Climate‐driven shifts in the distribution of koala‐browse species from the Last Interglacial to the near future

The koala's Phascolarctos cinereus distribution is currently restricted to eastern and south‐eastern Australia. However, fossil records dating from 70 ± 4 ka (ka = 10³ yr) from south‐western Australia and the Nullarbor Plain are evidence of subpopulation extinctions in the southwest at least after the Last Interglacial (~128–116 ka). We hypothesize that koala sub‐population extinctions resulted from the eastward retraction of the koala's main browse species in response to unsuitable climatic conditions. We further posit a general reduction in the distribution of main koala‐browse trees in the near future in response climate change. We modelled 60 koala‐browse species and constructed a set of correlative species distribution models for five time periods: Last Interglacial (~128–116 ka), Last Glacial Maximum (~23–19 ka), Mid‐Holocene (~7–5 ka), present (interpolations of observed data, representative of 1960–1990), and 2070. We based our projections on five hindcasts and one forecast of climatic variables extracted from WorldClim based on two general circulation models (considering the most pessimistic scenario of high greenhouse‐gas emissions) and topsoil clay fraction. We used 17 dates of koala fossil specimens identified as reliable from 70 (± 4) to 535 (± 49) ka, with the last appearance of koalas at 70 ka in the southwest. The main simulated koala‐browse species were at their greatest modelled extent of suitability during the Last Glacial Maximum, with the greatest loss of koala habitat occurring between the Mid‐Holocene and the present. We predict a similar habitat loss between the present and 2070. The spatial patterns of habitat change support our hypothesis that koala extinctions in the southwest, Nullarbor Plain and central South Australia resulted from the eastward retraction of the dominant koala‐browse species in response to long‐term climate changes. Future climate patterns will likely increase the extinction risk of koalas in their remaining eastern ranges.     

Modelling time‐varying low‐temperature‐induced mortality rates for pupae of Tuta absoluta (Gelechiidae,Lepidoptera)

In a series of laboratory experiments, acclimated pupae of Tuta absoluta were exposed to various constant low temperatures in order to estimate their maximum survival times (Kaplan–Meier, Lt_99.99). A Weibull function was fitted to the data points, describing maximum survival time as a function of temperature. In another experiment at ?6°C, the progress of mortality increasing with exposure time was identified. These values were fitted by a sigmoidal function converging asymptotically to 100% mortality for very long exposure times. Analysing mortality data from the maximum survival experiment by a generalized linear model showed a significant common slope parameter (p < .001) that reveals parallelism of the survival curves at each temperature if a log time axis is used. These curves appear stretched (time scaled) if plotted with a nonlogarithmic time axis. By combining these mathematical relations, it was possible to calculate a species‐specific ‘mortality surface’ which exhibits mortalities, depending on temperature and duration of exposure. In order to accumulate hourly mortalities for courses of varying temperatures, an algorithm was developed which yields mortality values from that surface taking into account the attained mortality level. In validation experiments, recorded mortalities were compared against modelled mortalities. Prediction of mortality was partially supported by the model, but pupae experiencing intensely fluctuating temperatures showed decreased mortality, probably caused by rapid cold hardening during exposure. Despite this observation, mortality data converged to distinct levels very close to 100% depending on the intensity of temperature fluctuations that were characteristic for different types of experiments. The highest mortality limit occurred at intensely fluctuating temperatures in laboratory experiments. This constituted a benchmark that was not reached under various field conditions. Thus, it was possible to identify temperature limits for the extinction of field populations of Tuta absoluta pupae.     

Regulation of cell behaviour by plant receptor kinases: Pattern recognition receptors as prototypical models

In this review we focus on pattern recognition receptors in plants that detect extracellular signals indicative for pathogen attack and injury. We start out with a discussion on FLS2, which binds and responds to bacterial flagellin, and then concentrate on ligand–receptor interactions as initial steps in the molecular receptor activation process. Comparison with other receptor kinases, whether involved in plant immunity or regulation of other cellular programs, might indicate common principles of receptor activation.     

Conserved tertiary base pairing ensures proper RNA folding and efficient assembly of the signal recognition particle Alu domain

Proper folding of the RNA is an essential step in the assembly of functional ribonucleoprotein complexes. We examined the role of conserved base pairs formed between two distant loops in the Alu portion of the mammalian signal recognition particle RNA (SRP RNA) in SRP assembly and functions. Mutations disrupting base pairing interfere with folding of the Alu portion of the SRP RNA as monitored by probing the RNA structure and the binding of the protein SRP9/14. Complementary mutations rescue the defect establishing a role of the tertiary loop–loop interaction in RNA folding. The same mutations in the Alu domain have no major effect on binding of proteins to the S domain suggesting that the S domain can fold independently. Once assembled into a complete SRP, even particles that contain mutant RNA are active in arresting nascent chain elongation and translocation into microsomes, and, therefore, tertiary base pairing does not appear to be essential for these activities. Our results suggest a model in which the loop–loop interaction and binding of the protein SRP9/14 play an important role in the early steps of SRP RNA folding and assembly.     

Infrared video thermography: a technique for assessing cold adaptation in insects

Insects can survive subzero temperatures by two main strategies: freeze tolerance and freeze avoidance. An array of techniques have been used to investigate the physiological limits of insects to low temperatures, such as differential scanning calorimetry, temperature-controlled cooling apparatus, thermocouples, and computer-controlled chart recording equipment. However, these techniques require animals to be stationary, precluding behavioral data. We used infrared video thermography to investigate cold adaptation in an alpine insect, expanding such investigations to include behavioral response as an indicator of physiological stress. This technique is noninvasive and provides a large amount of physiological information, such as supercooling points, lower lethal temperatures, and hemolymph melting points. Insect supercooling points in response to a constant cooling rate were variable; however, temperatures at the initiation of behavioral stress response were less variable. Assessments of supercooling points and lower lethal temperatures obtained in this way are more biologically meaningful because allowing unhindered movement of insects more closely resembles natural environments.     

Experimentally elevated testosterone increases status signalling in male Greylag geese (<Emphasis Type="Italic">Anser anser</Emphasis>)

Testosterone modulates male vertebrates sexual and social behaviour. We experimentally investigated the testosterone-sensitive behaviours in male greylag geese (Anser anser) by implanting silastic tubes containing crystalline testosterone during the mating season (February; 5 implanted and 5 control males) and in the early winter (November; 7 and 7). Focal animals were part of a semi-tame, unrestrained flock with fully intact social relationships. Excreted testosterone and corticosterone immunoreactive metabolites (TM, BM) were determined by enzyme immunoassay. Individual faecal samples and behavioural protocols were collected daily over a period of 5 weeks, including 1 control week before implantation. In February, no significant behavioural effects of the supplemental testosterone were observed, which may be due to the naturally occurring high systemic androgen levels in spring. In November, however, implanted males had higher TM excretion rates and performed status signalling behaviour (beak up) more frequently than control males. No differences between implanted and control males were found with respect to BM, agonistic interactions or vigilance behaviour. Furthermore, during the second week after implantation, TM positively correlated with the frequency of beak up of implanted males, whilst their female partners were attacked with lower latency by other members of the flock than the females of control males. Hence, status signalling in greylag ganders seems to be testosterone-sensitive year-long and inappropriate status signalling of males may draw attacks towards their females.     

Peroxisomal localization of sulfite oxidase separates it from chloroplast-based sulfur assimilation

Recently, we isolated the sulfite oxidase (SO) gene from Arabidopsis thaliana and characterized the purified SO protein. The purpose of the present study was to determine the subcellular localization of this novel plant enzyme. Immunogold electron-microscopic analysis showed the gold labels nearly exclusively in the peroxisomes. To verify this finding, green fluorescent protein was fused to full-length plant SO including the putative peroxisomal targeting signal 1 (PTS1) 'SNL' and expressed in tobacco leaves. Our results showed a punctate fluorescence pattern resembling that of peroxisomes. Co-labelling with MitoTracker-Red excluded that the observed fluorescence was due to mitochondrial sorting. By investigation of deleted or mutated PTS1, no functional peroxisomal targeting signal 2 (PTS2) could be detected in plant SO. This conclusion is supported by expression studies in Pichia pastoris mutants with defined defects either in PTS1- or PTS2-mediated peroxisomal import.     

A nodule-specific dicarboxylate transporter from alder is a member of the peptide transporter family

Alder (Alnus glutinosa) and more than 200 angiosperms that encompass 24 genera are collectively called actinorhizal plants. These plants form a symbiotic relationship with the nitrogen-fixing actinomycete Frankia strain HFPArI3. The plants provide the bacteria with carbon sources in exchange for fixed nitrogen, but this metabolite exchange in actinorhizal nodules has not been well defined. We isolated an alder cDNA from a nodule cDNA library by differential screening with nodule versus root cDNA and found that it encoded a transporter of the PTR (peptide transporter) family, AgDCAT1. AgDCAT1 mRNA was detected only in the nodules and not in other plant organs. Immunolocalization analysis showed that AgDCAT1 protein is localized at the symbiotic interface. The AgDCAT1 substrate was determined by its heterologous expression in two systems. Xenopus laevis oocytes injected with AgDCAT1 cRNA showed an outward current when perfused with malate or succinate, and AgDCAT1 was able to complement a dicarboxylate uptake-deficient Escherichia coli mutant. Using the E. coli system, AgDCAT1 was shown to be a dicarboxylate transporter with a K(m) of 70 microm for malate. It also transported succinate, fumarate, and oxaloacetate. To our knowledge, AgDCAT1 is the first dicarboxylate transporter to be isolated from the nodules of symbiotic plants, and we suggest that it may supply the intracellular bacteria with dicarboxylates as carbon sources.     

Contribution of the cytoskeleton and the phospholipase C signaling pathway to fluid stream-induced membrane currents

A fluid stream induced by a concentration clamp system evokes in Xenopus oocytes a deformation of the membrane which results in transient chloride currents of high amplitude (stream-evoked inward current, I(i,st)) during calcium-activated chloride current oscillations. The involvement of cytoskeleton elements and of components of the phospholipase C-dependent signaling pathway on the generation of the I(i,st) were investigated. Incubation of the oocytes with cytoskeleton-disrupting agents exerted no effects on generation of the I(i,st), suggesting that the mechanotransduction is not mediated by these structures. The fluid stream induced an elevation of the submembraneous calcium concentration, as measured by an increase of Fluo-4-mediated fluorescence after the stimulus. Lowering the intracellular calcium concentration by injection of calcium chelators or depleting inositol 1,4,5-triphosphate (InsP(3))-sensitive calcium stores by blockers of the calcium pumps suppressed the generation of the I(i,st) in most cases. Furthermore, the phospholipase C inhibitor U73122 reversibly blocked the I(i,st). The results suggest that the fluid stream leads to a membrane stretch which modulates directly or indirectly the activity of a membrane-bound phospholipase C. The phospholipase C transiently elevates the InsP(3) concentration, in turn releasing calcium from InsP(3)-sensitive internal calcium stores, thus evoking an enhanced calcium-sensitive chloride current.     

Purification of polyethylenimine polyplexes highlights the role of free polycations in gene transfer

BACKGROUND: Nonviral vectors based on polyethylenimine (PEI) usually contain an excess of PEI that is not complexed to DNA. Since unbound PEI contributes to cellular and systemic toxicity, purification of polyplexes from unbound PEI is desirable. METHODS: Size exclusion chromatography (SEC) was used to purify PEI polyplexes of free PEI. Transfection properties of purified polyplexes and the effect of free PEI on gene delivery were studied in vitro and in vivo after systemic application into mice. RESULTS: SEC did not change the size and zeta-potential of polyplexes. Independent of the amount of PEI used for complex formation, purified PEI polyplexes had the same final PEI nitrogen/DNA phosphate ratio of 2.5. Notably, purified PEI polyplexes demonstrated low cellular and systemic toxicity. High transfection efficiency was achieved with purified polyplexes at high DNA concentrations (8-15 microg/ml). At low DNA concentrations (2-4 microg/ml) gene transfer with purified particles was less efficient than with polyplexes containing free PEI both in vitro and in vivo. Mechanistic studies showed that free PEI partly blocked cellular association of DNA complexes but was essential for the following intracellular gene delivery. Adding free PEI to cells treated with purified particles with a delay of up to 4 h resulted in significantly enhanced transfection efficiency compared with non-purified particles or purified particles without free PEI. CONCLUSIONS: This study presents an efficient method to remove free PEI from PEI polyplexes by SEC. Our results from transfection experiments demonstrate that free PEI substantially contributes to efficient gene expression but also mediates toxic effects in a dose-dependent manner. Purified polyplexes without free PEI have to be applied at increased concentrations to achieve high transfection levels, but exhibit a greatly improved toxicity profile.