Functional polymorphism of CYCLE underlies the diapause variation in moths.

Diapause is a common seasonal adaptive strategy that regulates annual timing in insects. Very few causal loci underlying diapause variation have yet been identified. By leveraging cross-mapping and genome-wide association analysis, we identified the N terminus of the clock protein CYCLE as a major causal effector underlying embryonic diapause differences in the silk moth. We found that the nondiapause phenotype in polyvoltine strains results from a specific deletion that disrupts an alternative isoform of CYCLE. We further demonstrated that different CYCLE isoforms contribute to a functional diversity in modulating circadian rhythms and diapause, which has been preserved in Lepidoptera for at least 110 million years. Our study proposes a model that explains how adaptive phenotypes can evolve rapidly without affecting related essential functions. Editor's summary: Akin to hibernation in other species, diapause allows insects to remain dormant through harsh seasons. Zheng et al. examined the genetic basis of this trait in silk moths (Bombyx mori), the strains of which vary in their diapause timing and expression. Several genomic loci associated with this variation, including a peak encompassing the gene Cycle. This transcription factor is well known for its importance in circadian rhythms across species. Strains lacking the diapause phenotype altogether were homozygous for a frameshift mutation that completely disrupted a single isoform of Cycle. Given the conservation of Cycle across Lepidoptera (moths and butterflies), this gene may control diapause more broadly. —Corinne Simonti INTRODUCTION: Seasonal adaptation is crucial for the survival of natural animals. As a specific form of dormancy, diapause halts development to endure unfavorable seasons and commonly serves as a seasonal adaptative strategy in insects. Diapause traits vary pronouncedly within and among species, contributing to life history diversification in response to latitude and climate changes. Elucidating the genetic basis of diapause variation enhances our understanding of how insects rapidly adapt to changing environments for speciation and range expansion and allows for prediction of their further adaptation under global climate change. Despite broad interest, the molecular bases underlying the diversity of diapause traits and annual timing remain largely unexplored in insects, particularly for specified causal alleles in nonmodel species. RATIONALE: The domestic silk moth (Bombyx mori) exhibits characteristic diapause phenotypes across strains. To synchronize their life cycles with human activities, most domestic strains produce one or two generations a year through embryonic diapause (univoltinism or bivoltinism), depending on the perception of environmental cues in the maternal generation. By contrast, local strains originating from the tropics do not enter diapause regardless of environmental conditions (polyvoltinism). The rich variety of resources and well-documented phenotypes enable in-depth studies in this species to characterize the molecular mechanisms underlying diapause traits in Lepidoptera (moths and butterflies). We aimed to combine canonical genetic approaches and high-throughput genome-wide investigations to (i) identify causal loci responsible for the diapause variation in B. mori and (ii) test whether these identified loci have analogous effects in other species of Lepidoptera. RESULTS: We generated cross-mapping progeny between a facultative diapause bivoltine strain and a nondiapause polyvoltine strain of silk moths that mapped the major locus responsible for diapause variation on chromosome Z. By integrating with genome-wide association of 255 parental strains exhibiting diapause polymorphism, we localized the most effecting locus to the 5′ region of a central clock gene, Cycle (an insect homolog of BMAL1 in vertebrates). We identified a 1–base pair deletion exclusive to nondiapause strains as the key causal allele that disrupts one specific isoform of CYC (CYC-C), while preserving the complete copies of alternative isoforms (CYC-A/B). Multidimensional lines of evidence supported the involvement of CYC-C in controlling diapause in the silk moth, linking it with multiple functional modules potentially related to diapause modulation, and showed that CYC-A/B may play the native role of CYC as a fundamental component of circadian regulation. We further showed that the expression of alternative CYC transcripts is subject to independent regulation and that this functional diversity has been reserved across a wide range of Lepidoptera taxa. On the basis of isoform-specific mutagenesis, we validated the effect of CYC-C in initiating larval diapause in the Asian corn borer (Ostrinia furnacalis) that diverged with the silk moth approximately 100 million years ago. CONCLUSION: In this study, we defined the additional function of the circadian protein CYC, through one of its alternative isoforms, in controlling the entry into diapause in Lepidoptera, and identified a newly derived mutation in this isoform, arising along with the domestication in tropical regions, which is responsible for the diapause variation in silk moths. The potential functional diversity of CYC is widespread in Lepidoptera, which may reconcile the flexibility of seasonal adaptation with the functional constraint on fundamental circadian regulation. Combined with previously documented examples in TIMELESS (TIM) and PERIOD (PER) in Drosophila, the diverse isoforms of central clock genes may serve as common targets of selection for seasonal adaptation in insects. A tale of two roles: Central clock genes maintain circadian rhythm and confer variable seasonal timing of diapause. Genetic analyses identify a causal deletion that disrupts an isoform of CYC (C), thereby affecting diapause incidence in domestic silk moths (the brown unhatched eggs). The isoform-based functional complexity of clock genes may widely contribute to seasonal adaptation in insects, supported by previously documented similar cases involving TIM and PER in Drosophila (circled in red). GWAS, genome-wide association study (a detailed explanation of this model is available in fig. S21). [ABSTRACT FROM AUTHOR]