CRISPR/Cas9-based split homing gene drive targeting doublesex for population suppression of the global fruit pest Drosophila suzukii.

Genetic-based methods offer environmentally friendly species-specific approaches for control of insect pests. One method, CRISPR homing gene drive that target genes essential for development, could provide very efficient and cost-effective control. While significant progress has been made in developing homing gene drives for mosquito disease vectors, little progress has been made with agricultural insect pests. Here, we report the development and evaluation of split homing drives that target the doublesex (dsx) gene in Drosophila suzukii, an invasive pest of soft-skinned fruits. The drive component, consisting of dsx single guide RNA and DsRed genes, was introduced into the female-specific exon of dsx, which is essential for function in females but not males. However, in most strains, hemizygous females were sterile and produced the male dsx transcript. With a modified homing drive that included an optimal splice acceptor site, hemizygous females from each of the four independent lines were fertile. High transmission rates of the DsRed gene (94 to 99%) were observed with a line that expressed Cas9 with two nuclear localization sequences from the D. suzukii nanos promoter. Mutant alleles of dsx with small in-frame deletions near the Cas9 cut site were not functional and thus would not provide resistance to drive. Finally, mathematical modeling showed that the strains could be used for suppression of lab cage populations of D. suzukii with repeated releases at relatively low release ratios (1:4). Our results indicate that the split CRISPR homing gene drive strains could potentially provide an effective means for control of D. suzukii populations.

Expansion of the genetic toolbox for manipulation of the global crop pest Drosophila suzukii: Isolation and assessment of eye colour mutant strains.

Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), commonly called spotted wing Drosophila, is an important agricultural pest recognised worldwide. D. suzukii is a pest of soft-skinned fruits as females can lay eggs in ripening fruit before harvest. While strains for genetic biocontrol of D. suzukii have been made, the development of transgenic D. suzukii strains and their further screening remain a challenge partly due to the lack of phenotypically trackable genetic-markers, such as those widely used with the model genetic organism D. melanogaster. Here, we have used CRISPR/Cas9 to introduce heritable mutations in the eye colour genes white, cinnabar and sepia, which are located on the X, second and third chromosomes, respectively. Strains were obtained, which were homozygous for a single mutation. Genotyping of the established strains showed insertion and/or deletions (indels) at the targeted sites. A strain homozygous for mutations in cinnabar and sepia showed a pale-yellow eye colour at eclosion but darkened to a sepia colour after a week. The fecundity and fertility of some of the cinnabar and sepia strains were comparable with the wild type. Although white mutant males were previously reported to be sterile, we found that sterility is not fully penetrant and we have been able to maintain white-eyed strains for over a year. The cinnabar, sepia and white mutant strains developed in this study should facilitate future genetic studies in D. suzukii and the development of strains for genetic control of this pest.

Clinical Characteristics of Corynebacterium ulcerans Infection, Japan.

Corynebacterium ulcerans is a closely related bacterium to the diphtheria bacterium C. diphtheriae, and some C. ulcerans strains produce toxins that are similar to diphtheria toxin. C. ulcerans is widely distributed in the environment and is considered one of the most harmful pathogens to livestock and wildlife. Infection with C. ulcerans can cause respiratory or nonrespiratory symptoms in patients. Recently, the microorganism has been increasingly recognized as an emerging zoonotic agent of diphtheria-like illness in Japan. To clarify the overall clinical characteristics, treatment-related factors, and outcomes of C. ulcerans infection, we analyzed 34 cases of C. ulcerans that occurred in Japan during 2001-2020. During 2010-2020, the incidence rate of C. ulcerans infection increased markedly, and the overall mortality rate was 5.9%. It is recommended that adults be vaccinated with diphtheria toxoid vaccine to prevent the spread of this infection.

Natural genetic variation in a dopamine receptor is associated with variation in female fertility in Drosophila melanogaster.

Fertility is a major component of fitness but its genetic architecture remains poorly understood. Using a full diallel cross of 50 Drosophila Genetic Reference Panel inbred lines with whole genome sequences, we found substantial genetic variation in fertility largely attributable to females. We mapped genes associated with variation in female fertility by genome-wide association analysis of common variants in the fly genome. Validation of candidate genes by RNAi knockdown confirmed the role of the dopamine 2-like receptor (Dop2R) in promoting egg laying. We replicated the Dop2R effect in an independently collected productivity dataset and showed that the effect of the Dop2R variant was mediated in part by regulatory gene expression variation. This study demonstrates the strong potential of genome-wide association analysis in this diverse panel of inbred strains and subsequent functional analyses for understanding the genetic architecture of fitness traits.

Limitations of Ribotyping as Genotyping Method for Corynebacterium ulcerans.

We conducted molecular typing of a Corynebacterium ulcerans isolate from a woman who died in Japan in 2016. Genomic DNA modification might have affected the isolate's ribotyping profile. Multilocus sequence typing results (sequence type 337) were more accurate. Whole-genome sequencing had greater ability to discriminate lineages at high resolution.

Genetic architecture of natural variation in visual senescence in Drosophila.

Senescence, i.e., functional decline with age, is a major determinant of health span in a rapidly aging population, but the genetic basis of interindividual variation in senescence remains largely unknown. Visual decline and age-related eye disorders are common manifestations of senescence, but disentangling age-dependent visual decline in human populations is challenging due to inability to control genetic background and variation in histories of environmental exposures. We assessed the genetic basis of natural variation in visual senescence by measuring age-dependent decline in phototaxis using Drosophila melanogaster as a genetic model system. We quantified phototaxis at 1, 2, and 4 wk of age in the sequenced, inbred lines of the Drosophila melanogaster Genetic Reference Panel (DGRP) and found an average decline in phototaxis with age. We observed significant genetic variation for phototaxis at each age and significant genetic variation in senescence of phototaxis that is only partly correlated with phototaxis. Genome-wide association analyses in the DGRP and a DGRP-derived outbred, advanced intercross population identified candidate genes and genetic networks associated with eye and nervous system development and function, including seven genes with human orthologs previously associated with eye diseases. Ninety percent of candidate genes were functionally validated with targeted RNAi-mediated suppression of gene expression. Absence of candidate genes previously implicated with longevity indicates physiological systems may undergo senescence independent of organismal life span. Furthermore, we show that genes that shape early developmental processes also contribute to senescence, demonstrating that senescence is part of a genetic continuum that acts throughout the life span.

Severe Pneumonia Caused by Toxigenic Corynebacterium ulcerans Infection, Japan.

Corynebacterium ulcerans infection was recently recognized as a zoonosis. We present 2 cases of severe pneumonia complicated by diffuse pseudomembrane formation on the bronchus caused by C. ulcerans-producing diphtheria toxin. Our purpose is to alert medical professionals to the virulence of Corynebacterium species other than C. diphtheriae.

Development of vaccine for Clostridium difficile infection using membrane fraction of nontoxigenic Clostridium difficile.

Although standard antibiotic therapy is performed for diarrhea and pseudomembranous colitis caused by Clostridium difficile, a high recurrence rate of C. difficile infection (CDI) remains a major problem. We previously showed that a membrane fraction of nontoxigenic C. difficile (ntCDMF) was effective as a vaccine antigen by in vitro experiments. In this study, we examined whether ntCDMF had an in vivo effect in animal challenge experiments. By intrarectal immunization with ntCDMF, the number of C. difficile cells in feces of mice was decreased approximately 99% compared to the control mice. In addition, survival rate of C. difficile-challenged hamsters was increased almost 30% by immunization with ntCDMF. These results showed that ntCDMF could be a practical vaccine candidate.

The Drosophila melanogaster Genetic Reference Panel.

A major challenge of biology is understanding the relationship between molecular genetic variation and variation in quantitative traits, including fitness. This relationship determines our ability to predict phenotypes from genotypes and to understand how evolutionary forces shape variation within and between species. Previous efforts to dissect the genotype-phenotype map were based on incomplete genotypic information. Here, we describe the Drosophila melanogaster Genetic Reference Panel (DGRP), a community resource for analysis of population genomics and quantitative traits. The DGRP consists of fully sequenced inbred lines derived from a natural population. Population genomic analyses reveal reduced polymorphism in centromeric autosomal regions and the X chromosome, evidence for positive and negative selection, and rapid evolution of the X chromosome. Many variants in novel genes, most at low frequency, are associated with quantitative traits and explain a large fraction of the phenotypic variance. The DGRP facilitates genotype-phenotype mapping using the power of Drosophila genetics.

Freeze-dried equine-derived redback spider antivenom: a local irritation study by intramuscular injection in rabbits and a repeated-dose toxicity study in rats.

The redback spider (Latrodectus hasseltii) is nonindigenous to Japan but has now spread throughout the country. Bites to humans are rare but can be fatal. We prepared freeze-dried redback spider antivenom for therapeutic use against bites in Japan by immunization of horse plasma. This study included two nonclinical tests of the antivenom: a local irritation study involving a single intramuscular administration to rabbits (with injections of physiological saline and an existing freeze-dried diphtheria antitoxin as control and comparison substances, respectively) and a 2-week repeated intermittent intravenous-dose toxicity study in rats. The irritation study showed the antivenom's irritancy to be comparable with that of the saline and the existing antitoxin preparations under the test conditions. In a repeated-dose toxicity study, no toxicity change was found in male or female rats, and the no-observed-adverse-effect level (NOAEL) was judged to be a dose volume of 20 mL/kg (1082 units/kg antivenom activity) in both male and female rats. In addition, there was no toxicological difference between proteinaceous diphtheria antitoxin and redback spider antivenom prepared to have the same protein content and the same additive composition. Based on these findings, we will further advance our research towards clinical application of the redback spider antivenom. This research was supported by the Research Program on Emerging and Re-emerging Infectious Disease of the Japan Agency for Medical Research and Development.

High-Sensitivity Rheo-NMR Spectroscopy for Protein Studies.

Shear stress can induce structural deformation of proteins, which might result in aggregate formation. Rheo-NMR spectroscopy has the potential to monitor structural changes in proteins under shear stress at the atomic level; however, existing Rheo-NMR methodologies have insufficient sensitivity to probe protein structure and dynamics. Here we present a simple and versatile approach to Rheo-NMR, which maximizes sensitivity by using a spectrometer equipped with a cryogenic probe. As a result, the sensitivity of the instrument ranks highest among the Rheo-NMR spectrometers reported so far. We demonstrate that the newly developed Rheo-NMR instrument can acquire high-quality relaxation data for a protein under shear stress and can trace structural changes in a protein during fibril formation in real time. The described approach will facilitate rheological studies on protein structural deformation, thereby aiding a physical understanding of shear-induced amyloid fibril formation.

Natural variation in genome architecture among 205 Drosophila melanogaster Genetic Reference Panel lines.

The Drosophila melanogaster Genetic Reference Panel (DGRP) is a community resource of 205 sequenced inbred lines, derived to improve our understanding of the effects of naturally occurring genetic variation on molecular and organismal phenotypes. We used an integrated genotyping strategy to identify 4,853,802 single nucleotide polymorphisms (SNPs) and 1,296,080 non-SNP variants. Our molecular population genomic analyses show higher deletion than insertion mutation rates and stronger purifying selection on deletions. Weaker selection on insertions than deletions is consistent with our observed distribution of genome size determined by flow cytometry, which is skewed toward larger genomes. Insertion/deletion and single nucleotide polymorphisms are positively correlated with each other and with local recombination, suggesting that their nonrandom distributions are due to hitchhiking and background selection. Our cytogenetic analysis identified 16 polymorphic inversions in the DGRP. Common inverted and standard karyotypes are genetically divergent and account for most of the variation in relatedness among the DGRP lines. Intriguingly, variation in genome size and many quantitative traits are significantly associated with inversions. Approximately 50% of the DGRP lines are infected with Wolbachia, and four lines have germline insertions of Wolbachia sequences, but effects of Wolbachia infection on quantitative traits are rarely significant. The DGRP complements ongoing efforts to functionally annotate the Drosophila genome. Indeed, 15% of all D. melanogaster genes segregate for potentially damaged proteins in the DGRP, and genome-wide analyses of quantitative traits identify novel candidate genes. The DGRP lines, sequence data, genotypes, quality scores, phenotypes, and analysis and visualization tools are publicly available.

Toxigenic Corynebacterium ulcerans isolated from a hunting dog and its diphtheria toxin antibody titer.

Toxigenic Corynebacterium ulcerans is a zoonotic pathogen that produces diphtheria toxin and causes a diphtheria-like illness in humans. The organism is known to infect and circulate among dogs, which can then transmit it to humans. Furthermore, previous studies have found that C. ulcerans is carried by wild animals, including game animals. In the present study, we tested hunting and companion dogs for the presence of toxigenic C. ulcerans and succeeded in isolating the bacterium from a hunting dog. Moreover, several hunting dogs had serum diphtheria antitoxin titers that were higher than the titers required for protection in humans, suggesting a history of exposure to toxigenic Corynebacterium strains. Notably, ribotyping, pulsed-field gel electrophoresis and tox gene sequencing demonstrated that the isolate from the hunting dog clustered with previously characterized C. ulcerans strains isolated from wild animals, as opposed to groups of isolates from humans and companion dogs. Interestingly, the wild animal cluster also contains an isolate from an outdoor breeding dog, which could have formed a bridge between isolates from wild animals and those from companion dogs. The results presented herein provide insight into the mechanism by which the zoonotic pathogen C. ulcerans circulates among wild animals, hunting and companion dogs, and humans.

Establishment of an equine tetanus antitoxin reference standard for veterinary use in Japan.

To establish the first National Veterinary Assay Laboratory (NVAL) equine tetanus antitoxin reference standard for veterinary use, we manufactured vials of a candidate antitoxin. These were quality tested for moisture content, vacuum, colour, clarity, and the presence of foreign objects. Ultimately, 115 quality-controlled vials were prepared. To estimate the antitoxin potency of the candidate standard, three different laboratories conducted parallel line assays alongside the existing antitoxin standard. These potency estimates ranged from 38 to 42 IU. This activity was maintained for two years after manufacture, as compared with a fresh vial. No statistically significant non-linearity or non-parallelism of the regression lines was observed (p > 0.05). Statistical assessment of inter- and intra-laboratory variability revealed acceptable coefficients of variation of 3.2% and 2.4-3.1%, respectively. Based on these results, the potency of the potential reference standard was calculated at 40 units of antitoxin activity per 1-mL vial. Vials of this preparation were distributed for use as the first equine tetanus antitoxin reference standard for veterinary use in September 2015.

Inhibition of adhesion of Clostridium difficile to human intestinal cells after treatment with serum and intestinal fluid isolated from mice immunized with nontoxigenic C. difficile membrane fraction.

Diarrhea and pseudomembrane colitis caused by Clostridium difficile infection is a global health concern because of the high recurrence rate after standard antibiotic therapy. Vaccination presents a powerful countermeasure against disease recurrence. In this study, mice vaccinated with the nontoxigenic C. difficile membrane fraction generated a marked immune response to the antigen, as demonstrated by the serum IgG and intestinal fluid IgA levels. Significantly, pretreatment with harvested IgG- and IgA-containing fluids was sufficient to prevent in vitro adhesion of C. difficile to human Caco-2 intestinal cells. These results highlight the potential of nontoxigenic C. difficile membrane fraction as a vaccine candidate for C. difficile infection.

Pleiotropic fitness effects of the Tre1-Gr5a region in Drosophila melanogaster.

The abundance of transposable elements and DNA repeat sequences in mammalian genomes raises the question of whether such insertions represent passive evolutionary baggage or may influence the expression of complex traits. We addressed this question in Drosophila melanogaster, in which the effects of single transposable elements on complex traits can be assessed in genetically identical individuals reared in controlled environments. Here we demonstrate that single P-element insertions in the intergenic region between the gustatory receptor 5a (Gr5a, also known as Tre) and trapped in endoderm 1 (Tre1), which encodes an orphan receptor, exert complex pleiotropic effects on fitness traits, including selective nutrient intake, life span, and resistance to starvation and heat stress. Mutations in this region interact epistatically with downstream components of the insulin signaling pathway. Transposon-induced sex-specific and sex-antagonistic effects further accentuate the complex influences that intergenic transposable elements can contribute to quantitative trait phenotypes.

Total synthesis of halichondrin A, the missing member in the halichondrin class of natural products.

A total synthesis of halichondrin A, the phantom member in the halichondrin class of natural products, is reported. The highlights of synthesis include: (1) synthesis of C1-C19 building block 6b via a catalytic asymmetric Cr-mediated coupling of 12 and 13b; (2) synthesis of the right-half of 19 via an asymmetric Ni/Cr-mediated coupling, followed by base-induced furan formation, and Shiina macrolactonization; (3) synthesis of enone 20 via Ni/Cr-mediated coupling of 5 with 19, followed by oxidation; (4) synthesis of halichondrin A from 20, with use of a newly discovered, highly selective TMSOTf-mediated equilibration of C38-epi-halichondrin A to halichondrin A. Two pieces of evidence are presented unambiguously to establish the structure of halichondrin A thus synthesized: one is the synthesis of norhalichondrin A (24) from 19 and 23, and the other is the study of the proton chemical shift difference between synthetic halichondrin A and known members of this class of natural products.

Genetic characterization and comparison of Clostridium botulinum isolates from botulism cases in Japan between 2006 and 2011.

Genetic characterization was performed for 10 group I Clostridium botulinum strains isolated from botulism cases in Japan between 2006 and 2011. Of these, 1 was type A, 2 were type B, and 7 were type A(B) {carrying a silent bont/B [bont/(B)] gene} serotype strains, based on botulinum neurotoxin (BoNT) production. The type A strain harbored the subtype A1 BoNT gene (bont/A1), which is associated with the ha gene cluster. The type B strains carried bont/B5 or bont/B6 subtype genes. The type A(B) strains carried bont/A1 identical to that of type A(B) strain NCTC2916. However, bont/(B) genes in these strains showed single-nucleotide polymorphisms (SNPs) among strains. SNPs at 2 nucleotide positions of bont/(B) enabled classification of the type A(B) strains into 3 groups. Pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem-repeat analysis (MLVA) also provided consistent separation results. In addition, the type A(B) strains were separated into 2 lineages based on their plasmid profiles. One lineage carried a small plasmid (5.9 kb), and another harbored 21-kb plasmids. To obtain more detailed genetic information about the 10 strains, we sequenced their genomes and compared them with 13 group I C. botulinum genomes in a database using whole-genome SNP analysis. This analysis provided high-resolution strain discrimination and enabled us to generate a refined phylogenetic tree that provides effective traceability of botulism cases, as well as bioterrorism materials. In the phylogenetic tree, the subtype B6 strains, Okayama2011 and Osaka05, were distantly separated from the other strains, indicating genomic divergence of subtype B6 strains among group I strains.

Reverse transcription polymerase chain reaction-based method for selectively detecting vegetative cells of toxigenic Clostridium difficile.

The laboratory diagnostic methods for Clostridium difficile infection (CDI) include toxigenic culture, enzyme immunoassays (EIAs) to detect the toxins of C. difficile, and nucleic acid amplification tests (NAATs) to detect C. difficile toxin genes, but each of these methods has disadvantages; toxigenic cultures require a long time to produce results, EIAs have low sensitivity, and NAATs that target DNA cannot distinguish vegetative cells from spores and dead cells. Here we report a new detection method that uses reverse transcription polymerase chain reaction to target the toxin-gene transcripts. This method was able to specifically detect the vegetative cells of toxigenic C. difficile in fecal samples in spike tests, with a minimum detection limit of 5 × 10(2) colony-forming units per 100 mg of stool specimen. The performance of this method was also demonstrated in a pilot scale evaluation using clinical fecal specimens, which showed that this method may be more sensitive than EIA and requires a shorter time than toxigenic culture. This method could potentially be applied in the clinical laboratory to detect C. difficile in fecal specimens. The ability of this method to discriminate the presence of vegetative cells from spores and dead cells could help to further the understanding of CDI.

Seroprevalence of tetanus toxoid antibody and booster vaccination efficacy in Japanese travelers.

Tetanus can be prevented by vaccination, which is especially important for overseas travelers. However, despite booster vaccination every 10 years being recommended, most Japanese adults do not receive it in the absence of physical injury or overseas travel. We aimed to investigate the level of protective immunity against tetanus among Japanese travelers, which may provide valuable information for formulating booster vaccination recommendations. 113 Japanese travelers given tetanus toxoid were recruited. The collected samples included paired samples prior to and 3-5 weeks after receiving the booster vaccination. Travelers who did not return and those lacking sample collection at the second visit were excluded. Finally, 96 paired blood samples were collected. History of immunization against tetanus, including DPT and DT vaccines, was determined from interviews or immunization records. The pre-vaccination geometric mean titer for the 96 participants was 1.07 IU/mL; 76% had a protective antitoxin level (>0.1 IU/mL), and 50% had a long-term protective antitoxin level (>1.0 IU/mL). Most participants <40 years old had protective immunity without receiving booster vaccination, whereas only 30.8% of those >50 years of age had protective immunity. Among the 23 participants without protective antitoxin levels (<0.1 IU/mL), booster vaccination was efficient in 100% of those <40 years but in only 28.6% of those >50 years of age. Although the tetanus antitoxin level decreases with age, booster vaccination helped to achieve an adequate protective antitoxin levels in Japanese travelers <40 years of age. Furthermore, the individuals who have never been vaccinated against tetanus especially in those >50 years old need to obtain protective immunity against tetanus according to a basic immunization schedule to prevent tetanus in travelers and residents of Japan.