The brown planthopper Nilaparvata lugens is one of the most economically important pests of cultivated rice in Southeast Asia. Extensive use of insecticide treatments, such as imidacloprid, fipronil and ethiprole, has resulted in the emergence of multiple resistant strains of N. lugens. Previous investigation of the mechanisms of resistance to imidacloprid and ethiprole demonstrated that overexpression and qualitative changes in the cytochrome P450 gene CYP6ER1 lead to enhanced metabolic detoxification of these compounds. Here, we present the identification of a secondary mechanism enhancing ethiprole resistance mediated by differential splicing and overexpression of CYP419A1, a planthopper-specific, mitochondrial P450 gene. Although metabolic resistance to insecticides is usually mediated by overexpression of P450 genes belonging to either CYP 3 or 4 clades, we validate the protective effect of over-expression of CYP419A1, in vivo, using transgenic Drosophila melanogaster. Additionally, we report some unusual features of both the CYP419A1 gene locus and protein, which include, altered splicing associated with resistance, a non-canonical heme-binding motif and an extreme 5' end extension of the open reading frame. These results provide insight into the molecular mechanisms underpinning resistance to insecticides and have applied implications for the control of a highly damaging crop pest.

In some aphid species, intraspecific variation in body colour is caused by differential carotenoid content: whilst green aphids contain only yellow carotenoids (β-, γ-, and β,γ-carotenes), red aphids additionally possess red carotenoids (torulene and 3,4-didehydrolycopene). Unusually, within animals who typically obtain carotenoids from their diet, ancestral horizontal gene transfer of carotenoid biosynthetic genes from fungi (followed by gene duplication), have imbued aphids with the intrinsic gene repertoire necessary to biosynthesise carotenoids. In the pea aphid, Acyrthosiphon pisum a lycopene (phytoene) desaturase gene (Tor) underpins the red/green phenotype, with this locus present in heterozygous form in red individuals but absent in green aphids, resulting in them being unable to convert lycopene into the red compounds 3,4-didehydrolycopene and torulene. The green peach aphid, Myzus persicae, separated from the pea aphid for ≈45MY also exists as distinct colour variable morphs, with both red and green individuals present. Here, we examined genomic data for both red and green morphs of M. persicae and identified an enlarged (compared to A. pisum) repertoire of 16 carotenoid biosynthetic genes (11 carotenoid desaturases and five carotenoid cyclase/synthase genes). From these, we identify the homolog of A. pisum Tor (here called carotene desaturase 2 or CDE-2) and show through 3D modelling that this homolog can accommodate the torulene precursor lycopene and, through RNA knockdown feeding experiments, demonstrate that disabling CDE-2 expression in red M. persicae clones results in green-coloured offspring. Unlike in A. pisum, we show that functional CDE-2 is present in the genomes of both red and green aphids. However, expression differences between the two colour morphs (350-700 fold CDE-2 overexpression in red clones), potentially driven by variants identified in upstream putative regulatory elements, underpin this phenotype. Thus, whilst aphids have a common origin of their carotenoid biosynthetic pathway, two aphid species separated for over 40MY have evolved very different drivers of intraspecific colour variation.

The evolution of resistance to insecticides threatens the sustainable control of many of the world's most damaging insect crop pests and disease vectors. To effectively combat resistance, it is important to understand its underlying genetic architecture, including the type and number of genetic variants affecting resistance and their interactions with each other and the environment. While significant progress has been made in characterizing the individual genes or mutations leading to resistance, our understanding of how genetic variants interact to influence its phenotypic expression remains poor. Here, we uncover a mechanism of insecticide resistance resulting from transposon-mediated insertional mutagenesis of a genetically dominant but insecticide-susceptible allele that enables the adaptive potential of a previously unavailable recessive resistance allele to be unlocked. Specifically, we identify clones of the aphid pest Myzus persicae that carry a resistant allele of the essential voltage-gated sodium channel (VGSC) gene with the recessive M918T and L1014F resistance mutations, in combination with an allele lacking these mutations but carrying a Mutator-like element transposon insertion that disrupts the coding sequence of the VGSC. This results in the down-regulation of the dominant susceptible allele and monoallelic expression of the recessive resistant allele, rendering the clones resistant to the insecticide bifenthrin. These findings are a powerful example of how transposable elements can provide a source of evolutionary potential that can be revealed by environmental and genetic perturbation, with applied implications for the control of highly damaging insect pests.

The diamondback moth, Plutella xylostella, is a damaging pest of cruciferous crops, and has evolved resistance to many of the insecticides used for control, including members of the diamide class. Previous work on the molecular basis of resistance to diamides has documented mutations in the target-site, the ryanodine receptor, in resistant populations of P. xylostella worldwide. In contrast the role of metabolic resistance to this insecticide class is significantly less clear. Here we show that overexpression of a flavin-dependent monooxgenase (FMO) confers resistance to the diamide chlorantraniliprole in P. xylostella. Transcriptome profiling of diamide resistant strains, with and without target-site resistance, revealed constitutive over-expression of several transcripts encoding detoxification enzymes compared to susceptible strains. Two of these, CYP6BG1, and PxFMO2 were particularly highly overexpressed (33,000 and 14,700-fold, respectively) in a resistant strain (HAW) lacking target-site resistance. After 17 generations without diamide selection the resistance of the HAW strain fell by 52-fold and the expression of PxFMO2 by > 1300-fold, however, the expression of CYP6BG1 declined by only 3-fold. Generation of transgenic Drosophila melanogaster expressing these genes demonstrated that PxFMO2, but not CYP6BG1, confers resistance in vivo. Overexpression of PxFMO2 in the HAW strain is associated with mutations, including a putative transposable element insertion, in the promoter of this gene. These enhance the expression of a reporter gene when expressed in a lepidopteran cell line suggesting they are, at least in part, responsible for the overexpression of PxFMO2 in the resistant strain. Our results provide new evidence that insect FMOs can be recruited to provide resistance to synthetic insecticides.

Suggestions that novel, non-food, dedicated biomass crops used to produce bioenergy may provide opportunities to diversify and reinstate biodiversity in intensively managed farmland have not yet been fully tested at the landscape scale. Using two of the largest, currently available landscape-scale biodiversity data sets from arable and biomass bioenergy crops, we take a taxonomic and functional trait approach to quantify and contrast the consequences for biodiversity indicators of adopting dedicated biomass crops on land previously cultivated under annual, rotational arable cropping. The abundance and community compositions of biodiversity indicators in fields of break and cereal crops changed when planted with the dedicated biomass crops, miscanthus and short rotation coppiced (SRC) willow. Weed biomass was consistently greater in the two dedicated biomass crops than in cereals, and invertebrate abundance was similarly consistently higher than in break crops. Using canonical variates analysis, we identified distinct plant and invertebrate taxa and trait-based communities in miscanthus and SRC willows, whereas break and cereal crops tended to form a single, composite community. Seedbanks were shown to reflect the longer term effects of crop management. Our study suggests that miscanthus and SRC willows, and the management associated with perennial cropping, would support significant amounts of biodiversity when compared with annual arable crops. We recommend the strategic planting of these perennial, dedicated biomass crops in arable farmland to increase landscape heterogeneity and enhance ecosystem function, and simultaneously work towards striking a balance between energy and food security.

Rust diseases caused by Melampsora spp. represent a major threat to the productivity of short rotation coppice (SRC) willows grown for biomass, causing yield losses of up to 40%. The routine use of fungicide in SRC plantations is not a viable option because of economic and environmental considerations; thus, breeding for rust resistance is a major target for willow breeding programmes. To characterise the genetic basis of rust resistance in willow and provide targets for use in future marker-assisted selections, quantitative trait analyses were performed using a large full-sib mapping population (K8) which segregates for rust resistance and several other important agronomic traits. Rust resistance in field conditions was assessed in three consecutive years. For a more detailed genetic dissection, laboratory inoculation tests using isolates of two distinct and prevalent pathotypes (LET1 and LET5) were also performed. For field-based resistance, a major quantitative resistance locus, designated SRR1 (Salix Rust Resistance 1), was detected in addition to several quantitative trait loci (QTL) of more modest effect. Inoculation test data also supported an important role for SRR1. Specific interactions between particular rust isolates and different QTL were detected, and QTL that only influenced resistance in field conditions were identified. The QTL reported here represent an important basis for the future development of markers for use in willow breeding programmes. As the linkage map for the K8 population is anchored to the Populus trichocarpa genome sequence, a more efficient marker development for future fine-scale mapping and candidate gene identification is possible.

We compared birds in a group of established and well-managed miscanthus (Miscanthus x giganteus) fields in Somerset and East Devon, southwestern England, with plots of short rotation coppice (SRC) willow, arable crops and grassland in two winters and one summer. Following early spring cutting, 19 miscanthus fields grew taller, initially produced greater cover and were less weedy than SRC. As stubble in May, the miscanthus contained broadly similar species at similar densities to arable and grassland comparison plots. By July, at 2-m-tall, miscanthus held higher densities of birds but of fewer species, most of them characteristic of woodland and scrub. SRC, previously identified as being a beneficial crop for many birds, always contained more species and individuals than miscanthus. Throughout each of two winters, 15 miscanthus plots remained unharvested and contained more wood/scrub species such as Blackbirds Turdus merula, tits, Reed Buntings Emberiza schoeniclus and Woodcock Scolopax rusticola than the comparison plots, which held more corvids and Skylarks Alauda arvensis amongst others. Similar overall mean densities of birds in the miscanthus and the comparison plots masked relatively low density variance in miscanthus and very high variance in the comparison plots. Unharvested miscanthus crops grown in place of habitat types supporting flocks of wintering birds would displace these flocks. Miscanthus plantations with open patches attracted more finches and waders in winter. The two previous studies of birds in miscanthus in the UK found more species and more individuals than we did in summer and winter. Both these studies documented high levels of weediness and patchy crop growth. In the context of this previous work our data suggest that bird use of miscanthus in summer and winter is likely to be variable, affected by region, weediness, crop structure and patchiness. While large-scale cropping of SRC in England is likely to have a positive overall impact on a suite of common farmland and woodland birds, our data suggest that miscanthus in the southwest of England may have an approximately neutral effect. However, some open farmland specialist species may be lost when planting either crop.

Concern about climate change and energy security is stimulating land-use change, which in turn precipitates social, economic and environmental responses. It is predicted that within 20 years in the UK, bioenergy crops could occupy significant areas of rural land. Among these, dedicated biomass crops, such as Miscanthus (Miscanthus spp.) grass and short rotation willow (Salix spp.) coppice, differ significantly from arable crops in their growth characteristics and management. It is important that the potential impacts of these differences are assessed before large-scale, long-term planting occurs.We used a Sustainability Appraisal Framework (SAF) approach to landscape planning in the UK to identify stakeholder aspirations (objectives) and associated criteria (indicators) for the planting of dedicated biomass crops.The use of environmental and physical constraints mapping allowed the SAF to focus only on environmentally-acceptable locations, thereby avoiding unsustainable trade-offs. The mapping identified 3.1 million ha of land in England as suitable for planting, suggesting the UK government target of 1.1 million ha by 2020 is feasible.Evaluation of the SAF identified that while biodiversity was of concern to stakeholders, some current indicators of biodiversity are not appropriate. Butterfly abundance proved the most appropriate indicator, and it was found that total abundance was greater in field margins of both willow and Miscanthus biomass crops than in arable field margins.Synthesis and applications. The potential conflicts of assuring food security, water availability, energy security and biodiversity conservation are recognized as a key challenge by governments worldwide. Methods with which decision-makers can compare the performance of different land-use scenarios against sustainability objectives will be crucial for achieving optimized and sustainable use of land-based resources to meet all four challenges. Using biomass crops planting as an example, this work illustrates the potential of a Sustainability Appraisal Framework, subject to identification and agreement of appropriate indicators, in securing a holistic understanding of the wide-ranging implications of large-scale, long-term changes to rural land-use in the wider context of sustainable land-use planning per se.

Angela Karp, Alison J. Haughton, David A. Bohan, Andrew A. Lovett, Alan J. Bond , Trudie Dockerty, Gilla Sünnenberg, Jon W. Finch, Rufus B. Sage, Katy J. Appleton, Andrew B. Riche, Mark D. Mallott, Victoria E. Mallott, Mark D. Cunningham, Suzanne J. Clark and Martin M. Turner Centre for Bioenergy and Climate Change and 1b Centre for Mathematical and Computational Biology, Rothamsted Research, West Common Harpenden, Hertfordshire, AL5 2JQ, UK; School of Environmental Sciences, University of East Anglia, Norwich, NR4 7TJ, UK; Centre for Ecology and Hydrology, Maclean Building, Benson Lane, Crowmarsh Gifford, Wallingford, Oxfordshire, OX10 8BB, UK; Game and Wildlife Conservation Trust, Fordingbridge, Hampshire, SP5 1EF, UK; Department of Geography, University of Exeter, Laver Building, New North Road, Exeter, Devon, EX4 4QE, UK

Poplars (genus Populus) and willows (genus Salix) are members of the Salicaceae, a family of catkin-bearing trees, shrubs and sub-shrubs. Poplar is considered the model system for biological studies in trees and considerable genetic and genomic resources have become available in recent years. The transfer of information to research studies in willow, for which fewer resources are currently available, would be highly beneficial. However, the extent of conservation between poplar and willow genomes has not yet been extensively studied. To address this, we have constructed a linkage map of willow based on a large mapping population derived from a cross between two Salix viminalis × (S. viminalis × S. schwerinii) hybrid sibs, and aligned this to the publicly available poplar genome sequence. A set of genome-wide, expressed poplar sequences was selected and used to design primer sets that efficiently amplified homeologous regions in willow. Direct sequencing of the willow products confirmed homology with the poplar target in the majority of instances and allowed identification of single nucleotide polymorphisms (SNPs) that were used to map these loci. In total, 202 amplified fragment length polymorphisms (AFLPs), 75 microsatellites and 79 SNPs were used to construct a willow consensus map that spanned 1,856.7 cM with an average interval between markers of 6.3 cM. Poplar sequences homologous to those of the mapped willow microsatellite loci were identified and used in addition to the SNP markers to putatively align all but two minor linkage groups to the poplar genome sequence. A high degree of macrosynteny was revealed.