Disentangling the effects of mating systems and mutation rates on cytoplamic diversity in gynodioecious Silene nutans and dioecious Silene otites

Many flowering plant species exhibit a variety of distinct sexual morphs, the two mostcommon cases being the co-occurrence of females and males (dioecy) or the co-occurrence ofhermaphrodites and females (gynodioecy). In this study, we compared DNA sequencevariability of the three genomes (nuclear, mitochondrial and chloroplastic) of agynodioecious species, Silene nutans, with that of a closely related dioeciousspecies, Silene otites. In the light of theoretical models, we expect cytoplasmicdiversity to differ between the two species due to the selective dynamics that acts oncytoplasmic genomes in gynodioecious species: under an epidemic scenario, thegynodioecious species is expected to exhibit lower cytoplasmic diversity than thedioecious species, while the opposite is expected in the case of balancing selectionmaintaining sterility cytoplasms in the gynodioecious species. We found no differencebetween the species for nuclear gene diversity, but, for the cytoplasmic loci, thegynodioecious S. nutans had more haplotypes, and higher nucleotide diversity,than the dioecious relative, S. otites, even though the latter has a relativelyhigh rate of mitochondrial synonymous substitutions, and therefore presumably a highermutation rate. Therefore, as the mitochondrial mutation rate cannot account for the highercytoplasmic diversity found in S. nutans, our findings support the hypothesisthat gynodioecy in S. nutans has been maintained by balancing selection ratherthan by epidemic-like dynamics.     

中红侧沟茧蜂对烟草挥发物的触角电生理及行为反应

中红侧沟茧峰 Microplitis mediator是棉铃虫 H elicoverpa armigera的主要内寄生蜂 ,是优良的蜂种 ,在生物防治上具有广阔的应用前景。但迄今 ,有关中红侧沟茧蜂的化学生态的研究甚少。利用触角电位技术 ,测定了中红侧沟茧蜂对烟草叶片粗提物和烟草中 10种挥发物的触角电生理反应 ,并测定了中红侧沟茧蜂的触角电位反应随日龄的变化。以嗅觉仪测定了中红侧沟茧蜂对烟草粗提物、烟草中 4种挥发物的行为反应。结果表明中红侧沟茧蜂利用烟草释放的气味向烟草定向 ,进而找到其寄主——棉铃虫 ,也佐证了烟草同样是棉铃虫的重要寄主植物的论点。雌蜂对脂肪族化合物反应最强 ,对芳香族化合物和糠醛 (杂环化合物 )的反应居中 ,对萜类化合物反应较弱或者不反应。表明烟草挥发性物质在中红侧沟茧蜂寻找寄主的过程中起着重要的作用 ,但挥发物各组分的作用存在着差异。雌性中红侧沟茧蜂对顺 - 3-己烯醇的触角电位反应随日龄的增加而变化。 1日龄的雌蜂的反应较弱 ,2日龄、3日龄的雌蜂反应较强 ,4日龄、5日龄的雌蜂的反应又减弱。这与该蜂产卵习性相符 :羽化当天的雌蜂产卵较少 ,羽化 2～ 3d之后的雌蜂产卵量较大     

The relationship between pollen grain size and progeny gender in dioecious Silene latifolia (Caryophyllaceae)

Summary Experiments and observations conducted during the past 90 years have provided conflicting evidence concerning the existence of a size difference between pollen grains containing an X chromosome (female-determining) and those containing a Y chromosome (maledetermining) in dioecious Silene latifolia. Were such a size difference to exist, this might explain, at least in part, the observation that X-bearing pollen tubes reach the ovary more quickly, on average, than Y-bearing pollen tubes. We tested for such a size difference by separating pollen collected from single anthers into three size classes: small, large, and random. Fruit set (number of pollinated flowers that set fruit) and seed set (number of seeds per capsule) did not differ for these three pollination treatments. Progeny sex ratios resulting from these three pollen size classes also did not differ significantly. Thus, pollen grain size is not affected by which of the two sex chromosomes is present. Our experiment is the first direct test of this relationship. Based on our results, size differences should not be invoked to explain competitive differences in male- and female-determining microgametophytes.     

Predation potential of three flatworm species (Platyhelminthes: Turbellaria) on mosquitoes (Diptera: Culicidae)

We conducted a field survey for flatworms to select species as potential biological control agents against Aedes aegypti and Culex pipiens (Diptera, Culicidae) breeding in artificial containers. Laboratory experiments were performed to determine the daily predation rate, differential predation on each mosquito larval instar, selective predation on either A. aegypti or C. pipiens, and predator tolerance to water from artificial containers. Girardia anceps (Tricladida, Paludicola, Dugesiidae), Mesostoma ehrenbergii and Bothromesostoma cf. evelinae (Rhabdocoela, Typhloplanoida, Typhloplanidae) were found in temporary puddles and permanent pools. In the laboratory, they killed between 52% and 100% of immature mosquitoes coexisting in the same habitat. No preference of flatworms for mosquito preys was detected. Predation rate was related to predator size and instar of preys. Girardia anceps and B. evelinae survived after a dry period and when re-flooding occurred, they laid eggs. Tolerance to water from artificial containers was highest in G. anceps and this species could be a suitable predator to reduce mosquito populations from artificial containers using an inoculative approach.     

Identification of immune-related protein kinases from mosquitoes (Aedes aegypti)

Protein kinases are known to be involved in signal transduction for numerous physiological events. However, little is known about the roles of protein kinases in insect immunity. A fragment around 150 bp was amplified by polymerase chain reaction using cDNA templates from bacterial inoculated mosquitoes and primers corresponding to the conserved domain of protein kinases. Based on sequence analysis, 11 groups of protein kinases were characterized including 3 nonreceptor tyrosine kinases, 3 receptor tyrosine kinases, 3 serine/threonine kinases, and 2 novel protein kinases. The most abundant kinase obtained in this study reveals a high degree of similarity to human cholinesterase-related cell division controller (CHED) protein kinase. The expression of this mosquito CHED-like kinase is not detectable in normal female mosquitoes, but induced only after bacterial inoculation and trauma. A mosquito protein kinase was demonstrated to share homology with a plant Tousled gene, but has not yet been characterized in the animal system. In addition, analysis of the sequences of several protein kinases cloned from mosquitoes suggests that they might be involved in the regulation of cellular or humoral immunity.     

Microplate assay of elevated esterase activity in individual pyrethroid-resistant mosquitoes

Involvement of esterase-mediated hydrolysis as a mechanism of pyrethroidresistance in three species of mosquitoes,viz., Aedes aegypti, Culex quinquefasciatus andAnopheles Stephensi was investigated by microplate assay of Β-esterases in individual larva and adult female and male mosquitoes. Assuming an absorbance value of 0.4 and above at 555 nm as the threshold level of elevated esterase activity which confers resistance, frequency distributions of such individual test mosquitoes were constructed in resistant and susceptible populations. The results indicate the involvement of ester hydrolysis of Pyrethroids as a predominant mechanism of pyrethroid-resistance in the larvae ofCulex quinquefasciatus but not inAedes aegypti. However, a marginal role of esterases is indicated in the larvae ofAnopheles stephensi.     

Comparative sensitivity of larval mosquitoes to vegetable polyphenols versus conventional insecticides

The larvicidal effects of polyphenols of natural crude decomposed alder leaf litter and commercially available tannic acid were experimentally compared with those of two common conventional insecticides (Bacillus thuringiensis ssp. israelensis: microbial insecticide; temephos: organophosphate insecticide). Comparative standard bioassays using third instar larval Aedes aegypti, A. albopictus, Culex pipiens and Coquillettidia richiardii as references indicated that Aedes and Culex taxa are far more sensitive to alder leaf litter than to tannic acid and conventional insecticides. C. richiardii is far more resistant to conventional insecticides than Aedes and Culex taxa, but its sensitivity to tannic acid is close to that of those taxa. Dietary vegetable polyphenols are thus proposed as new, practical, alternative chemicals for mosquito control when conventional insecticides are difficult and costly to be used (e.g., in the management of Aedes and Culex populations in man-made breeding sites and Coquillettidia control strategy).     

Experimental infection of Aedes (Stegomyia) albopictus and Culex pipiens mosquitoes with Bluetongue virus

Bluetongue disease (BT), caused by Bluetongue virus (BTV), infects wild and domestic ruminants, causing severe economic damage in the cattle and sheep industry. Proven vectors of BTV are biting midges belonging to the Culicoides genus, but other arthropods are considered potential vectors, such as ticks, mosquitoes, wingless flies, and sand flies. The present study represents the first attempt to evaluate the vectorial capacity of Culex pipiens and Aedes albopictus for BTV. Mosquitoes were artificially fed with blood containing BTV serotype 1. Infection, dissemination and transmission rates were evaluated at 0, 3, 7, 14 and 21 days after an infected blood meal. Viral RNA was only detected up to 3 days post infection in the bodies of both species. This study indicates that the two Italian populations of Cx. pipiens and Ae. albopictus are not susceptible to BTV infection.     

Callus induction and plant regeneration in the metallophyte <Emphasis Type="Italic">Silene vulgaris</Emphasis> (Caryophyllaceae)

Zinc tolerant and non-tolerant ecotypes of Silene vulgaris (Moench) Garcke were examined for their suitability to provide an efficient and reproducible callus formation and regeneration system. Successful and rapid regeneration of adventitious shoots from callus was achieved in leaf tissue but not root or apical meristematic tissue using concentrations of plant growth regulators that spanned a concentration range of (0.05–1 mg l^–1) NAA and (0.5–10 mg l^–1) BAP respectively. Large differences were observed between ecotypes regarding both callus formation and shoot regeneration on the different hormone concentrations. Leaf explants incubated on basal media with different concentrations of auxin/cytokinin demonstrated initial callus formation after 3 weeks of incubation. Callus initiation was seen to develop from the wounded margins of the leaf explants and, after 2 weeks the initially dark callus became more swollen and green. A mean of 6–8 shoots per leaf explant was observed and the survival rate of these regenerates was seen to be 90%. All regenerated plants that were transferred to soil after the emergence of roots, were seen to have no disturbed morphological characteristics. This study demonstrates the stability of the zinc tolerance traits in the regenerated explants and the potential use of this calli formation and regeneration system in Silene vulgaris. Further, this study is a necessary pre-requite for the development of a genetic transformation system with which to study the genetic basis of zinc and, other heavy metal tolerances in a species with a naturally selected high-level tolerance.     

A comparative study of allozyme variation of peripheral and central populations of Silene nutans L. (Caryophyllaceae) from Western Europe: implications for conservation

In Belgium, at the western border of its continental distribution range, the perennial herb S. nutans has evolved two parapatric ecotypes (calcicolous or silicicolous), which show contrasted allozyme, morphological and adaptive patterns, and isolating reproductive barriers. We examined allozyme variation in 21 populations close to Belgium (from France and Luxemburg) in relation to their peripheral or central geographical position, and investigated their genetic relationships with the Belgian ecotypes. Both peripheral and central populations showed high genetic variation. They were differentiated from each other, but not in relation to the substrate (calcareous or siliceous). The peripheral and central populations were related to the Belgian silicicolous and calcicolous ecotype, respectively. This suggests an ancient differentiation and different past histories for the Belgian ecotypes, and parapatric speciation processes. This study exemplifies the high evolutionary potential of populations at range peripheries and the need for considering them in conservation strategies.     

Electrophysiological and behavioural responses of the pollen beetle, Meligethes aeneus, to volatiles from a non-host plant, lavender, Lavandula angustifolia (Lamiaceae)

A semiochemical based push-pull strategy for control of oilseed rape pests is being developed at Rothamsted Research. This strategy uses insect and plant derived semiochemicals to manipulate pests and their natural enemies. An important element within this strategy is an understanding of the importance of non-host plant cues for pest insects and how such signals could be used to manipulate their behaviour. Previous studies using a range of non-host plants have shown that, for the pollen beetle Meligethes aeneus (Coleoptera: Nitidulidae), the essential oil of lavender, Lavandula angustifolia (Lamiaceae), was the most repellent. The aim of this study was to identify the active components in L. angustifolia oil, and to investigate the behaviour of M. aeneus to these chemicals, to establish the most effective use of repellent stimuli to disrupt colonisation of oilseed rape crops. Coupled gas chromatography-electroantennography (GC-EAG) and gas chromatography-mass spectrometry (GC-MS) resulted in the identification of seven active compounds which were tested for behavioural activity using a 4-way olfactometer. Repellent responses were observed with (±)-linalool and (±)-linalyl acetate. The use of these chemicals within a push-pull pest control strategy is discussed. Handling Editor: Yvan Rahbe.     

Central projections of olfactory receptor neurons from single antennal and palpal sensilla in mosquitoes

In insects, olfactory receptor neurons (ORNs) are located in cuticular sensilla, that are present on the antennae and on the maxillary palps. Their axons project into spherical neuropil, the glomeruli, which are characteristic structures in the primary olfactory center throughout the animal kingdom. ORNs in insects often respond specifically to single odor compounds. The projection patterns of these neurons within the primary olfactory center, the antennal lobe, are, however, largely unknown.We developed a method to stain central projections of intact receptor neurons known to respond to host odor compounds in the malaria mosquito, Anopheles gambiae. Terminal arborizations from ORNs from antennal sensilla had only a few branches apparently restricted to a single glomerulus. Axonal arborizations of the different neurons originating from the same sensillum did not overlap.ORNs originating from maxillary palp sensilla all projected into a dorso-medial area in both the ipsi- and contralateral antennal lobe, which received in no case axon terminals from antennal receptor neurons. Staining of maxillary palp receptor neurons in a second mosquito species (Aedes aegypti) revealed unilateral arborizations in an area at a similar position as in An. gambiae.     

Electrophysiological and behavioural responses of Scyphophorus acupunctatus (Col., Curculionidae) to Agave tequilana volatiles

Abstract:  The weevil Scyphophorus acupunctatus Gyllenhal (Col., Curculionidae) is the most important insect pest of cultivated agaves in Mexico. The objective of this study was to search for potential attractants for this weevil species from the freshly cut fleshy leaves of healthy Agave tequilana Weber var. Blue plants. Combined gas chromatography-electroantennography (GC-EAD) analysis of A. tequilana volatile extracts showed that seven peaks elicited antennal responses from males and females. Five of these peaks were identified by gas chromatography-mass spectrometry (GC-MS) as o -xylene, α -pinene, 3-carene, γ -terpinene and linalool. Electroantennogram (EAG) records of both sexes to 1-, 10- and 100- μ g-stimulus load of these five compounds showed that there was a dose effect on the EAG responses for linalool, 3-carene and α -pinene, but not for γ -terpinene and o -xylene. There were no sexual differences between male and female EAG responses to any of the compounds tested. Y-tube olfactometer bioassays of the compounds showed that males and females were attracted to α -pinene, 3-carene, γ -terpinene and/or linalool at doses of 1 and 10  μ g, while weevils were repelled by linalool at a dose of 100  μ g.     

Cytogenetic analysis of metaphase chromosomes from pupal testes of four mosquito species using fluorescence <Emphasis Type="Italic">in situ</Emphasis> hybridization technique (FISH)

The DNA probes, P1887, P2405, P2056 (being specific tags for Aedes aegypti genes coding for ribosomal RNA) and a centric heterochromatin probe, K20-1A5, were chosen to hybridize the metaphase chromosomes from the testes of four mosquito species, Culex pipiens, Aedes aegypti, Aedes albopictus and Aedes triseriatus. In addition, a single plasmid, P2392, which contained the three probes, P1887, P2405 and P2056, was also used as chromosome landmark in aedine species. Only the Aedes aegypti metaphase chromosome 1-specific tag, P1887, was conserved in Aedes albopictus, Aedes triseriatus, and Culex pipiens metaphase chromosomes. Aedes triseriatus exhibited two gene loci, on chromosomes 1 and 3, coding for ribosomal RNA per haploid genome. When the specific probes for chromosomes 2 and 3, 2405 and 2056, were used in the fluorescence in situ hybridization technique against the metaphase chromosomes the fluorescent signals were not seen in Aedes albopictus, Aedes triseriatus or Culex pipiens. Also the centric heterochromatin probe, K20-1A5, exhibited strong fluorescent signals on chromosomes 1, 2 and 3 of Aedes aegypti. These fluorescent signals were not observed in metaphase chromosomes derived from the other aedine species, indicating that the centromere sequence can vary within the species.This paper was presented at the Second Arab Conference on Biotechnology and Genetic Engineering, held in the Kingdom of Bahrain, 15–17 April, 2002 and is published here with the endorsement of the Co-ordinator of the Scientific Committee, Professor Essam H. Ghanem, University of Bahrain. Its publication has been delayed because of the ill health of the senior author. Other papers from this conference were published in the July 2003 issue (vol. 19, no. 5).     

Differentiation of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) with egg surface morphology and morphometrics using scanning electron microscopy

Aedes aegypti and Aedes albopictus are potential arboviral vectors leading to high human fatality worldwide. Efforts in the present study were made to differentiate the eggs of A. aegypti and A. albopictus morphologically and morphometrically using scanning electron microscopy (SEM). Morphometrically, these species’ eggs were 48.48% significantly different of the 33 attributes including egg dimensions, micropylar apparatus, dimensions and density of outer chorionic cells (OCCs), tubercles and width of exochorionic network. In comparison to A. aegypti eggs, A. albopictus eggs were significantly smaller and more tapered at the posterior end; however, the micropylar disc of A. aegypti was wider and had incomplete circular sectors whereas it was a narrower polygon without sectors in A. albopictus. These species were also significantly different with regards to OCC which enclose both large central and small peripheral tubercles. Specifically, the exochorionic networks in A. aegypti were interwoven, reticulated and extensively wide whereas they were narrow, prominent and solid-wall-like in A. albopictus. This feature may strengthen A. albopictus eggs against desiccation, when they are laid in containers. The morphometrical and morphological analysis of the egg’s attributes of A. aegypti and A. albopictus may be helpful in understanding egg biology as well as in species confirmation.     

Insecticide susceptibility of Culex pipiens pallens (L.) and Aedes albopictus (Skuke) to five commonly used pesticides in the Republic of Korea

Field collected populations of two mosquito species, Culex pipiens pallens and Aedes albopictus, were tested for susceptibility to five pyrethroid insecticides using a topical application bioassay. The laboratory strain of Cx. pipiens pallens (KS‐CP) demonstrated the most and the lowest susceptibility to deltamethrin and d‐cis/trans prallethrine with LD₅₀ values of 0.00022 μg/♀ and 0.00178 μg/♀, respectively, while Ae. albopictus (KS‐AL) showed the greatest and the lowest susceptibility to phthalethrin and deltamethrin with LD₅₀ values of 0.00015 μg/♀ and 0.00085 μg/♀, respectively. Cx. pipiens pallens (JF‐CP) collected at a nearby rice field in Jeonju showed the greatest susceptibility to deltamethrin (LD₅₀ value of 0.0473 μg/♀) and the resistance ratios (RRs) and compared to the laboratory strain were 215 fold greater. JF‐CP demonstrated the lowest susceptibility to d‐cis/trans phenothrin (0.7697 μg/♀) with RRs 711.0 fold greater than the KS‐CP. An Ae. albopictus (JF‐AL) collected at a nearby forest in Jeonju showed the highest susceptibility to deltamethrin (0.00203 μg/♀) with RRs 2.4 fold greater than the KS‐AL. JF‐AL demonstrated the lowest susceptibility to d‐cis/trans prallethrine (LD₅₀ value 0.00646 μg/♀) with RRs 9.4 fold greater than the KS‐AL. JF‐AL demonstrated the greatest resistance to phthalethrin with RRs 39.3 fold greater than the KS‐AL. Resistance ratios of Cx. pipiens pallens between field and laboratory strains ranged from 215.0 to 711.0 and Ae. albopictus ranged from 2.4 to 39.3. These results indicate that the choice of effective insecticides to selected species of mosquito populations will result in more effective control against field strains of mosquito vectors and decrease environmental pollution.     

Transient expression of the Drosophila melanogaster cinnabar gene rescues eye color in the white eye (WE) strain of Aedes aegypti

The lack of eye pigment in the Aedes aegypti WE (white eye) colony was confirmed to be due to a mutation in the kynurenine hydroxylase gene, which catalyzes one of the steps in the metabolic synthesis of ommochrome eye pigments. Partial restoration of eye color (orange to red phenotype) in pupae and adults occurred in both sexes when first or second instar larvae were reared in water containing 3-hydroxykynurenine, the metabolic product of the enzyme kynurenine hydroxylase. No eye color restoration was observed when larvae were reared in water containing kynurenine sulfate, the precursor of 3-hydroxykynurenine in the ommochrome synthesis pathway. In addition, a plasmid clone containing the wild type Drosophila melanogaster gene encoding kynurenine hydroxylase, cinnabar (cn), was also able to complement the kynurenine hydroxylase mutation when it was injected into embryos of the A. aegypti WE strain. The ability to complement this A. aegypti mutant with the transiently expressed D. melanogaster cinnabar gene supports the value of this gene as a transformation reporter for use with A. aegypti WE and possibly other Diptera with null mutations in the kynurenine hydroxylase gene.     

Structure of Silenan, a Pectic Polysaccharide from Campion Silene vulgaris (Moench) Garcke

A pectic polysaccharide named silenan, [alpha]D20 +148.6 degrees (c 0.1; H2O), was isolated earlier from the aerial part of campion, Silene vulgaris (Moench) Garcke. Silenan has been shown to contain homogalacturonan segments as "smooth regions" and rhamnogalacturonan fragments as "hairy regions". The present study reveals a generalization of structural features of silenan. Silenan was subjected to enzymic digestion with pectinase, to Smith degradation, and to lithium-degradation to determine the conforming poly- and oligosaccharide fragments of "hairy regions" of silenan. The NMR-spectral data and mass-spectrometry confirmed that the core of the ramified region of silenan consisted of residues of alpha-rhamnopyranose 2-O-glycosylated with the residues of alpha-1,4-D-galactopyranosyl uronic acid. The part of the alpha-rhamnopyranose residues of the backbone are branched at O-4. On the basis of the data, the hairy regions of silenan proved to contain mainly linear chains of beta-1,3-, beta-1,4-, and beta-1,6-galactopyranan and alpha-1,5-arabinofuranan. The side chains of the ramified region were shown to have branching points represented 2,3-, 3,6-, 4,6-di-O-substituted beta-galactopyranose residues.     

Analysis of the clinal variation pattern inSilene latifolia (Caryophyllaceae) pollen morphology

Reticulate and microechinate pollen forms withinSilene latifolia (S. alba, S. pratensis) were first described from N. America, where the species is an introduced weed. A previous study showed that the two forms also exist in Europe and intergrade along a zone roughly congruent with intergradation zones in seed morphology and flavone glycosylation genotypes. The present survey of pollen from herbarium specimens is more extensive and covers localities from nearly the whole of the species' native Eurasian and North African range. The first axis of a detrended correspondence analysis (DCA) of qualitative characterstate data for 11 pollen morphological characters shows a gradient between the two extreme exine types, reticulate and microechinate. When mapped, the co-ordinates of pollen samples on this axis indicate a broad zone of intergradation which coincides approximately with the somewhat sharper transition between low- and high-tubercle seeds in C. and N. Europe, but becomes diffuse in Italy and the Mediterranean region and diverges from the seed transition zone in SW. Asia and the Middle East. The biological significance of the pollen morphs is unknown; the variation pattern in pollen morphology, unlike that in seeds, is not consistently correlated with macroclimate.     

Innate responses of the parasitoidsCotesia glomerata andC. rubecula (Hymenoptera: Braconidae) to volatiles from different plant-herbivore complexes

To determine and compare innate preferences of the parasitoid speciesCotesia glomerata andC. rubecula for different plant-herbivore complexes, long-range (1-m) foraging behavior was studied in dual-choice experiments in a wind tunnel. In this study we tested the hypothesis that naive females of the specialistC. rubecula should show more pronounced preferences for different plant-herbivore complexes than females of the generalistC. glomerata. The herbivore species used were the pieridsPieris brassicae, P. rapae, P. Napi, andAporia crataegi and the nonhostsPlutella xylostella andMamestra brassicae. All herbivore species feed mainly on cabbage and wild crucifers, exceptAporia crataegi, which feeds on species of Rosaceae. Both parasitoid species preferred herbivore-damaged plants over nondamaged plants. NeitherC. rubecula norC. glomerata discriminated between plants infested by different caterpillar species, not even between plants infested by host-and nonhost species. Both parasitoid species showed preferences for certain cabbage cultivars and plant species. No differences were found in innate host-searching behavior betweenC. glomerata andC. rubecula. The tritrophic system cabbage-caterpillars-Cotesia sp. seems to lack specificity on the herbivore level, whereas on the plant level differences in attractiveness to parasitoids were found.