Analyses of isoamylase gene activity in wild-type barley indicate its involvement in starch synthesis

The notion of debranching enzyme activity as a participant in starch synthesis is gaining acceptance. Inconsistent reports from mutant analyses implicate either isoamylase or pullulanase as a determinant in amylopectin formation and whether wild-type plants utilize one or the other, or both, of these debranching enzymes in starch synthesis is unclear. Recent results on the su1 mutant in maize suggest that both forms of debranching enzymes might be involved in amylopectin formation. We wished to find out if isoamylase takes part in starch synthesis by comparing isoamylase gene activity under three conditions: (1) during starch accumulation in developing sink tissues; (2) during starch degradation in germinating seeds; (3) in ectopic expression after applying sucrose, a starch precursor. We isolated the gene for barley isoamylase, iso1, and analysed its expression and regulation in germinating seeds, developing endosperm and vegetative tissues, and compared the isoamylase gene expression in sink tissues from three different species. Our results indicate that isoamylase gene activity is involved in starch synthesis in wild-type plants and is modulated by sucrose.     

Reidentification of the female sex pheromone of the Indian meal moth, Plodia interpunctella: evidence for a four-component pheromone blend

Pheromone gland extracts from calling female Plodia interpunctella contained at least seven compounds that consistently elicited electroantennographic responses from male antennae upon gas chromatographic analysis. Three of these compounds were found to be the previously identified gland constituents, i.e., (Z,E)-9,12-tetradecadienyl acetate (Z9,E12-14:OAc), (Z,E)-9,12-tetradecadienal (Z9,E12-14:Ald) and (Z,E)-9,12-tetradecadienol (Z9,E12-14:OH). A fourth EAD-active compound was identified as (Z)-9-tetradecenyl acetate (Z9-14:OAc). The homologue (Z)-11-hexadecenyl acetate (Z11-16:OAc) was also identified in the extracts, but showed no EAD activity. The identity of all five compounds was confirmed by comparison of GC retention times and mass spectra with those of synthetic standards. In flight tunnel tests there were no significant differences in response of male P. interpunctella to the bait containing all four EAD-active compounds and the responses to female gland extacts. A behavioural assay of different two-compound blends in the flight tunnel showed that only addition of the corresponding aldehyde to the major pheromone component Z9,E12-14:OAc raised the male response. A subtractive assay, however, revealed that the exclusion of any of the compounds from the complete four-compound blend reduced its activity significantly. We thus conclude that the female-produced sex pheromone of P. interpunctella consists of at least four components, i.e., Z9,E12-14:OAc, Z9,E12-14:Ald, Z9,E12-14:OH and Z9-14:OAc.In a field trapping test performed in a storage facility, the four-component blend attracted significantly more males of P. interpunctella than traps baited with Z9,E12-14:OAc alone. In contrast, the highest number of Ephestia kuehniella males was found in the traps baited with this major component, suggesting that the secondary pheromone components contribute to the species specificity of the blend.     

A test of monophyly of the gutless Phallodrilinae (Oligochaeta, Tubificidae) and the use of a 573-bp region of the mitochondrial cytochrome oxidase I gene in analysis of annelid phylogeny

A 573-bp region of the mitochondrial gene cytochrome c oxidase subunit I (COI) of two species of Inanidrilus Erséus and four species of Olavius Erséus (Phallodrilinae, Tubificidae) is used in a parsimony analysis together with a selection of 35 other annelids (including members of Polychaeta, Pogonophora, Aphanoneura, and the clitellate taxa Tubificidae, Enchytraeidae, Naididae, Lumbriculidae, Haplotaxidae, Lumbricidae, Criodrilidae, Branchiobdellida and Hirudinea), and with two molluscs as outgroups. The data support the monophyly of the Olavius and Inanidrilus group, with a monophyletic Inanidrilus . However, parsimony jackknife analyses show that most of the other groups are unsupported by the data set, thus revealing a large amount of homoplasy in the selected gene region. Practically no information is given of within/between family relationships except for a few, closely related species. This suggests that the analysed COI region is not useful, when used alone, for inferring higher level relationships among the annelids.     

Real-time measurement of pheromone release from individual female moths and synthetic dispensers in a wind tunnel by recording of single receptor-neurone responses

Measurements of single neurone activity in the peripheral pheromone receptors of male Agrotis segetum (Denis & Schiffermüller) (Lepidoptera: Noctuidae) were performed in a wind tunnel using a portable electrophysiological recording unit. Filter paper and rubber septa loaded with synthetic sex pheromone, as well as individual conspecific female glands, were used as pheromone sources. Recordings, up to 3 h long, were analysed for temporal variation in spiking activity. The recordings were performed 2 m downwind of the source, where the pheromone plume had a width of approximately 12 cm, as could be measured with the single cell preparations. The system allowed reliable measurements of relative pheromone concentration with a 20-s time resolution. The release rate from rubber septa loaded with pheromone was more or less constant over time, whereas the release rate from filter paper loaded with pheromone decreased to one tenth of the initial value within 6 min from the application of the pheromone. The release of pheromone from female pheromone glands was pulsed with an interval of 2–10 min between bursts. This pulsing was not caused by retraction of the gland, as the glands were forcibly extruded during the entire experiment, but should reflect variation in transport of pheromone to the gland surface and subsequent release. The demonstrated stability of the preparations using tungsten electrodes, the reliable monitoring of female-produced pheromone plumes at several metres distance, and the time resolution obtained are important steps towards field monitoring of natural pheromone plumes, as well as pheromone concentration and distribution in applications for mating disruption.     

Regulation of Synaptotagmin I Phosphorylation by Multiple Protein Kinases

Synaptotagmin I has been suggested to function as a low-affinity calcium sensor for calcium-triggered exocytosis from neurons and neuroendocrine cells. We have studied the phosphorylation of synaptotagmin I by a variety of protein kinases in vitro and in intact preparations. SyntagI, the purified, recombinant, cytoplasmic domain of rat synaptotagmin I, was an effective substrate in vitro for Ca2+/calmodulin-dependent protein kinase II (CaMKII), protein kinase C (PKC), and casein kinase II (caskII). Sequencing of tryptic phosphopeptides from syntagI revealed that CaMKII and PKC phosphorylated the same residue, corresponding to Thr112, whereas caskII phosphorylated two residues, corresponding to Thr125 and Thr128. Endogenous synaptotagmin I was phosphorylated on purified synaptic vesicles by all three kinases. In contrast, no phosphorylation was observed on clathrin-coated vesicles, suggesting that phosphorylation of synaptotagmin I in vivo occurs only at specific stage(s) of the synaptic vesicle life cycle. In rat brain synaptosomes and PC12 cells, K+-evoked depolarization or treatment with phorbol ester caused an increase in the phosphorylation state of synaptotagmin I at Thr112. The results suggest the possibility that the phosphorylation of synaptotagmin I by CaMKII and PKC contributes to the mechanism(s) by which these two kinases regulate neurotransmitter release.     

Identifying and correcting spatial bias in opportunistic citizen science data for wild ungulates in Norway

Many publications make use of opportunistic data, such as citizen science observation data, to infer large‐scale properties of species’ distributions. However, the few publications that use opportunistic citizen science data to study animal ecology at a habitat level do so without accounting for spatial biases in opportunistic records or using methods that are difficult to generalize. In this study, we explore the biases that exist in opportunistic observations and suggest an approach to correct for them. We first examined the extent of the biases in opportunistic citizen science observations of three wild ungulate species in Norway by comparing them to data from GPS telemetry. We then quantified the extent of the biases by specifying a model of the biases. From the bias model, we sampled available locations within the species’ home range. Along with opportunistic observations, we used the corrected availability locations to estimate a resource selection function (RSF). We tested this method with simulations and empirical datasets for the three species. We compared the results of our correction method to RSFs obtained using opportunistic observations without correction and to RSFs using GPS‐telemetry data. Finally, we compared habitat suitability maps obtained using each of these models. Opportunistic observations are more affected by human access and visibility than locations derived from GPS telemetry. This has consequences for drawing inferences about species’ ecology. Models naïvely using opportunistic observations in habitat‐use studies can result in spurious inferences. However, sampling availability locations based on the spatial biases in opportunistic data improves the estimation of the species’ RSFs and predicted habitat suitability maps in some cases. This study highlights the challenges and opportunities of using opportunistic observations in habitat‐use studies. While our method is not foolproof it is a first step toward unlocking the potential of opportunistic citizen science data for habitat‐use studies.     

New specific primers for amplification of the Internal Transcribed Spacer region in Clitellata (Annelida)

Nuclear molecular evidence, for example, the rapidly evolving Internal Transcribed Spacer region (ITS), integrated with maternally inherited (mitochondrial) COI barcodes, has provided new insights into the diversity of clitellate annelids. PCR amplification and sequencing of ITS, however, are often hampered by poor specificity of primers used. Therefore, new clitellate‐specific primers for amplifying the whole ITS region (ITS: 29F/1084R) and a part of it (ITS2: 606F/1082R) were developed on the basis of a collection of previously published ITS sequences with flanking rDNA coding regions. The specificity of these and other ITS primers used for clitellates were then tested in silico by evaluating their mismatches with all assembled and annotated sequences (STD, version r127) from EMBL, and the new primers were also tested in vitro for a taxonomically broad sample of clitellate species (71 specimens representing 11 families). The in silico analyses showed that the newly designed primers have a better performance than the universal ones when amplifying clitellate ITS sequences. In vitro PCR and sequencing using the new primers were successful, in particular, for the 606F/1082R pair, which worked well for 65 of the 71 specimens. Thus, using this pair for amplifying the ITS2 will facilitate further molecular systematic investigation of various clitellates. The other pair (29F/1084R), will be a useful complement to existing ITS primers, when amplifying ITS as a whole.     

Starvation Response of Saccharomyces cerevisiae Grown in Anaerobic Nitrogen- or Carbon-Limited Chemostat Cultures

Anaerobic starvation conditions are frequent in industrial fermentation and can affect the performance of the cells. In this study, the anaerobic carbon or nitrogen starvation response of Saccharomyces cerevisiae was investigated for cells grown in anaerobic carbon or nitrogen-limited chemostat cultures at a dilution rate of 0.1 h⁻¹ at pH 3.25 or 5. Lactic or benzoic acid was present in the growth medium at different concentrations, resulting in 16 different growth conditions. At steady state, cells were harvested and then starved for either carbon or nitrogen for 24 h under anaerobic conditions. We measured fermentative capacity, glucose uptake capacity, intracellular ATP content, and reserve carbohydrates and found that the carbon, but not the nitrogen, starvation response was dependent upon the previous growth conditions. All cells subjected to nitrogen starvation retained a large portion of their initial fermentative capacity, independently of previous growth conditions. However, nitrogen-limited cells that were starved for carbon lost almost all their fermentative capacity, while carbon-limited cells managed to preserve a larger portion of their fermentative capacity during carbon starvation. There was a positive correlation between the amount of glycogen before carbon starvation and the fermentative capacity and ATP content of the cells after carbon starvation. Fermentative capacity and glucose uptake capacity were not correlated under any of the conditions tested. Thus, the successful adaptation to sudden carbon starvation requires energy and, under anaerobic conditions, fermentable endogenous resources. In an industrial setting, carbon starvation in anaerobic fermentations should be avoided to maintain a productive yeast population.     

Competition and facilitation within and between a snail and a mayfly larva and the effect on the grazing process

We studied the competitive effects within and between two taxonomically distant freshwater herbivores, a snail and a mayfly, common in Swedish lakes, Lymnaea peregra and Cloeon dipterum, respectively, and their effect on grazing in a laboratory experiment. The experimental set-up consisted of 2-l aquaria, each containing a periphyton covered tile. Intra- and interspecific effects were tested by increasing the density of one species at a time in four different treatments, (1) snails (intraspecific treatment), (2) mayflies (intraspecific treatment), (3) mixed-snails (interspecific treatments, snails kept constant) and (4) mixed-mayflies (interspecific treatments, mayflies kept constant). Intraspecific competition affected both snails and mayflies negatively, i.e. increasing mortality with increasing con-specific density. Furthermore, there was a decrease in snail growth with increasing snail density. In the mixed-species treatments both species changed their microhabitat use indicating interspecific competition. Despite this, we also found a positive effect of mayfly density on snail growth, most likely due to indirect commensalism. No density-dependent effect of grazing on periphyton was found, probably due to interference competition between grazers. However, there was a significant difference in periphyton biomass, due to species composition of grazers. Irrespective of their densities, if they co-existed, the two grazer species decreased the periphyton biomass significantly compared with both single-species treatments. We considered this as a joint action of facilitation and interaction. Our results suggest that competition can be an important structuring factor in macroinvertebrate communities and that species composition can be significant for ecosystem processes within lentic environments.