suggests that these resistances, which can either be constitutive or inducible, are associated with metabolic costs. They should therefore be impaired under conditions of energy limitation, but evidence for this prediction is scarce. In two subsequent experiments, we tested whether a reduction of light availability is changing feeding rates of the two mesoherbivores Idotea ochotensis and Lacuna smithii on the red alga Chondrus yendoi. Algal individuals were kept in outdoor mesocosm facilities for 10 days, during which we manipulated the amount of incoming sunlight at 6 levels (0%-99% reduction, i.e. 2000-20 μmol s^− 1 m^− 2). Orthogonal to this, we established the presence or absence of one of the herbivores to test whether C. yendoi can generate a defence. Algal palatability was investigated afterwards in no-choice feeding assays using naïve grazer individuals. The consumption of algal tissue in L. smithii increased with decreasing light, while this was not the case for I. ochotensis. However, we found a defence induced as a reaction to herbivory only by the highly mobile isopod but not when the slowly moving snail grazed on C. yendoi. Isopod total consumption rates in our experiments were 40 times higher than those of the gastropods. We therefore suggest that C. yendoi exhibits grazer-specific reactions to herbivory, depending on the mobility and voracity of the consumers. Interestingly, only for one of the grazers, i.e. the snail, short-term light reduction influenced the palatability of algal tissue. We discuss different but not mutually exclusive models that could explain this pattern. In conclusion, we view this three-species system as an illustrative example for specificity in grazer-algal interactions and their modification by environmental stress.     

Stage dependent habitat use under conflicting predation pressure: An experimental test with larval and juvenile two-spotted gobies, Gobiusculus flavescens Fabricius

The effect of size, predator types and presence of multiple predators on the microhabitat use of larvae and juveniles of a sublittoral, semipelagically schooling fish, the two-spotted goby (Gobiusculus flavescens), was tested in two experiments. Larvae (15 and 25 days old, Experiment I) and juveniles (mean ± 1 S.E.: small, 15.9 mm ± 1.28; medium, 19.2 mm ± 1.43; and large, 23.4 mm ± 1.67, Experiment II) were allowed to choose between two sections of the tanks; an upper, representing a water column habitat, and a lower, artificially vegetated, representing the hyperbenthic habitat. Position of larvae or juveniles and the activity level of juveniles were recorded. Predator treatments were: (I) no predators (control), (II) a pelagic predator, the jellyfish Aurelia aurita L., (III) a hyperbenthic predator, the mysid Praunus flexuosus O.F. Müller or (VI) both predator types simultaneously. In Experiment I predators were restricted to the habitat which they were chosen to represent, while goby larvae could move freely. In Experiment II both predators and juvenile gobies were allowed to move freely between compartments.Increasing age caused larval gobies, but not juveniles to shift downwards. Only 25-day-old larvae and small juveniles avoided the mysid by shifting upwards. Larval response to A. aurita was also size dependant: 25-day-old larvae avoided medusae by shifting downwards, while 15 day olds did not. Emergent multiple predator effects were found for the vertical distribution of 15-day-old larvae and small juveniles. Larger juveniles were more active than smaller, both in the upper and the lower sections of tanks. P. flexuosus caused juvenile gobies in their vicinity (i.e. in the lower section) to increase their activity level, while small juveniles (but not medium-sized or large) increased their activity level even when further away (i.e. in the upper section). The presence of A. aurita led to a reduction in activity of small juveniles in its vicinity (i.e. in the upper section), while no response was observed among older juveniles or juveniles further away from the predator (i.e. in the lower section). Emergent multiple predator effects on the activity level of juveniles were not observed.     

Annesorhiza asparagoides (Apiaceae), a new species from the Cederberg Mountains, Western Cape Province, South Africa

A highly localised new species from the Cederberg Mountains near Wuppertal in the Western Cape Province is described. Annesorhiza asparagoides B.-E. Van Wyk, collected for the first time in 2009, differs from all other species of Annesorhiza (and the closely related Chamarea) in the unusual leaf structure, with crowded, subsessile, acicular leaf segments, resulting in dense, bottlebrush-like pinnae. The new species has a cluster of 10 or more slender roots, small (< 150 mm long), sparsely hairy leaves and small (± 8 mm long), oblong, conspicuously ribbed, homomericarpic fruits.