Assessment of interleukin 1 and interleukin 2 effects on cycling and noncycling murine thymocytes

When mouse thymocytes are stimulated with PHA, the proliferative response is very low, unless the culture medium is enriched with interleukin 1 (IL-1)- or interleukin 2 (IL-2)-containing supernatants. Cytofluorometric analyses show, however, that PHA stimulation generates a significant number of cells with increased RNA content (transition from the G₀ to G₁ phase of the cell cycle). If IL-2 is added to such cultures, the activated cells complete their process of RNA synthesis and then enter the S phase. The use of IL-2-containing culture medium thus permits one to obtain a high correlation between the number of g₁ cells and [³H]thymidine incorporation (r = 0.97). Enrichment with IL-1-containing supernatants also results in a statistically significant correlation (r = 0.68), but the regression lines are markedly different for the two interleukins (s = 20.3 for IL-2 and s = 9.2 for IL-1), when analyzed after 48 hr of incubation. These observations suggest that the G₁ phase must be divided into two subcompartments, G_1a and G_1b, the G_1a-G_1b transition being an IL-2-dependent event. If the number of G_1b cells is used to establish correlations with [³H]thymidine incorporation, all values fall on the same regression line, regardless of culture conditions and of the addition of interleukins. It is concluded that IL-2 regulates lymphocyte proliferation at the level of RNA synthesis (G_1a-G_1b transition) rather than that of DNA synthesis (G₁-S transition).     

Phylogeny and phylogeography of the genus Geothelphusa (Crustacea: Decapoda, Brachyura, Potamidae) in southwestern Taiwan based on two mitochondrial genes

Eleven species of Geothelphusa have been reported from southwestern Taiwan (Tainan, Kaohsiung and the northern part of Pingtung counties): G. albogilva Shy, Ng, and Yu, 1994; G. ancylophallus Shy, Ng, and Yu, 1994; G. caesia Shy, Ng, and Yu, 1994; G. lili Chen, Cheng, and Shy, 2005; G. nanhsi Shy, Ng, and Yu, 1994; G. neipu Chen, Cheng, and Shy, 1998; G. olea Shy, Ng, and Yu, 1994; G. pingtung Tan and Liu, 1998; G. shernshan Chen, Cheng, and Shy, 2005; G. tsayae Shy, Ng, and Yu, 1994 and G. wutai Shy, Ng, and Yu, 1994. Comparisons of DNA sequences encoding parts of the mitochondrial large subunit (16S) rRNA and cytochrome oxidase subunit I (COI) genes revealed three major clades, of which one is the species G. ancylophallus, and the other two are species groups here referred to as the G. olea and G. pingtung clades. Geothelphusa ancylophallus is geographically restricted and adapted to an ecologically challenging habitat with an unstable water supply and uneven topology. The G. olea clade (G. olea, G. caesia, G. nanhsi, G. tsayae, and G. wutai) is widely distributed throughout central-western and southwestern Taiwan. The G. pingtung clade (G. pingtung, G. neipu and G. shernshan) is confined to southwestern Taiwan between the previously defined southernmost clades of G. tawu, G. albogilva, and G. ferruginea, and the G. olea clade to the north. It includes an isolated population on distant Chaishan Mountain near Taiwan Strait, which probably dispersed from the peripheral hills of the Central Range during the early Pleistocene. The available genetic evidence indicates that the differential coloration observed in members of the G. olea and G. pingtung clades is not reflected in mtDNA, appears to be dependent on environmental conditions, food, etc., and has little value as a taxonomic character. Possible geological events and climatic factors responsible for the historic isolation of the different freshwater crab clades in southwestern Taiwan are discussed in detail.     

Flight initiation distance and escape behavior in the black redstart (Phoenicurus ochruros)

There are many anti‐predatory escape strategies in animals. A well‐established method to assess escape behavior is the flight initiation distance (FID), which is the distance between prey and predator at which an animal flees. Previous studies in various species throughout the animal kingdom have shown that group size, urbanization, and distance to refuge and body mass affect FID. In most species, FID increases if body mass, group size or distance to refuge decreases. However, how age and sexual dimorphism affect FID is rather unknown. Here, we assess the escape behavior and FID of the black redstart (Phoenicurus ochruros), a small turdid passerine. When approached by a human, males initiated flights later, that is allowing a closer approach than females. Males of this species are more conspicuous, and therefore, may exhibit aposematism to deter potential predators or are less fearful than females. Additionally, juveniles fled at shorter distances and fled to lower heights than adults. Lastly, concerning escape strategy, black redstarts, unless other passerine birds, fled less often into cover, but rather onto open or elevated spots. Black redstarts are especially prone to predation by ambushing predators that might hide in cover. Hence, this species most likely has a higher chance of escaping by fleeing to an open spot rather than to a potentially risky cover.     

Effects of Heat Shock on Resistance to Parasitoids and on Life History Traits in an Aphid/Endosymbiont System

Temperature variation is an important factor determining the outcomes of interspecific interactions, including those involving hosts and parasites. This can apply to variation in average temperature or to relatively short but intense bouts of extreme temperature. We investigated the effect of heat shock on the ability of aphids (Aphis fabae) harbouring protective facultative endosymbionts (Hamiltonella defensa) to resist parasitism by Hymenopteran parasitoids (Lysiphlebus fabarum). Furthermore, we investigated whether heat shocks can modify previously observed genotype-by-genotype (G x G) interactions between different endosymbiont isolates and parasitoid genotypes. Lines of genetically identical aphids possessing different isolates of H. defensa were exposed to one of two heat shock regimes (35°C and 39°C) or to a control temperature (20°C) before exposure to three different asexual lines of the parasitoids. We observed strong G x G interactions on parasitism rates, reflecting the known genetic specificity of symbiont-conferred resistance, and we observed a significant G x G x E interaction induced by heat shocks. However, this three-way interaction was mainly driven by the more extreme heat shock (39°C), which had devastating effects on aphid lifespan and reproduction. Restricting the analysis to the more realistic heat shock of 35°C, the G x G x E interaction was weaker (albeit still significant), and it did not lead to any reversals of the aphid lines'' susceptibility rankings to different parasitoids. Thus, under conditions feasibly encountered in the field, the relative fitness of different parasitoid genotypes on hosts protected by particular symbiont strains remains mostly uncomplicated by heat stress, which should simplify biological control programs dealing with this system.     

Carbon source utilization of soil extracted microorganisms as a tool to detect the effects of soil supplemented with genetically engineered and non-engineered Corynebacterium glutamicum and a recombinant peptide at the community level

Abstract: Substrate utilization of microbial cells extracted from soil with a 0.85% aqueous sodium chloride solution, was determined to estimate effects on soil microorganisms at the community level with microtiter plates (Biolog GN®) containing 95 different sources of organic carbon. A consistent pattern of utilized substrates was obtained after 24 h of microtiter plate incubation at 28°C. The absorbance values (OD₅₉₀) obtained from a microtiter plate reader after background correction were transformed by using the average absorbance values of oxidized substrates as a threshold to distinguish between well utilized and poorly or non-utilized substrates and thereby reduce variances between replicates. Doubling times of the extracted soil microorganisms in the microtiter plates were tested with 12 substrates and ranged from 1.96 h to 3.23 h, depending on the carbon source. The carbon source utilization assay was used to assess the effects of soil inoculation with Corynebacterium glutamicum with and without a genetically engineered plasmid (pUN1; 6.3 kb), which encoded for the synthesis of the mammalian protease inhibiting peptide, aprotinin. Additionally, aprotinin itself was added at two concentrations to soil samples. An identical decrease in the number of carbon sources utilized, especially carbohydrates, occurred upon soil inoculation with both C. glutamicum strains after inoculation with 10⁶ cells g⁻¹ soil. This effect was only detectable during the first three weeks of incubation, as long as cell numbers of C. glutamicum (pUN1) were above 10⁵ cfu g⁻¹. Soil amendment with aprotinin resulted in utilization of additional substrates, most of them carbohydrates. With 0.1 mg aprotinin g⁻¹ soil this stimulation lasted 2 days and with 10 mg g⁻¹ it lasted for 7 days.     

Forager age and foraging state,but not cumulative foraging activity,affect biogenic amine receptor gene expression in the honeybee mushroom bodies

Foraging behavior is crucial for the development of a honeybee colony. Biogenic amines are key mediators of learning and the transition from in-hive tasks to foraging. Foragers vary considerably in their behavior, but whether and how this behavioral diversity depends on biogenic amines is not yet well understood. For example, forager age, cumulative foraging activity or foraging state may all be linked to biogenic amine signaling. Furthermore, expression levels may fluctuate depending on daytime. We tested if these intrinsic and extrinsic factors are linked to biogenic amine signaling by quantifying the expression of octopamine, dopamine and tyramine receptor genes in the mushroom bodies, important tissues for learning and memory. We found that older foragers had a significantly higher expression of Amdop1, Amdop2, AmoctαR1, and AmoctβR1 compared to younger foragers, whereas Amtar1 showed the opposite pattern. Surprisingly, our measures of cumulative foraging activity were not related to the expression of the same receptor genes in the mushroom bodies. Furthermore, we trained foragers to collect sucrose solution at a specific time of day and tested if the foraging state of time-trained foragers affected receptor gene expression. Bees engaged in foraging had a higher expression of Amdop1 and AmoctβR3/4 than inactive foragers. Finally, the expression of Amdop1, Amdop3, AmoctαR1, and Amtar1 also varied with daytime. Our results show that receptor gene expression in forager mushroom bodies is complex and depends on both intrinsic and extrinsic factors.     

De novo indol-3-ylmethyl glucosinolate biosynthesis,and not long-distance transport,contributes to defence of Arabidopsis against powdery mildew

Powdery mildew is a fungal disease that affects a wide range of plants and reduces crop yield worldwide. As obligate biotrophs, powdery mildew fungi manipulate living host cells to suppress defence responses and to obtain nutrients. Members of the plant order Brassicales produce indole glucosinolates that effectively protect them from attack by non-adapted fungi. Indol-3-ylmethyl glucosinolate is constitutively produced in the phloem and transported to epidermal cells for storage. Upon attack, indol-3-ylmethyl glucosinolate is activated by CYP81F2 to provide broad-spectrum defence against fungi. How de novo biosynthesis and transport contribute to defence of powdery mildew-attacked epidermal cells is unknown. Bioassays and glucosinolate analysis demonstrate that GTR glucosinolate transporters are not involved in antifungal defence. Using quantitative live-cell imaging of fluorophore-tagged markers, we show that accumulation of the glucosinolate biosynthetic enzymes CYP83B1 and SUR1 is induced in epidermal cells attacked by the non-adapted barley powdery mildew Blumeria graminis f.sp. hordei. By contrast, glucosinolate biosynthesis is attenuated during interaction with the virulent powdery mildew Golovinomyces orontii. Interestingly, SUR1 induction is delayed during the Golovinomyces orontii interaction. We conclude that epidermal de novo synthesis of indol-3-ylmethyl glucosinolate contributes to CYP81F2-mediated broad-spectrum antifungal resistance and that adapted powdery mildews may target this process.     

Parasitoids as vectors of facultative bacterial endosymbionts in aphids

Heritable bacterial endosymbionts play an important role in aphid ecology. Sequence-based evidence suggests that facultative symbionts such as Hamiltonella defensa or Regiella insecticola also undergo horizontal transmission. Other than through male-to-female transfer during the sexual generation in autumn, the routes by which this occurs remain largely unknown. Here, we tested if parasitoids or ectoparasitic mites can act as vectors for horizontal transfer of facultative symbionts. Using symbiont-specific primers for diagnostic PCR, we demonstrate for the first time, to our knowledge, that parasitoids can indeed transfer H. defensa and R. insecticola by sequentially stabbing infected and uninfected individuals of their host, Aphis fabae, establishing new, heritable infections. Thus, a natural route of horizontal symbiont transmission is also available during the many clonal generations of the aphid life cycle. No transmissions by ectoparasitic mites were observed, nor did parasitoids that emerged from symbiont-infected aphids transfer any symbionts in our experiments.     

A site accessible to extracellular TEA+ and K+ influences intracellular Mg2+ block of cloned potassium channels

The members of the RCK family of cloned voltage-dependent K⁺ channels are quite homologous in primary structure, but they are highly diverse in functional properties. RCK4 channels differ from RCK1 and RCK2 channels in inactivation and permeation properties, the sensitivity to external TEA, and to current modulation by external K⁺ ions. Here we show several other interesting differences: While RCK1 and RCK2 are blocked in a voltage and concentration dependent manner by internal Mg²⁺ ions, RCK4 is only weakly blocked at very high potentials. The single-channel current-voltage relations of RCK4 are rather linear while RCK2 exhibits an inwardly rectifying single-channel current in symmetrical K⁺ solutions. The deactivation of the channels, measured by tail current protocols, is faster in RCK4 by a factor of two compared with RCK2. In a search for the structural motif responsible for these differences, point mutants creating homology between RCK2 and RCK4 in the pore region were tested. The single-point mutant K533Y in the background of RCK4 conferred the properties of Mg²⁺ block, tail current kinetics, and inward ion permeation of RCK2 to RCK4. This mutant was previously shown to be responsible for the alterations in external TEA sensitivity and channel regulation by external K⁺ ions. Thus, this residue is expected to be located at the external side of the pore entrance. The data are consistent with the idea that the mutation alters the channel occupancy by K⁺ and thereby indirectly affects internal Mg²⁺ block and channel closing.Abbreviations TEA tetraethylammonium - EGTA Ethylene glycol-bis (-aminoethyl ether) N,N,N,N-tetraacetic acid - 2S3B model 2-site 3-barrier model Correspondence to: S. H. Heinemann