The effect of pre-acquisition starvation on aphid transmission of potyviruses during observed and electrically recorded stylet penetrations

The effect of pre-acquisition starvation on stylet penetration behaviour by the aphidMyzus persicae (Sulz.) and the consequent non-persistent transmission of the potyviruses beet mosaic virus (BMV) and potato virus Y (PVY) were investigated. Visual observations indicated that starved aphids initiated penetrations earlier and penetrated for shorter periods than non-starved insects. Tethering with a fine gold wire did not affect these observations with either starved or non-starved aphids, but starvation caused increased PVY and BMV acquisition efficiency, regardless of tethering. Tethered aphids were then made part of an electrical circuit and their stylet activities investigated in detail. Electrically recorded aphids also acquired and inoculated both potyviruses more efficiently when starved, and these acquisitions and inoculations were associated with stylet punctures of plant cell membranes. However, starvation did not affect the occurrence of electrically recorded membrane puncture, suggesting that non-behavioural factors may contribute to the enhancement of virus transmission by pre-acquisition starvation.     

Cell membrane punctures during epidermal penetrations by aphids: consequences for the transmission of two potyviruses

Electrical penetration graphs (EPGs) were used to investigate aphid stylet activities during brief penetrations and the importance of these events for the nonpersistent transmission of two potyviruses, beet mosaic virus (BMV) and potato virus Y (PVY). Stylet puncture of a cell membrane, recorded as a potential drop, was correlated with the acquisition and inoculation of PVY and acquisition of BMV by Myzus persicae.     

Effects of the antifeedant polygodial on plant penetration by aphids, assessed by video and electrical recording

Adult apterousMyzus persicae (Sulz.) discriminated within 2 min between mature Chinese cabbage (Brassica pekinensis L.) leaf halves treated with (±)-polygodial solution and solvent alone, and walked off leaf areas treated with polygodial faster than off the solvent-treated areas. However, when aphids were attached to a fine gold wire and stylet penetration of cabbage leaves was recorded electrically, polygodial treatment did not affect the number or duration of electrically-recorded penetrations, time taken to initiate a first penetration, or total penetration time. As the tethering of aphids causes behavioural restrictions which may negate the response to polygodial, indications of stylet penetration by freely-moving insects were sought. Simultaneous electrical recording and video monitoring showed that stylet penetration duration could be accurately inferred from antennal and body movements, enabling assessment of penetration times without attachment to the wire tether. When freely-movingM. persicae were video recorded during 15 min access to cabbage seedlings, polygodial treatment again had no apparent effect on stylet penetration. However, when aphids were presented with a choice of polygodial- and solvent-treated sides of floating mature cabbage leaf discs, video recordings revealed that the insects spent less time and made fewer penetrations on the polygodial-treated side. In addition to this rapid repellent effect, prolonged exposure to polygodial also produced behavioural changes. After being held for 24 h on polygodial-treated leaves or green paper prior to behavioural examination, aphids penetrated seedlings fewer times but for longer periods. The relevance of the results to virus transmission studies is discussed.     

The effect of mineral oil on stylet activities and potato virus Y transmission by aphids

Mineral oil sprayed onto potato virus Y (PVY) infected tobacco plants reduced acquisition of this potyvirus by Myzus persicae (Sulz.). Although the pre-penetration activities of aphids were longer on oil treated leaves, the inhibitory effect of the oil could not be attributed to differences in the duration of stylet penetration. Aphids were therefore made part of a DC circuit in order to investigate their stylet activities during penetration of PVY infected source plants and healthy test plants. Both acquisition and inoculation of the virus were reduced by the presence of oil on the plant surface, but these reductions could not be related to electrically recorded differences in plant penetration behaviour. In particular, stylet punctures of plant cell membranes were not reduced by mineral oil. Non-behavioural reasons are suggested to explain the mode of action of the oil.     

The combination of electronic monitoring and video-assisted observations of plant penetration by aphids and behavioural effects of polygodial

Simultaneous electronic and close-up video recordings were made of behaviour during the initial 15 min of plant contact by adult, apterous Aphis fabae Scopoli on tick bean seedlings (Vicia faba Moench). Electronic techniques accurately determined stylet penetration of plant tissue and there was a close correlation between penetration and periods during which the insect antennae and body were immobile (r=0.994, n=60). Video techniques were then used alone to infer stylet penetration and the behaviour of aphids after various treatments was monitored. In particular, the time to first penetration, the number of penetrations, the mean duration of penetrations and the total time of penetration were observed. Behavioural differences were recorded between tethered (as required for electronic recording) and freelymoving insects as well as between fed and starved insects. The behaviour of starved aphids placed on beans treated with the plant-derived antifeedant, polygodial could not be distinguished from aphids on solvent-treated control beans. However, there were significant differences in behaviour of aphids which had previously been exposed to polygodial on plant or green/yellow paper surfaces for 24 h when compared with insects exposed to solvent alone. The possible modes of action of polygodial are discussed.     

Seedling resistance of wheat to the powdery mildew fungus,Erysiphe graminis f. sp. tritici. I. Resistance of first leaves

During primary infection by conidia ofErysiphe graminis f. sp.tritici, three mechanisms of resistance operate in first leaves of 8-day-old seedlings of both resistant and susceptible wheats. The first mechanism, operating at the penetration site, is responsible for the failure of penetrations attempted by primary germ tubes (PGT). The second mechanism is concerned with the abortion of haustoria in normal-appearing host cells. The third mechanism relates to the abortion of haustoria and the hypersensitivity of the penetrated host cells.With the inoculum-level of 19–24 conidia/mm², the three mechanisms together prevented 89.3 % of the attempted penetrations by PGT from producing normal haustoria in resistant wheat Purdue 5752C1-7-5-1 and 37.4 % in the susceptible wheat Vermillion. The first mechanism accounted for the prevention of 73.3 % of the attempted PGT penetrations on Purdue 5752C1-7-5-1 and 36 % on Vermillion. The second mechanism was responsible for stopping 19 % of all the successful penetrations in Purdue 5752C1-7-5-1 and 0.8 % in Vermillion. The third mechanism accounted for the failure of 41 % of all the successful penetrations in Purdue 5752C1-7-5-1 and 1.4% in Vermillion. Thirty-six hours after inoculation, 10.7% of all the attempted PGT penetrations appeared to be developing normally in first leaves of 8-day-old seedlings of resistant wheat Purdue 5752C1-7-5-1 as compared to 62.6 % in the susceptible wheat Vermillion.This appears to be the first report showing the relative effectiveness of various mechanisms of resistance concerning any powdery mildew fungus.     

Isolation and characterization of membrane-associated proteoglycans from normal and malignant human mammary epithelial cells

The plasma membrane-associated proteoglycans of a malignant human breast cell line (MDA-MB-231) were compared with the corresponding proteoglycans from a normal cell line (HBL-100). The labeled proteoglycans were isolated from the plasma membranes of cells grown in the presence of [³H]glucosamine and [³⁵S]Na₂SO₄ by extraction with guanidine hydrochloride and subsequently purified by DEAE-ion exchange chromatography. Their structural properties were established by treatment with nitrous acid, heparitinase and chondroitinase ABC, and by gel filtration before and after alkaline -elimination. About 18% of the proteoglycans synthesized by these cell lines were associated with the plasma membranes. The HBL plasma membranes contained 80% heparan sulfate and 20% chondroitin sulfate proteoglycans whereas MDA plasma membranes had 50% heparan sulfate and 50% chondroitin sulfate proteoglycans. The MDA plasma membrane contained two heparan sulfate proteoglycans, both having nearly the same molecular size as the two species secreted into the medium by these cells. The HBL plasma membrane also contained two hydrodynamic size heparan sulfate proteoglycans. The larger hydrodynamic size species has a slightly lower molecular size than that secreted into the medium, and the smaller hydrodynamic size species was not detectable in the medium. Even though the major chondroitin sulfate proteoglycans from MDA plasma membranes were smaller in size than those from HBL plasma membrane, a larger proportion of the glycosaminoglycan chains of the former were bigger than those from the latter.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate - Di-OS 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-d-galactose - Di-4S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-4-O-sulfo-d-galactose - Di-6S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-6-O-sulfo-d-galactose - Gdn-HCl guanidine hydrochloride - WGA wheat germ agglutinin     

Comparison of two methods for detection of Mollicutes (Mycoplasmatales and Acholeplasmatales) in cell cultures in The Netherlands

A total of 1949 cell cultures was tested for contamination with mollicutes by cultivation on and in mycoplasma media, 25.7% of the cell cultures was positive, 243 strains of Mycoplasma hyorhinis were isolated. Furthermore, mainly M. arginini and M. orale were detected, less often Acholeplasma laidlawii, M. fermentans and M. pneumoniae. Optimal conditions for isolation were discussed. About one third of 217 hybridoma cultures and two third of 57 myeloma cultures proved to be contaminated, all with M. hyorhinis. A DNA fluorochrome staining method (DAPI-test) was compared to cultivation for testing 1039 cell cultures. The efficiency of the DAPI-test could be estimated to be about 96% that of cultivation about 89%, but cultivation is more specific. The highest assurance is obtained when both methods are applied.     

Electrical recording of plant penetration by western flower thrips

Plant penetration by western flower thrips (Frankliniella occidentalis (Pergande)) was analysed with the electrical penetration graph technique (EPG, DC-system). Thrips attached to a gold wire were included in an electrical circuit to record EPGs when penetrating the plant tissues with their stylets. Three basic EPG waveforms have been distinguished, correlated with stylet penetration into cells, salivation, and ingestion, respectively. The main difference with EPGs of Homoptera is the occurrence of continued separate penetrations that are not necessarily followed by ingestion. Insertion of the stylets causes strong voltage fluctuations in the EPG. We could confirm earlier evidence that penetration of cells and subsequent ingestion of (part of) the protoplast takes less than 20 seconds. Repeated short penetrations can be followed by a continuous feeding pattern during which the stylets are not withdrawn. The same sequence of waveforms is produced on other plant parts such as fruits or pollen grains. The specific waveforms are mainly caused by electromotive force (emf). The emf component was recorded with high resolution and the correlation of waveform details with activities of the cibarial muscle system is discussed.     

Multi-parameter flow cytometry as a tool to monitor heterotrophic microalgal batch fermentations for oil production towards biodiesel

Multi-parameter flow cytometry was used to monitor cell intrinsic light scatter, viability, and lipid content of Chlorella protothecoides cells grown in shake flasks. Changes in the right angle light scatter (RALS) and forward angle light scatter (FALS) were detected during the microalgal growth, which were attributed to the different microalgal cell cycle stages. The proportion of cells not stained with PI (cells with intact cytoplasmic membrane) was high (> 90%) during the microalgal growth, even in the latter stationary phase, suggesting that the microalgal cells built-up storage materials which allowed them to survive under nutrient starvation, maintaining their cytoplasmic membranes intact. A high correlation between the Nile Red fluorescence intensity measured by flow cytometry and total lipid content assayed by the traditional lipid extraction method was found for this microalga, making this method a suitable and quick technique for the screening of microalgal strains for lipid production, optimization of biofuel production bioprocesses, and scale-up studies. The highest oil content (∼28% w/w dry cell weight, estimated by flow cytometry) was observed in the latter stationary phase. In addition, C. protothecoides oil also depicted the adequate fatty acid methyl ester composition for biodiesel purposes at this growth phase, suggesting that the microalgal oil produced during the latter stationary phase could be an adequate substitute for diesel fuel. Medium growth optimization for enhancement of microalgal oil production is now in progress, using the multi-parameter approach.     

Freeze-fracture observations on membranes of dry and hydrated pollen from Collomia,Phoenix and Zea

Pollen from Collomia grandiflora Dougl. ex Lindl., Phoenix dactylifera L. and Zea mays L. was examined by freeze-fracture electron microscopy. Particular attention was paid to the organization of the cell membranes in the naturally dehydrated, as compared to the fully hydrated, state. All membranes examined had a normal bilayer organization similar to that seen in the hydrated cells of these and other plants. This organization of dry pollen membranes is discussed as it relates to physiological studies (e.g., leakage of ions during hydration), and to biophysical properties of biological and model membranes under various conditions of hydration and dehydration.Abbreviations EF, PF exoplasmic and protoplasmic fracture, respectively - H_II hexagonal II - IMPs intramembranous particles     

Protoplast responses in the epidermis of Allium cepa induced by penetration by Botrytis allii

Penetration of Allium cepa epidermal cells (white, yellow, and red varieties) by Botrytis allii induced a response by host protoplasts in normal tissue which was not observed when penetrations were made in protoplast-free host cell walls. Callose and auto-fluorescing substances (possibly phenolic compounds) were located at the penetration sites only in normal host cells containing protoplasts. Lignin tests were negative. Halos were clearly visible in both types of tissue. Autofluorescence was observed at penetration sites in normal cells of all cultivars but general wall background autofluorescence was not observed in white onions. Autofluorescence was generally yellow green and when treated with ammonium hydroxide became green. Treatment with sodium hydroxide abolished autofluorescence. No attempt was made to isolate the autofluorescing material.     

Parasites of the spotted stalk borer,Chilo partellus [Lepidoptera: Pyralidae] in South Africa

One egg parasite, 7 larval parasites, 2 pupal parasites and 3 larval hyperparasites were recorded parasitizing the spotted stalk borer,Chilo partellus (Swinhoe) on maize and grain sorghum in South Africa.Trichogrammatoidea lutea Girault [Trichogrammatidae] parasitized eggs ofC. partellus mainly in mid-summer. The larval parasites were active throughout the season with occasional peaks of up to 75% parasitism.Apanteles sesamiae Cameron [Braconidae], proved to be the most abundant larval parasite. It was recorded fromca. 93% of parasitized larvae but its efficiency was reduced by the hyperparasite,Aphanogmus fijiensis (Ferriére) [Ceraphronidae], which reached sometimes up to 100% parasitism on cocoons ofA. sesamiae. The efficiency ofIphiaulax sp. [Braconidae], the 2^nd most abundant larval parasite, was also hindered by the hyperparasite,Eurytoma sp. [Eurytomidae] Pupal parasites were sometimes very abundant reaching up to 100% parasitism without any interference by hyperparasites. The most abundant pupal parasites wereDentichasmias busseolae Heinrich [Ichneumonidae] andPediobius furvus (Gahan) [Eulophidae].      

Microscopic observations on the interaction of the mycoparasite Verticillium biguttatum with Rhizoctonia solani and other soil-borne fungi

The mycoparasitic interactions of Verticillium biguttatum with Rhizoctonia solani and with a variety of other soil-borne fungi were investigated in dual cultures. V. biguttatum interacted with various soil fungi by appressed growth along the host hyphae and infrequent penetrations. Intracellular growth and subsequent sporulation, however, only occurred with R. solani, a few binucleate Rhizoctonia and Ceratobasidium spp., and Sclerotinia sclerotiorum. Effective mycoparasitism on sclerotia was restricted to those belonging to R. solani.Electron-microscopic observations revealed that V. biguttatum can penetrate the host cell with infection tubes. This process is probably mediated by enzymatic hydrolysis of the cell wall. Subsequently, trophic hyphae develop within the host cytoplasm, ultimately resulting in death of the host cell.     

Photosynthetic efficiency as a function of thermal stratification and phytoplankton size structure in an oligotrophic alpine lake

Allometric relationships of phytoplankton communities were studied on the basis of a five-year data-set in a deep oligotrophic alpine lake in Austria. The seasonal phytoplankton succession in Mondsee is characterised by diatoms during winter mixing and a distinct metalimnetic population of Planktothrix rubescens during stratification in summer. The variation of phytoplankton photosynthetic efficiency between seasons was assessed using in situ carbon-uptake rates (5 years data) and Fast Repetition Rate Fluorometry (FRRF) (2 years data). The light-saturated, chlorophyll-specific rate of photosynthesis (P*_max), irradiance at the onset of saturation (E _k) and maximum light-utilisation efficiency (α*) were determined for winter mixing and summer stratification. Fluorescence-based parameters as the functional absorption cross section of Photosystem II (σ _PSII) and the photochemical quantum yield (F _v/F _m) were additionally analysed in 2003 and 2004 to study the underlying physiological mechanisms for the variability in photosynthetic performance. Beyond their sensitivity to changing environmental conditions like thermal stratification, phytoplankton populations differ in their photosynthetic behaviour according to their size structure. Therefore Photosynthesis vs. Irradiance (P/E)-relationships were analysed in detail within a 1-year period from size fractionated cell counts, chlorophyll-a and carbon-uptake.     

Field persistence of the entomopathogenic nematode <Emphasis Type="Italic">Heterorhabditis bacteriophora</Emphasis> in different crops

To investigate nematode establishment and persistence, dauer juveniles (DJs) of Heterorhabditis bacteriophora were applied at 50 cm^-2 in different crops in June and July with conventional spraying equipment and 420 l water ha^-1. Application hardly had any effects on survival and infectivity. The number of DJs reaching the soil was assessed and the establishment and persistence recorded by baiting soil samples with larvae of the wax moth Galleria mellonella. The better the plant canopy was developed the fewer DJs reached the soil during application. Whereas in pasture 77% and in potatoes 78% of the applied nematodes reached the soil, in wheat and peas little less than 50%, in oil-seed rape only 5% and in lupine 6% were recorded. Between 50 and 60% of the soil samples contained H. bacteriophora a month after application with the exception of wheat (>90%) and potatoes (<5%) indicating that the number of nematodes reaching the soil during application had no influence on their establishment in the soil. Probably DJs can survive in the plant canopy and reach the soil later after application. The percentage of nematode-positive soil samples dropped considerably after tillage. In potatoes no nematodes were recovered after two months, which probably was also due to the intensive movement of the soil. Although nematodes are susceptible to freezing, temperatures below 0°C during the winter did not extinguish the H. bacteriophora population. In field crops EPN usually persisted not much longer than one year. The longest persistence of H. bacteriophora was detected 23 months after release in beans followed in rotation by wheat with red clover as cover crop. In this field larvae of the pea weevil Sitona lineatus (Coleoptera: Curculionidae) were detected in soil samples and found infected with the released nematode population. In the laboratory the field soils were tested for persistence of H. bacteriophora at 8°C and a half-life of 24.8 days was recorded in the absence of host insects and plants. Thus long-term persistence in the field was a result of recycling in host insects, which could not be detected in other crops than beans and clover. As H. bacteriophora seems to be restricted in its host potential, this species disappears after release once the host population is not available anymore.     

Microbial pathogens of the coconut pest Oryctes rhinoceros: influence of weather factors on their infectivity and study of their coincidental ecology in Kerala,India

The rhinoceros beetle, Oryctes rhinoceros L., is an economically important pest of the coconut palm. Management of this pest has been accomplished using microbial agents viz., Oryctes virus (OrV) and an entomofungal pathogen Metarhizium anisopliae. Recently an opportunistic bacterial pathogen Pseudomonas alcaligenes has also been noticed to cause septicaemia in the grubs when under stress. To unravel the influence of abiotic weather factors and the interactions amongst these microbial pathogens, a 3 year study was conducted from September 1996 to August 1999 in three of the southern districts of Kerala, India. Of the 6627 grubs and 307 adults collected from various breeding sites of the pest, 5% of the grubs and 22% of the adults had natural virus infection, 3% larvae died of M. anisopliae mycosis and 20% larvae succumbed to bacterial septicaemia. Oryctes virus infection in grubs and adults was negatively correlated to minimum temperature (correlation co-efficient, r = –0.4, and –0.6 respectively, sample size, n = 0). Increase in relative humidity increased the fungal activity (r = 0.8) whereas, maximum temperature had a negative impact (r = –0.7). Occurrence of virus infection in grubs and adults was positively correlated (r = 0.6), supporting the contention of active transmission of the virus pathogen between these two stages. The bacterial septicaemia in the grubs was marginally correlated with virus infection and P. alcaligenes undermined the efficiency of the virus pathogen.     

Transfer of organelles of the alga Chlamydomonas reinhardii into carrot cells by protoplast fusion

A mutant of Chlamydomonas reinhardii which lacks a cell wall was fused with Daucus carota protoplasts using polyethylene glycol and the resulting fusion products were cultured. Fusion involved integration of Chlamydomonas and carrot plasma membranes and the release of algal organelles into the carrot cytoplasm. Chlamydomonas basal bodies, nuclei and chloroplasts were frequently observed in the fusion products. Cultured fusion products regenerated cell walls and divided; most Chlamydomonas organelles degenerated during culture but chloroplasts were still recognizable in the carrot cytoplasm after 10.Abbreviations PEG polyethylene glycol - TEM transmission electron microscopy - SEM scanning electron microscopy This study was undertaken during sabbatical leave in The Research School of Biological Sciences. Australian National University     

Regulatory properties of the ADPglucose pyrophosphorylase from Rhodopseudomonas sphaeroides and from Rhodopseudomonas gelatinosa

Highly purified fractions of chlorosomes and cytoplasmic membranes were isolated from Chloroflexus aurantiacus Ok-70-fl and Chlorobium limicola 6230. These fractions were comparatively analyzed for their pigmentation, phospholipid, glycolipid, and cytochrome c content as well as for their specific activities of succinate dehydrogenase and NADH-oxidase. The data showed that there are some differences in pigmentation and phospholipid content between the isolated fractions of Chloroflexus and Chlorobium. Chlorosomes of Chloroflexus contained a specific BChl a-complex with a characteristic absorption maximum at about 790 nm. This BChl a-complex could not be detected in spectra of chlorosomes from Chlorobium. The near infrared region of the spectra of the isolated cytoplasmic membranes of both organisms revealed considerable differences: The BChl a-complexes of Chloroflexus membranes exhibited peaks at 806 and 868 nm whereas the membranes of Chlorobium had a single BChl a-peak at 710 nm. In contrast to the findings with Chlorobium the chlorosomes of Chloroflexus contained at least twice as much phospholipids as did the cytoplasmic membranes. In Chlorobium the phospholipid content of cytoplasmic membranes is three times that of their chlorosomes. The distribution of all other components (carotenoid composition, enzyme activities, cytochrome c content, and glycolipids) was about the same in both strains. From the data it was concluded that differences in the organization of the photosynthetic apparatus are mainly based on differences of the organization of the photosynthetic units in the cytoplasmic membrane and probably the kind of linkage of the light harvesting system in the chlorosomes with the reaction center in the cytoplasmic membranes.Abbreviations BChl c bacteriochlorophyll c - BChl a bacteriochlorophyll a - DSM Deutsche Sammlung von Mikrorganismen     

Identification of b,c, and d cytochromes in the membrane of Vitreoscilla

Cytochromes b, c, d, and o were identified by spectroscopic analysis of respiratory membrane fragments from Vitreoscilla sp., strain C1. Carbon monoxide difference spectra of the reduced membranes had absorption maxima at 416, 534, and 571 nm (ascribed to cytochrome o) and 632 nm (cytochrome d). Derivative spectra of the pyridine hemochromogen spectra of the membranes identified the presence of b- and c-type cytochromes in Vitreoscilla. The cyanide binding curve of the membranes was biphasic with dissociation constants of 2.14 mM and 10.7 mM which were assigned to cytochrome o and cytochrome d, respectively. Membranes bound carbon monoxide with dissociation constant 3.9 M, which was assigned to cytochrome o. Cytochrome c ₅₅₆ and a NADH-p-iodonitrotetrazolium violet reductase component were partially purified from Vitreoscilla membranes.Abbreviations INT p-iodonitrotetrazolium violet - RMF respiratory membrane fragments - K _d dissociation constant - CHAPS 3-[(3-cholamido propyl) dimethylammonio]-1-propanesulfonate - DOC sodium deoxycholate - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate