Precise construction of oligonucleotide–Fab fragment conjugate for homogeneous immunoassay using HaloTag technology

The use of oligonucleotide–protein conjugates enables the development of novel types of bioanalytical assays. However, convenient methods for producing covalent and stoichiometric oligonucleotide–protein conjugates are still rare. Here we demonstrate, for the first time, covalent conjugation of DNA oligonucleotide to Fab fragments with a 1:1 ratio using HaloTag self-labeling technology. The oligonucleotide coupling was carried out while the Fab was attached to protein G matrix, thereby enabling straightforward production of covalent conjugates. Furthermore, it allowed convenient purification of the product because the unreacted components were easily removed before the elution of the high-purity conjugate. The prepared conjugate was employed in a homogeneous immunoassay where prostate-specific antigen was used as a model analyte. Switchable lanthanide luminescence was used for detection, and the obtained limit of detection was 0.27 ng/ml. In the future, the developed method for covalent conjugation and successive purification in protein G column could also be applied for introducing other kinds of modifications to Fab fragments in a simple and site-specific manner.     

Biogenic volatile organic compound emissions in four vegetation types in high arctic Greenland

Biogenic volatile organic compounds (BVOCs) emitted from terrestrial vegetation participate in oxidative reactions in the atmosphere, leading to the formation of secondary organic aerosols and longer lifetime of methane. Global models of BVOC emissions have assumed minimal emissions from the high latitudes. However, measurements from this region are lacking, and studies from the high arctic are yet to be published. This study aimed to obtain estimates for BVOC emissions from the high arctic, and hereby to add new knowledge to the understanding of global BVOC emissions. Measurements were conducted in four vegetation types dominated by Cassiope tetragona, Salix arctica, Vaccinium uliginosum and a mixture of Kobresia myosuroides, Dryas spp. and Poa arctica. Emissions were measured by an enclosure technique and collection of volatiles into adsorbent cartridges in August. Volatiles were analyzed by gas chromatography–mass spectrometry following thermal desorption. Isoprene showed highest emissions in S. arctica heath. Monoterpene and sesquiterpene emissions were especially associated with C. tetragona heath. Total observed emissions were comparable in magnitude to emissions previously found in the subarctic, whereas isoprene emissions were lower. This study shows that considerable amounts of BVOCs are emitted from the high arctic. The results are also of importance as the emissions from this region are expected to increase in the future as a result of the predicted climate warming in the high arctic. We suggest further studies to assess the effects of climate changes in the region in order to gain new knowledge and understanding of future global BVOC emissions.     

Transcriptome Analysis Reveals Signature of Adaptation to Landscape Fragmentation

We characterize allelic and gene expression variation between populations of the Glanville fritillary butterfly (Melitaea cinxia) from two fragmented and two continuous landscapes in northern Europe. The populations exhibit significant differences in their life history traits, e.g. butterflies from fragmented landscapes have higher flight metabolic rate and dispersal rate in the field, and higher larval growth rate, than butterflies from continuous landscapes. In fragmented landscapes, local populations are small and have a high risk of local extinction, and hence the long-term persistence at the landscape level is based on frequent re-colonization of vacant habitat patches, which is predicted to select for increased dispersal rate. Using RNA-seq data and a common garden experiment, we found that a large number of genes (1,841) were differentially expressed between the landscape types. Hexamerin genes, the expression of which has previously been shown to have high heritability and which correlate strongly with larval development time in the Glanville fritillary, had higher expression in fragmented than continuous landscapes. Genes that were more highly expressed in butterflies from newly-established than old local populations within a fragmented landscape were also more highly expressed, at the landscape level, in fragmented than continuous landscapes. This result suggests that recurrent extinctions and re-colonizations in fragmented landscapes select a for specific expression profile. Genes that were significantly up-regulated following an experimental flight treatment had higher basal expression in fragmented landscapes, indicating that these butterflies are genetically primed for frequent flight. Active flight causes oxidative stress, but butterflies from fragmented landscapes were more tolerant of hypoxia. We conclude that differences in gene expression between the landscape types reflect genomic adaptations to landscape fragmentation.     

An improved amplified fragment length polymorphism (AFLP) protocol for discrimination of Listeria isolates

Nine restriction enzyme combinations and 108 different primer combinations were initially tested for suitability for amplified fragment length polymorphism (AFLP) analysis of Listeria monocytogenes; the combination of HindIII and HpyCH4IV showed consistently strong signals on gels, amplified an adequate number of DNA fragments and detected polymorphism among closely related strains based on AscI macrorestriction profiles. AFLP also distinguished between L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri, L. welshimeri and L. grayi species. All Listeria species showed species-specific clusters, with less than 33% similarity between different species. A total of 34 L. monocytogenes strains were characterised by using both AFLP and pulsed-field gel electrophoresis (PFGE). The results of AFLP analysis of L. monocytogenes strains were in concordance with those obtained by PFGE. Both methods identified 29 different genotypes of L. monocytogenes and had a high discrimination index (> 0.999). By combining the results of AFLP and PFGE, subtype discrimination was further improved. Numerical analysis of both AFLP and PFGE profiles yielded three genomic groups of L. monocytogenes strains. AFLP was found to be faster and less labour-intensive than PFGE. We conclude that the AFLP protocol is a highly discriminatory, reproducible and valuable tool in characterisation of Listeria strains and may also be suitable for Listeria species identification.     

Trade-off between floor level and floor material in farmed silver foxes

Farmed silver foxes (Vulpes vulpes) were allowed to balance their known preference for an elevated floor against their presumed preference for a sand floor. In Experiment 1, foxes had to choose between two identical cages, connected with an opening. One cage had a wire floor and the other had a sand floor, but the cages either were on the same (low or elevated) or on different levels (one cage 40 cm higher than the other). In Experiment 2, the cage pairs were connected with a 1.2 m long wire-mesh tunnel, one cage was always on a higher level (50 cm) than the other. In Experiment 1, foxes always preferred the sand floor during their active time. They also preferred the sand floor for resting, if it was on the same level as wire floor, but did not show any genuine preference if the floors were on different levels. In Experiment 2, foxes never preferred the lower floor. They preferred the elevated sand floor for activity and the elevated wire floor for lying. When two floors were identical they preferred the elevated one. Their rest consisted of 11-22 bouts, a major part of these being spent in the preferred cage. They also preferred a previous lying site to a new one, often exclusively and independently of floor material. In Experiment 1 foxes preferred the sand floor whereas in Experiment 2 they preferred the elevated floor indicating that the ability of a trade-off situation to rank resources depends on the method it is inflicted.     

Identification of mutations causing temperature-sensitive defects in Semliki Forest virus RNA synthesis

We have sequenced the nonstructural protein coding region of Semliki Forest virus temperature-sensitive (ts) mutant strains ts1, ts6, ts9, ts10, ts11, ts13, and ts14. In each case, the individual amino acid changes uncovered were transferred to the prototype strain background and thereby identified as the underlying cause of the altered RNA synthesis phenotype. All mutations mapping to the protease domain of nonstructural protein nsP2 caused defects in nonstructural polyprotein processing and subgenomic RNA synthesis, and all mutations in the helicase domain of nsP2 affected subgenomic RNA production. These types of defects were not associated with mutations in other nonstructural proteins.     

16S rDNA PCR-denaturing gradient gel electrophoresis in determining proportions of coexisting Actinobacillus actinomycetemcomitans strains

Certain serotypes of Actinobacillus actinomycetemcomitans seem to prefer coexistence in vivo. The 16S rDNA PCR-denaturing gradient gel electrophoresis (DGGE) was tested for its capability to distinguish coexisting A. actinomycetemcomitans strains of different serotypes or genetic lineages and to determine their proportions in vitro. The migration pattern of the PCR amplicon from serotype c differed from those of the other serotypes. Contrary to the strains of serotypes c, d, and e, strains of serotypes a, b, and f consistently demonstrated intra-serotype migration patterns similar to each other. Since the migration patterns differed between serotype c and b strains a strain of each was used to determine their proportional representation in a strain mixture. The strains were distinguishable from each other above the 5% PCR-DGGE detection level (12.5 ng DNA/1.5 x 10(6) cells). DGGE provides a promising tool for in vitro studies on the coexistence of different genetic lineages of A. actinomycetemcomitans.     

A parasitoid wasp uses landmarks while monitoring potential resources

Social insects and insects that provision nests are well known to have complex foraging behaviour involving repeated visits to learned locations. Other insects do not forage from a central location and are generally assumed to respond to resources by simple attraction without spatial memory. This simple response to resource cues is generally taken as giving rise to patterns of resource use that correspond directly to resource distribution. By contrast, the solitary parasitoid wasp Hyposoter horticola monitors the locations of multiple potential hosts (butterfly eggs) for up to several weeks, until the hosts become susceptible to parasitism. Essentially all hosts in the landscape are found, and one-third of them are parasitized, independent of host density. Here, we show that the wasps do not relocate hosts using odour markers previously left by themselves or other foragers, nor do they find the eggs anew repeatedly. Instead, the wasps relocate host eggs by learning the position of the eggs relative to visual landmarks. The anticipatory foraging behaviour presented here is a key to the wasp's exceptionally stable population dynamics.     

Variation of lignans in Norway spruce (Picea abies [L.] Karst.) knotwood: within-stem variation and the effect of fertilisation at two experimental sites in Finland

Lignan concentrations in Norway spruce (Picea abies [L.] Karst.) knotwood were studied in two long-term nitrogen fertilised experimental sites: a boreal site (66°5′N, 27°07′E) close to the Arctic tree line in northern Finland and a boreal site (61°10′N, 26°03′E) in southern Finland. Altogether 20 mature trees, representing five different size classes, were felled in the autumn 2002 and knots of the largest and smallest diameter in each whorl at the height of 4 m were analysed. Within-stem variation of knotwood lignans was studied at the 4 m intervals from stump height to the top from five control trees in the southern site. The following knotwood lignans were identified with GC-MS: hydroxymatairesinol (two isomers), secoisolariciresinol, α-conidendrin, α-conidendric acid, isohydroxymatairesinol, lariciresinol, lignan A, matairesinol, nortrachelogenin, todolactol A and isoliovil. The predominant lignan in Norway spruce knotwood was hydroxymatairesinol comprising approximately 77% of the total concentration of lignans. In mature trees in the southern site, the lignan concentrations were highest close to the starting point of the living branches. In the northern site, the total concentration of knotwood lignans was significantly higher (approximately 14% of the dry mass) than in the southern site (approximately 5.4% of the dry mass). In the northern site in control trees, a negative linear correlation was detected between branch diameter and hydroxymatairesinol concentration, but the similar correlation was not detected in fertilised trees or in the trees of the southern site. The possible reasons for higher hydroxymatairesinol concentrations in the northern knots were discussed.     

First insights into female sperm storage duration in tardigrades

Female sperm storage is ubiquitous in the animal kingdom and it has been shown to be linked to several evolutionary processes, from postcopulatory sexual selection to dispersal. Here we report, for the first time, long‐term sperm storage in females of the tardigrade Macrobiotus polonicus. Females, isolated after a short contact with a male, were able to use the stored sperm for up to 5 weeks (mean of 2 weeks), which translates to a considerable proportion of female post‐mating longevity under controlled laboratory conditions (60% on average). Our study provides the first insights into the duration of sperm storage, an underexplored feature of the reproductive biology of tardigrades. Additionally, we discuss important considerations for reproductive studies on these non‐model animals.