Scaling up single-cell RNA-seq data analysis with CellBridge workflow.

SUMMARY: Single-cell RNA sequencing (scRNA-seq) has revolutionized the study of gene expression at the individual cell level, unraveling unprecedented insights into cellular heterogeneity. However, the analysis of scRNA-seq data remains a challenging and time-consuming task, often demanding advanced computational expertise, rendering it impractical for high-volume environments and applications. We present CellBridge, an automated workflow designed to simplify the standard procedures entailed in scRNA-seq data analysis, eliminating the need for specialized computational expertise. CellBridge utilizes state-of-the-art computational methods, integrating a range of advanced functionalities, covering the entire process from raw unaligned sequencing reads to cell type annotation. Hence, CellBridge accelerates the pace of discovery by seamlessly enabling insights into vast volumes of scRNA-seq data, without compromising workflow control and reproducibility. AVAILABILITY AND IMPLEMENTATION: The source code, detailed documentation, and materials required to reproduce the results are available on GitHub and archived in Zenodo. For the CellBridge pre-processing step (v1.0.0), access the GitHub repository at https://github.com/Sanofi-Public/PMCB-ToBridge and the Zenodo archive at https://zenodo.org/records/10246161. For the CellBridge processing step (v1.0.0), visit the GitHub repository at https://github.com/Sanofi-Public/PMCB-CellBridge and the Zenodo archive at https://zenodo.org/records/10246046.

A marker gene-based method for identifying the cell-type of origin from single-cell RNA sequencing data.

Single-cell RNA sequencing (scRNA-seq) experiments provide opportunities to peer into complex tissues at single-cell resolution. However, insightful biological interpretation of scRNA-seq data relies upon precise identification of cell types. The ability to identify the origin of a cell quickly and accurately will greatly improve downstream analyses. We present Sargent, a transformation-free, cluster-free, single-cell annotation algorithm for rapidly identifying the cell types of origin based on cell type-specific markers. We demonstrate Sargent's high accuracy by annotating simulated datasets. Further, we compare Sargent performance against expert-annotated scRNA-seq data from human organs including PBMC, heart, kidney, and lung. We demonstrate that Sargent retains both the flexibility and biological interpretability of cluster-based manual annotation. Additionally, the automation eliminates the labor intensive and potentially biased user annotation, producing robust, reproducible, and scalable outputs.•Sargent is a transformation-free, cluster-free, single-cell annotation algorithm for rapidly identifying the cell types of origin based on cell type-specific markers.•Sargent retains both the flexibility and biological interpretability of cluster-based manual annotation.•Automation eliminates the labor intensive and potentially biased user annotation, producing robust, reproducible, and scalable outputs.

DiSiR: fast and robust method to identify ligand-receptor interactions at subunit level from single-cell RNA-sequencing data.

Most cell-cell interactions and crosstalks are mediated by ligand-receptor interactions. The advent of single-cell RNA-sequencing (scRNA-seq) techniques has enabled characterizing tissue heterogeneity at single-cell level. In the past few years, several methods have been developed to study ligand-receptor interactions at cell type level using scRNA-seq data. However, there is still no easy way to query the activity of a specific user-defined signaling pathway in a targeted way or to map the interactions of the same subunit with different ligands as part of different receptor complexes. Here, we present DiSiR, a fast and easy-to-use permutation-based software framework to investigate how individual cells are interacting with each other by analyzing signaling pathways of multi-subunit ligand-activated receptors from scRNA-seq data, not only for available curated databases of ligand-receptor interactions, but also for interactions that are not listed in these databases. We show that, when utilized to infer ligand-receptor interactions from both simulated and real datasets, DiSiR outperforms other well-known permutation-based methods, e.g. CellPhoneDB and ICELLNET. Finally, to demonstrate DiSiR's utility in exploring data and generating biologically relevant hypotheses, we apply it to COVID lung and rheumatoid arthritis (RA) synovium scRNA-seq datasets and highlight potential differences between inflammatory pathways at cell type level for control versus disease samples.

Trans-driven variation in expression is common among detoxification genes in the extreme generalist herbivore Tetranychus urticae.

The extreme adaptation potential of the generalist herbivore Tetranychus urticae (the two-spotted spider mite) to pesticides as well as diverse host plants has been associated with clade-specific gene expansions in known detoxifying enzyme families, and with extensive and rapid transcriptional responses. However, how this broad transcriptional potential is regulated remains largely unknown. Using a parental/F1 design in which four inbred strains were crossed to a common inbred strain, we assessed the genetic basis and inheritance of gene expression variation in T. urticae. Mirroring known phenotypic variation in the progenitor strains of the inbreds, we confirmed that the inbred strains we created were genetically distinct, varied markedly in pesticide resistance, and also captured variation in host plant fitness as is commonly observed in this species. By examining differences in gene expression between parents and allele-specific expression in F1s, we found that variation in RNA abundance was more often explained in trans as compared to cis, with the former associated with dominance in inheritance. Strikingly, in a gene ontology analysis, detoxification genes of the cytochrome P450 monooxygenase (CYP) family, as well as dioxygenases (DOGs) acquired from horizontal gene transfer from fungi, were specifically enriched at the extremes of trans-driven up- and downregulation. In particular, multiple CYPs and DOGs with broad substrate-specificities for pesticides or plant specialized compounds were exceptionally highly upregulated as a result of trans-regulatory variation, or in some cases synergism of cis and trans, in the most multi-pesticide resistant strains. Collectively, our findings highlight the potential importance of trans-driven expression variation in genes associated with xenobiotic metabolism and host plant use for rapid adaptation in T. urticae, and also suggests modular control of these genes, a regulatory architecture that might ameliorate negative pleiotropic effects.

Maize Inbred Line B96 Is the Source of Large-Effect Loci for Resistance to Generalist but Not Specialist Spider Mites.

Maize (Zea mays subsp. mays) yield loss from arthropod herbivory is substantial. While the basis of resistance to major insect herbivores has been comparatively well-studied in maize, less is known about resistance to spider mite herbivores, which are distantly related to insects and feed by a different mechanism. Two spider mites, the generalist Tetranychus urticae, and the grass-specialist Oligonychus pratensis, are notable pests of maize, especially during drought conditions. We assessed resistance (antibiosis) to both mites of 38 highly diverse maize lines, including several previously reported to be resistant to one or the other mite species. We found that line B96, as well as its derivatives B49 and B75, were highly resistant to T. urticae. In contrast, neither these three lines, nor any others included in our study, were notably resistant to the specialist O. pratensis. Quantitative trait locus (QTL) mapping with replicate populations from crosses of B49, B75, and B96 to susceptible B73 identified a QTL in the same genomic interval on chromosome 6 for T. urticae resistance in each of the three resistant lines, and an additional resistance QTL on chromosome 1 was unique to B96. Single-locus genotyping with a marker coincident with the chromosome 6 QTL in crosses of both B49 and B75 to B73 revealed that the respective QTL was large-effect; it explained ∼70% of the variance in resistance, and resistance alleles from B49 and B75 acted recessively as compared to B73. Finally, a genome-wide haplotype analysis using genome sequence data generated for B49, B75, and B96 identified an identical haplotype, likely of initial origin from B96, as the source of T. urticae resistance on chromosome 6 in each of the B49, B75, and B96 lines. Our findings uncover the relationship between intraspecific variation in maize defenses and resistance to its major generalist and specialist spider mite herbivores, and we identified loci for use in breeding programs and for genetic studies of resistance to T. urticae, the most widespread spider mite pest of maize.

Adaptive divergence and post-zygotic barriers to gene flow between sympatric populations of a herbivorous mite.

Plant-herbivore interactions promote the generation and maintenance of both plant and herbivore biodiversity. The antagonistic interactions between plants and herbivores lead to host race formation: the evolution of herbivore types specializing on different plant species, with restricted gene flow between them. Understanding how ecological specialization promotes host race formation usually depends on artificial approaches, using laboratory experiments on populations associated with agricultural crops. However, evidence on how host races are formed and maintained in a natural setting remains scarce. Here, we take a multidisciplinary approach to understand whether populations of the generalist spider mite Tetranychus urticae form host races in nature. We demonstrate that a host race co-occurs among generalist conspecifics in the dune ecosystem of The Netherlands. Extensive field sampling and genotyping of individuals over three consecutive years showed a clear pattern of host associations. Genome-wide differences between the host race and generalist conspecifics were found using a dense set of SNPs on field-derived iso-female lines and previously sequenced genomes of T. urticae. Hybridization between lines of the host race and sympatric generalist lines is restricted by post-zygotic breakdown, and selection negatively impacts the survival of generalists on the native host of the host race. Our description of a host race among conspecifics with a larger diet breadth shows how ecological and reproductive isolation aid in maintaining intra-specific variation in sympatry, despite the opportunity for homogenization through gene flow. Our findings highlight the importance of explicitly considering the spatial and temporal scale on which plant-herbivore interactions occur in order to identify herbivore populations associated with different plant species in nature. This system can be used to study the underlying genetic architecture and mechanisms that facilitate the use of a large range of host plant taxa by extreme generalist herbivores. In addition, it offers the chance to investigate the prevalence and mechanisms of ecological specialization in nature.

High-resolution genetic mapping reveals cis-regulatory and copy number variation in loci associated with cytochrome P450-mediated detoxification in a generalist arthropod pest.

Chemical control strategies are driving the evolution of pesticide resistance in pest populations. Understanding the genetic mechanisms of these evolutionary processes is of crucial importance to develop sustainable resistance management strategies. The acaricide pyflubumide is one of the most recently developed mitochondrial complex II inhibitors with a new mode of action that specifically targets spider mite pests. In this study, we characterize the molecular basis of pyflubumide resistance in a highly resistant population of the spider mite Tetranychus urticae. Classical genetic crosses indicated that pyflubumide resistance was incompletely recessive and controlled by more than one gene. To identify resistance loci, we crossed the resistant population to a highly susceptible T. urticae inbred strain and propagated resulting populations with and without pyflubumide exposure for multiple generations in an experimental evolution set-up. High-resolution genetic mapping by a bulked segregant analysis approach led to the identification of three quantitative trait loci (QTL) linked to pyflubumide resistance. Two QTLs were found on the first chromosome and centered on the cytochrome P450 CYP392A16 and a cluster of CYP392E6-8 genes. Comparative transcriptomics revealed a consistent overexpression of CYP392A16 and CYP392E8 in the experimental populations that were selected for pyflubumide resistance. We further corroborated the involvement of CYP392A16 in resistance by in vitro functional expression and metabolism studies. Collectively, these experiments uncovered that CYP392A16 N-demethylates the toxic carboxamide form of pyflubumide to a non-toxic compound. A third QTL coincided with cytochrome P450 reductase (CPR), a vital component of cytochrome P450 metabolism. We show here that the resistant population harbors three gene copies of CPR and that this copy number variation is associated with higher mRNA abundance. Together, we provide evidence for detoxification of pyflubumide by cytochrome P450s that is likely synergized by gene amplification of CPR.

Trait mapping in diverse arthropods by bulked segregant analysis.

Bulked segregant analysis (BSA) is a cross-based method for genetic mapping in sexually reproducing organisms. The method's use of bulked (pooled) samples markedly reduces the genotyping effort associated with traditional linkage mapping studies. Further, it can be applied to species with life histories or physical attributes (as for micro-insects) that render genetic mapping with other methods impractical. Recent studies in both insects and mites have revealed that advanced BSA experimental designs can resolve causal loci to narrow genomic intervals, facilitating follow-up investigations. As high-quality genomes become more widely available, BSA methods are poised to become an increasingly important tool for the rapid mapping of both monogenic and polygenic traits in diverse arthropod species.

Convergent evolution of cytochrome P450s underlies independent origins of keto-carotenoid pigmentation in animals.

Keto-carotenoids contribute to many important traits in animals, including vision and coloration. In a great number of animal species, keto-carotenoids are endogenously produced from carotenoids by carotenoid ketolases. Despite the ubiquity and functional importance of keto-carotenoids in animals, the underlying genetic architectures of their production have remained enigmatic. The body and eye colorations of spider mites (Arthropoda: Chelicerata) are determined by ß-carotene and keto-carotenoid derivatives. Here, we focus on a carotenoid pigment mutant of the spider mite Tetranychus kanzawai that, as shown by chromatography, lost the ability to produce keto-carotenoids. We employed bulked segregant analysis and linked the causal locus to a single narrow genomic interval. The causal mutation was fine-mapped to a minimal candidate region that held only one complete gene, the cytochrome P450 monooxygenase CYP384A1, of the CYP3 clan. Using a number of genomic approaches, we revealed that an inactivating deletion in the fourth exon of CYP384A1 caused the aberrant pigmentation. Phylogenetic analysis indicated that CYP384A1 is orthologous across mite species of the ancient Trombidiformes order where carotenoids typify eye and body coloration, suggesting a deeply conserved function of CYP384A1 as a carotenoid ketolase. Previously, CYP2J19, a cytochrome P450 of the CYP2 clan, has been identified as a carotenoid ketolase in birds and turtles. Our study shows that selection for endogenous production of keto-carotenoids led to convergent evolution, whereby cytochrome P450s were independently co-opted in vertebrate and invertebrate animal lineages.

High-resolution QTL mapping in Tetranychus urticae reveals acaricide-specific responses and common target-site resistance after selection by different METI-I acaricides.

Arthropod herbivores cause dramatic crop losses, and frequent pesticide use has led to widespread resistance in numerous species. One such species, the two-spotted spider mite, Tetranychus urticae, is an extreme generalist herbivore and a major worldwide crop pest with a history of rapidly developing resistance to acaricides. Mitochondrial Electron Transport Inhibitors of complex I (METI-Is) have been used extensively in the last 25 years to control T. urticae around the globe, and widespread resistance to each has been documented. METI-I resistance mechanisms in T. urticae are likely complex, as increased metabolism by cytochrome P450 monooxygenases as well as a target-site mutation have been linked with resistance. To identify loci underlying resistance to the METI-I acaricides fenpyroximate, pyridaben and tebufenpyrad without prior hypotheses, we crossed a highly METI-I-resistant strain of T. urticae to a susceptible one, propagated many replicated populations over multiple generations with and without selection by each compound, and performed bulked segregant analysis genetic mapping. Our results showed that while the known H92R target-site mutation was associated with resistance to each compound, a genomic region that included cytochrome P450-reductase (CPR) was associated with resistance to pyridaben and tebufenpyrad. Within CPR, a single nonsynonymous variant distinguished the resistant strain from the sensitive one. Furthermore, a genomic region linked with tebufenpyrad resistance harbored a non-canonical member of the nuclear hormone receptor 96 (NHR96) gene family. This NHR96 gene does not encode a DNA-binding domain (DBD), an uncommon feature in arthropods, and belongs to an expanded family of 47 NHR96 proteins lacking DBDs in T. urticae. Our findings suggest that although cross-resistance to METI-Is involves known detoxification pathways, structural differences in METI-I acaricides have also resulted in resistance mechanisms that are compound-specific.

Long-Term Population Studies Uncover the Genome Structure and Genetic Basis of Xenobiotic and Host Plant Adaptation in the Herbivore Tetranychus urticae.

Pesticide resistance arises rapidly in arthropod herbivores, as can host plant adaptation, and both are significant problems in agriculture. These traits have been challenging to study as both are often polygenic and many arthropods are genetically intractable. Here, we examined the genetic architecture of pesticide resistance and host plant adaptation in the two-spotted spider mite, Tetranychus urticae, a global agricultural pest. We show that the short generation time and high fecundity of T. urticae can be readily exploited in experimental evolution designs for high-resolution mapping of quantitative traits. As revealed by selection with spirodiclofen, an acetyl-CoA carboxylase inhibitor, in populations from a cross between a spirodiclofen-resistant and a spirodiclofen-susceptible strain, and which also differed in performance on tomato, we found that a limited number of loci could explain quantitative resistance to this compound. These were resolved to narrow genomic intervals, suggesting specific candidate genes, including acetyl-CoA carboxylase itself, clustered and copy variable cytochrome P450 genes, and NADPH cytochrome P450 reductase, which encodes a redox partner for cytochrome P450s. For performance on tomato, candidate genomic regions for response to selection were distinct from those responding to the synthetic compound and were consistent with a more polygenic architecture. In accomplishing this work, we exploited the continuous nature of allele frequency changes across experimental populations to resolve the existing fragmented T. urticae draft genome to pseudochromosomes. This improved assembly was indispensable for our analyses, as it will be for future research with this model herbivore that is exceptionally amenable to genetic studies.

Disruption of a horizontally transferred phytoene desaturase abolishes carotenoid accumulation and diapause in Tetranychus urticae.

Carotenoids underlie many of the vibrant yellow, orange, and red colors in animals, and are involved in processes ranging from vision to protection from stresses. Most animals acquire carotenoids from their diets because de novo synthesis of carotenoids is primarily limited to plants and some bacteria and fungi. Recently, sequencing projects in aphids and adelgids, spider mites, and gall midges identified genes with homology to fungal sequences encoding de novo carotenoid biosynthetic proteins like phytoene desaturase. The finding of horizontal gene transfers of carotenoid biosynthetic genes to three arthropod lineages was unprecedented; however, the relevance of the transfers for the arthropods that acquired them has remained largely speculative, which is especially true for spider mites that feed on plant cell contents, a known source of carotenoids. Pigmentation in spider mites results solely from carotenoids. Using a combination of genetic approaches, we show that mutations in a single horizontally transferred phytoene desaturase result in complete albinism in the two-spotted spider mite, Tetranychus urticae, as well as in the citrus red mite, Panonychus citri Further, we show that phytoene desaturase activity is essential for photoperiodic induction of diapause in an overwintering strain of T. urticae, consistent with a role for this enzyme in provisioning provitamin A carotenoids required for light perception. Carotenoid biosynthetic genes of fungal origin have therefore enabled some mites to forgo dietary carotenoids, with endogenous synthesis underlying their intense pigmentation and ability to enter diapause, a key to the global distribution of major spider mite pests of agriculture.

Ixodes pacificus ticks maintain embryogenesis and egg hatching after antibiotic treatment of Rickettsia endosymbiont.

Rickettsia is a genus of intracellular bacteria that causes a variety of diseases in humans and other mammals and associates with a diverse group of arthropods. Although Rickettsia appears to be common in ticks, most Rickettsia-tick relationships remain generally uncharacterized. The most intimate of these associations is Rickettsia species phylotype G021, a maternally and transstadially transmitted endosymbiont that resides in 100% of I. pacificus in California. We investigated the effects of this Rickettsia phylotype on I. pacificus reproductive fitness using selective antibiotic treatment. Ciprofloxacin was 10-fold more effective than tetracycline in eliminating Rickettsia from I. pacificus, and quantitative PCR results showed that eggs from the ciprofloxacin-treated ticks contained an average of 0.02 Rickettsia per egg cell as opposed to the average of 0.2 in the tetracycline-treated ticks. Ampicillin did not significantly affect the number of Rickettsia per tick cell in adults or eggs compared to the water-injected control ticks. We found no relationship between tick embryogenesis and rickettsial density in engorged I. pacificus females. Tetracycline treatment significantly delayed oviposition of I. pacificus ticks, but the antibiotic's effect was unlikely related to Rickettsia. We also demonstrated that Rickettsia-free eggs could successfully develop into larvae without any significant decrease in hatching compared to eggs containing Rickettsia. No significant differences in the incubation period, egg hatching rate, and the number of larvae were found between any of the antibiotic-treated groups and the water-injected tick control. We concluded that Rickettsia species phylotype G021 does not have an apparent effect on embryogenesis, oviposition, and egg hatching of I. pacificus.