Genetics of flight in spongy moths (Lymantria dispar ssp.): functionally integrated profiling of a complex invasive trait.

BACKGROUND: Flight can drastically enhance dispersal capacity and is a key trait defining the potential of exotic insect species to spread and invade new habitats. The phytophagous European spongy moths (ESM, Lymantria dispar dispar) and Asian spongy moths (ASM; a multi-species group represented here by L. d. asiatica and L. d. japonica), are globally invasive species that vary in adult female flight capability-female ASM are typically flight capable, whereas female ESM are typically flightless. Genetic markers of flight capability would supply a powerful tool for flight profiling of these species at any intercepted life stage. To assess the functional complexity of spongy moth flight and to identify potential markers of flight capability, we used multiple genetic approaches aimed at capturing complementary signals of putative flight-relevant genetic divergence between ESM and ASM: reduced representation genome-wide association studies, whole genome sequence comparisons, and developmental transcriptomics. We then judged the candidacy of flight-associated genes through functional analyses aimed at addressing the proximate demands of flight and salient features of the ecological context of spongy moth flight evolution. RESULTS: Candidate gene sets were typically non-overlapping across different genetic approaches, with only nine gene annotations shared between any pair of approaches. We detected an array of flight-relevant functional themes across gene sets that collectively suggest divergence in flight capability between European and Asian spongy moth lineages has coincided with evolutionary differentiation in multiple aspects of flight development, execution, and surrounding life history. Overall, our results indicate that spongy moth flight evolution has shaped or been influenced by a large and functionally broad network of traits. CONCLUSIONS: Our study identified a suite of flight-associated genes in spongy moths suited to exploration of the genetic architecture and evolution of flight, or validation for flight profiling purposes. This work illustrates how complementary genetic approaches combined with phenotypically targeted functional analyses can help to characterize genetically complex traits.

Potential Benefit of Add-on Δ9-Tetrahydrocannabinol in Pediatric Drug-Resistant Epilepsy: A Case Series.

We present five cases of pediatric drug-resistant epilepsy (DRE) that failed management using high cannabidiol (CBD) doses, but had significant reduction in seizure frequency with reintroduction or increasing doses of tetrahydrocannabinol (THC). There is growing evidence supporting the use of whole-plant CBD-rich extracts (containing THC and other cannabinoids) in the treatment of pediatric DRE. Based on our experiences and reports in the literature, we propose that, in patients who fail management with an initial trial of high-dose CBD-focused therapy, there may be a role for add-on THC-focused formulations.

Host behaviour alteration by its parasite: from brain gene expression to functional test.

Many parasites with complex life cycles modify their intermediate hosts' behaviour, presumably to increase transmission to their final host. The threespine stickleback (Gasterosteus aculeatus) is an intermediate host in the cestode Schistocephalus solidus life cycle, which ends in an avian host, and shows increased risky behaviours when infected. We studied brain gene expression profiles of sticklebacks infected with S. solidus to determine the proximal causes of these behavioural alterations. We show that infected fish have altered expression levels in genes involved in the inositol pathway. We thus tested the functional implication of this pathway and successfully rescued normal behaviours in infected sticklebacks using lithium exposure. We also show that exposed but uninfected fish have a distinct gene expression profile from both infected fish and control individuals, allowing us to separate gene activity related to parasite exposure from consequences of a successful infection. Finally, we find that selective serotonin reuptake inhibitor-treated sticklebacks and infected fish do not have similarly altered gene expression, despite their comparable behaviours, suggesting that the serotonin pathway is probably not the main driver of phenotypic changes in infected sticklebacks. Taken together, our results allow us to predict that if S. solidus directly manipulates its host, it could target the inositol pathway.

Transcriptomic signatures of social experience during early development in a highly social cichlid fish.

The social environment encountered early during development can temporarily or permanently influence life history decisions and behaviour of individuals and correspondingly shape molecular pathways. In the highly social cichlid fish Neolamprologus pulcher, deprivation of brood care permanently affects social behaviour and alters the expression of stress axis genes in juveniles and adults. It is unclear when gene expression patterns change during early life depending on social experience, and which genes are involved. We compared brain gene expression of N. pulcher at two time points during the social experience phase when juveniles were reared either with or without brood care, and one time point shortly afterwards. We compared (a) whole transcriptomes and (b) expression of 79 genes related to stress regulation, in order to define a neurogenomic state of stress for each fish. At developmental day 75, that is, after the social experience phase, 43 genes were down-regulated in fish having experienced social deprivation, while two genes involved in learning and memory and in post-translational modifications of proteins (PTM), respectively, were up-regulated. Down-regulated genes were mainly associated with immunity, PTM and brain function. In contrast, during the experience phase no genes were differentially expressed when assessing the whole transcriptome. When focusing on the neurogenomic state associated with the stress response, we found that individuals from the two social treatments differed in how their brain gene expression profiles changed over developmental stages. Our results indicate that the early social environment influences the transcriptional activation in fish brains, both during and after an early social experience, possibly affecting plasticity, immune system function and stress axis regulation.

Divergent brain gene expression profiles between alternative behavioural helper types in a cooperative breeder.

Juveniles of the cooperatively breeding cichlid fish Neolamprologus pulcher either consistently provide help in form of alloparental egg care ("cleaners") or consistently abstain from helping ("noncleaners"). These phenotypes are not based on heritable genetic differences. Instead, they arise during ontogeny, which should lead to differences in brain structure or physiology, a currently untested prediction. We compared brain gene expression profiles of cleaners and noncleaners in two experimental conditions, a helping opportunity and a control condition. We aimed to identify (a) expression differences between cleaners and noncleaners in the control, (b) changes in gene expression induced by the opportunity and (c) differences in plasticity of gene expression between cleaners and noncleaners. Control cleaners and noncleaners differed in the expression of a single gene, irx2, which regulates neural differentiation. During the opportunity, cleaners and noncleaners had three upregulated genes in common, which were implicated in neuroplasticity, hormonal signalling and cell proliferation. Thus, the stimulus in the opportunity was sufficiently salient. Cleaners also showed higher expression of seven additional genes that were unique to the opportunity. One of these cleaner-specific genes is implicated in neuropeptide metabolism, indicating that this process is associated with cleaning performance. This suggests that the two types employed different pathways to integrate social information, preparing them for accelerated reaction to future opportunities. Interestingly, three developmental genes were downregulated between the control and the opportunity in cleaners only. Our results indicate that the two behavioural types responded differently to the helping opportunity and that only cleaners responded by downregulating developmental genes.

Major host transitions are modulated through transcriptome-wide reprogramming events in Schistocephalus solidus, a threespine stickleback parasite.

Parasites with complex life cycles have developed numerous phenotypic strategies, closely associated with developmental events, to enable the exploitation of different ecological niches and facilitate transmission between hosts. How these environmental shifts are regulated from a metabolic and physiological standpoint, however, still remain to be fully elucidated. We examined the transcriptomic response of Schistocephalus solidus, a trophically transmitted parasite with a complex life cycle, over the course of its development in an intermediate host, the threespine stickleback, and the final avian host. Results from our differential gene expression analysis show major reprogramming events among developmental stages. The final host stage is characterized by a strong activation of reproductive pathways and redox homoeostasis. The attainment of infectivity in the fish intermediate host-which precedes sexual maturation in the final host and is associated with host behaviour changes-is marked by transcription of genes involved in neural pathways and sensory perception. Our results suggest that un-annotated and S. solidus-specific genes could play a determinant role in host-parasite molecular interactions required to complete the parasite's life cycle. Our results permit future comparative analyses to help disentangle species-specific patterns of infection from conserved mechanisms, ultimately leading to a better understanding of the molecular control and evolution of complex life cycles.

Can the behaviour of threespine stickleback parasitized with Schistocephalus solidus be replicated by manipulating host physiology?

Sticklebacks infected by the parasitic flatworm Schistocephalus solidus show dramatic changes in phenotype, including a loss of species-typical behavioural responses to predators. The timing of host behaviour change coincides with the development of infectivity of the parasite to the final host (a piscivorous bird), making it an ideal model for studying the mechanisms of infection-induced behavioural modification. However, whether the loss of host anti-predator behaviour results from direct manipulation by the parasite, or is a by-product (e.g. host immune response) or side effect of infection (e.g. energetic loss), remains controversial. To understand the physiological mechanisms that generate these behavioural changes, we quantified the behavioural profiles of experimentally infected fish and attempted to replicate these in non-parasitized fish by exposing them to treatments including immunity activation and fasting, or by pharmacologically inhibiting the stress axis. All fish were screened for the following behaviours: activity, water depth preference, sociability, phototaxis, anti-predator response and latency to feed. We were able to change individual behaviours with certain treatments. Our results suggest that the impact of S. solidus on the stickleback might be of a multifactorial nature. The behaviour changes observed in infected fish might result from the combined effects of modifying the serotonergic axis, lack of energy and activation of the immune system.

Ecological genomics of host behavior manipulation by parasites.

Among the vast array of niche exploitation strategies exhibited by millions of different species on Earth, parasitic lifestyles are characterized by extremely successful evolutionary outcomes. Some parasites even seem to have the ability to 'control' their host's behavior to fulfill their own vital needs. Research efforts in the past decades have focused on surveying the phylogenetic diversity and ecological nature of these host-parasite interactions, and trying to understand their evolutionary significance. However, to understand the proximal and ultimate causes of these behavioral alterations triggered by parasitic infections, the underlying molecular mechanisms governing them must be uncovered. Studies using ecological genomics approaches have identified key candidate molecules involved in host-parasite molecular cross-talk, but also molecules not expected to alter behavior. These studies have shown the importance of following up with functional analyses, using a comparative approach and including a time-series analysis. High-throughput methods surveying different levels of biological information, such as the transcriptome and the epigenome, suggest that specific biologically-relevant processes are affected by infection, that sex-specific effects at the level of behavior are recapitulated at the level of transcription, and that epigenetic control represents a key factor in managing life cycle stages of the parasite through temporal regulation of gene expression. Post-translational processes, such as protein-protein interactions (interactome) and post translational modifications (e.g. protein phosphorylation, phosphorylome), and processes modifying gene expression and translation, such as interactions with microRNAs (microRNAome), are examples of promising avenues to explore to obtain crucial insights into the proximal and ultimate causes of these fascinating and complex inter-specific interactions.

Absence of Evidence for Sustained Effects of Daily Cannabidiol Administration on Anandamide Plasma Concentration in Individuals with Cocaine Use Disorder: Exploratory Findings from a Randomized Controlled Trial.

Background: Cannabidiol (CBD) has been proposed to have a therapeutic potential over a wide range of neuropsychiatric disorders, including substance use disorders. Pre-clinical evidence suggests that CBD can increase anandamide (AEA) plasma concentration, possibly mediating some of its therapeutic properties. Whether CBD exerts such an effect on AEA in individuals with cocaine use disorder (CUD) remains unknown. Aims: To explore the sustained effects of daily CBD administration on AEA plasma concentrations compared with placebo in CUD. Methods: We used data from a randomized, double-blind, placebo-controlled trial evaluating CBD's efficacy in CUD. Seventy-eight individuals were randomized to receive a daily oral dose of 800 mg CBD (n = 40) or a placebo (n = 38). Participants stayed in an inpatient detoxification setting for 10 days, after which they were followed in an outpatient setting for 12 weeks. AEA plasma concentration was measured at baseline and at 23-h post CBD ingestion on day 8 and week 4. A generalized estimating equation model was used to assess CBD's effects on AEA, and sensitivity analyses were computed using Bayesian linear regressions. Results: Sixty-four participants were included in the analysis. Similar mean AEA plasma concentrations in both treatment groups (p = 0.357) were observed. At day 8, mean AEA plasma concentrations (± standard deviation) were 0.26 (± 0.07) ng/mL in the CBD group and 0.29 (± 0.08) ng/mL in the placebo group (p = 0.832; Bayes factor [BF] = 0.190). At week 4, they were 0.27 (± 0.09) ng/mL in the CBD group and 0.30 (± 0.09) ng/mL in the placebo group (p = 0.181; BF = 0.194). Conclusion: While not excluding any potential acute and short-term effect, daily CBD administration did not exert a sustained impact on AEA plasma concentrations in individuals with CUD compared with placebo. Registration: clinicaltrials.gov (NCT02559167).

Targeted sequence capture and resequencing implies a predominant role of regulatory regions in the divergence of a sympatric lake whitefish species pair (Coregonus clupeaformis).

Latest technological developments in evolutionary biology bring new challenges in documenting the intricate genetic architecture of species in the process of divergence. Sympatric populations of lake whitefish represent one of the key systems to investigate this issue. Despite the value of random genotype-by-sequencing methods and decreasing cost of sequencing technologies, it remains challenging to investigate variation in coding regions, especially in the case of recently duplicated genomes as in salmonids, as this greatly complicates whole genome resequencing. We thus designed a sequence capture array targeting 2773 annotated genes to document the nature and the extent of genomic divergence between sympatric dwarf and normal whitefish. Among the 2728 genes successfully captured, a total of 2182 coding and 10,415 noncoding putative single-nucleotide polymorphisms (SNPs) were identified after applying a first set of basic filters. A genome scan with a quality-refined selection of 2203 SNPs identified 267 outlier SNPs in 210 candidate genes located in genomic regions potentially involved in whitefish divergence and reproductive isolation. We found highly heterogeneous FST estimates among SNP loci. There was an overall low level of coding polymorphism, with a predominance of noncoding mutations among outliers. The heterogeneous patterns of divergence among loci confirm the porous nature of genomes during speciation with gene flow. Considering that few protein-coding mutations were identified as highly divergent, our results, along with previous transcriptomic studies, imply that changes in regulatory regions most likely had a greater role in the process of whitefish population divergence than protein-coding mutations. This study is the first to demonstrate the efficiency of large-scale targeted resequencing for a nonmodel species with such a large and unsequenced genome.

Nonparallelism in MHCIIß diversity accompanies nonparallelism in pathogen infection of lake whitefish (Coregonus clupeaformis) species pairs as revealed by next-generation sequencing.

Major histocompatibility (MHC) immune system genes may evolve in response to pathogens in the environment. Because they also may affect mate choice, they are candidates for having great importance in ecological speciation. Here, we use next-generation sequencing to test the general hypothesis of parallelism in patterns of MHCIIß diversity and bacterial infections among five dwarf and normal whitefish sympatric pairs. A second objective was to assess the functional relationships between specific MHCIIß alleles and pathogens in natural conditions. Each individual had between one and four alleles, indicating two paralogous loci. In Cliff Lake, the dwarf ecotype was monomorphic for the most common allele. In Webster Lake, the skew in the allelic distribution was towards the same allele but in the normal ecotype, underscoring the nonparallel divergence among lakes. Our signal of balancing selection matched putative peptide binding region residues in some cases, but not in others, supporting other recent findings of substantial functional differences in fish MHCIIß compared with mammals. Individuals with fewer alleles were less likely to be infected; thus, we found no evidence for the heterozygote advantage hypothesis. MHCIIß alleles and pathogenic bacteria formed distinct clusters in multivariate analyses, and clusters of certain alleles were associated with clusters of pathogens, or sometimes the absence of pathogens, indicating functional relationships at the individual level. Given that patterns of MHCIIß and bacteria were nonparallel among dwarf and normal whitefish pairs, we conclude that pathogens driving MHCIIß evolution did not play a direct role in their parallel phenotypic evolution.

Differential effect of cannabis use on opioid agonist treatment outcomes: Exploratory analyses from the OPTIMA study.

INTRODUCTION: Conflictual evidence exists regarding the effects of cannabis use on the outcomes of opioid agonist therapy (OAT). In this exploratory analysis, we examined the effect of recent cannabis use on opioid use, craving, and withdrawal symptoms, in individuals participating in a trial comparing flexible buprenorphine/naloxone (BUP/NX) take-home dosing model to witnessed ingestion of methadone. METHODS: We analyzed data from a multi-centric, pragmatic, 24-week, open label, randomized controlled trial in individuals with prescription-type opioid use disorder (n = 272), randomly assigned to BUP/NX (n = 138) or methadone (n = 134). The study measured last week cannabis and opioid use via timeline-follow back, recorded at baseline and every two weeks during the study. Craving symptoms were measured using the Brief Substance Craving Scale at baseline, and weeks 2, 6, 10, 14, 18 and 22. The study measured opioid withdrawal symptoms via Clinical Opiate Withdrawal Scale at treatment initiation and weeks 2, 4, and 6. RESULTS: The mean maximum dose taken during the study was 17.3 mg/day (range = 0.5-32 mg/day) for BUP/NX group and 67.7 mg/day (range = 10-170 mg/day) in the methadone group. Repeated measures generalized linear mixed models demonstrated that cannabis use in the last week (mean of 2.3 days) was not significantly associated with last week opioid use (aß ± standard error (SE) = -0.06 ± 0.04; p = 0.15), craving (aß ± SE = -0.05 ± 0.08, p = 0.49), or withdrawal symptoms (aß ± SE = 0.09 ± 0.1, p = 0.36). Bayes factor (BF) for each of the tested models supported the null hypothesis (BF < 0.3). CONCLUSIONS: The current study did not demonstrate a statistically significant effect of cannabis use on outcomes of interest in the context of a pragmatic randomized-controlled trial. These findings replicated previous results reporting no effect of cannabis use on opioid-related outcomes.

Genome-Wide Characterization of the MLO Gene Family in Cannabis sativa Reveals Two Genes as Strong Candidates for Powdery Mildew Susceptibility.

Cannabis sativa is increasingly being grown around the world for medicinal, industrial, and recreational purposes. As in all cultivated plants, cannabis is exposed to a wide range of pathogens, including powdery mildew (PM). This fungal disease stresses cannabis plants and reduces flower bud quality, resulting in significant economic losses for licensed producers. The Mildew Locus O (MLO) gene family encodes plant-specific proteins distributed among conserved clades, of which clades IV and V are known to be involved in susceptibility to PM in monocots and dicots, respectively. In several studies, the inactivation of those genes resulted in durable resistance to the disease. In this study, we identified and characterized the MLO gene family members in five different cannabis genomes. Fifteen Cannabis sativa MLO (CsMLO) genes were manually curated in cannabis, with numbers varying between 14, 17, 19, 18, and 18 for CBDRx, Jamaican Lion female, Jamaican Lion male, Purple Kush, and Finola, respectively (when considering paralogs and incomplete genes). Further analysis of the CsMLO genes and their deduced protein sequences revealed that many characteristics of the gene family, such as the presence of seven transmembrane domains, the MLO functional domain, and particular amino acid positions, were present and well conserved. Phylogenetic analysis of the MLO protein sequences from all five cannabis genomes and other plant species indicated seven distinct clades (I through VII), as reported in other crops. Expression analysis revealed that the CsMLOs from clade V, CsMLO1 and CsMLO4, were significantly upregulated following Golovinomyces ambrosiae infection, providing preliminary evidence that they could be involved in PM susceptibility. Finally, the examination of variation within CsMLO1 and CsMLO4 in 32 cannabis cultivars revealed several amino acid changes, which could affect their function. Altogether, cannabis MLO genes were identified and characterized, among which candidates potentially involved in PM susceptibility were noted. The results of this study will lay the foundation for further investigations, such as the functional characterization of clade V MLOs as well as the potential impact of the amino acid changes reported. Those will be useful for breeding purposes in order to develop resistant cultivars.

Sequencing, assembly and annotation of the whole-insect genome of Lymantria dispar dispar, the European gypsy moth.

The European gypsy moth, Lymantria dispar dispar (LDD), is an invasive insect and a threat to urban trees, forests and forest-related industries in North America. For use as a comparator with a previously published genome based on the LD652 pupal ovary-derived cell line, as well as whole-insect genome sequences obtained from the Asian gypsy moth subspecies L. dispar asiatica and L. dispar japonica, the whole-insect LDD genome was sequenced, assembled and annotated. The resulting assembly was 998 Mb in size, with a contig N50 of 662 Kb and a GC content of 38.8%. Long interspersed nuclear elements constitute 25.4% of the whole-insect genome, and a total of 11,901 genes predicted by automated gene finding encoded proteins exhibiting homology with reference sequences in the NCBI NR and/or UniProtKB databases at the most stringent similarity cutoff level (i.e., the gold tier). These results will be especially useful in developing a better understanding of the biology and population genetics of L. dispar and the genetic features underlying Lepidoptera in general.

Expansion of LINEs and species-specific DNA repeats drives genome expansion in Asian Gypsy Moths.

Two subspecies of Asian gypsy moth (AGM), Lymantria dispar asiatica and L. dispar japonica, pose a serious alien invasive threat to North American forests. Despite decades of research on the ecology and biology of this pest, limited AGM-specific genomic resources are currently available. Here, we report on the genome sequences and functional content of these AGM subspecies. The genomes of L.d. asiatica and L.d. japonica are the largest lepidopteran genomes sequenced to date, totaling 921 and 999 megabases, respectively. Large genome size in these subspecies is driven by the accumulation of specific classes of repeats. Genome-wide metabolic pathway reconstructions suggest strong genomic signatures of energy-related pathways in both subspecies, dominated by metabolic functions related to thermogenesis. The genome sequences reported here will provide tools for probing the molecular mechanisms underlying phenotypic traits that are thought to enhance AGM invasiveness.

Direct In Vivo Microbial Transcriptomics During Infection.

The challenge in infectious diseases is monitoring infection in the host. Omics-based genomics and transcriptomics can define microbial genes expressed during infection and treatment with antimicrobials. Recent studies pinpoint a direct in situ in vivo approach revolutionizing infection monitoring and optimizing antimicrobial therapy using machine learning.

Transcriptome sequences spanning key developmental states as a resource for the study of the cestode Schistocephalus solidus, a threespine stickleback parasite.

BACKGROUND: Schistocephalus solidus is a well-established model organism for studying the complex life cycle of cestodes and the mechanisms underlying host-parasite interactions. However, very few large-scale genetic resources for this species are available. We have sequenced and de novo-assembled the transcriptome of S. solidus using tissues from whole worms at three key developmental states - non-infective plerocercoid, infective plerocercoid and adult plerocercoid - to provide a resource for studying the evolution of complex life cycles and, more specifically, how parasites modulate their interactions with their hosts during development. FINDINGS: The de novo transcriptome assembly reconstructed the coding sequence of 10,285 high-confidence unigenes from which 24,765 non-redundant transcripts were derived. 7,920 (77 %) of these unigenes were annotated with a protein name and 7,323 (71 %) were assigned at least one Gene Ontology term. Our raw transcriptome assembly (unfiltered transcripts) covers 92 % of the predicted transcriptome derived from the S. solidus draft genome assembly currently available on WormBase. It also provides new ecological information and orthology relationships to further annotate the current WormBase transcriptome and genome. CONCLUSION: This large-scale transcriptomic dataset provides a foundation for studies on how parasitic species with complex life cycles modulate their response to changes in biotic and abiotic conditions experienced inside their various hosts, which is a fundamental objective of parasitology. Furthermore, this resource will help in the validation of the S solidus gene features that have been predicted based on genomic sequence.

Identification of candidate mimicry proteins involved in parasite-driven phenotypic changes.

BACKGROUND: Endoparasites with complex life cycles are faced with several biological challenges, as they need to occupy various ecological niches throughout their development. Host phenotypes that increase the parasite's transmission rate to the next host have been extensively described, but few mechanistic explanations have been proposed to describe their proximate causes. In this study we explore the possibility that host phenotypic changes are triggered by the production of mimicry proteins from the parasite by using an ecological model system consisting of the infection of the threespine stickleback (Gasterosteus aculeatus) by the cestode Schistocephalus solidus. METHOD: Using RNA-seq data, we assembled 9,093 protein-coding genes from which ORFs were predicted to generate a reference proteome. Based on a previously published method, we built two complementary analysis pipelines to i) establish a general classification of protein similarity among various species (pipeline A) and ii) identify candidate mimicry proteins showing specific host-parasite similarities (pipeline B), a key feature underlying the possibility of molecular mimicry. RESULTS: Ninety-four tapeworm proteins showed high local sequence homology with stickleback proteins. Four of these candidates correspond to secreted or membrane proteins that could be produced by the parasite and eventually be released in or be in contact with the host to modulate physiological pathways involved in various phenotypes (e.g. behaviors). One of these candidates belongs to the Wnt family, a large group of signaling molecules involved in cell-to-cell interactions and various developmental pathways. The three other candidates are involved in ion transport and post-translational protein modifications. We further confirmed that these four candidates are expressed in three different developmental stages of the cestode by RT-PCR, including the stages found in the host. CONCLUSION: In this study, we identified mimicry candidate peptides from a behavior-altering cestode showing specific sequence similarity with host proteins. Despite their potential role in modulating host pathways that could lead to parasite-induced phenotypic changes and despite our confirmation that they are expressed in the developmental stage corresponding to the altered host behavior, further investigations will be needed to confirm their mechanistic role in the molecular cross-talk taking place between S. solidus and the threespine stickleback.