Low-input PacBio sequencing generates high-quality individual fly genomes and characterizes mutational processes.

Long-read sequencing, exemplified by PacBio, revolutionizes genomics, overcoming challenges like repetitive sequences. However, the high DNA requirement ( > 1 µg) is prohibitive for small organisms. We develop a low-input (100 ng), low-cost, and amplification-free library-generation method for PacBio sequencing (LILAP) using Tn5-based tagmentation and DNA circularization within one tube. We test LILAP with two Drosophila melanogaster individuals, and generate near-complete genomes, surpassing preexisting single-fly genomes. By analyzing variations in these two genomes, we characterize mutational processes: complex transpositions (transposon insertions together with extra duplications and/or deletions) prefer regions characterized by non-B DNA structures, and gene conversion of transposons occurs on both DNA and RNA levels. Concurrently, we generate two complete assemblies for the endosymbiotic bacterium Wolbachia in these flies and similarly detect transposon conversion. Thus, LILAP promises a broad PacBio sequencing adoption for not only mutational studies of flies and their symbionts but also explorations of other small organisms or precious samples.

Draft genome assemblies of the ponerine ant Odontoponera transversa and the carpenter ant Camponotus friedae (Hymenoptera: Formicidae).

OBJECTIVES: Ants are ecologically dominant insects in most terrestrial ecosystems, with more than 14,000 extant species in about 340 genera recorded to date. However, genomic resources are still scarce for most species, especially for species endemic in East or Southeast Asia, limiting the study of phylogeny, speciation and adaptation of this evolutionarily successful animal lineage. Here, we assemble and annotate the genomes of Odontoponera transversa and Camponotus friedae, two ant species with a natural distribution in China, to facilitate future study of ant evolution. DATA DESCRIPTION: We obtained a total of 16 Gb and 51 Gb PacBio HiFi data for O. transversa and C. friedae, respectively, which were assembled into the draft genomes of 339 Mb for O. transversa and 233 Mb for C. friedae. Genome assessments by multiple metrics showed good completeness and high accuracy of the two assemblies. Gene annotations assisted by RNA-seq data yielded a comparable number of protein-coding genes in the two genomes (10,892 for O. transversa and 11,296 for C. friedae), while repeat annotations revealed a remarkable difference of repeat content between these two ant species (149.4 Mb for O. transversa versus 49.7 Mb for C. friedae). Besides, complete mitochondrial genomes for the two species were assembled and annotated.

Genome organization regulates nuclear pore complex formation and promotes differentiation during Drosophila oogenesis.

Genome organization can regulate gene expression and promote cell fate transitions. The differentiation of germline stem cells (GSCs) to oocytes in Drosophila involves changes in genome organization mediated by heterochromatin and the nuclear pore complex (NPC). Heterochromatin represses germ cell genes during differentiation, and NPCs anchor these silenced genes to the nuclear periphery, maintaining silencing to allow for oocyte development. Surprisingly, we found that genome organization also contributes to NPC formation, mediated by the transcription factor Stonewall (Stwl). As GSCs differentiate, Stwl accumulates at boundaries between silenced and active gene compartments. Stwl at these boundaries plays a pivotal role in transitioning germ cell genes into a silenced state and activating a group of oocyte genes and nucleoporins (Nups). The upregulation of these Nups during differentiation is crucial for NPC formation and further genome organization. Thus, cross-talk between genome architecture and NPCs is essential for successful cell fate transitions.

A Naturally Active Spy Transposon Discovered from the Insect Genome of Colletes gigas as a Promising Novel Gene Transfer Tool.

Novel active DNA transposons, such as Spy transposons from the PHIS superfamily, are identified through bioinformatics in this study. The native transposases cgSpy and cvSpy displayed transposition activities of approximately 85% and 35% compared to the hyperactive piggyBac transposase (hyPB). The cgSpy transposon showed unique characteristics, including a lack of overproduction inhibition and reduced efficiency for insertion sizes between 3.1 to 8.5 kb. Integration preferences of cgSpy are found in genes and regulatory regions, making it suitable for genetic manipulation. Evaluation in T-cell engineering demonstrated that cgSpy-mediated chimeric antigen receptor (CAR) modification is comparable to the PB system, indicating its potential utility in cell therapy. This study unveils the promising application of the active native transposase, Spy, from Colletes gigas, as a valuable tool for genetic engineering, particularly in T-cell manipulation.

Protocol for preparing Drosophila genomic DNA to create chromosome-level de novo genome assemblies.

De novo genome assemblies are common tools for examining novel biological phenomena in non-model organisms. Here, we present a protocol for preparing Drosophila genomic DNA to create chromosome-level de novo genome assemblies. We describe steps for high-molecular-weight DNA preparation with phenol or Genomic-tips, quality control, long-read nanopore sequencing, short-read DNA library preparation, and sequencing. We then detail procedures of genome assembly, annotation, and assessment that can be used for downstream comparison and functional analysis. For complete details on the use and execution of this protocol, please refer to Sperling et al.1.

Hybrid speciation driven by multilocus introgression of ecological traits.

Hybridization allows adaptations to be shared among lineages and may trigger the evolution of new species1,2. However, convincing examples of homoploid hybrid speciation remain rare because it is challenging to demonstrate that hybridization was crucial in generating reproductive isolation3. Here we combine population genomic analysis with quantitative trait locus mapping of species-specific traits to examine a case of hybrid speciation in Heliconius butterflies. We show that Heliconius elevatus is a hybrid species that is sympatric with both parents and has persisted as an independently evolving lineage for at least 180,000 years. This is despite pervasive and ongoing gene flow with one parent, Heliconius pardalinus, which homogenizes 99% of their genomes. The remaining 1% introgressed from the other parent, Heliconius melpomene, and is scattered widely across the H. elevatus genome in islands of divergence from H. pardalinus. These islands contain multiple traits that are under disruptive selection, including colour pattern, wing shape, host plant preference, sex pheromones and mate choice. Collectively, these traits place H. elevatus on its own adaptive peak and permit coexistence with both parents. Our results show that speciation was driven by introgression of ecological traits, and that speciation with gene flow is possible with a multilocus genetic architecture.

Unsupervised AI reveals insect species-specific genome signatures.

Insects are a highly diverse phylogeny and possess a wide variety of traits, including the presence or absence of wings and metamorphosis. These diverse traits are of great interest for studying genome evolution, and numerous comparative genomic studies have examined a wide phylogenetic range of insects. Here, we analyzed 22 insects belonging to a wide phylogenetic range (Endopterygota, Paraneoptera, Polyneoptera, Palaeoptera, and other insects) by using a batch-learning self-organizing map (BLSOM) for oligonucleotide compositions in their genomic fragments (100-kb or 1-Mb sequences), which is an unsupervised machine learning algorithm that can extract species-specific characteristics of the oligonucleotide compositions (genome signatures). The genome signature is of particular interest in terms of the mechanisms and biological significance that have caused the species-specific difference, and can be used as a powerful search needle to explore the various roles of genome sequences other than protein coding, and can be used to unveil mysteries hidden in the genome sequence. Since BLSOM is an unsupervised clustering method, the clustering of sequences was performed based on the oligonucleotide composition alone, without providing information about the species from which each fragment sequence was derived. Therefore, not only the interspecies separation, but also the intraspecies separation can be achieved. Here, we have revealed the specific genomic regions with oligonucleotide compositions distinct from the usual sequences of each insect genome, e.g., Mb-level structures found for a grasshopper Schistocerca americana. One aim of this study was to compare the genome characteristics of insects with those of vertebrates, especially humans, which are phylogenetically distant from insects. Recently, humans seem to be the "model organism" for which a large amount of information has been accumulated using a variety of cutting-edge and high-throughput technologies. Therefore, it is reasonable to use the abundant information from humans to study insect lineages. The specific regions of Mb length with distinct oligonucleotide compositions have also been previously observed in the human genome. These regions were enriched by transcription factor binding motifs (TFBSs) and hypothesized to be involved in the three-dimensional arrangement of chromosomal DNA in interphase nuclei. The present study characterized the species-specific oligonucleotide compositions (i.e., genome signatures) in insect genomes and identified specific genomic regions with distinct oligonucleotide compositions.

GC Content Across Insect Genomes: Phylogenetic Patterns, Causes and Consequences.

The proportions of A:T and G:C nucleotide pairs are often unequal and can vary greatly between animal species and along chromosomes. The causes and consequences of this variation are incompletely understood. The recent release of high-quality genome sequences from the Darwin Tree of Life and other large-scale genome projects provides an opportunity for GC heterogeneity to be compared across a large number of insect species. Here we analyse GC content along chromosomes, and within protein-coding genes and codons, of 150 insect species from four holometabolous orders: Coleoptera, Diptera, Hymenoptera, and Lepidoptera. We find that protein-coding sequences have higher GC content than the genome average, and that Lepidoptera generally have higher GC content than the other three insect orders examined. GC content is higher in small chromosomes in most Lepidoptera species, but this pattern is less consistent in other orders. GC content also increases towards subtelomeric regions within protein-coding genes in Diptera, Coleoptera and Lepidoptera. Two species of Diptera, Bombylius major and B. discolor, have very atypical genomes with ubiquitous increase in AT content, especially at third codon positions. Despite dramatic AT-biased codon usage, we find no evidence that this has driven divergent protein evolution. We argue that the GC landscape of Lepidoptera, Diptera and Coleoptera genomes is influenced by GC-biased gene conversion, strongest in Lepidoptera, with some outlier taxa affected drastically by counteracting processes.

Genomes from historical Drosophila melanogaster specimens illuminate adaptive and demographic changes across more than 200 years of evolution.

The ability to perform genomic sequencing on long-dead organisms is opening new frontiers in evolutionary research. These opportunities are especially notable in the case of museum collections, from which countless documented specimens may now be suitable for genomic analysis-if data of sufficient quality can be obtained. Here, we report 25 newly sequenced genomes from museum specimens of the model organism Drosophila melanogaster, including the oldest extant specimens of this species. By comparing historical samples ranging from the early 1800s to 1933 against modern-day genomes, we document evolution across thousands of generations, including time periods that encompass the species' initial occupation of northern Europe and an era of rapidly increasing human activity. We also find that the Lund, Sweden population underwent local genetic differentiation during the early 1800s to 1933 interval (potentially due to drift in a small population) but then became more similar to other European populations thereafter (potentially due to increased migration). Within each century-scale time period, our temporal sampling allows us to document compelling candidates for recent natural selection. In some cases, we gain insights regarding previously implicated selection candidates, such as ChKov1, for which our inferred timing of selection favors the hypothesis of antiviral resistance over insecticide resistance. Other candidates are novel, such as the circadian-related gene Ahcy, which yields a selection signal that rivals that of the DDT resistance gene Cyp6g1. These insights deepen our understanding of recent evolution in a model system, and highlight the potential of future museomic studies.

Highly accurate long reads are crucial for realizing the potential of biodiversity genomics.

BACKGROUND: Generating the most contiguous, accurate genome assemblies given available sequencing technologies is a long-standing challenge in genome science. With the rise of long-read sequencing, assembly challenges have shifted from merely increasing contiguity to correctly assembling complex, repetitive regions of interest, ideally in a phased manner. At present, researchers largely choose between two types of long read data: longer, but less accurate sequences, or highly accurate, but shorter reads (i.e., >Q20 or 99% accurate). To better understand how these types of long-read data as well as scale of data (i.e., mean length and sequencing depth) influence genome assembly outcomes, we compared genome assemblies for a caddisfly, Hesperophylax magnus, generated with longer, but less accurate, Oxford Nanopore (ONT) R9.4.1 and highly accurate PacBio HiFi (HiFi) data. Next, we expanded this comparison to consider the influence of highly accurate long-read sequence data on genome assemblies across 6750 plant and animal genomes. For this broader comparison, we used HiFi data as a surrogate for highly accurate long-reads broadly as we could identify when they were used from GenBank metadata. RESULTS: HiFi reads outperformed ONT reads in all assembly metrics tested for the caddisfly data set and allowed for accurate assembly of the repetitive ~ 20 Kb H-fibroin gene. Across plants and animals, genome assemblies that incorporated HiFi reads were also more contiguous. For plants, the average HiFi assembly was 501% more contiguous (mean contig N50 = 20.5 Mb) than those generated with any other long-read data (mean contig N50 = 4.1 Mb). For animals, HiFi assemblies were 226% more contiguous (mean contig N50 = 20.9 Mb) versus other long-read assemblies (mean contig N50 = 9.3 Mb). In plants, we also found limited evidence that HiFi may offer a unique solution for overcoming genomic complexity that scales with assembly size. CONCLUSIONS: Highly accurate long-reads generated with HiFi or analogous technologies represent a key tool for maximizing genome assembly quality for a wide swath of plants and animals. This finding is particularly important when resources only allow for one type of sequencing data to be generated. Ultimately, to realize the promise of biodiversity genomics, we call for greater uptake of highly accurate long-reads in future studies.

Strong Positive Selection in Aedes aegypti and the Rapid Evolution of Insecticide Resistance.

Aedes aegypti vectors the pathogens that cause dengue, yellow fever, Zika virus, and chikungunya and is a serious threat to public health in tropical regions. Decades of work has illuminated many aspects of Ae. aegypti's biology and global population structure and has identified insecticide resistance genes; however, the size and repetitive nature of the Ae. aegypti genome have limited our ability to detect positive selection in this mosquito. Combining new whole genome sequences from Colombia with publicly available data from Africa and the Americas, we identify multiple strong candidate selective sweeps in Ae. aegypti, many of which overlap genes linked to or implicated in insecticide resistance. We examine the voltage-gated sodium channel gene in three American cohorts and find evidence for successive selective sweeps in Colombia. The most recent sweep encompasses an intermediate-frequency haplotype containing four candidate insecticide resistance mutations that are in near-perfect linkage disequilibrium with one another in the Colombian sample. We hypothesize that this haplotype may continue to rapidly increase in frequency and perhaps spread geographically in the coming years. These results extend our knowledge of how insecticide resistance has evolved in this species and add to a growing body of evidence suggesting that Ae. aegypti has an extensive genomic capacity to rapidly adapt to insecticide-based vector control.

Drosophila p53 isoforms have overlapping and distinct functions in germline genome integrity and oocyte quality control.

p53 gene family members in humans and other organisms encode a large number of protein isoforms whose functions are largely undefined. Using Drosophila as a model, we find that a p53B isoform is expressed predominantly in the germline where it colocalizes with p53A into subnuclear bodies. It is only p53A, however, that mediates the apoptotic response to ionizing radiation in the germline and soma. In contrast, p53A and p53B are both required for the normal repair of meiotic DNA breaks, an activity that is more crucial when meiotic recombination is defective. We find that in oocytes with persistent DNA breaks p53A is also required to activate a meiotic pachytene checkpoint. Our findings indicate that Drosophila p53 isoforms have DNA lesion and cell type-specific functions, with parallels to the functions of mammalian p53 family members in the genotoxic stress response and oocyte quality control.

Chromosome-level genome assembly of Bactrocera dorsalis reveals its adaptation and invasion mechanisms.

Bactrocera dorsalis is an invasive polyphagous pest causing considerable ecological and economic damage worldwide. We report a high-quality chromosome-level genome assembly and combine various transcriptome data to explore the molecular mechanisms of its rapid adaptation to new environments. The expansions of the DDE transposase superfamily and key gene families related to environmental adaptation and enrichment of the expanded and unique gene families in metabolism and defence response pathways explain its environmental adaptability. The relatively high but not significantly different expression of heat-shock proteins, regardless of the environmental conditions, suggests an intrinsic mechanism underlying its adaptation to high temperatures. The mitogen-activated protein kinase pathway plays a key role in adaptation to new environments. The prevalence of duplicated genes in its genome explains the diversity in the B. dorsalis complex. These findings provide insights into the genetic basis of the invasiveness and diversity of B. dorsalis, explaining its rapid adaptation and expansion.

The Complete Genome of Chelonus insularis Reveals Dynamic Arrangement of Genome Components in Parasitoid Wasps That Produce Bracoviruses.

Bracoviruses (BVs) are endogenized nudiviruses in parasitoid wasps of the microgastroid complex (family Braconidae). Microgastroid wasps have coopted nudivirus genes to produce replication-defective virions that females use to transfer virulence genes to parasitized hosts. The microgastroid complex further consists of six subfamilies and ∼50,000 species but current understanding of BV gene inventories and organization primarily derives from analysis of two wasp species in the subfamily Microgastrinae (Microplitis demolitor and Cotesia congregata) that produce M. demolitor BV (MdBV) and C. congregata BV (CcBV). Notably, several genomic features of MdBV and CcBV remain conserved since divergence of M. demolitor and C. congregata ∼53 million years ago (MYA). However, it is unknown whether these conserved traits more broadly reflect BV evolution, because no complete genomes exist for any microgastroid wasps outside the Microgastrinae. In this regard, the subfamily Cheloninae is of greatest interest because it diverged earliest from the Microgastrinae (∼85 MYA) after endogenization of the nudivirus ancestor. Here, we present the complete genome of Chelonus insularis, which is an egg-larval parasitoid in the Cheloninae that produces C. insularis BV (CinsBV). We report that the inventory of nudivirus genes in C. insularis is conserved but are dissimilarly organized compared to M. demolitor and C. congregata. Reciprocally, CinsBV proviral segments share organizational features with MdBV and CcBV but virulence gene inventories exhibit almost no overlap. Altogether, our results point to the functional importance of a conserved inventory of nudivirus genes and a dynamic set of virulence genes for the successful parasitism of hosts. Our results also suggest organizational features previously identified in MdBV and CcBV are likely not essential for BV virion formation. IMPORTANCE Bracoviruses are a remarkable example of virus endogenization, because large sets of genes from a nudivirus ancestor continue to produce virions that thousands of wasp species rely upon to parasitize hosts. Understanding how these genes interact and have been coopted by wasps for novel functions is of broad interest in the study of virus evolution. This work characterizes bracovirus genome components in the parasitoid wasp Chelonus insularis, which together with existing wasp genomes captures a large portion of the diversity among wasp species that produce bracoviruses. Results provide new information about how bracovirus genome components are organized in different wasps while also providing additional insights on key features required for function.

InsectBase 2.0: a comprehensive gene resource for insects.

Insects are the largest group of animals on the planet and have a huge impact on human life by providing resources, transmitting diseases, and damaging agricultural crop production. Recently, a large amount of insect genome and gene data has been generated. A comprehensive database is highly desirable for managing, sharing, and mining these resources. Here, we present an updated database, InsectBase 2.0 (http://v2.insect-genome.com/), covering 815 insect genomes, 25 805 transcriptomes and >16 million genes, including 15 045 111 coding sequences, 3 436 022 3'UTRs, 4 345 664 5'UTRs, 112 162 miRNAs and 1 293 430 lncRNAs. In addition, we used an in-house standard pipeline to annotate 1 434 653 genes belonging to 164 gene families; 215 986 potential horizontally transferred genes; and 419 KEGG pathways. Web services such as BLAST, JBrowse2 and Synteny Viewer are provided for searching and visualization. InsectBase 2.0 serves as a valuable platform for entomologists and researchers in the related communities of animal evolution and invertebrate comparative genomics.

Hymenoptera Genome Database: new genomes and annotation datasets for improved go enrichment and orthologue analyses.

We report an update of the Hymenoptera Genome Database (HGD; http://HymenopteraGenome.org), a genomic database of hymenopteran insect species. The number of species represented in HGD has nearly tripled, with fifty-eight hymenopteran species, including twenty bees, twenty-three ants, eleven wasps and four sawflies. With a reorganized website, HGD continues to provide the HymenopteraMine genomic data mining warehouse and JBrowse/Apollo genome browsers integrated with BLAST. We have computed Gene Ontology (GO) annotations for all species, greatly enhancing the GO annotation data gathered from UniProt with more than a ten-fold increase in the number of GO-annotated genes. We have also generated orthology datasets that encompass all HGD species and provide orthologue clusters for fourteen taxonomic groups. The new GO annotation and orthology data are available for searching in HymenopteraMine, and as bulk file downloads.

The chromosome-level genome of Chinese praying mantis Tenodera sinensis (Mantodea: Mantidae) reveals its biology as a predator.

BACKGROUND: The Chinese praying mantis, Tenodera sinensis (Saussure), is a carnivorous insect that preys on a variety of arthropods and small vertebrates, including pest species. Several studies have been conducted to understand its behavior and physiology. However, there is limited knowledge about the genetic information underlying its genome evolution, digestive demands, and predatory behaviors. FINDINGS: Here we have assembled the chromosome-level genome of T. sinensis, representing the first sequenced genome of the family Mantidae, with a genome size of 2.54 Gb and scaffold N50 of 174.78 Mb. Our analyses revealed that 98.6% of BUSCO genes are present, resulting in a well-annotated assembly compared to other insect genomes, containing 25,022 genes. The reconstructed phylogenetic analysis showed the expected topology placing the praying mantis in an appropriate position. Analysis of transposon elements suggested the Gypsy/Dirs family, which belongs to long terminal repeat (LTR) transposons, may be a key factor resulting in the larger genome size. The genome shows expansions in several digestion and detoxification associated gene families, including trypsin and glycosyl hydrolase (GH) genes, ATP-binding cassette (ABC) transporter, and carboxylesterase (CarE), reflecting the possible genomic basis of digestive demands. Furthermore, we have found 1 ultraviolet-sensitive opsin and 2 long-wavelength-sensitive (LWS) opsins, emphasizing the core role of LWS opsins in regulating predatory behaviors. CONCLUSIONS: The high-quality genome assembly of the praying mantis provides a valuable repository for studying the evolutionary patterns of the mantis genomes and the gene expression profiles of insect predators.

A strategy for a high enrichment of insect mitochondrial DNA for mitogenomic analysis.

Next-generation sequencing has dramatically fostered insect mitogenomic research in recent years. However, studies on the insect mitochondrial genome (mitogenome) assembly mainly rely on the sequencing data from total DNA, which is not cost-effective as a huge data from nuclear DNA are wasted. Besides, many mitogenomic studies require genomic information from individual organisms, whereas the DNA yield from small individual insects is too low to meet the sequencing requirements. Here, we describe a strategy for a high enrichment of insect mitochondrial DNA (mtDNA) using rolling circle amplification (RCA) technique. This strategy consists of standard DNA extraction, RCA enrichment, next-generation sequencing and mitogenome assembly. We have evaluated the performance of this strategy on nine insect species representing eight families of insecta, three other invertebrates, and even two vertebrate specimens. Results show that our strategy is especially suitable for insects, which allows almost all tested insect mtDNA contents to reach 80% and above. A further examination of enrichment efficiency of our strategy among different taxa shows that it is also applicable to other invertebrates and even some vertebrates such as Rhacophorus and ptyas species, although its enrichment efficiency in these groups is lower than that of insects. After treatment with our strategy, small flux sequencing data can realize the assembly of mitogenome with deep coverage, providing a solid base for subsequent mitogenome-based studies.

Genome evolution in an agricultural pest following adoption of transgenic crops.

Replacing synthetic insecticides with transgenic crops for pest management has been economically and environmentally beneficial, but these benefits erode as pests evolve resistance. It has been proposed that novel genomic approaches could track molecular signals of emerging resistance to aid in resistance management. To test this, we quantified patterns of genomic change in Helicoverpa zea, a major lepidopteran pest and target of transgenic Bacillus thuringiensis (Bt) crops, between 2002 and 2017 as both Bt crop adoption and resistance increased in North America. Genomic scans of wild H. zea were paired with quantitative trait locus (QTL) analyses and showed the genomic architecture of field-evolved Cry1Ab resistance was polygenic, likely arising from standing genetic variation. Resistance to pyramided Cry1A.105 and Cry2Ab2 toxins was controlled by fewer loci. Of the 11 previously described Bt resistance genes, 9 showed no significant change over time or major effects on resistance. We were unable to rule out a contribution of aminopeptidases (apns), as a cluster of apn genes were found within a Cry-associated QTL. Molecular signals of emerging Bt resistance were detectable as early as 2012 in our samples, and we discuss the potential and pitfalls of whole-genome analysis for resistance monitoring based on our findings. This first study of Bt resistance evolution using whole-genome analysis of field-collected specimens demonstrates the need for a more holistic approach to examining rapid adaptation to novel selection pressures in agricultural ecosystems.

Modulation of Global Gene Expression by Aneuploidy and CNV of Dosage Sensitive Regulatory Genes.

Aneuploidy, which disrupts the genetic balance due to partial genome dosage changes, is usually more detrimental than euploidy variation. To investigate the modulation of gene expression in aneuploidy, we analyzed the transcriptome sequencing data of autosomal and sex chromosome trisomy in Drosophila. The results showed that most genes on the varied chromosome (cis) present dosage compensation, while the remainder of the genome (trans) produce widespread inverse dosage effects. Some altered functions and pathways were identified as the common characteristics of aneuploidy, and several possible regulatory genes were screened for an inverse dosage effect. Furthermore, we demonstrated that dosage changes of inverse regulator Inr-a/pcf11 can produce a genome-wide inverse dosage effect. All these findings suggest that the mechanism of genomic imbalance is related to the changes in the stoichiometric relationships of macromolecular complex members that affect the overall function. These studies may deepen the understanding of gene expression regulatory mechanisms.