CRISPR-based editing of the ω- and γ-gliadin gene clusters reduces wheat immunoreactivity without affecting grain protein quality.

Wheat immunotoxicity is associated with abnormal reaction to gluten-derived peptides. Attempts to reduce immunotoxicity using breeding and biotechnology often affect dough quality. Here, the multiplexed CRISPR-Cas9 editing of cultivar Fielder was used to modify gluten-encoding genes, specifically focusing on ω- and γ-gliadin gene copies, which were identified to be abundant in immunoreactive peptides based on the analysis of wheat genomes assembled using the long-read sequencing technologies. The whole-genome sequencing of an edited line showed mutation or deletion of nearly all ω-gliadin and half of the γ-gliadin gene copies and confirmed the lack of editing in the α/ß-gliadin genes. The estimated 75% and 64% reduction in ω- and γ-gliadin content, respectively, had no negative impact on the end-use quality characteristics of grain protein and dough. A 47-fold immunoreactivity reduction compared to a non-edited line was demonstrated using antibodies against immunotoxic peptides. Our results indicate that the targeted CRISPR-based modification of the ω- and γ-gliadin gene copies determined to be abundant in immunoreactive peptides by analysing high-quality genome assemblies is an effective mean for reducing immunotoxicity of wheat cultivars while minimizing the impact of editing on protein quality.

Improved Apis mellifera reference genome based on the alternative long-read-based assemblies.

Apis mellifera L., the western honey bee is a major crop pollinator that plays a key role in beekeeping and serves as an important model organism in social behavior studies. Recent efforts have improved on the quality of the honey bee reference genome and developed a chromosome-level assembly of 16 chromosomes, two of which are gapless. However, the rest suffer from 51 gaps, 160 unplaced/unlocalized scaffolds, and the lack of 2 distal telomeres. The gaps are located at the hard-to-assemble extended highly repetitive chromosomal regions that may contain functional genomic elements. Here, we use de novo re-assemblies from the most recent reference genome Amel_HAv_3.1 raw reads and other long-read-based assemblies (INRA_AMelMel_1.0, ASM1384120v1, and ASM1384124v1) of the honey bee genome to resolve 13 gaps, five unplaced/unlocalized scaffolds and, the lacking telomeres of the Amel_HAv_3.1. The total length of the resolved gaps is 848,747 bp. The accuracy of the corrected assembly was validated by mapping PacBio reads and performing gene annotation assessment. Comparative analysis suggests that the PacBio-reads-based assemblies of the honey bee genomes failed in the same highly repetitive extended regions of the chromosomes, especially on chromosome 10. To fully resolve these extended repetitive regions, further work using ultra-long Nanopore sequencing would be needed. Our updated assembly facilitates more accurate reference-guided scaffolding and marker/sequence mapping in honey bee genomics studies.

New record of Agrisius Walker (Lepidoptera, Erebidae, Arctiinae) from Cambodia, with description of a new species.

The genus Agrisius Walker (1855) is reported for the first time in Cambodia, for the new species Agrisius nigripunctata sp. n. Additionally, Agrisius fuliginosus is reported for the first time in Laos, Myanmar, China, Thailand and Vietnam. Illustrations of the adult and genitalia of Agrisius nigripunctata sp. n. and A. fuliginosus Moore are presented.

Comparison of Biochemical Constituents and Contents in Floral Nectar of Castanea spp.

Pollination is essential for efficient reproduction in pollinator-dependent crops that rely on the attraction of pollinators to flowers. Especially, floral nectar is considered to be an important factor attracting pollinator like honey bees, but differences among major chestnut species (Castanea crenata, C. mollissima, C. dentata, and C. sativa) are still little explored. This study aims to evaluate the value of honey source by analyzing floral nectar characteristics and comparing the composition of volatile organic compounds (VOCs) that mediate plant-pollinator interaction. In this study, we analyzed nectar samples obtained from male flowers using HPLC and HS-SPME/GC-MS. The five chestnuts showed significant differences between the volume of secreted nectar, free sugar composition, amino acid content and VOCs composition. Furthermore, C. crenata (Japanese cultivar 'Ungi') was revealed to emit the highest total amounts of VOCs and high levels of benzenoid compounds that are generally associated with flower-visiting insects. The sugar content per catkin, which is used to determine the honey yield, was the highest in C. crenata, suggesting that C. crenata 'Ungi' can be highly valued as a honey tree. Therefore, a better understanding of the relationship between pollinator and nectar characteristics of C. crenara could contribute to a prospective honey plant.

Estimation of C-derived introgression into A. m. mellifera colonies in the Russian Urals using microsatellite genotyping.

BACKGROUND: Marker-assisted selection is well established in animal breeding method of selecting individuals with desirable traits in a breeding scheme based on DNA molecular marker patterns. OBJECTIVE: Genetic diversity and C-derived admixture into local purebred gene pool of A. m. mellifera colonies was assessed using polymorphism of nine microsatellite loci in order to provide further marker-assisted selection of desired honey bee colonies. METHODS: The genetic diversity and the level of C-derived introgression into A. m. mellifera colonies in the Shulgan-Tash Nature Reserve (Russia) was assessed based on nine microsatellite loci (ap243, 4a110, A24, A8, A43, A113, A88, Ap049, A28), which were analized using the fragment analysis of the PCR products in Applied Biosystems 3130 DNA Analyzer. Phylogenetic relationship of colonies was evaluated using Neighbor-Joining methods with Cavalli-Sforza and Edwards genetic distance using the PHYLIP 3.68. The model-based Bayesian clustering algorithm implemented in STRUCTURE 2.3.3 was employed to infer membership and introgression proportions (Q-value). RESULTS: In the Shulgan-Tash Nature Reserve colonies of A. m. mellifera subdivided into four groups by level of C-derived introgression. Only five colonies of A. m. mellifera had C-derived introgression which varied from 0.5 to 2%. The genetic diversity in colonies of A. m. mellifera varied from 0.12 to 0.40. The Neighbor-Joining tree demonstrates the genetic relationship of A. m. mellifera colonies, which subdivided into three groups with different levels of C-derived introgression. Group 1 combined five honey bee colonies Bort_1, Bort_2, Bort_3, Baisalyan_1, and Kush_7 with a fraction of introgression close to 0.000 and genetic diversity from 0.20 to 0.25. CONCLUSION: The results showed the excellence of nine microsatellite loci genotyping in estimation of genetic diversity, distinguishing the two European evolutionary lineages M and C and estimating C-derived introgression. These genetic parameters can be applied further to perform the marker-assisted selection of purebred dark European honey bees.

Recent effective population size in Eastern European plain Russians correlates with the key historical events.

Effective population size reflects the history of population growth, contraction, and structuring. When the effect of structuring is negligible, the inferred trajectory of the effective population size can be informative about the key events in the history of a population. We used the IBDNe and DoRIS approaches, which exploit the data on IBD sharing between genomes, to reconstruct the recent effective population size in two population datasets of Russians from Eastern European plain: (1) ethnic Russians sampled from the westernmost part of Russia; (2) ethnic Russians, Bashkirs, and Tatars sampled from the Volga-Ural region. In this way, we examined changes in effective population size among ethnic Russians that reside in their historical area at the West of the plain, and that expanded eastward to come into contact with the indigenous peoples at the East of the plain. We compared the inferred demographic trajectories of each ethnic group to written historical data related to demographic events such as migration, war, colonization, famine, establishment, and collapse of empires. According to IBDNe estimations, 200 generations (~6000 years) ago, the effective size of the ancestral populations of Russians, Bashkirs, and Tatars hovered around 3,000, 30,000, and 8,000 respectively. Then, the ethnic Russians exponentially grew with increasing rates for the last 115 generations and become the largest ethnic group of the plain. Russians do not show any drop in effective population size after the key historical conflicts, including the Mongol invasion. The only exception is a moderate drop in the 17th century, which is well known in Russian history as The Smuta. Our analyses suggest a more eventful recent population history for the two small ethnic groups that came into contact with ethnic Russians in the Volga-Ural region. We found that the effective population size of Bashkirs and Tatars started to decrease during the time of the Mongol invasion. Interestingly, there is an even stronger drop in the effective population size that coincides with the expansion of Russians to the East. Thus, 15-20 generations ago, i.e. in the 16-18th centuries in the trajectories of Bashkirs and Tatars, we observe the bottlenecks of four and twenty thousand, respectively. Our results on the recent effective population size correlate with the key events in the history of populations of the Eastern European plain and have importance for designing biomedical studies in the region.

Abdominal contact of fluvalinate induces olfactory deficit in Apis mellifera.

τ-Fluvalinate (fluvalinate) is a highly selective pyrethroid insecticide compound used for controlling ectoparasitic mites that cause major damages in honey bee colonies. Although honey bees have resistance and low toxicity to this xenobiotic chemical, little is known about the effects of this chemical on sensory modulation and behaviors in honey bees. Here we addressed the effect on olfactory cognition at the behavioral, molecular, and neurophysiological levels. First, we found that topical application of fluvalinate to honeybee abdomen elicited somewhat severe toxicity to honey bees. Furthermore, honeybees treated with sublethal doses of fluvalinate showed a significant decrease in olfactory responses. At the molecular level, there was no change in gene expression levels of odorant receptor co-receptor (Orco), which is important for electrical conductivity induced by odorant binding in insects. Rather, small neuropeptide F (sNPF) signaling pathway was involved in olfactory fluctuation after treatment of fluvalinate. This indicates that olfactory deficits by abdominal contact of fluvalinate may stem from various internal molecular pathways in honey bees.

Author Correction: Characterization and its implication of a novel taste receptor detecting nutrients in the honey bee, Apis mellifera.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Characterization and its implication of a novel taste receptor detecting nutrients in the honey bee, Apis mellifera.

Umami taste perception indicates the presence of amino acids, which are essential nutrients. Although the physiology of umami perception has been described in mammals, how insects detect amino acids remains unknown except in Drosophila melanogaster. We functionally characterized a gustatory receptor responding to L-amino acids in the western honey bee, Apis mellifera. Using a calcium-imaging assay and two-voltage clamp recording, we found that one of the honey bee's gustatory receptors, AmGr10, functions as a broadly tuned amino acid receptor responding to glutamate, aspartate, asparagine, arginine, lysine, and glutamine, but not to other sweet or bitter compounds. Furthermore, the sensitivity of AmGr10 to these L-amino acids was dramatically enhanced by purine ribonucleotides, like inosine-5'-monophosphate (IMP). Contact sensory hairs in the mouthpart of the honey bee responded strongly to glutamate and aspartate, which house gustatory receptor neurons expressing AmGr10. Interestingly, AmGr10 protein is highly conserved among hymenopterans but not other insects, implying unique functions in eusocial insects.

Reconstructing recent population history while mapping rare variants using haplotypes.

Haplotype-based methods are a cost-effective alternative to characterize unobserved rare variants and map disease-associated alleles. Moreover, they can be used to reconstruct recent population history, which shaped distribution of rare variants and thus can be used to guide gene mapping studies. In this study, we analysed Illumina 650 k genotyped dataset on three underrepresented populations from Eastern Europe, where ancestors of Russians came into contact with two indigenous ethnic groups, Bashkirs and Tatars. Using the IBD mapping approach, we identified two rare IBD haplotypes strongly enriched in asthma patients of distinct ethnic background. We reconstructed recent population history using haplotype-based methods to reconcile this contradictory finding. Our ChromoPainter analysis showed that these haplotypes each descend from a single ancestor coming from one of the ethnic groups studied. Next, we used DoRIS approach and showed that source populations for patients exchanged recent (<60 generations) asymmetric gene flow, which supported the ChromoPainter-based scenario that patients share haplotypes through inter-ethnic admixture. Finally, we show that these IBD haplotypes overlap with asthma-associated genomic regions ascertained in European population. This finding is consistent with the fact that the two donor populations for the rare IBD haplotypes: Russians and Tatars have European ancestry.

Treatment efficacy of drug-resistant tuberculosis in Bashkortostan, Russia: A retrospective cohort study.

BACKGROUND: Russia, together with other former Soviet Union countries, is characterized by one of the highest burdens of drug-resistant tuberculosis. Published data on the drug-resistant tuberculosis for these countries are limited, and it is not clear whether current treatment regimens remain effective against constantly evolving drug-resistant strains. OBJECTIVES: The aim of the study was to evaluate treatment efficacy of patients with multidrug-resistant (MDR), extensively drug-resistant (XDR) and drug-susceptible (DSTB) tuberculosis in the most populous region of Russia (Bashkortostan) that borders with Central Asia. METHODS: A retrospective cohort study was performed on 436 patients with pulmonary tuberculosis who were enrolled between January 1, 2016, and February 28, 2018, and received treatment according to WHO recommendations. Altogether, 369 patients completed the full course of chemotherapy. Clinical characteristics and treatment outcomes of DSTB, MDR, and XDR-TB patients were analyzed. RESULTS: Of 436 patients, 169 (39%) had XDR-TB, 94 (22%) had MDR-TB and 173 (40%) had DSTB. Half of the MDR-TB patients (44%) and 82% of XDR-TB patients failed treatment. Patients with DSTB had unexpectedly poor treatment efficacy: only 67% had treatment success. We found that most of the MDR isolates from our patients were resistant to all first-line drugs, and a majority of the XDR isolates were resistant to more than 6-7 anti-TB drugs. While this can explain poor treatment efficacy in drug-resistant cases, causes of poor treatment efficacy in DSTB patients remain unclear. Finally, a considerable fraction (46%) of newly diagnosed patients had MDR-TB (27%) and XDR-TB (19%), suggesting that drug-resistant Mtb is being transmitted in the general population. To our best knowledge, this study is the first one to report XDR-TB prevalence in Russia in recent years (2016-2018). CONCLUSIONS: MDR and XDR-TB became more common in recent years and treatment efficacy is declining at the face of more extensive drug resistance. There is evidence for the transmission of resistant strains in the general population, which calls for urgent changes not only in clinical practice but also in measures to prevent spread in the general population.