Some gene families associated with plant detoxifcation and insecticide resistance have rapidly expanded , whereas others have contracted . Finally, gene families associated with immune defense exhibit expansions that may be linked to defense against pathogens and parasitoids that commonly attack exposed herbivores. A substantial proportion of the rapidly evolving gene families include proteins with transposable element domains , while other important functional groups include DNA and protein binding , nuclease activity, protein processing, and cellular transport. Diversification of transcription factor families potentially signals greater complexity of gene regulation, including enhanced cell specificity and refined spatiotemporal signalin. Notably, several TF families melanogaster , but also from species as distant as human and mouse . Motifs were inferred for a substantial proportion of the TFs in the largest TF families, including Homeodomain , bHLH , and Forkhead box . We could only infer a small number of C2H2 zinc finger motifs ,growing blueberries in containers which is expected as these sequences evolve quickly by shufing zinc finger arrays, resulting in largely dissimilar DNA-binding domains across metazoans.
Collectively, the almost 200 inferred DNA binding motifs for L. decemlineata TFs provide a unique resource to begin unraveling gene regulatory networks in this organism. To identify genes active in mid-gut tissues, life-stages, and sex diferences, we examined differential transcript expression levels using RNA sequencing data. Comparison of significantly differentially expressed genes with >100-fold change, after Bonferroni correction, indicated higher expression of digestive enzymes in mid-gut versus whole larval tissues, while cuticular proteins were largely expressed at lower levels . Comparison of an adult male and female showed higher expression of testes and sperm related genes in males, while genes involved in egg production and sterol biosynthesis are more highly expressed in females . Comparisons of larvae to both an adult male and an adult female showed higher expression of larval-specific cuticle proteins, and lower expression of odorant binding and chemosensory proteins. The adults, both drawn from a pesticide resistant population, showed higher constitutive expression of cytochrome p450 genes compared to the larval population, which is consistent with the results from previous studies of neonicotinoid resistance in this population.Transposable elements are ubiquitous mobile elements within most eukaryotic genomes and play critical roles in both genome architecture and the generation of genetic variatio.
Trough insertional mutagenesis and recombination, TEs are a major contributor to the generation of novel mutations , and are increasingly thought to generate much of the genetic diversity that contributes to rapid evolution. In addition to finding that genes with TE domains comprise 25% of the rapidly evolving gene families within L. decemlineata, we found that at least 17% of the genome consists of TEs . This is substantially greater than the 6% found in T. castaneum, but less than some Lepidoptera . LINEs were the largest TE class, comprising ~10% of the genome, while SINEs were not detected. Curation of the TE models with intact protein domains resulted in 334 current models of potentially active TEs, meaning that these TEs are capable of transposition and excision. Within the group of active TEs, we found 191 LINEs, 99 DNA elements, 38 LTRs, and 5 Helitrons. Given that TEs have been associated with the ability of species to rapidly adapt to novel selection pressures, particularly via alterations of gene expression patterns in neighboring genomic regions, we scanned gene rich regions for active TE elements. Genes with active neighboring TEs have functions that include transport, protein digestion, diapause, and metabolic detoxifcation . Because TE elements have been implicated in conferring insecticide resistance in other insects, future work should investigate the role of these TE insertions on rapid evolutionary changes within pest populations of L. decemlineata.To understand the propensity for L. decemlineata pest populations to rapidly evolve across a range of environmental conditions, we examined geographical patterns of genomic variability and the evolutionary history of L. decemlineata .
We identified 1.34 million biallelic single nucleotide polymorphisms from pooled RNAseq datasets, or roughly 1 variable site for every 22 base pairs of coding DNA. This rate of polymorphism is exceptionally high when compared to vertebrates , and is 8-fold higher than other beetles and 2 to 5-fold higher than some dipterans. It is likely that these values simply scale with efective population size, although the dipterans, with the largest known population sizes, have reduced variation due to widespread selective sweeps and genetic bottlenecks.Evolutionary relationships and the amount of genetic drif among Midwestern USA, Northeastern USA, and European L. decemlineata populations were estimated based on genome-wide allele frequency differences using a population graph. A substantial amount of local genetic structure and high genetic drif is evident among all populations, although both the reference lab strain from New Jersey and European populations appear to have undergone more substantial drif, suggestive of strong inbreeding . Population genetic divergence values range from 0.035 to 0.182 . Te allele frequency spectrum was calculated for populations in Wisconsin, Michigan, and Europe to estimate the population genetic parameter θ, or the product of the mutation rate and the ancestral effective population size, and the ratio of contemporary to ancestral population size in models that allowed for single or multiple episodes of population size change. Estimates of ancestral θ are much higher for Wisconsin and Michigan than Europe , providing support for a single introduction into Europe following a large genetic bottleneck. In all three populations, a model of population size growth is supported, in agreement with historical accounts of the beetles expanding from the Great Plains into the Midwestern U.S. and Europe, but the dynamics of each population appear independent, with the population from Michigan apparently undergoing a very recent decline in contemporary population size .To interact with their environment, insects have evolved neurosensory organs to detect environmental signals, including tactile, auditory, chemical and visual cues.
We examined neural receptors, olfactory genes, and light sensory genes to understand the sensory ecology and host-plant specializations of L. decemlineata. We found high sequence similarity in the neuroreceptors of L. decemlineata compared to other Coleoptera. The transient receptor potential channels are permeable transmembrane proteins that respond to temperature, touch, pain, osmolarity, pheromones, taste, hearing, smell and visual cues of insects. In most insect genomes, there are typically 13–14 TRP genes located in insect stretch receptor cells and several are targeted by commercial insecticides. We found 12 TRP genes present in the L. decemlineata genome, including the two TRPs that are targeted by commercial insecticides, representing a complete set of one-to-one orthologs with T. castaneum. Similarly, the 20 known amine neurotransmitter receptors in T. castaneum are present as one-to-one orthologs in L. decemlineata. Amine receptors are G-protein-coupled receptors that interact with biogenic amines, such as octopamine, dopamine and serotonin. These neuroactive substances regulate behavioral and physiological traits in animals by acting as neurotransmitters,square pots neuromodulators and neurohormones in the central and peripheral nervous systems. The majority of phytophagous insects are restricted to feeding on several plant species within a genus, or at least restricted to a particular plant family. Thus, to fnd their host plants within heterogeneous landscapes, insect herbivores detect volatile organic compounds through olfaction, which utilizes several families of chemosensory gene families, such as the odorant binding proteins , odorant receptors , gustatory receptors , and ionotropic receptors . OBPs directly bind with volatile organic compounds emitted from host plants and transport the ligands across the sensillar lymph to activate the membrane-bound ORs in the dendrites of the olfactory sensory neurons. The ORs and GRs are 7-transmembrane proteins related within a superfamily of ligand-gated ion channels. The ionotropic receptors are related to ionotropic glutamate receptors and function in both smell and taste. These four gene families are commonly large in insect genomes, consisting of tens to hundreds of members. We compared the number of genes found in L. decemlineata in the four chemosensory gene families to T. castaneum and A. glabripennis . While the OBP family is slightly enlarged, the three receptor families are considerably smaller in L. decemlineata than in either A. glabripennis or T. castaneum, consistent with the specialization of this beetle on one genus of plants. However, each beetle species exhibits species-specifc gene subfamily expansions ; in particular, some lineages of GRs related to bitter taste are expanded in L. decemlineata relative to A. glabripennis and other beetles. Among the OBPs, we identified a major L. decemlineata-specifc expansion of proteins belonging to the Minus-C class that appear unrelated to the ‘traditional’ Minus-C subfamily in Coleoptera, indicating that coleopteran OBPs have lost cysteines on at least two occasions.
To understand the visual acuity of L. decemlineata, we examined the G-protein-coupled transmembrane receptor opsin gene family. We found five opsins, three of which are members of rhabdomeric opsin subfamilies expressed in the retina of insects. Specifically, the L. decemlineata genome contains one member of the long wavelength-sensitive R-opsin and two short wavelength UV-sensitive R-opsins. Te latter were found to be closely linked in a range of less than 20,000 bp, suggestive of recent tandem gene duplication. Overall, the recovered repertoire of retinally-expressed opsins in L. decemlineata is consistent with the beetle’s attraction to yellow light and to the yellow flowers of its ancestral host plant, S. rostratum, and is consistent with the beetle’s sensitivity in the UV- and LW-range. In addition, we found a member of the Rh7 R-opsin subfamily, which is broadly conserved in insects including other beetle species , although it is missing from T. castaneum. Finally, L. decemlineata has a single ortholog of the c-opsin subfamily shared with T. castaneum, which is absent in A. glabripennis and has an unclear role in photoreception.Insect herbivores are fundamentally limited by nitrogen availability, and thus need to efficiently break down plant proteins in order to survive and develop on host plants. Leptinotarsa decemlineata has serine and cysteine digestive peptidases , as well as aspartic and metallo peptidases, for protein digestion. For the vast majority of plant-eating beetles , cysteine peptidases contribute most strongly to proteolytic activity in the gut. In response to herbivory, plants produce a wide range of proteinase inhibitors to prevent insect herbivores from digesting plant proteins. Coleopteran peptidases are differentially susceptible to plant peptidase inhibitors, and our annotation results suggest that gene duplication and selection for inhibitor insensitive genotypes may have contributed to the success of leaf-feeding beetles on different plants. We found that gene expansion of cysteine cathepsins from the C1 family in L. decemlineata correlates with the acquisition of greater digestive function by this group of peptidases, which is supported by gene expression activity of these genes in mid-gut tissue . The gene expansion may be explained by an evolutionary arms race between insects and plants that favors insects with a variety of digestive peptidases in order to resist plant peptidase inhibitors and allows for functional specialization. Cysteine peptidases of the C1 family were represented by more than 50 genes separated into four groups with different structure and functional characteristics : cathepsin L subfamily, cathepsin B subfamily, TINAL-like genes, and cysteine peptidase inhibitor domains . Cathepsin L subfamily cysteine peptidases are endopeptidases that can be distinguished by the cathepsin propeptide inhibitor domain I29. Within the cathepsin L subfamily, we found sequences that were similar to classical cathepsin L, cathepsin F, and cathepsin O. However, there were 28 additional predicted peptidases of this subfamily that could not be assigned to any of the “classical” cathepsin types, and most of these were grouped into two gene expansions according to their phylogenetic and structural characteristics. Cathepsin B subfamily cysteine peptidases are distinguished by the specifc peptidase family C1 propeptide domain . Within the cathepsin B subfamily, there was one gene corresponding to typical cathepsin B peptidases and 14 cathepsin B-like genes. According to the structure of the occluding loop, only the typical cathepsin B may have typical endo- and exopeptidase activities, while a large proportion of cathepsin B-like peptidases presumably possesses only endopeptidase activity due to the absence of a His-His active subsite in the occluding loop, which is responsible for exopeptidase activity. Only one gene corresponding to a TINAL-like-protein was present, which has a domain similar to cathepsin B in the C-terminus, but lacks peptidase activity due to the replacement of the active site Cys residue with Ser. Cysteine peptidase inhibitor domain genes encode the I29 domain of cysteine peptidases without the mature peptidase domain.