Epi- and intracuticular lipids and cuticular transpiration rates of primary leaves of eight barley (Hordeum vulgare) cultivars

The major constituents of the epi- and intracuticular lipids of primary leaves of 8 cultivars of barley ( Hordeum vulgare L.) have been studied together with cuticular transpiration rates. The total amount of analysed cuticular lipids ranged from 9.6 to 13.4 μg cm⁻² and was dominated by the epicuticular fraction, which made up 73–84% of the total. There were variations in the percentages of the analysed lipid classes, alkanes, esters, aldehydes, β-diketones and alcohols, between epi- and intracuticular lipids among individual cultivars, but no clear tendency in these variations, except for the aldehydes, was found. The epicuticular lipids were richer in aldehydes than the intracuticular lipids. The cuticular transpiration rates were poorly correlated with the levels or composition of epi-, intra- or total cuticular lipids. The cuticular transpiration rates were considerably altered as a response to a water stress treatment, but these changes could not be correlated with any changes in amount or composition of the cuticular lipids. From these results it is concluded that some property other than amount or composition of cuticular lipids is the most important in regulation of water diffusion through the cuticle.     

Leaf cuticular waxes are arranged in chemically and mechanically distinct layers: evidence from Prunus laurocerasus L.

The composition and spatial arrangement of cuticular waxes on the leaves of Prunus laurocerasus were investigated. In the wax mixture, the triterpenoids ursolic acid and oleanolic acid as well as alkanes, fatty acids, aldehydes, primary alcohols and alcohol acetates were identified. The surface extraction of upper and lower leaf surfaces yielded 280 mg m ^{? 2} and 830 mg m ^{? 2}, respectively. Protocols for the mechanical removal of waxes from the outermost layers of the cuticle were devised and evaluated. With the most selective of these methods, 130 mg m ^{? 2} of cuticular waxes could be removed from the adaxial surface before a sharp, physically resistant boundary was reached. Compounds thus obtained are interpreted as ‘epicuticular waxes’ with respect to their localization in a distinct layer on the surface of the cutin matrix. The epicuticular wax film can be transferred onto glass and visualized by scanning electron microscopy. Prunus laurocerasus epicuticular waxes consisted entirely of aliphatic compounds, whereas the remaining intracuticular waxes comprised 63% of triterpenoids. The ecological relevance of this layered structure for recognition by phytotrophic fungi and herbivorous insects that probe the surface composition for sign stimuli is discussed.     

Effect of alterations in cuticular wax biosynthesis on the physicochemical properties and topography of maize leaf surfaces

The leaf surface properties of 11 cuticular wax mutants of maize were characterized, and this information was used to identify the quantitative relations among distinct leaf surface traits. Compared with the wild‐type maize, these mutants were reduced 3–24% in their leaf surface hydrophobicity, 20–88% in the mass of cuticular waxes on their leaves, and 52–94% in the percentage of planar leaf surface area covered with epicuticular crystalline waxes. They also differed in the presence and abundance of the epicuticular crystalline waxes in each of seven structural classes. With the exception of one mutant, the mass of cuticular waxes produced by these mutants was positively correlated with the number of epicuticular crystalline waxes per unit area on their leaves. Furthermore, an increase of 0·4 mg of cuticular wax per gram of leaf (dry weight) was associated with a 1% increase in leaf surface area covered by epicuticular crystalline waxes, and this 1% increase was associated with a 2° increase in the contact angle of a water droplet on the leaf surface. Linear differences in the leaf surface hydrophobicity were associated with exponential differences in the mass of the cuticular waxes produced. Quantitative knowledge of these leaf surface properties is highly relevant to the interactions of leaves with environmental factors such as microbes, insects, agricultural chemicals, and pollutants.     

Linkage of autophagy to fungal development,lipid storage and virulence in Metarhizium robertsii

Autophagy is a highly conserved process that maintains intracellular homeostasis by degrading proteins or organelles in all eukaryotes. The effect of autophagy on fungal biology and infection of insect pathogens is unknown. Here, we report the function of MrATG8, an ortholog of yeast ATG8, in the entomopathogenic fungus Metarhizium robertsii. MrATG8 can complement an ATG8-defective yeast strain and deletion of MrATG8 impaired autophagy, conidiation and fungal infection biology in M. robertsii. Compared with the wild-type and gene-rescued mutant, Mratg8Δ is not inductive to form the infection-structure appressorium and is impaired in defense response against insect immunity. In addition, accumulation of lipid droplets (LDs) is significantly reduced in the conidia of Mratg8Δ and the pathogenicity of the mutant is drastically impaired. We also found that the cellular level of a LD-specific perilipin-like protein is significantly lowered by deletion of MrATG8 and that the carboxyl terminus beyond the predicted protease cleavage site is dispensable for MrAtg8 function. To corroborate the role of autophagy in fungal physiology, the homologous genes of yeast ATG1, ATG4 and ATG15, designated as MrATG1, MrATG4 and MrATG15, were also deleted in M. robertsii. In contrast to Mratg8Δ, these mutants could form appressoria, however, the LD accumulation and virulence were also considerably impaired in the mutant strains. Our data showed that autophagy is required in M. robertsii for fungal differentiation, lipid biogenesis and insect infection. The results advance our understanding of autophagic process in fungi and provide evidence to connect autophagy with lipid metabolism.     

Changes in cuticular transpiration rate and cuticular lipids of oat (Avena sativa) seedlings induced by water stress

Two cultivars of oat ( Avena sativa L. cvs Pendek and Stormogul II) were exposed to short periods of water-deficit stress on five consecutive days. The plants responded to the stress by decreasing their cuticular transpiration rate. After two stress periods the cuticular transpiration rate was reduced by 30% for Pendek and by 47% for Stormogul II, and after another three stress periods by 30% and 20%, respectively. These reductions were correlated neither to changes in the total amount of what is generally called epicuticular lipids, nor to changes in any of the major individual constituents of the epicuticular lipids (alkanes, free and esterified fatty acids or free primary alcohols). After removal of the epicuticular lipids the long chain free primary alcohols of the leaves were extracted and determined. The amount of these presumably intracuticular alcohols increased after stress and changed to shorter chain length. From these results it is concluded that the intra- as well as the epicuticular lipids must be taken into consideration when discussing leaf surface lipids as protecting agents against water loss.     

Susceptibility of Metarhizium spp. and other entomopathogenic fungi to dodine-based selective media

The fungicide dodine has been widely used in selective media to isolate entomopathogenic fungi (EF) from contaminating microorganisms, primarily bacteria and non-entomopathogenic fungi. In order to isolate the fungus Metarhizium acridum from soil for grasshopper and Mormon cricket control in the western USA, the susceptibility of M. acridum was compared with two Metarhizium spp. and other EF species. The isolates were inoculated onto mycological media with concentrations of dodine ranging from 0.0001 to 0.03% active ingredient (A.I.). In addition, susceptibilities of five Metarhizium spp. isolates to two sources of dodine, Syllit® commercial fungicide (65% A.I.) and Sigma® reagent grade (99% A.I.), were compared using Czapek agar medium. Responses to the two dodine sources were virtually identical. Accordingly, subsequent experiments used the less expensive Syllit dodine. Three media [Czapek, potato dextrose agar plus yeast extract (PDAY) and oatmeal agar] were evaluated for appropriateness as the base in selective media. Germination of all three of the M. acridum isolates tested was almost completely inhibited by dodine concentrations of 0.002% A.I. in Czapek or 0.006% A.I. in PDAY. On the other hand, M. robertsii and M. anisopliae isolates were considerably more tolerant, with germination not being inhibited until 0.010% A.I. in Czapek or 0.030% A.I. in PDAY. The higher vulnerability of the isolates to low concentrations of dodine in Czapek medium suggests that this medium would be less effective than PDAY in a selective medium. Oatmeal agar greatly improved fungal growth, but the levels of inhibition were lower. Therefore, PDAY was selected as the best selective basal medium. The lowest concentration that inhibited a common soil-inhabiting fungus, Aspergillus nidulans, was 0.001% A.I. Dodine tolerances were highest with M. robertsii, M. anisopliae, and Beauveria bassiana, followed by Isaria fumosorosea and Lecanicillium spp. The least tolerant EF isolates were M. acridum.     

Bartonella henselae in eastern Poland: the relationship between tick infection rates and the serological response of individuals occupationally exposed to tick bites

To explore the potential role of Ixodes ricinus as the presumed vector of Bartonella henselae in eastern Poland, ticks collected in various geographic locations were examined for the presence of B. henselae, and the results were matched against the prevalence of anti‐B. henselae antibodies in individuals occupationally exposed to tick bites. The presence of Bartonella DNA was investigated by PCR in a total of 1,603 unfed Ixodes ricinus ticks. The presence of IgG antibodies against B. henselae was investigated in serum samples from 332 people occupationally exposed to tick bites (94 farmers and 238 forestry workers). The total prevalence of B. henselae in ticks was 1.7%; the infection rates in males (3.1%) and females (2.7%) were nearly ten times greater than in nymphs (0.3%). The prevalence of seropositive results in the risk group (30.4%), farmers (27.7%) and forestry workers (31.5%), was significantly greater compared to the control group (8.9%). The results showed a weak positive correlation between the degree of infection of ticks and humans living in the same geographic region. The lack of a direct relationship indicates that exposure to tick bites is only one of the factors contributing to the significant preponderance of a seropositive response to B. henselae in the forestry workers and farmers over the control group. Other factors must be considered, such as contact with cats, which are popular domestic animals in Polish villages, and exposure to cat fleas.     

Sex- and age-related changes in the biophysical properties of cuticular lipids of the housefly,Musca domestica

We examined the biophysical properties of cuticular lipids isolated from the housefly, Musca domestica. Melting temperatures (T_m) of surface lipids isolated from female houseflies decreased from 39.3 °C to 35.3 °C as the females attained sexual maturity and produced sex pheromone, whereas those prepared from males did not change with age. Lipids melted over a 10–25 °C temperature range, and their physical properties were a complex function of the properties of the component lipids. The T_m of total cuticular lipids was slightly below that of cuticular hydrocarbons (HC), the predominant lipid fraction. Hydrocarbons were further fractionated into saturated, unsaturated, and methyl-branched components. The order of decreasing T_m was total alkanes > total HCs > methyl-branched alkanes > alkenes. For 1-day-old flies, measured T_ms of hydrocarbons were 1.3–5.5 °C lower than T_ms calculated from a weighted average of T_ms for saturated and unsaturated components. For 4-day-old flies, calculated T_ms underestimated T_m by 11–14 °C. © 1995 Wiley-Liss, Inc.     

马尾松毛虫感染绿僵菌后某些生化指标的变化

3～ 4龄马尾松毛虫Dendrolimuspunctatus幼虫被金龟子绿僵菌Metarhiziumanisopliae感染后 1～ 4d,血淋巴中血细胞总数和可溶性蛋白浓度均显著高于同期未感染的幼虫。利用聚丙烯酰胺凝胶电泳技术 ,分析了感染后 3 ,4,6d幼虫体壁组织中可溶性蛋白和过氧化物酶 ,以及血淋巴中过氧化物酶的变化。结果显示 ,金龟子绿僵菌的侵染对马尾松毛虫幼虫体壁组织蛋白质代谢产生影响 ,过氧化物酶活性有减弱趋势     

Variability in cuticular hydrocarbons and phenotypic discrimination of Ixodes ricinus populations (Acarina: Ixodidae) from Europe

The cuticular hydrocarbon composition and a stepwise discriminant analysis are used to elucidate the phenotypic relationships of 66 populations of Ixodes ricinus in Europe. The method correctly allocates Ixodes persulcatus (outgroup) populations away from the main cluster of I. ricinus samples and separates the samples into ten relatively defined clusters of specimens. Populations from Poland are inseparable from samples collected in Germany, Switzerland and the Italian Alps, while individuals from Slovakia and the Czech Republic come into separate groups of phenotypic similarity. Irish and British specimens are separated but highly related and Spanish populations show an unexpectedly high distance from the remaining clusters.     

不同寄主及地理来源的16株绿僵菌对桃蚜的毒力比较

害虫生防真菌绿僵菌的不同种及变种被广泛应用于害虫微生物防治,但罕见以蚜虫等同翅目刺吸式害虫作为靶标。从两种绿僵菌的4个变种中精选16个不同寄主及地理来源的菌株,用喷塔接种桃蚜(Myzus persicae)无翅成蚜并在25±1℃和12L∶12D条件下饲养观察,所获生物测定数据进行时间-剂量-死亡率模型模拟分析。结果显示,高接种剂量(~1000个孢子/mm2)下7d内死亡率达67%~100%的10个菌株均为金龟子绿僵菌Metarhiziumanisopliae(Ma)及其金龟子变种M.anisopliaevar.anisopliae(Maa);而高剂量处理下仅感染致死个别或少数蚜虫的菌株包括金龟子绿僵菌及其金龟子变种、大孢变种M.anisopliaevar.majus和蝗变种M.anisopliaevar.acridum以及黄绿绿僵菌小孢变种M.flavoviridevar.minus。杀蚜活性优异的2个菌株分别为Ma 456和Maa 3332,接种后第4天的LC50分别为113和260个孢子/mm2,第5天为32和43个孢子/mm2,第6天为17和26个孢子/mm2,第7天仅11.4和19.9个孢子/mm2。这两个菌株具有用于蚜虫微生物防治的良好开发潜力。     

椰心叶甲CYP4基因的克隆及绿僵菌侵染后的诱导表达研究

根据细胞色素P450家族4（CYP4）的氨基酸保守序列设计1对简并引物,从椰心叶甲Brontispa longissima成虫总RNA中扩增得到5个cDNA片段（GenBank登录号: DQ238840－DQ238844）。以3′-RACE法获得片段BLWH4的3′端序列,推导的氨基酸序列表明其结构中含有CYP家族的特征性保守序列： 螺旋K区的ETLR和血红素结合区的F××G×××C×G。以18S 为对照的RT-PCR分析表明,BLWH4在成虫的mRNA表达量远大于幼虫。绿僵菌Metarhizium anisopliae菌株MA-3和MA-4侵染椰心叶甲成虫及5龄幼虫后,BLWH4的mRNA表达增强,提示BLWH4可能具有增强椰心叶甲抵抗绿僵菌侵染的作用。     

Isolation of Beauveria bassiana and Metarhizium anisopliae var. anisopliae from Boophilus microplus tick (Canestrini, 1887), in Rio de Janeiro State,Brazil

The objective of this work were to isolate and identify strains of entomopathogenic fungi from ingurgitated female Boophilus microplus ticks, collected from the soil in the municipality of Paracambi, Rio de Janeiro State, Brazil. The ingurgitated females were inoculated in the selective medium oat dodine agar (oda), where 49 colonies of Beauveria bassiana (71%) and 20 of Metarhizium anisopliae var. anisopliae (29%) were isolated. These isolated strains characterize for the first time in Brazil the natural occurrence of these species of fungi in this tick, and will be used to conduct bioassays to evaluate the pathogenicity and virulence of these strains for ticks of the genus Boophilus microplus. This revised version was published online in June 2006 with corrections to the Cover Date.     

红火蚁Serpin家族基因鉴定及其在绿僵菌侵染下的表达模式分析

丝氨酸蛋白酶抑制剂（Serine protease inhibitor, Serpin）在昆虫Toll通路和PPO级联反应的防御机制中起着至关重要的作用。本研究利用现代生物信息学工具预测和分析了红火蚁Solenopsis invicta中Serpin家族的结构和功能多样性。染色体定位表明,红火蚁基因组中7个Serpin基因家族成员不均匀的分布在4条染色体上。结构域分析表明,SiSerpin蛋白具有Serpin基因的保守结构,即典型的反应中心环（Reactive central loop, RCL）结构与Serpin结构域,不同成员蛋白拥有多样化的基序。系统进化分析表明,红火蚁Serpin蛋白与其他的昆虫Serpin蛋白有较高亲缘关系,SiSerpin7与烟草天蛾Manduca sexta的Serpin3a亲缘关系最近,SiSerpin12与黄粉虫Tenebrio molitor Serpin55亲缘关系最近,SiSerpin13与烟草天蛾Serpin6亲缘关系最近,SiSerpin4与家蚕Serpin27亲缘关系最近。为了进一步明确红火蚁Serpin家族在绿僵菌Metarhizium侵染后的表达模式,用绿僵菌侵染不同体型的工蚁,荧光定量PCR（RT-qPCR）分析表明,红火蚁Serpin基因家族成员能在早期响应绿僵菌的侵染;除SiSerpin7基因外,大型和小型两种工蚁的表达模式存在较大的差异。结果表明红火蚁Serpin家族作为免疫调控蛋白,能够响应绿僵菌的侵染,且在红火蚁不同体型工蚁之间存在着不同的免疫调控模式。这些发现不仅为Serpins的功能分析提供了理论基础,而且有可能有助于绿僵菌作为一种有效的生防剂的开发。     

Concurrence of losing a chromosome and the ability to produce destruxins in a mutant of Metarhizium anisopliae

In a previous study, a spontaneous subtilisin pr1A and pr1B gene-deficient mutant of the entomopathogenic fungus Metarhizium anisopliae strain V275 has been identified [Wang, C.-S. et al. (2002) FEMS Microbiol. Lett. 213, 251-255]. The insecticidal metabolites of this mutant were studied further. High-performance liquid chromatography (HPLC) analysis indicated that the mutant isolate lost the ability to produce cyclic peptide toxins, destruxins, both in vitro and in vivo. Pulsed-field gel electrophoresis revealed that the mutant concurrently lost a 1.05 Mb (approximately) chromosome, demonstrating for the first time that a conditionally dispensable (CD) chromosome exists in the insect pathogenic fungus, M. anisopliae. Concurrence of losing the ability to produce destruxins and a CD chromosome in the mutant suggests that the toxin synthetase genes of M. anisopliae are located on this CD chromosome, as similarly described for plant pathogenic fungi. Semi-quantitative api ZYM analysis showed more biochemical disparities between the mutant and the wild-type strain.     

Role of small mammals in the persistence of Louping-ill virus: field survey and tick co-feeding studies

Louping-ill (LI) is a tick-borne viral disease of red grouse, Lagopus lagopus scoticus Lath. (Tetraonidae: Galliformes), and sheep, Ovis aries L. (Bovidae: Artiodactyla), that causes economic loss to upland farms and sporting estates. Unvaccinated sheep, grouse and mountain hares, Lepus timidus L. (Leporidae: Lagomorpha), are known to transmit LI virus, whereas red deer, Cenrus elaphus L. (Cervidae: Artiodactyla), and rabbits, Oryctolagus cuniculus L. (Leporidae: Lagomorpha), do not. However, the role of small mammals is unknown. Here, we determine the role of small mammals, in particular field voles, Microtus agrestis L. (Muridae: Rodentia), in the persistence of LI virus on upland farms and sporting estates in Scotland, using field sampling and non-viraemic transmission trials. Small mammals were not abundant on the upland sites studied, few ticks were found per animal and none of the caught animals tested seropositive to LI virus. Laboratory trials provided no evidence that small mammals (field voles, bank voles, Clethrionomys glareolus L. (Muridae: Rodentia), and wood mice, Apodemus sylvaticus L. (Muridae: Rodentia), can transmit LI virus between cofeeding ticks and, in the field, LI virus was prevalent only in areas with known LI virus competent hosts (grouse, mountain hares or unvaccinated sheep) and absent elsewhere. In contrast to the case of tick-borne encephalitis (TBE) virus in Europe, it is concluded that small mammals seem to be relatively unimportant in LI virus persistence.     

Sexual dimorphism of arrestment and gregariousness in the bed bug (Cimex lectularius) in response to cuticular extracts from nymphal exuviae

Releaser pheromones have direct behavioural effects to arrest, attract or disperse insects, whereas interactions within groups of social insects are often influenced by primer pheromones. The behaviour of insects displaying intermediate levels of sociality is largely unexplored in this context. In the present study, both the gregariousness and arrestment (settling near the odour source) of bed bugs Cimex lectularius L. (Hemiptera: Cimicidae) in response to conspecific exuvial extracts are described. Adult males are arrested on filter papers with extracts derived from exuviae of fifth‐instar nymphs. Adult females and nymphs display no significant evidence for such behaviour. Adults of both sexes show no preference for extracts of male versus female fifth‐instar exuviae. Arrestment of adult males does not occur on papers treated with fourth‐instar exuvial extracts. Because the insects are assayed behaviourally in groups, an index is calculated describing how far bugs are away from being located independently of one another, as a measure of gregariousness. Adult males have lower values for this index (i.e. locations are closer to independence). Adult females, nymph cohorts and mixed age groups all have higher values for this index, which tend to increase over time. Females exhibit a clear increasing dose‐dependent relationship for this index. It is concluded that the extracts of fifth‐instar nymphal exuvia arrest males on refuges that possess the odour source. However, gregariousness is induced in females, without evidence of a tendency to assemble near the odour source.     

A proteomic approach to identifying proteins differentially expressed in conidia and mycelium of the entomopathogenic fungus Metarhizium acridum

Metarhizium spp. is an important worldwide group of entomopathogenic fungi used as an interesting alternative to chemical insecticides in programs of agricultural pest and disease vector control. Metarhizium conidia are important in fungal propagation and also are responsible for host infection. Despite their importance, several aspects of conidial biology, including their proteome, are still unknown. We have established conidial and mycelial proteome reference maps for Metarhizium acridum using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF MS). In all, 1130±102 and 1200±97 protein spots were detected in ungerminated conidia and fast-growing mycelia, respectively. Comparison of the two protein-expression profiles reveled that only 35% of the protein spots were common to both developmental stages. Out of 94 2-DE protein spots (65 from conidia, 25 from mycelia and two common to both) analyzed using mass spectrometry, seven proteins from conidia, 15 from mycelia and one common to both stages were identified. The identified protein spots exclusive to conidia contained sequences similar to known fungal stress-protector proteins (such as heat shock proteins (HSP) and 6-phosphogluconate dehydrogenase) plus the fungal allergen Alt a 7, actin and the enzyme cobalamin-independent methionine synthase. The identified protein spots exclusive to mycelia included proteins involved in several cell housekeeping biological processes. Three proteins (HSP 90, 6-phosphogluconate dehydrogenase and allergen Alt a 7) were present in spots in conidial and mycelial gels, but they differed in their locations on the two gels.     

Effects of vanadium-containing compounds on membrane lipids and on microdomains used in receptor-mediated signaling

There is increasing evidence for the involvement of plasma membrane microdomains in insulin receptor function. Moreover, disruption of these structures, which are typically enriched in sphingomyelin and cholesterol, results in insulin resistance. Treatment strategies for insulin resistance include the use of vanadium (V) compounds which have been shown in animal models to enhance insulin responsiveness. One possible mechanism for insulin-enhancing effects might involve direct effects of V compounds on membrane lipid organization. These changes in lipid organization promote the partitioning of insulin receptors and other receptors into membrane microdomains where receptors are optimally functional. To explore this possibility, we have used several strategies involving V complexes such as [VO(2)(dipic)](-) (pyridin-2,6-dicarboxylatodioxovanadium(V)), decavanadate (V(10)O(28)(6-), V(10)), BMOV (bis(maltolato)oxovanadium(IV)), and [VO(saltris)](2) (2-salicylideniminato-2-(hydroxymethyl)-1,3-dihydroxypropane-oxovanadium(V)). Our strategies include an evaluation of interactions between V-containing compounds and model lipid systems, an evaluation of the effects of V compounds on lipid fluidity in erythrocyte membranes, and studies of the effects of V-containing compounds on signaling events initiated by receptors known to use membrane microdomains as signaling platforms.