A new assay for quantifying brown algal phlorotannins and comparisons to previous methods

Quantitative measurement of phlorotannins (polyphenolics) in brown algae (Phaeophyta) by colorimetric assays can be confounded because: (1) most such assays also react to nonphlorotannin substances (interferences) and (2) the appropriate reference compound for such assays is not always clear, although phloroglucinol is typically used. We developed a new assay in which 2,4-dimethoxybenzaldehyde (DMBA) reacts specifically with 1,3-and 1,3,5-substituted phenols (e.g., phlorotannins) to form a colored product. This new assay, as well as eliminating the problem of measuring interferences, is inexpensive, rapid, and can be used with small sample volumes. We recommend it for all assays of phlorotannins from one or a set of closely related species where the structural types of phlorotannins present are likely to be similar among samples. It is also appropriate for broader surveys of phlorotannin levels across many species, but in this case a reference must be chosen with care. We also compared the DMBA assay to existing assays, including the Folin-Denis [both before and after the samples were mixed with polyvinylpolypyrrolidone (PVPP)] and the Prussian blue assays. PVPP was not 100% efficient (and often much less) at removing phlorotannins from solution, and its effectiveness varied among different phlorotannins. Thus, in contrast to previous studies, measuring phenolic levels in extracts before and after treatment with PVPP will not necessarily result in an interference-free measure of phlorotannins. Based on an analysis of reactive substances in red and green algae (which do not contain phlorotannins) in the Folin-Denis and Prussian blue assays, we estimate that the average level of interferences (nonphlorotannins) in brown algae measured in these two assays is on the order of 0.5% by dry weight.     

Sequestration of lichen compounds by three species of terrestrial snails

Three species of lichen-grazing snails,Balea perversa, Chondria clienta, andHelicigona lapicida, all from the Swedish island of Öland, were found to sequester lichen compounds when feeding on the crustous lichen speciesAspicila calcarea, Caloplaca flavovirescens, Lecanora muralis, Physcia adscendens, Tephromela atra, andXanthoria parietina. The lichen compounds detected in the soft bodies of the snail species analyzed included the anthraquinone parietin, the depside atranorin, as well as a presumable degradation product of the latter. Other lichen compounds such as (+)-usnic acid or -collatolic acid were not found in the soft bodies but were only detected in the feces, suggesting selective uptake of lichen compounds by the snails. In individuals ofC. clienta initially fed on the lichenX. parietina, the amount of sequestered parietin decreased over time on a parietin-free diet but was still detectable in the soft bodies after 28 days. In the ovoviviparous land snail,B. perversa, sequestered parietin was transferred from the mother to the eggs in the reproductive tract.Dedicated to Prof. Dr. F.-C. Czygan on the occasion of his 60th birthday.     

CONTENTS OF SOLUBLE,CELL-WALL-BOUND AND EXUDED PHLOROTANNINS IN THE BROWN ALGA <Emphasis Type="Italic">Fucus vesiculosus</Emphasis>, WITH IMPLICATIONS ON THEIR ECOLOGICAL FUNCTIONS

Phlorotannins are ubiquitous secondary metabolites in brown algae that are phenotypically plastic and suggested to have multiple ecological roles. Traditionally, phlorotannins have been quantified as total soluble phlorotannins. Here, we modify a quantification procedure to measure, for the first time, the amount of cell-wall-bound phlorotannins. We also optimize the quantification of soluble phlorotannins. We use these methods to study the responses of soluble and cell-wall-bound phlorotannin to nutrient enrichment in growing and nongrowing parts of the brown alga Fucus vesiculosus. We also examine the effects of nutrient shortage and herbivory on the rate of phlorotannin exudation. Concentrations of cell-wall-bound phlorotannins were much lower than concentrations of soluble phlorotannins; we also found that nutrient treatment over a period of 41 days affected only soluble phlorotannins. Concentrations of each phlorotannin type correlated positively between growing and nongrowing parts of individual seaweeds. However, within nongrowing thalli, soluble and cell-wall-bound phlorotannins were negatively correlated, whereas within growing thalli there was no correlation. Phlorotannins were exuded from the thallus in all treatments. Herbivory increased exudation, while a lack of nutrients had no effect on exudation. Because the amount of cell-wall-bound phlorotannins is much smaller than the amount of soluble phlorotannins, the major function of phlorotannins appears to be a secondary one.     

Comparison of techniques for extracting volatile compounds from conifer needles

Five extraction techniques were used to obtain quantitative measurements of monoterpenes from blue spruce and grand fir needles. Multivariate analysis of variance indicated that there was a significant difference in the amount of each monoterpene extracted by using different techniques. For both species, an 8-hr steam distillation of 10 g of material yielded the greatest amount of each monoterpene. This technique was also the most reproducible in that the coefficients of variation were lowest. A rapid steam distillation was relatively effective for grand fir, but not for blue spruce. A CO₂ extraction was unreliable for both species.     

Biocidal activity of algal toxins against immature mosquitoes

Extracts of two species of green algae, filamentousRhizoclonium hieroglyphicum Kütz and a phytoplankton,Chlorella ellipsoidea Gerneck, obtained with solvents in the laboratory were assayed againstAedes aegypti L.,Culex quinquefasciatus Say, andCuliseta incidens (Thomson). On extraction with petroleum ether, groundR. hieroglyphicum yielded an active crude extract which was chromatographed on a neutral alumina column and eluted consecutively with petroleum ether, benzene, and methanol. All three eluted fractions were found to induce significant mortality in test mosquito species. The benzene-eluted fraction was the least toxic. The methanol-eluted fraction was the most toxic to all species and exhibited juvenile hormone-like activity; it also caused morphogenetic changes in emerging adults. All three fractions delayed the rate of development of mosquito larvae by 2–5 days. Three supernatants ofC. ellipsoidea obtained on different occasions were tested against first instars ofC. quinquefasciatus. After the confirmation of their activity, all supernatants were extracted with diethyl ether, combined, and assayed against first- and fourth-stage larvae of the three mosquito species. The first-stage larvae ofC. quinquefasciatus andC. incidens were approximately three times more suspectible than those ofA. aegypti. However, fourth-stage larvae of the former two species were about twice as susceptible to the extract as those of the latter species. Dead first-stage larvae of all the species had a shrunken appearance. In general,C. ellipsoidea extract was quicker acting than that ofR. hieroglyphicum.     

Response of brown tree snakes (Boiga irregularis) to human blood

Ten specimens ofBoiga irregularis were presented with clean or bloody tampons. The latter were used by women during menses. Trial duration was 60 sec, intertrial interval was 24 hr, and the dependent variable was rate of tongue flicking (a measure of chemosensory investigation). Bloody tampons elicited significantly more tongue flicking than did control tampons. An additional snake is shown attacking and ingesting a soiled tampon, confirming that chemosensory interest was associated with predatory behavior.     

Predatory response of brown tree snakes to chemical stimuli from human skin

The brown tree snake (Boiga irregularis) is an exotic pest species on Pacific islands, most notably on Guam where it has caused considerable ecological and economic damage. On Guam, the snake commonly associates with people and can be found near or in human habitations. Bites are common, approximately 1 of 1200 emergency room visits to Guam hospitals were reported to be the result of B. irregularis bites; 80% of these victims were attacked while sleeping. Most of the attacks occurred on fingers and hands and the attacks appeared to be predatory, rather than defensive, in nature. In order to characterize the mechanism releasing this unusual behavior, we measured the predatory response of B. irregularis to chemical stimuli from humans and controls using a lab population that originated from Guam and a wild population from the species' native range in Queensland, Australia. To quantify behavior we measured the proportion of snakes displaying predatory behavior to each of the stimuli, the latency to attack, and the number of tongue-flicks displayed. We quantified predatory behavior using the tongue-flick attack score for repeated measures [TFAS(R)], a common method for quantifying predatory behavior in squamate reptiles. Captive brown tree snakes responded to human skin stimuli with feeding behavior, including predatory attacks, at the same frequency as they did to prey stimuli derived from mice, while never responding to controls with such behavior. Captive snakes also responded to human skin stimuli and prey stimuli with significantly higher TFAS(R) scores than to controls, although there were no differences between the human and mouse stimuli. Wild-caught animals in Australia also responded with predatory attacks to human skin stimuli, while not showing predatory behavior to a blank control and with higher TFAS(R) scores to human skin stimuli than to the control. As B. irregularis is a generalized predator that relies heavily on chemical signals to recognize prey, we hypothesize that the snakes recognize compounds on human skin that may be shared with other prey.     

Extraction of phenolic compounds from fresh leaves: A comparison of methods

The conventional sonicator/shaker bath method for phenolic extraction was compared with a less traditional one using a homogenizer. The homogenizer proved to be both more efficient and consistent in extracting phenolics from tender, as well as tough, leaves. We propose that adoption of the homogenizer technique will increase phenolic yield and efficiency.     

Feeding-stimulant activity of algal glycerolipids for marine herbivorous gastropods

Phagostimulant activity of glycerolipids such as digalactosyldiacylglycerol (DGDG), 6-sulfoquinovosyldiacylglycerol (SQDG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), etc., have been examined using the Avicel plate method for three kinds of marine herbivorous gastropods, the abaloneHaliotis discus, the turban shellTurbo cornutus, and the topshellOmphalius pfeifferi. DGDG showed strong activity for all the test animals. SQDG was much less active than the other glycerolipids for abalone. The turban shell and the top shell responded more or less sensitively to all tested glycerolipids at doses of 10–20g/sample zone.Chemical Studies on Phagostimulants for Marine Gastropods. Part VIII. For Part VII, see Sakata et al. (1988a).     

Effect of steam distillate extract of resistant wild riceOryza officinalis on behavior of brown planthopperNilaparvata lugens (Stal) (Homoptera: Delphacidae)

Rice plant volatiles extracted as steam distillates significantly affected the behavior of the brown planthopperNilaparvata lugens (Stal). In a choice test, significantly more females settled and fed on tillers of a susceptible rice variety, Taichung Native 1 (TN 1), sprayed with acetone or untreated TN 1 plants than on TN 1 tillers sprayed with the extracts of resistant wild riceOryza officinalis. O. officinalis steam distillate was highly toxic to first-instar nymphs ofN. lugens. Ingestion and assimilation of food by females was significantly reduced on TN 1 plants sprayed withO. officinalis distillate compared to TN 1 plants sprayed with acetone. Application of0. officinalis distillate on TN 1 plants adversely affected hatchability of eggs ofN. lugens.     

Photocatalytic inactivation of algal growth in eutrophic water with hollow glass beads

Photocatalytic inactivation of algae,Anabaena, Microcystis, andMelosira, was performed with TiO₂-coated pyrex glass beads under the illumination of UV light (370 nm wavelength). After being irradiated with UV light in the presence of the TiO₂-coated pyrex glass beads,Anabaena andMicrocystis, known as typical cyanobacteria, lost their photosynthetic activity, and the string ofAnabaena cells and the colonies ofMicrocystis cells were completely separated into individual spherical ones. In the case ofMelosira, which is a typical diatom, however, somewhat lower photocatalytic inactivation efficiency was obtained, which was believed to be due to the presence of the inorganic siliceous wall surrounding the cells ofMelosira.     

Role of plant volatiles in resistance of selected rice varieties to brown planthopper,Nilaparvata lugens (Stål) (Homoptera: Delphacidae)

Rice plant volatiles extracted as steam distillates significantly affected the behavior and biology of the brown planthopper,Nilaparvata lugens (Stål). In a multichoice test, more females settled and fed on tillers of a susceptible rice variety Taichung Native 1 (TN1), sprayed with its own extract or acetone than on TN1 tillers sprayed with the extract of the resistant variety ARC6650 or Ptb33. In another test,N. lugens females ingested significantly more of a 10% sucrose solution mixed with TN1 steam distillate extract than of plain sucrose solution or that mixed with extracts of resistant varieties. Topical application of the extracts of resistant varieties Mudgo, ASD7, Rathu Heenati, Babawee, Ptb33, and ARC6650 caused significantly higher mortality of females than did the TN1 extract. Likewise, significantly more first-instar nymphs died when they were caged on susceptible TN1 plants sprayed with the extracts of resistant varieties than on plants sprayed with TN1 extract. The extract of 60-day-old resistant plants was more toxic than the extract of 30-, 45-, or 100-day-old plants. However, toxicity of the extract from susceptible TN1 remained low at all plant growth stages.Principal Research Scientist (ICIPE), based at International Rice Research Institute (IRRI), P.O. Box 933, Manila, Philippines.     

Effects of three phenolic compounds onLemna gibba G3

Lemna gibba L. G3, (duckweed) was used as a bioassay organism to test the allelochemical effects of salicylic acid (SA), ferulic acid (FA), and umbelliferone (UM). Growth rate (K), dry weight (DW) and total chlorophyll (CHL) production were measured after seven days of growth. The bioassay procedure used 50 ml of E medium with and without sucrose in 125-ml Erlenmeyer flasks plus the selected concentration of allelochemical. At concentrations of 50 M and greater, SA caused inhibition of K and DW production inL. gibba G3, while the threshold for CHL reduction was 20 M. FA inhibited the DW and CHL production at 100 M when the compound was auto-claved in E medium containing sucrose. Treatments of UM were least toxic with an inhibition threshold of 500 M for K and DW production in medium without sucrose. UM did not reduce CHL production until 750 M. In some cases, different thresholds of inhibition were observed depending on the presence or absence of sucrose and tartaric acid in the medium, and whether or not the chemicals were autoclaved with the medium.This research was presented by the senior author as partial fulfillment for the Master of Arts degree, Biology Department, Hood College, Frederick, Maryland. Mention of a trade name or proprietary product does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products that may be suitable.     

Behavioral correlates for minor volatile compounds from stink bugs (Heteroptera: Pentatomidae)

Many terrestrial Heteroptera have small, but functional, dorsal abdominal glands as adults. The chemistry, and associated intra- and inter-specific behavior, for dorsal abdominal gland secretions from 10 species representing four genera of Pentatomidae was investigated. Eighteen volatile compounds were identified in species-specific blends from the dorsal abdominal gland secretions ofEuschistus, Acrosternum, andEurydema adults, including aliphatic, aromatic, and terpenoid constituents. Evidence from bioassays is presented that parasitic Tachinidae (Diptera) and Scelionidae (Hymenoptera) use these secretions as kairomones. A field experiment was performed to test the hypothesis that minor volatiles increase the specificity of the main pheromone component from NearcticEuschistus species, methyl (2E,4Z)-decadienoate. However, significantly fewer individuals ofE. tristigmus were captured in traps baited with the complete blend for this species than in traps baited with methyl (2E,4Z)-decadienoate alone. Thus, at the concentrations tested, these kinds of dorsal abdominal gland secretions may be epideictic, promoting spacing in the natural habitat.     

Effect of drying methods on bioactive compounds,nutritional, antioxidant,and antidiabetic potential of brown alga Durvillaea antarctica

Abstract

The effects of vacuum, solar, convective, infrared, and freeze drying (VD, SD, CD, IRD, FD) on Durvillaea antarctica were evaluated in terms of nutritional composition, color, bioactive components, and antioxidant and antidiabetic activities. Nutritional composition of the dried seaweeds scarcely varied, while a tendency to lose the reddish tone occurred. CD-seaweed had highest phytochemical and vitamin contents, while FD-samples showed best retention of pigments. Both samples also exhibited the strongest antioxidant and antidiabetic activities. The highest essential amino acids and polyunsaturated fatty acids contents were found in IRD- and SD-samples, respectively. In general, FD and CD were proved to be viable drying options for D. antarctica.     

Comparison of NO adsorbing ability and process on Pt/Mg/Al oxide catalysts prepared by different methods

Pt/Mg/Al metal oxide catalysts were prepared by impregnation and co-precipitation methods, respectively. These samples were characterized by BET, XRD and NO-TPD; their NO _X storage property and adsorbing intermediate species were investigated with NSC and FTIR. The results showed that the prepared methods exert significant influence on the physical structure properties and the adsorption abilities of NO. (Pt)/Mg/Al samples prepared by impregnation (IM) have larger specific areas and higher NO _X storage capacity than (Pt)/Mg/Al catalysts prepared by co-precipitation (CP). The intermediate species of NO adsorbing process indicated that NO was firstly adsorbed as bridged nitrites both on Pt/Mg/Al (IM) and on Pt/Mg/Al (CP), then on Pt/Mg/Al (IM) the nitrites transferred into monodentate and bidentate nitrate species while on Pt/Mg/Al (CP) the nitrites only transferred into monodentate nitrate species.     

Influence of antifungal compounds from a soil-borne actinomycete onFusarium spp. in asparagus

Asparagus decline syndrome is caused by fungal infection of asparagus roots and crowns byFusarium oxysporum f.sp.asparagi (FOA) andF. moniliforme (FM). Several soil-borne microorganisms have been found inhibitory toFusarium pathogens in other crops. A novelStreptomyces spp. (ME2-27-19A) was isolated from asparagus field soil and found to be inhibitory to FOA and FM in vitro. Solvent extraction of ME2-27-19A and Chromatographic purification of the extract yielded compound(s) that were inhibitory to FOA and FM at 40g/ml. ME2-27-19A extract produced variable control of FOA and FM in vitro, and was phytotoxic at 1000 (g/ml. In soil, ME2-27-19A extract reduced theFusarium population at 100g/ml, but also reduced the asparagus shoot length.     

3种支原体检测方法的比较

目的比较PCR法、酶法和DNA荧光染色法3种支原体检测方法的灵敏度。方法支原体阳性的BSR细胞培养上清10倍系列稀释后(10-1~10-8),接种至支原体阴性的Vero细胞上,盲传培养5代,每代每个稀释度的样品分别采用PCR法、酶法和DNA荧光染色法检测支原体,并以支原体阴性的Vero细胞作为阴性对照。结果 10倍系列稀释的阳性样品盲传1代,PCR法能检测到10-4,酶法和DNA荧光染色法能检测到10-3;盲传2代,3种方法均能检测到10-4;盲传3代后,3种方法均能检测到10-5,且检测的支原体滴度不随盲传代次的增加而增加。结论待检样本盲传3代后的支原体用3种方法均可检出,检测灵敏度一致。PCR法与酶法检测支原体准确、快速、简便易行,可作为DNA荧光染色法(支原体检测的金标准)的补充手段。     

Inhibitory effects of terpene alcohols and aldehydes on growth of green algaChlorella pyrenoidosa

The growth of the green algaChlorella pyrenoidosa was inhibited by terpene alcohols and the terpene aldehyde citral. The strongest activity was shown by citral. Nerol, geraniol, and citronellol also showed pronounced activity. Strong inhibition was linked to acyclic terpenes containing a primary alcohol or aldehyde function. Inhibition appeared to be taking place through the vapor phase rather than by diffusion through the agar medium from the terpene-treated paper disks used in the system. Inhibition through agar diffusion was shown by certain aged samples of terpene hydrocarbons but not by recently purchased samples.     

Action of antialgal compounds fromJuncus effusus L. onSelenastrum capricornutum

Twenty-nine free and glucosylated dihydrophenanthrenes and tetrahydropyrenes isolated fromJuncus effusus have been tested against the unicellular green algaSelenastrum capricornutum. The free compounds showed a strong inhibitory activity, while the glucosides were inactive or had a slight stimulating effect on growth. Tetrahydropyrene33, obtained by hydrolysis of the natural-glucosides29 and30 or by hemisynthesis from1, is the most active compound, causing 90% inhibition of the growth at 2.5 × 10^–5 M concentration. Synthetic -glucosides28, 31, and32 showed comparable activity to those of29 and30.