Engineering novel traits in plants through RNA interference

RNA interference (RNAi) is a homology-dependent gene silencing technology that involves double-stranded RNA directed against a target gene or its promoter region. Using hairpin constructs, double-stranded RNA can be expressed in plants relatively easily, enabling this technology to be applied to a wide range of species to silence the expression of both specific endogenous genes and genes of invading pathogens. RNAi has also been used to engineer metabolic pathways to overproduce secondary products with health, yield or environmental benefits. The application of tissue-specific or inducible gene silencing, with the use of appropriate promoters, and the ability to silence several genes simultaneously should enhance our ability to create novel traits in plants.     

Moss Responses to Elevated CO2 and Variation in Hydrology in a Temperate Lowland Peatland

We studied the effects of elevated CO₂ (180–200 ppmv above ambient) on growth and chemistry of three moss species (Sphagnum palustre, S. recurvum and Polytrichum commune) in a lowland peatland in the Netherlands. Thereto, we conducted both a greenhouse experiment with both Sphagnum species and a field experiment with all three species using MiniFACE (Free Air CO₂ Enrichment) technology during 3 years. The greenhouse experiment showed that Sphagnum growth was stimulated by elevated CO₂ in the short term, but that in the longer term (≥1 year) growth was probably inhibited by low water tables and/or down-regulation of photosynthesis. In the field experiment, we did not find significant changes in moss abundance in response to elevated CO₂, although CO₂ enrichment appeared to reduce S. recurvum abundance. Both Sphagnum species showed stronger responses to spatial variation in hydrology than to increased atmospheric CO₂ concentrations. Polytrichum was insensitive to changes in hydrology. Apart from the confounding effects of hydrology, the relative lack of growth response of the moss species may also have been due to the relatively small increase in assimilated CO₂ as achieved by the experimentally added CO₂. We calculated that the added CO₂ contributed at most 32% to the carbon assimilation of the mosses, while our estimates based on stable C isotope data even suggest lower contributions for Sphagnum (24–27%). Chemical analyses of the mosses showed only small elevated CO₂ effects on living tissue N concentration and C/N ratio of the mosses, but the C/N ratio of Polytrichum was substantially lower than those of the Sphagnum species. Continuing expansion of Polytrichum at the expense of Sphagnum could reduce the C sink function of this lowland Sphagnum peatland, and similar ones elsewhere, as litter decomposition rates would probably be enhanced. Such a reduction in sink function would be driven mostly by increased atmospheric N deposition, water table regulation for agricultural purposes and land management to preserve the early successional stage (mowing, tree and shrub removal), since these anthropogenic factors will probably exert a greater control on competition between Polytrichum and Sphagnum than increased atmospheric CO₂ concentrations.     

Evolutionary Connectionism: Algorithmic Principles Underlying the Evolution of Biological Organisation in Evo-Devo,Evo-Eco and Evolutionary Transitions

The mechanisms of variation, selection and inheritance, on which evolution by natural selection depends, are not fixed over evolutionary time. Current evolutionary biology is increasingly focussed on understanding how the evolution of developmental organisations modifies the distribution of phenotypic variation, the evolution of ecological relationships modifies the selective environment, and the evolution of reproductive relationships modifies the heritability of the evolutionary unit. The major transitions in evolution, in particular, involve radical changes in developmental, ecological and reproductive organisations that instantiate variation, selection and inheritance at a higher level of biological organisation. However, current evolutionary theory is poorly equipped to describe how these organisations change over evolutionary time and especially how that results in adaptive complexes at successive scales of organisation (the key problem is that evolution is self-referential, i.e. the products of evolution change the parameters of the evolutionary process). Here we first reinterpret the central open questions in these domains from a perspective that emphasises the common underlying themes. We then synthesise the findings from a developing body of work that is building a new theoretical approach to these questions by converting well-understood theory and results from models of cognitive learning. Specifically, connectionist models of memory and learning demonstrate how simple incremental mechanisms, adjusting the relationships between individually-simple components, can produce organisations that exhibit complex system-level behaviours and improve the adaptive capabilities of the system. We use the term “evolutionary connectionism” to recognise that, by functionally equivalent processes, natural selection acting on the relationships within and between evolutionary entities can result in organisations that produce complex system-level behaviours in evolutionary systems and modify the adaptive capabilities of natural selection over time. We review the evidence supporting the functional equivalences between the domains of learning and of evolution, and discuss the potential for this to resolve conceptual problems in our understanding of the evolution of developmental, ecological and reproductive organisations and, in particular, the major evolutionary transitions.     

Soundscape diversity in the Great Barrier Reef: Lizard Island,a case study

Passive acoustic monitoring can provide valuable information on coral reefs, and examining the acoustic attributes of these ecosystems has the potential to provide an insight into their status and condition. From 2014 to 2016, a series of underwater recordings were taken at field sites around Lizard Island in the Great Barrier Reef, Australia. Six individual fish choruses were identified where each chorus displayed distinct acoustic characteristics. Choruses exhibited diurnal activity and some field sites displayed consistently higher diversity of choruses and levels than others, suggesting that particular locations are important aggregation areas for soniferous fish species. During peak activity, choruses were a prominent component of reef soundscapes, where received levels of a chorus reached upwards of 120 dB re 1μPa rms over the 450–650 Hz band, equating to a 40 dB increase above ambient noise levels of ≈80 dB re 1μPa rms. Three out of the six detected choruses exhibited spectral and temporal characteristics similar to choruses previously documented at these sites and elsewhere, produced by planktivorous fish species. Three of these choruses appear to be undocumented and could hold information on the presence, abundance and dispersal patterns of important fish species, which may have potential long-term management applications. Future research should focus on extricating the temporal patterns associated with bioacoustic activity and determining the potential environmental drivers of biological choruses. Additionally, developing appropriate techniques for direct identification of vocalizing species would strongly increase the management applicability of passive acoustic monitoring.     

Use of an ungulate‐specific feed structure as a potential tool for controlling feral goats in Australian forest ecosystems

The feral Goat (Capra hircus) has successfully exploited a range of landscapes around the world with occurrences of overabundance resulting in significant damage to ecological values. In forested ecosystems in Australia, there are currently limited means to control the species when compared to the range of management techniques available for other pest animals. To redress this deficiency, we designed a feed structure combined with commercially available salt blocks to attract goats to set locations in a forested study area. Structures that exploited differences in the pedal morphology (foot size and shape) of native herbivores (kangaroos and wallabies) and ungulates (feral goats and deer) were found to be highly target‐specific, with feral goats freely able to access salt blocks, whilst nontarget native species were effectively excluded. Other introduced ungulate species, Fallow Deer (Dama dama) and Red Deer (Cervus elaphus), successfully accessed salt blocks in feed structures but at a considerably lower rate than feral goats. The capacity to present a range of bait types within a target‐specific feed structure, once matched with a humane toxicant, could provide land managers with an additional cost‐effective lethal control tool for future management of feral ungulates, particularly goats.     

A Thermostable Salmonella Phage Endolysin,Lys68, with Broad Bactericidal Properties against Gram-Negative Pathogens in Presence of Weak Acids

Resistance rates are increasing among several problematic Gram-negative pathogens, a fact that has encouraged the development of new antimicrobial agents. This paper characterizes a Salmonella phage endolysin (Lys68) and demonstrates its potential antimicrobial effectiveness when combined with organic acids towards Gram-negative pathogens. Biochemical characterization reveals that Lys68 is more active at pH 7.0, maintaining 76.7% of its activity when stored at 4°C for two months. Thermostability tests showed that Lys68 is only completely inactivated upon exposure to 100°C for 30 min, and circular dichroism analysis demonstrated the ability to refold into its original conformation upon thermal denaturation. It was shown that Lys68 is able to lyse a wide panel of Gram-negative bacteria (13 different species) in combination with the outer membrane permeabilizers EDTA, citric and malic acid. While the EDTA/Lys68 combination only inactivated Pseudomonas strains, the use of citric or malic acid broadened Lys68 antibacterial effect to other Gram-negative pathogens (lytic activity against 9 and 11 species, respectively). Particularly against Salmonella Typhimurium LT2, the combinatory effect of malic or citric acid with Lys68 led to approximately 3 to 5 log reductions in bacterial load/CFUs after 2 hours, respectively, and was also able to reduce stationary-phase cells and bacterial biofilms by approximately 1 log. The broad killing capacity of malic/citric acid-Lys68 is explained by the destabilization and major disruptions of the cell outer membrane integrity due to the acidity caused by the organic acids and a relatively high muralytic activity of Lys68 at low pH. Lys68 demonstrates good (thermo)stability properties that combined with different outer membrane permeabilizers, could become useful to combat Gram-negative pathogens in agricultural, food and medical industry.     

Selective enrichment and sequencing of whole mitochondrial genomes in the presence of nuclear encoded mitochondrial pseudogenes (numts)

Numts are an integral component of many eukaryote genomes offering a snapshot of the evolutionary process that led from the incorporation of an α-proteobacterium into a larger eukaryotic cell some 1.8 billion years ago. Although numt sequence can be harnessed as molecular marker, these sequences often remain unidentified and are mistaken for genuine mtDNA leading to erroneous interpretation of mtDNA data sets. It is therefore indispensable that during the process of amplifying and sequencing mitochondrial genes, preventive measures are taken to ensure the exclusion of numts to guarantee the recovery of genuine mtDNA. This applies to mtDNA analyses in general but especially to studies where mtDNAs are sequenced de novo as the launch pad for subsequent mtDNA-based research. By using a combination of dilution series and nested rolling circle amplification (RCA), we present a novel strategy to selectively amplify mtDNA and exclude the amplification of numt sequence. We have successfully applied this strategy to de novo sequence the mtDNA of the Black Field Cricket Teleogryllus commodus, a species known to contain numts. Aligning our assembled sequence to the reference genome of Teleogryllus emma (GenBank EU557269.1) led to the identification of a numt sequence in the reference sequence. This unexpected result further highlights the need of a reliable and accessible strategy to eliminate this source of error.