Differential gene expression of varroa-tolerant and varroa-susceptible honey bees (Apis mellifera) in response to Varroa destructor infestation

The honey bee is one of the most familiar insects in the world, and plays an important role in the global economy providing essential pollination services to crops, fruit trees and vegetables. However, honey bee health is severely threatened by the ectoparasitic mite Varroa destructor, which feeds on the hemolymph of pupal and adult bees, resulting in loss of nutrients and circulatory fluids, decreased overall body weight and eventually the death of the bees. To investigate the molecular defense mechanisms of the honey bee against varroa mite infestation, we employed DNA microarray analysis to compare gene expression of two contrasting honey bee colony phenotypes selected from the Saskatraz breeding program. One designated as G4 is susceptible to the varroa mite, while the other designated as S88 is highly tolerant to the varroa. Total RNAs were isolated from bees at two different stages, dark-eyed pupa and adult worker, infected or non-infected with varroa mites, and used for DNA microarray analysis. The results showed that distinct sets of genes were differentially regulated in the varroa-tolerant and varroa-susceptible honey bee phenotypes, with and without varroa infestation. In both phenotypes, there were more differentially-expressed genes identified at the pupal stage than at the adult stage, indicating that at the pupal stage honey bees are more responsive to the varroa infestation than adult bees. In the phenotype comparisons, substantially more differentially-expressed genes were found in the tolerant than susceptible line, indicating that the tolerant phenotype has an increased capacity to mobilize the expression of the genes in response to varroa mite infestation. Based on function, the differentially-expressed genes could be classified into groups that are involved in olfactory signal transduction, detoxification, metabolism and exoskeleton formation, implying several possible mechanisms for the host-parasite interaction and resistance. Quantitative RT-PCR was used to confirm the data obtained from the DNA microarray hybridization. Eleven out of twelve genes selected based on the microarray data showed consistent expression patterns measured by both methods. Overall, comprehensive evaluation of the gene expression of honey bees in response to the mite infestation by DNA microarray has revealed several possible molecular mechanisms for the host defense against the pest. Identification of highly differentially expressed genes between the two phenotypes provides potential biomarkers that can be used for breeding honey bees resistant to the varroa mite

Draft genomes of two Artocarpus plants, jackfruit (A. heterophyllus) and breadfruit (A. altilis)

Two of the most economically important plants in the Artocarpus genus are jackfruit (A. heterophyllus Lam.) and breadfruit (A. altilis (Parkinson) Fosberg). Both species are long-lived trees that have been cultivated for thousands of years in their native regions. Today they are grown throughout tropical to subtropical areas as an important source of starch and other valuable nutrients. There are hundreds of breadfruit varieties that are native to Oceania, of which the most commonly distributed types are seedless triploids. Jackfruit is likely native to the Western Ghats of India and produces one of the largest tree-borne fruit structures (reaching up to 45 kg). To-date, there is limited genomic information for these two economically important species. Here, we generated 273 Gb and 227 Gb of raw data from jackfruit and breadfruit, respectively. The high-quality reads from jackfruit were assembled into 162,440 scaffolds totaling 982 Mb with 35,858 genes. Similarly, the breadfruit reads were assembled into 180,971 scaffolds totaling 833 Mb with 34,010 genes. A total of 2822 and 2034 expanded gene families were found in jackfruit and breadfruit, respectively, enriched in pathways including starch and sucrose metabolism, photosynthesis, and others. The copy number of several starch synthesis-related genes were found to be increased in jackfruit and breadfruit compared to closely-related species, and the tissue-specific expression might imply their sugar-rich and starch-rich characteristics. Overall, the publication of high-quality genomes for jackfruit and breadfruit provides information about their specific composition and the underlying genes involved in sugar and starch metabolism

A CpG Methylation Signature as a Potential Marker for Early Diagnosis of Hepatocellular Carcinoma From HBV-Related Liver Disease Using Multiplex Bisulfite Sequencing.

BACKGROUND: Aberrant methylation of CpG sites served as an epigenetic marker for building diagnostic, prognostic, and recurrence models for hepatocellular carcinoma (HCC). METHODS: Using Illumina 450K and EPIC Beadchip, we identified 34 CpG sites in peripheral blood mononuclear cell (PBMC) DNA that were differentially methylated in early HCC versus HBV-related liver diseases (HBVLD). We employed multiplex bisulfite sequencing (MBS) based on next-generation sequencing (NGS) to measure methylation of 34 CpG sites in PBMC DNA from 654 patients that were divided into a training set (n = 442) and a test set (n = 212). Using the training set, we selected and built a six-CpG-scorer (namely, cg14171514, cg07721852, cg05166871, cg18087306, cg05213896, and cg18772205), applying least absolute shrinkage and selection operator (LASSO) regression. We performed multivariable analyses of four candidate risk predictors (namely, six-CpG-scorer, age, sex, and AFP level), using 20 times imputation of missing data, non-linearly transformed, and backwards feature selection with logistic regression. The final model's regression coefficients were calculated according to "Rubin's Rules". The diagnostic accuracy of the model was internally validated with a 10,000 bootstrap validation dataset and then applied to the test set for validation. RESULTS: The area under the receiver operating characteristic curve (AUROC) of the model was 0.81 (95% CI, 0.77-0.85) and it showed good calibration and decision curve analysis. Using enhanced bootstrap validation, adjusted C-statistics and adjusted Brier score were 0.809 and 0.199, respectively. The model also showed an AUROC value of 0.84 (95% CI 0.79-0.88) of diagnosis for early HCC in the test set. CONCLUSIONS: Our model based on the six-CpG-scorer was a reliable diagnosis tool for early HCC from HBVLD. The usage of the MBS method can realize large-scale detection of CpG sites in clinical diagnosis of early HCC and benefit the majority of patients

The signature of HBV-related liver disease in peripheral blood mononuclear cell DNA methylation.

BACKGROUND: Hepatitis B virus (HBV)-related liver disease induces liver damage by hepatic immune and inflammatory response. The association between aberrant peripheral blood mononuclear cell (PBMC) DNA methylation and progression of liver disease and fibrosis remains unclear. RESULTS: Here we applied Infinium 450 K BeadChip investigating PBMC genome-wide methylation profiling of 48 HBV-related liver disease patients including 24 chronic hepatitis B (CHB), 14 compensated liver cirrhosis (LC), and 10 decompensated liver cirrhosis (DLC). In total, there were 7888 differentially methylated CpG sites (36.06% hypermethylation, 63.94% hypomethylation) correlate with liver disease progression. LC was difficult to be diagnosed, intermediating between CHB and DLC. We used least absolute shrinkage and selection operator (LASSO)-logistic regression method to perform a LC predictive model. The predicted probability (P) of having LC was estimated by the combined model: P = 1/(1 - e-x), where X = 11.52 - 2.82 × (if AST within the normal range - 0.19 × (percent methylation of cg05650055) - 0.21 × (percent methylation of cg17149911 ). Pyrosequencing validation and confusion matrix analysis was used for internal testing, area under receiver operating characteristic curve (AUROC) of model was 0.917 (95% CI, 0.80-0.977). On the fibrosis progress, there were 1705 genes in LC compared with CHB, whose differentially methylated CpG sites loading within the "promoter" regions (including TSS1500, TSS200, 5'UTR, and the 1st exon of genes) subject into the enrichment analysis using Ingenuity Pathway Analysis (IPA). There were 113 enriched immune-related pathways indicated that HBV-related liver fibrosis progression caused epigenetic reprogramming of the immune and inflammatory response. CONCLUSIONS: These data support idea that development of HBV-related chronic liver disease is linked with robust and broad alteration of methylation in peripheral immune system. CpG methylation sites serve as relevant biomarker candidates to monitor and diagnose LC, providing new insight into the immune mechanisms understanding the progression of HBV-related liver fibrosis and cirrhosis

Virus Infection of Plants Alters Pollinator Preference: A Payback for Susceptible Hosts?

Plant volatiles play important roles in attraction of certain pollinators and in host location by herbivorous insects. Virus infection induces changes in plant volatile emission profiles, and this can make plants more attractive to insect herbivores, such as aphids, that act as viral vectors. However, it is unknown if virus-induced alterations in volatile production affect plant-pollinator interactions. We found that volatiles emitted by cucumber mosaic virus (CMV)-infected tomato (Solanum lycopersicum) and Arabidopsis thaliana plants altered the foraging behaviour of bumblebees (Bombus terrestris). Virus-induced quantitative and qualitative changes in blends of volatile organic compounds emitted by tomato plants were identified by gas chromatography-coupled mass spectrometry. Experiments with a CMV mutant unable to express the 2b RNA silencing suppressor protein and with Arabidopsis silencing mutants implicate microRNAs in regulating emission of pollinator-perceivable volatiles. In tomato, CMV infection made plants emit volatiles attractive to bumblebees. Bumblebees pollinate tomato by 'buzzing' (sonicating) the flowers, which releases pollen and enhances self-fertilization and seed production as well as pollen export. Without buzz-pollination, CMV infection decreased seed yield, but when flowers of mock-inoculated and CMV-infected plants were buzz-pollinated, the increased seed yield for CMV-infected plants was similar to that for mock-inoculated plants. Increased pollinator preference can potentially increase plant reproductive success in two ways: i) as female parents, by increasing the probability that ovules are fertilized; ii) as male parents, by increasing pollen export. Mathematical modeling suggested that over a wide range of conditions in the wild, these increases to the number of offspring of infected susceptible plants resulting from increased pollinator preference could outweigh underlying strong selection pressures favoring pathogen resistance, allowing genes for disease susceptibility to persist in plant populations. We speculate that enhanced pollinator service for infected individuals in wild plant populations might provide mutual benefits to the virus and its susceptible hosts.Major funding for this project was provided to JPC by the Leverhulme Trust (Grant numbers RPG-2012-667 and F/09741/F: https://www. leverhulme.ac.uk/). Additional funding to JPC and studentships to support JHW and SCG came from the Biotechnological and Biological Sciences Research Council (Grant number BB/J011762/1: http://www.bbsrc.ac.uk/). Other additional funding was obtained from the Isaac Newton Trust (http://www. newtontrust.cam.ac.uk/: grant number 12.07/I to AMM).This is the final version of the article. It first appeared from the Public Library of Science via http://dx.doi.org/:10.1371/journal.ppat.100579

Single cell atlas for 11 non-model mammals, reptiles and birds.

The availability of viral entry factors is a prerequisite for the cross-species transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Large-scale single-cell screening of animal cells could reveal the expression patterns of viral entry genes in different hosts. However, such exploration for SARS-CoV-2 remains limited. Here, we perform single-nucleus RNA sequencing for 11 non-model species, including pets (cat, dog, hamster, and lizard), livestock (goat and rabbit), poultry (duck and pigeon), and wildlife (pangolin, tiger, and deer), and investigated the co-expression of ACE2 and TMPRSS2. Furthermore, cross-species analysis of the lung cell atlas of the studied mammals, reptiles, and birds reveals core developmental programs, critical connectomes, and conserved regulatory circuits among these evolutionarily distant species. Overall, our work provides a compendium of gene expression profiles for non-model animals, which could be employed to identify potential SARS-CoV-2 target cells and putative zoonotic reservoirs

CMV Infection Affects Bumblebee Pollination Behaviour and Plant Reproductive Success

Viruses can affect plant-insect interactions by altering emission of plant volatile organic compounds (VOCs). Previous work in the lab suggested that VOCs emitted by tomato (Solanum lycopersicum) plants infected with cucumber mosaic virus (CMV) were more attractive to bumblebees (Bombus terrestris) in free choice assays. I extended this work using Arabidopsis thaliana mutants with lesions in genes encoding factors in RNA silencing. In conditioning assays, I confirmed that plant VOC emission is controlled in part by the microRNA regulatory pathway. I used gas chromatography coupled to mass spectrometry and principal component analysis to confirm that CMV infection caused changes in VOC emission by tomato. VOCs collected from non-flowering mock-inoculated and CMV-infected plants were qualitatively distinct from each other. CMV-infected plants also released greater quantities of VOCs than mock-inoculated plants. CMV appears to be both ‘turning up the volume’ of plant volatile emission, whilst ‘tuning’ volatile blend composition so as to diminish levels of a repellant signal. These data are likely to explain how bumblebees can discriminate between VOCs emitted by mock-inoculated and CMV-infected plants. To determine if CMV infection of tomato plants affects plant reproductive success, I carried out a series of bumblebee pollination experiments. Bumblebees pollinate tomato by ‘buzzing’ (sonicating) the flowers, which releases pollen and enhances self-fertilization and seed production as well as pollen export. First, I established that CMV-infected tomato plants produced fruits with a lower seed yield than mock-inoculated plants. When single bumblebees were allowed to buzz-pollinate flowers in a small flying arena, the fruit that developed from buzz-pollinated flowers on virus infected plants had significantly more seeds than fruit from non-visited flowers. Subsequent experiments were performed in a large flying arena. Bumblebees consistently spent longer foraging on the mock-inoculated tomato plants but seed yield was increased by bumblebee pollination in both mock-inoculated as well as virus-infected tomato plants. However, although buzz-pollination significantly enhanced seed yield from CMV-infected plants compared to fruit from non-buzz-pollinated flowers, the yield was higher in buzz-pollinated fruits from mock-inoculated plants. Similar experiments were carried out utilizing a transgenic line of tomato that constitutively expresses the green fluorescent protein in order to estimate the level of cross-pollination from either CMV-infected plants to mock-inoculated plants or vice versa. More pollen from virus-infected plants was transferred to mock-inoculated plants than the reciprocal cross. However, some caution is needed in the interpretation of the larger scale experiments because the tomato plants were affected by a fungal infection. I investigated if the defensive plant hormone salicylic acid (SA) affects bee-perceivable VOC emission. Exogenous SA treatment renders non-flowering tomato plants more attractive to bumblebees in free choice experiments in which bees could only perceive VOCs, but bumblebees spent less time visiting SA-treated flowering tomato plants in the glasshouse (when the bumblebees were allowed unimpeded access to the flowers). Taken together, these data provide evidence that virus infection can affect host-pollinator interactions. Speculatively, CMV infection may change the fitness of susceptible plants via changes in production of pollinator-attractive VOCs and this may affect the balance of resistant or susceptible plants within the host population

Cut&tag: a powerful epigenetic tool for chromatin profiling

ABSTRACTAnalysis of transcription factors and chromatin modifications at the genome-wide level provides insights into gene regulatory processes, such as transcription, cell differentiation and cellular response. Chromatin immunoprecipitation is the most popular and powerful approach for mapping chromatin, and other enzyme-tethering techniques have recently become available for living cells. Among these, Cleavage Under Targets and Tagmentation (CUT&Tag) is a relatively novel chromatin profiling method that has rapidly gained popularity in the field of epigenetics since 2019. It has also been widely adapted to map chromatin modifications and TFs in different species, illustrating the association of these chromatin epitopes with various physiological and pathological processes. Scalable single-cell CUT&Tag can be combined with distinct platforms to distinguish cellular identity, epigenetic features and even spatial chromatin profiling. In addition, CUT&Tag has been developed as a strategy for joint profiling of the epigenome, transcriptome or proteome on the same sample. In this review, we will mainly consolidate the applications of CUT&Tag and its derivatives on different platforms, give a detailed explanation of the pros and cons of this technique as well as the potential development trends and applications in the future