A total of 1,237 putative transcription factors were identified in the L. decemlineata predicted proteome

The heart of new agricultural paradigms for a hotter and more populous world must be systems that close the loop of nutrient flows from microorganisms and plants to animals and back, powered and irrigated as much as possible by sunlight and seawater. This has the potential to decrease the land, energy, and freshwater demands of agriculture, while at the same time ameliorating the pollution currently associated with agricultural chemicals and animal waste. The design and large scale implementation of farms based on nontraditional species in arid places will undoubtedly pose new research, engineering, monitoring, and regulatory challenges, with respect to food safety and ecological impacts as well as control of pests and pathogens. But if we are to resume progress toward eliminating hunger, we must scale up and further build on the innovative approaches already under development, and we must do so immediately.The Colorado potato beetle, Leptinotarsa decemlineata Say 1824 , is widely considered one of the world’s most successful globally-invasive insect herbivores, with costs of ongoing management reaching tens of millions of dollars annually and projected costs if unmanaged reaching billions of dollars. This beetle was first identified as a pest in 1859 in the Midwestern United States, after it expanded from its native host plant, Solanum rostratum , onto potato . As testimony to the difficulty in controlling L. decemlineata, the species has the dubious honor of starting the pesticide industry, when Paris Green acetoarsenite was first applied to control it in the United States in 1864 . Leptinotarsa decemlineata is now widely recognized for its ability to rapidly evolve resistance to insecticides, as well as a wide range of abiotic and biotic stresses ,plastic gutter and for its global expansion across 16 million km2 to cover the entire Northern Hemisphere within the 20th century .

Over the course of 150 years of research, L. decemlineata has been the subject in more than 9,700 publications ranging from molecular to organismal biology from the fields of agriculture, entomology, molecular biology, ecology, and evolution. In order to be successful, L. decemlineata evolved to exploit novel host plants, to inhabit colder climates at higher latitudes , and to cope with a wide range of novel environmental conditions in agricultural landscapes. Genetic data suggest the potato-feeding pest lineage directly descended from populations that feed on S. rostratum in the U.S. Great Plains. This beetle subsequently expanded its range northwards, shifing its life history strategies to exploit even colder climates, and steadily colonized potato crops despite substantial geographical barriers. Leptinotarsa decemlineata is an excellent model for understanding pest evolution in agroecosystems because, despite its global spread, individuals disperse over short distances and populations often exhibit strong genetic diferentiation, providing an opportunity to track the spread of populations and the emergence of novel phenotypes. The development of genomic resources in L. decemlineata will provide an unparalleled opportunity to investigate the molecular basis of traits such as climate adaptation, herbivory, host expansion, and chemical detoxifcation. Perhaps most significantly, understanding its ability to evolve rapidly would be a major step towards developing sustainable methods to control this widely successful pest in agricultural settings. Given that climate is thought to be the major factor in structuring the range limits of species, the latitudinal expansion of L. decemlineata, spanning more than 40° latitude from Mexico to northern potato-producing countries such as Canada and Russia, warrants further investigation.

Harsh winter climates are thought to present a major barrier for insect range expansions, especially near the limits of a species’ range. To successfully overwinter in temperate climates, beetles need to build up body mass, develop greater amounts of lipid storage, have a low resting metabolism, and respond to photoperiodic keys by initiating diapause. Although the beetle has been in Europe for less than 100 years, local populations have demonstrating remarkably rapid evolution in life history traits linked to growth, diapause and metabolism. Understanding the genetic basis of these traits, particularly the role of specific genes associated with metabolism, fatty acid synthesis, and diapause induction, could provide important information about the mechanism of climate adaptation. Although Leptinotarsa decemlineata has long-served as a model for the study of host expansion and herbivory due to its rapid ability to host switch, a major outstanding question is what genes and biological pathways are associated with herbivory in this species? While >35,000 species of Chrysomelidae are well-known herbivores, most species feed on one or a few host species within the same plant family. Within Leptinotarsa, the majority of species feed on plants within Solanaceae and Asteraceae, while L. decemlineata feeds exclusively on solanaceous species. It has achieved the broadest host range amongst its congeners , potato , eggplant , silverleaf nightshade , horsenettle , bittersweet nightshade , tomato , and tobacco, and exhibits geographical variation in the use of locally abundant Solanum species. Another major question is what are the genes that underlie the beetle’s remarkable capacity to detoxify plant secondary compounds and are these the same biological pathways used to detoxify insecticidal compounds? Solanaceous plants are considered highly toxic to a wide range of insect herbivore species, because they contain steroidal alkaloids and glycoalkaloids, nitrogen-containing compounds that are toxic to a wide range of organisms, including bacteria, fungi, humans, and insects, as well as glandular trichomes that contain additional toxic compounds.

In response to beetle feeding, potato plants upregulate pathways associated with terpenoid, alkaloid, and phenylpropanoid biosynthesis, as well as a range of protease inhibitors. A complex of digestive cysteine proteases is known to underlie L. decemlineata’s ability to respond to potato-induced defenses. There is evidence that larvae excrete and perhaps even sequester toxic plant-based compounds in the hemolymph. Physiological mechanisms involved in detoxifying plant compounds, as well as other xenobiotics, have been proposed to underlie pesticide resistance. To date, while cornerstone of L. decemlineata management has been the use of insecticides, the beetle has evolved resistance to over 50 compounds and all of the major classes of insecticides. Some of these chemicals have even failed to control L. decemlineata within the first year of release, and notably, regional populations of L. decemlineata have demonstrated the ability to independently evolve resistance to pesticides and to do so at different rates. Previous studies have identified target site mutations in resistance phenotypes and a wide range of genes involved in metabolic detoxifcation, including carboxylesterase genes, cytochrome P450s,blueberry container and glutathione S-transferase genes. To examine evidence of rapid evolutionary change underlying L. decemlineata’s extraordinary success utilizing novel host plants, climates, and detoxifying insecticides, we evaluated structural and functional genomic changes relative to other beetle species, using whole-genome sequencing, transcriptome sequencing, and a large community-driven bio-curation effort to improve predicted gene annotations. We compared the size of gene families associated with particular traits against existing available genomes from related species, particularly those sequenced by the i5k project , an initiative to sequence 5,000 species of Arthropods. While efforts have been made to understand the genetic basis of phenotypes in L. decemlineata , previous work has been limited to candidate gene approaches rather than comparative genomics. Genomic data can not only illuminate the genetic architecture of a number of phenotypic traits that enable L. decemlineata to continue to be an agricultural pest, but can also be used to identify new gene targets for control measures. For example, recent efforts have been made to develop RNAi-based pesticides targeting critical metabolic pathways in L. decemlineata. With the extensive wealth of biological knowledge and a newly-released genome, this beetle is well-positioned to be a model system for agricultural pest genomics and the study of rapid evolution.A single female L. decemlineata from Long Island, NY, USA, a population known to be resistant to a wide range of insecticides, was sequenced to a depth of ~140x coverage and assembled with ALLPATHS followed by assembly improvement with ATLAS . The average coleopteran genome size is 760 Mb , while most of the beetle genome assemblies have been smaller . The draf genome assembly of L. decemlineata is 1.17 Gb and consists of 24,393 scafolds, with a N50 of 414 kb and a contig N50 of 4.9 kb. This assembly is more than twice the estimated genome size of 460 Mb, with the presence of gaps comprising 492 Mb, or 42%, of the assembly.

As this size might be driven by underlying heterozygosity, we also performed scafolding with REDUNDANS, which reduced the assembly size to 642 Mb, with gaps reduced to 1.3% of the assembly. However, the REDUNDANS assembly increased the contig N50 to 47.4 kb, the number of scafolds increased to 90,205 and the N50 declined to 139 kb. By counting unique 19 bp kmers and adjusting for ploidy, we estimate the genome size as 816.9 Mb. Using just the small-insert 100 bp PE reads, average coverage was 24X for the ALLPATHS assembly and 26X for the REDUNDANS assembly. For all downstream analyses, the ALLPATHS assembly was used due to its increased scafold length and reduced number of scafolds. The number of genes in the L. decemlineata genome predicted based on automated annotation using MAKER was 24,671 gene transcripts, with 93,782 predicted exons, which surpasses the 13,526–22,253 gene models reported in other beetle genome projects. This may be in part due to fragmentation of the genome, which is known to infate gene number estimates. To improve our gene models, we manually annotated genes using expert opinion and additional mRNA resources . A total of 1,364 genes were manually curated and merged with the unedited MAKER annotations to produce an official gene set of 24,850 transcripts, comprised of 94,859 exons. A total of 12 models were curated as pseudogenes.The predicted number of TFs is similar to some beetles, such as Anoplophora glabripennisand Hypothenemus hampei , but substantially greater than others, such as Tribolium castaneum , Nicrophorus vespilloides , and Dendroctonus ponderosae. We assessed the completeness of both the ALLPATHS and REDUNDANS assemblies, and the OGS separately, using bench marking sets of universal single-copy orthologs based on 35 holometabolous insect genomes, as well as manually assessing the completeness and co-localization of the homeodomain transcription factor gene clusters in the ALLPATHS assembly. Using the reference set of 2,442 BUSCOs, the ALLPATHS genome assembly, REDUNDANS genome assembly, and OGS were 93.0%, 91.9%, and 71.8% complete, respectively. We found an additional 4.1%, 5.4%, and 17.9% of the BUSCOs present but fragmented in each dataset, respectively. For the highly conserved Hox and Iroquois Complex clusters, we located and annotatedcomplete gene models in the ALLPATHS genome assembly for all 12 expected orthologs, but these were split across six different scafolds . All linked Hox genes occurred in the expected order and with the expected, shared transcriptional orientation, suggesting that the current draf assembly was correct but incomplete . Assuming direct concatenation of scafolds, the Hox cluster would span a region of 3.7 Mb, similar to the estimated 3.5 Mb Hox cluster of A. glabripennis. While otherwise highly conserved with A. glabripennis, we found a tandem duplication for Hox3/zen and an Antennapedia-class homeobox gene with no clear ortholog in other arthropods. We also assessed the ALLPATHS genome assembly for evidence of contamination using a Blobplot , which identified a small proportion of the reads as putative contaminants .We estimated a phylogeny among six coleopteran genomes using a conserved set of single copy orthologs and compared the official gene set of each species to understand how gene families evolved the branch representing Chysomelidae. Leptinotarsa decemlineata and A. glabripennisare sister taxa , as expected for members of the same superfamily Chrysomeloidea. We found 166 rapidly evolving gene families along the L. decemlineata lineage , 142 of which are rapid expansions and the remaining 24 rapid contractions . Among all branches of our coleopteran phylogeny, L. decemlineata has the highest average expansion rate , the highest number of genes gained, and the greatest number of rapidly evolving gene families. Examination of the functional classification of rapidly evolving families in L. decemlineata indicates that a subset of families are clearly associated with herbivory. Te peptidases, comprising several gene families that play a major role in plant digestion, displayed a significant expansion in genes . While olfactory receptor gene families have rapidly contracted , subfamilies of odorant binding proteins and gustatory receptors have grown . The expansion of gustatory receptor subfamilies are associated with bitter receptors, likely reflecting host plant detection of nightshades .