Purinergic and cholinergic agonists induce exocytosis from the same granule pool in HT29-Cl.16E monolayers

Severalsecretagogues induce mucin secretion in epithelial monolayers, asdetermined by measuring released granule contents. To assess whetherdifferent agonists act on the same granule pool, capacitance changes inintact monolayers of the goblet cell line HT29-Cl.16E were measured bya novel impedance method. Apical ATP (purinergic agonist) andbasolateral carbachol (cholinergic agonist) induce rapid exocytosiswith maximal capacitance changes within 3 min. The maximal levels ofexocytosis that can be induced by optimal concentrations of eitheragonist are the same and produce a 30-40% increase in totalmonolayer capacitance. When ATP and carbachol are appliedsimultaneously, the magnitude of exocytosis is unchanged from thesingle-secretagogue level. The recovery of capacitance to baseline(endocytosis) is significantly faster after ATP stimulation than aftercarbachol stimulation. When ATP and carbachol are applied sequentiallyat doses that give maximal exocytosis, the magnitude of the capacitanceincrease produced by the second secretagogue is less than or equal tothat of the capacitance decrease during the recovery period. Together,these data suggest that purinergic and cholinergic agonists act on the same granule pool.     

Frequency-dependent membrane impedance inChara corallina estimated by Fourier analysis

Summary The white noise method of measuring membrane impedance has been applied to internodal cells ofChara corallina. Fourier analysis of a white noise transmembrane current signal and the voltage response has been used to obtain the frequency-dependent impedance of the in-series combination of the plasmalemma and tonoplast membranes. The results are similar to those of other workers who have measured membrane impedances by different techniques. At very low frequencies the equivalent capacitance of the membrane treated as an RC-circuit becomes negative, indicating a pseudoinductive effect.Membrane impedance has been measured over a range of pH values from pH 5.2 to pH 11; impedance magnitude reaches a maximum at pH 7. At interesting effect of fusicoccin at pH 11 has been observed, in which a decrease in membrane conductance occurs simultaneously with a small hyperpolarization of membrane PD.     

Interdigitated capacitive biosensor based on molecularly imprinted polymer for rapid detection of Hev b1 latex allergen

Allergen protein detection was performed by a surface imprinted layer combined with an interdigitated capacitance (IDC) transducer that allowed label-free measurements. The immobilized imprinted polymers are the probes that bind to rubber allergen proteins extracted from products such as rubber gloves. Copolymers made from methacrylic acid–vinylpyrrolidone–dihydroxyethylene-bisacrylamide (MAA–NVP–DHEBA) are soluble in aqueous solution and eliminate the denaturation of protein. When deposited as a coating onto an IDC microelectrode transduction system, such materials lead to sensors that produce capacitance responses that are clearly dependent on the concentration of the latex protein (10–900 ng ml⁻¹) in pH 7.4 buffer. The biosensor can detect Hev b1 within minutes and with a detection limit of 10 ng ml⁻¹. Different but related hevein allergenic proteins isolated from natural rubber latex from the rubber tree (Hev b1, Hev b2, and Hev b3) were distinguished by the imprinted material, depending on the dimension and conformation of these proteins with a selectivity factor of 4. They recognized Hevea latex proteins better than non-Hev b proteins, such as lysozyme, ovalbumin, and bovine serum albumin, by a factor of 2. Moreover, the sensor exhibited good operational stability of up to 180 days when used continuously at room temperature.     

Complex admittance of Na+ conduction in squid axon

Summary The complex admittance,Y(p), of squid axon was measured (4-1000 Hz) during step voltage clamp to obtain linear data on Na⁺ conduction.Y(p) is used as a spectroscopic tool to identify Na⁺ and K⁺ conduction, which dominateY(p) at low frequencies and can be separated from each other and from the static capacitance. Na⁺ conduction is readily distinguishable from K⁺ conduction in that it produces a steady-state negative conductance. The admittance of the Na⁺ system can show an anomalous resonance or an antiresonance depending on whether the net shunt conductance is negative or positive. Use of the Na⁺ negative conductance to neutralize leakage yields a measurement of dielectric capacitance at low frequency. A 90^o phase angle suggests that the capacitance is ideal.     

Influence of induced water stress in ponderosa pine on pine sawflies

Summary Three levels of water stress were induced on pole-size ponderosa pine (Pinus ponderosa) to determine the influence of plant moisture stress on oviposition, survival, and growth of two species of pine sawfly (Neodiprion fulviceps and N. autumnalis). It was found that water stress affected oviposition and subsequent egg survival but not larval development or survival. Stress had a negative effect on early season oviposition (N. fulviceps) and a positive effect on late season oviposition (N. autumnalis). Egg hatch was different between species and years and among treatment levels. Larval development, feeding, and survival were not affected by water stress. Overall, the effect of stress was not sufficient to explain population outbreaks of sawflies. Several hypotheses are presented as possible explanations for the observed experimental results.     

Alanine-stimulated exocytosis in<Emphasis Type="Italic"> Aplysia</Emphasis> enterocytes: effect of Na<Superscript>+</Superscript> transport and requirement for actin filaments

We used the Aplysia californica intestinal epithelium to investigate the effect of alanine-stimulated Na⁺ absorption on apical membrane exocytosis and whether stimulated exocytosis requires intact actin filaments. The fluid-phase marker fluorescein dextran was used to determine rates of apical membrane exocytosis. L-alanine significantly increased apical exocytosis by ~30% compared to controls, and there is a modest, positive correlation between alanine-stimulated exocytosis and short-circuit current (I _SC). Thus, apical exocytosis is modulated to some extent by the magnitude of Na⁺ and alanine entry across the apical membrane. Apical exocytosis is also responsive to virtually any increase in Na⁺ and alanine entry because increments in alanine-stimulated I _SC as small as 1 A/cm² stimulated exocytosis. We used D-alanine to determine which parameter (sensitivity to transport vs. magnitude of transport) was most important in activation of apical exocytosis. D-alanine-stimulated I _SC was one-sixth that of L-alanine, but stimulated exocytosis was only 29% less than that of L-alanine. Therefore, the apical exocytic system is more responsive to small increases in transport than to the magnitude of transport. Latrunculin A (Lat-A) disrupts the actin cytoskeleton and reduced constitutive apical exocytosis by ~65% and completely abolished alanine-stimulated exocytosis. Hence, constitutive exocytosis and alanine-stimulated exocytosis require actin filaments for recruitment of vesicles to the apical membrane. During nutrient absorption, actin filament-regulated apical exocytosis may represent a negative feedback system that modulates apical membrane tension.Abbreviations alaASW ASW containing alanine - C _m membrane capacitance - ASW artificial seawater - ETOH ethanol - fCa apical membrane fractional capacitance - FD fluorescein dextran - G _K plasma membrane potassium conductance - G _K,a apical membrane potassium conductance - HRP horseradish peroxidase - I _SC short-circuit current - J _Na transcellular net sodium flux - K _D dissociation constant - Lat-A latrunculin A - manASW ASW containing mannitol - PT proximal tubule - RFU relative fluorescence units - V _a apical membrane potential Communicated by L.C.-H. Wang     

Alternative camouflage strategies mediate predation risk among closely related co-occurring kelp crabs

Although camouflage is a common predator defense strategy across a wide variety of organisms, direct tests of the adaptive and ecological consequences of camouflage are rare. In this study, we demonstrated that closely related crabs in the family Epialtidae coexist in the same algal environment but use alternative forms of camouflage––decoration and color change––to protect themselves from predation. Decoration and color change are both plastic camouflage strategies in that they can be changed to match different habitats: decoration occurs on a short timescale (hours to days), while color change accompanies molting and occurs on longer timescales (months). We found that the species that decorated the most had the lowest magnitude of color change (Pugettia richii); the species that decorated the least showed the highest magnitude of color change (Pugettia producta), and a third species (Mimulus foliatus) was intermediate in both decoration and color change, suggesting a negative correlation in utilization of these strategies. This negative correlation between color change and decoration camouflage utilization mirrored the effectiveness of these camouflage strategies in reducing predation in different species. Color camouflage primarily reduced predation on P. producta, while decoration camouflage (but not color camouflage) reduced predation on P. richii. These results indicate there might be among-species trade-offs in utilization and/or effectiveness of these two forms of plastic camouflage, with important consequences for distribution of these species among habitats and the evolution of different camouflage strategies in this group.     

Explaining stasis: microevolutionary studies in natural populations

Microevolution, defined as a change in the genetic constitution of a population over time, is considered to be of commonplace occurrence in nature. Its ubiquity can be inferred from the observation that quantitative genetic divergence among populations usually exceeds that to be expected due to genetic drift alone, and from numerous observations and experiments consistent with local adaptation. Experimental manipulations in natural populations have provided evidence that rapid evolutionary responses may occur in the wild. However, there are remarkably few cases where direct observations of natural populations have revealed microevolutionary changes occurring, despite the frequent demonstration of additive genetic variation and strong directional selection for particular traits. Those few cases where responses congruent with expectation have been demonstrated are restricted to changes over one generation. In this article we focus on possible explanations as to why heritable traits under apparently strong directional selection often fail to show the expected evolutionary response. To date, few of these explanations for apparent stasis have been amenable to empirical testing. We describe new methods, derived from procedures developed by animal breeding scientists, which can be used to address these explanations, and illustrate the approach with examples from long-term studies of collared flycatchers (Ficedula albicollis) and red deer (Cervus elaphus). Understanding why most intensively studied natural populations do not appear to be evolving is an important challenge for evolutionary biology.     

披碱草属3组植物叶片解剖特征及其系统关系

采用石蜡切片法对披碱草属中小颖组、宽颖组和长颖组主要代表种的叶片横切面形态学特征进行观察。结果显示:(1)披碱草属3个组植物的叶片均为等面叶,由表皮、叶肉和维管束三部分构成,表现为典型的狐茅型,即表皮细胞形状、大小和排列不均,叶肉无栅栏组织和海绵组织之分,具有双层维管束鞘,周围叶肉细胞呈不规则排列,厚壁组织与表皮相接;但3个组植物在上表皮细胞形状、大小、沟的深浅,以及大型导管数目等叶片横切面特征上存在明显差异。(2)根据3个组植物叶片横切面性状的演化趋势,对各组的演化关系和系统位置分析表明,小颖组最原始,宽颖组较进化,长颖组最高级;小颖组可能直接派生了较进化的宽颖组,并在宽颖组的基础上进而产生了最高级的长颖组;小颖组、宽颖组和长颖组的这一系统关系与利用外部形态特征所获得的演化趋势基本一致。     

A co-transformation system to produce transgenic grapevines free of marker genes

A co-transformation system was developed to produce grapevines free of selectable marker genes. This was achieved by transforming Vitis vinifera L. ‘Thompson Seedless’ somatic embryos with a mixture of two Agrobacterium strains. The first strain contained a binary plasmid with an egfp gene of interest between the T-DNA borders. The second strain harbored the neomycin phosphotransferase (nptII) gene for positive selection and the cytosine deaminase (codA) gene for negative selection, linked together by a bi-directional dual promoter complex. Our technique included a short positive selection phase on medium containing 100 mg l⁻¹ kanamycin before subjecting cultures to prolonged negative selection on medium containing 250 mg l⁻¹ 5-fluorocytosine. We regenerated 25 stable EGFP expressing transgenic lines. PCR analysis confirmed 18 lines contained only the egfp gene, whereas the remaining contained both egfp and codA/nptII genes. Presumably, the 18 monogenic lines arose through cross protection by being in close proximity to cells that expressed nptII and thus detoxified kanamycin in the immediate vicinity. This is the first report for grapevine using a combination of positive and negative selection to produce transgenic plants that do not contain marker genes.     

Virulence is context-dependent in a vertically transmitted aquatic host–microparasite system

Recent work has suggested that the outcomes of host–symbiont interactions can shift between positive, neutral and negative depending on both biotic and abiotic conditions. Even organisms traditionally defined as parasites can have positive effects on hosts under some conditions. For a given host–parasite system, the effects of infection on host fitness can depend on host vigour, route of transmission and environmental conditions. We monitored sublethal microsporidian infections in populations of Gammarus pseudolimnaeus (Amphipoda: Gammaridae) from four cool water streams in southwestern Michigan, USA. Our objectives were to: (i) infer the mechanism of transmission (horizontal, vertical or mixed) from observed effects of infection on host fitness, (ii) determine if the magnitude of the effects on host fitness is a function of parasite load (infection intensity) compared with simple presence or absence of infection, and (iii) determine if there is variation in parasite effects on host fitness in isolated populations. PCR and DNA sequence analyses revealed that there were two microsporidia present among the four host populations: Dictyocoela sp. and Microsporidium sp. PCR screening of a subset of infected hosts showed that Dictyocoela sp. accounted for 90% of infections and was present in all four G. pseudolimnaeus populations, while Microsporidium sp. was found in two populations but was only relatively common in one. We found very low prevalence in males (∼5%), but high prevalence in females (range: 37–85%). Female fitness was positively associated with infection in two streams, resulting from either higher fecundity or more reproductive bouts. Infection had a negative effect on the number of reproductive bouts in a third population, and no effect on fecundity in a fourth population. Infection intensity explained additional variation in fecundity in one population; females with intermediate infection intensity had higher fecundity than females with either light or heavy infection intensity. Given the high prevalence of infection in females compared with males and the generally weak negative fitness effects coupled with some positive fitness effects, it is likely that both Dictyocoela sp. and Microsporidium sp. are primarily vertically transmitted, feminizing microsporidia. Our results suggest that microsporidian effects on G. pseudolimnaeus fitness were context-dependent and varied with host sex and local environment.     

Adaptation in the Asexual False Spider Mite <Emphasis Type="Italic">Brevipalpus phoenicis</Emphasis>: Evidence for Frozen Niche Variation

Because asexual species lack recombination, they have little opportunity to produce genetically variable offspring and cannot adapt to changes in their environment. However, a number of asexual species are very successful and appear to contradict this general view. One such species is the phytophagous mite Brevipalpus phoenicis (Geijskes), a species that is found in a wide range of environments. There are two general explanations for this pattern, the General Purpose Genotype (GPG) and Frozen Niche Variation (FNV). According to the GPG model, an asexual species consists of clones that can all survive and reproduce in all the different niches. Alternatively, the FNV model postulates that different clones are specialized to different niches. We have performed a test to distinguish between these models in B. phoenicis. Mites from three populations from three different host plant species (citrus, hibiscus and acerola) were transplanted to their own and the two alternative host plants and mite survival and egg production were measured. Additionally, the mite populations were genotyped using microsatellites. Fitness was seriously reduced when mites were transplanted to the alternative host plant species, except when the alternative host was acerola. We concluded that B. phoenicis clones are specialized to different niches and thus the FNV best describes the broad ecological niche of this species but that there is also some evidence for host plant generalization. This conclusion was strengthened by the observations that on each host plant species the native mite population performed better than the introduced ones, and that three microsatellite markers showed that the mite populations are genetically distinct.     

Patterns of <Emphasis Type="Italic">Azteca</Emphasis> ants’ defence of <Emphasis Type="Italic">Cecropia</Emphasis> trees in a tropical rainforest: support for optimal defence theory

Optimal defence theory (ODT) predicts that, whereas high risk of herbivory should select for high constitutive levels of defence, induced defences should be more advantageous in environments with a low probability of herbivory. In the present field study, conducted on the Azteca–Cecropia ant–plant system in a Neotropical rainforest, we evaluated whether the constitutive and induced ant defence of leaves are directly and inversely related to an estimate of herbivory risk, respectively. To assess the constitutive level of Azteca defence in Cecropia obtusifolia trees, we recorded the number of ants patrolling undamaged leaves. To evaluate the induced level of Azteca defence, the same leaves were subjected to simulated herbivory by punching circular holes in them. We recorded the maximum number of ants patrolling the damaged leaves from 2 to 15 min after damage. Past herbivory (% defoliation of old leaves) was assumed to indicate a risk of herbivory. Regression analyses showed that, whereas the constitutive level of ant patrolling was positively associated with the magnitude of herbivory on old leaves, there was a negative association between the magnitude of induced ant defence and past herbivory. These preliminary results lend support to ODT.     

Reproductive investiment in the scorpion Centruroides vittatus

Among invertebrates, scorpions possess a relatively unique set of reproductive traits. The interrelationships of these traits may have important implications for life history theory, yet there have been few studies of these traits in scorpions. Our data indicate that larger female Centruroides vittatus produce more offspring and have a higher total litter mass than smaller females. There was, however, no significant relationship between offspring size and female or litter size. Mean offspring mass increased with increases in total litter mass and within litter variation in offspring size (coefficients of variation) decreased with increasing total litter mass. These results suggest that large female scorpions with a larger investment in reproduction produced more offspring that were more uniform in size, but not significantly larger, than small females with less investment. The fractional clutch principle and physiological and functional constraints on size and number of offspring are suggested as possible explanations for the relationships we found among offspring size, variation in offspring size and total investment in offspring in C. vittatus.     

Male recombination with single and homologous P elements in Drosophila melanogaster

It has previously been shown that, in the presence of a source of P element transposase, male recombination in Drosophila melanogaster is induced at a rate of about 1% in the region of a single P[CaSpeR] element. This paper shows that recombinant chromosomes retain unaltered P[CaSpeR] elements at the original site in a high proportion of cases. This result is incompatible with a simple model in which recombination occurs by resolution of a Holliday junction following P element excision and repair. It has also previously been shown that homozygous regions containing a P element produce male recombination levels of 10–20%, an order of magnitude higher than that given by a single element. This paper shows that reciprocal recombinant chromosomes retaining P[CaSpeR] elements can be combined to produce similarly high levels of recombination. This result potentially allows for recombinant chromosomes from homologous recombination to be analysed at the molecular level in the region of the inserted element.     

The distribution of <Emphasis Type="Italic">Echinococcus granulosus</Emphasis> in moose: evidence for parasite-induced vulnerability to predation by wolves?

The role of parasites in influencing the trophic dynamics of hosts is becoming increasingly recognized in the ecological literature. Echinococcus granulosus is a tapeworm that relies on the predator-prey relationship between the definitive host (wolf, Canis lupus) and the intermediate host, (moose, Alces alces) to complete its life cycle. Heavy infection by E. granulosus may predispose moose to increased risk of predation by wolves. Theory predicts that parasite-induced vulnerability to predation will reduce the degree of aggregation of parasites in a host population. We tested for different levels of aggregation of E. granulosus in moose in areas of low, moderate, and high levels of wolf predation using Greens coefficient of dispersion. Parasite aggregation was lower in an area with high predation rate, thus we hypothesize that heavy infection by E. granulosus predisposes moose to predation by wolves. This increase in predation rate due to parasite infection may influence the role of wolves in regulating moose populations. We discuss alternative explanations for the negative correlation between predation rate and parasite aggregation.An erratum to this article can be found at     

Trade-offs between sexual and asexual reproduction in the genus Mimulus

Summary Six estimates of resource allocation to sexual reproduction (nectar concentration, nectar volume, sugar production, pollinator visitation rates, fruit-set, and seed production) and a single estimate of resource allocation to asexual reproduction (the rate of rooted-branch production) were examined for five species of Mimulus. There were significant interspecific differences for all paramaters. With the exception of nectar concentration, there was 1) a consistent positive correlation among the parameters measuring allocation to sexual reproduction, and 2) a significant negative correlation between parameters measuring allocation to sexual reproduction and the rate of rooted-branch production (asexual reproduction). The results indicate that Mimulus species which produce the highest volumes of nectar 1) receive the most pollinator visits, 2) have the highest fruit-set, 3) produce the most seeds, and 4) produce the fewest rooted branches.     

Fouling of gastropods: a role for parasites?

A sample of mud snails Hydrobia ulvae (Prosobranchia) from an intertidal population revealed that the shells of trematode-infected specimens were especially likely to be fouled with epibionts. Experimentally trematode-infected Biomphalaria glabrata (Pulmonata) appeared to be especially prone to develop epigrowth in comparison with uninfected conspecifics as well. These findings suggest an interaction between trematode infections and epibiosis in aquatic gastropods. The two most likely explanations for this are (1) that trematode infections weakens the snails' natural defences against epibionts, or (2) that the defences against epibionts also are effective against invading trematodes, causing snail specimens with a particularly good fouling defence to be less likely to become infected.     

Herbivory-induced extrafloral nectar increases native and invasive ant worker survival

Ascertaining the costs and benefits of mutualistic interactions is important for predicting their stability and effect on community dynamics. Despite widespread designation of the interaction between ants and extrafloral nectaries (EFNs) as a mutualism and over 100 years of studies on ant benefits to plants, the benefits to ants have never been experimentally quantified. The success of invasive ants is thought to be linked to the availability of carbohydrate-rich resources, though reports of invasive ant visits to EFNs are mixed. In two laboratory experiments, we compared worker survival of one native (Iridomyrmex chasei) and two invasive ant species (Linepithema humile and Pheidole megacephala) exposed to herbivorized or non-herbivorized EFN-bearing plants (Acacia saligna) or positive and negative controls. We found that non-herbivorized plants did not produce any measurable extrafloral nectar, and ants with access to non-herbivorized plants had the same survival as ants with access to an artificial plant and water (unfed ants). Ants given herbivorized plants had 7–11 times greater worker survival relative to unfed ants, but there were no differences in survival between native and invasive ants exposed to herbivorized plants. Our results reveal that ants cannot induce A. saligna extrafloral nectar production, but workers of both native and invasive ant species can benefit from extrafloral nectar as much as they benefit from sucrose.     

Protease excretion during pineapple micropropagation in temporary immersion bioreactors

Summary Biotechnology has become an important tool to produce plant secondary metabolites and proteases are among them. Although pineapple plants have been found to produce proteases, most of the biotechnological investigations on this crop have been focused on propagation. The procedure involving the use of temporary immersion bioreactors is one of the most outstanding because of its high multiplication rate. We previously recorded specific protease activity in the culture medium during the pre-elongation step of this protocol. Therefore we decided to modify this phase, looking for an increase of protease excretion. Three independent experiments were performed to evaluate the effects of culture duration, and levels of gibberellic acid (GA) and 6-benzyladenine (BA). The following indicators were recorded: shoot fresh mass per bioreactor; and protein concentration, proteolytic activity, and specific protease activity in culture media. As happens in investigations focused on protease production, the specific protease activity was the most important indicator recorded here. It maximized at 21 d of culture. Moreover, GA (4.2 μM) increased specific activity in the culture medium while BA produced a negative effect. Results shown here demonstrate that conditions adquate for propagation purposes (15-d pre-elongation phase; 2.8 μM GA; 2.2 μM BA) are not necessarily adequate for protease excretion.