Introduction
In seasonal environments, organisms are faced with abiotic stressors and limited resources. In order to cope with seasonal challenges, many organisms enter some form of dormancy (Wilsterman, Ballinger, & Williams, 2021). For insects, this dormancy comes in the form of diapause, which is a pre-programmed endogenous dormancy that is associated with cessation of development, increased stress tolerance, and decreased metabolic expenditure (Denlinger, 2022). Diapause is not a single process, as it is composed of several stages associated with different physiological and cellular processes (V. Koštál, 2006). Due to the complex nature of diapause there is a high order of physiological coordination required to initiate and successfully complete diapause (Denlinger, 2002; V. Koštál, Štětina, Poupardin, Korbelová, & Bruce, 2017). Although our understanding of the hormonal regulation of diapause is becoming more clear, much remains unknown about the mechanisms giving rise to relevant hormonal changes (Denlinger, 2022; Denlinger, Yocum, & Rinehart, 2012). Here we investigate the role of post-transcriptional modification throughout diapause by exploring microRNA (miRNA) expression across important diapause transitions. Ultimately our goal is to gain sufficient understanding of the environmentally induced gene regulatory network controlling diapause (Lehmann et al., 2018; Lehmann et al., 2016; Pruisscher, Lehmann, Nylin, Gotthard, & Wheat, 2022), that such information via intersection with among population, heritable variation in diapause phenotypes (Lees & Archer, 1980; Pruisscher, Nylin, Wheat, & Gotthard, 2021), will begin to reveal how diapause as a phenotype evolves.
miRNAs work as a posttranscriptional gene-regulatory network influencing diverse phenotypes and likely play an important role in adaptation (Biggar & Storey, 2018; Fruciano, Franchini, & Jones, 2021; Lucas, Zhao, Liu, & Raikhel, 2015). miRNAs are 18-22 nucleotides (nt) long segments of RNA that have a characteristic hairpin structure (Wienholds & Plasterk, 2005). They are produced in the nucleus, exported to the cytosol where they are processed into mature sequences that then eventually bind to mRNA 3’-UTR to direct post-transcriptional repression (Yates, Norbury, & Gilbert, 2013). A single miRNA can target several hundred mRNAs, making them an interesting set of candidate loci that can have large impacts across diverse physiological processes (Schnall-Levin, Zhao, Perrimon, & Berger, 2010). Due to the multifaceted nature of diapause, which involves the coordination of the aforementioned diverse physiological processes, there is a need for understanding more about miRNA’s role as they are an understudied functional layer that could have major impacts on regulating diapause progression (Reynolds, 2019).
To date, only a handful of studies have investigated the role of miRNAs in the diapause phenotypes of insects. These have either had a direct focus upon specific miRNAs and their correlated expression change across diapause or non-diapause states, or they have been global analyses, looking at all the detectable miRNAs and how their expression changes, but again, these have been limited to a comparison of only diapause vs. non-diapause states (Batz, Goff, & Armbruster, 2017; Meuti, Bautista-Jimenez, & Reynolds, 2018; Reynolds, Peyton, & Denlinger, 2017). Additionally, there have been several studies focusing on miRNA expression patterns during aestivation (T. Duan, Li, Tan, Li, & Pang, 2021; T. F. Duan, Li, Wang, & Pang, 2023). Together, these studies have found a range of miRNAs that are differentially expressed during dormancy, suggesting that miRNA likely play an important role in diapause. Nevertheless, more detailed study of miRNA expression temporal dynamics across diapause, and their role in transitions between diapause stages, is needed given the dynamic expression changes expected and seen at the mRNA level (V. Koštál et al., 2017; Pruisscher et al., 2022). Ultimately, such studies will result in candidate miRNA genes and targets, whose contribution to diapause can be functionally validated (Gudmunds, Wheat, Khila, & Husby, 2022), and whose variation among populations used to reveal how diapause phenotypes evolve.
Here, we focus our efforts on Pieris napi (Pieridae, Lepidoptera), an emerging model for understanding the ecological drivers, physiological mechanisms and molecular underpinnings of insect diapause. Pieris napi is broadly distributed across Eurasia, spanning a wide range of seasonal environments, with among population variation in critical photoperiod induction and termination length (Lees & Archer, 1980; Pruisscher et al., 2021). In highly seasonal environments pupae can facultatively diapause when cued by a short photoperiod (Forsberg & Wiklund, 1988). There is an extensive understanding of the timing of diapause stages and how sensitive they are to environmental influences (Lehmann, Van Der Bijl, Nylin, Wheat, & Gotthard, 2017), as well as the mechanism of diapause progression via the prothoracicotropic hormone (PTTH)-ecdysone axis (Süess et al., 2022). There is also extensive data on gene expression, metabolome, and lipidome, making it a valuable resource for linking miRNA targets with known physiological function (Lehmann et al., 2018; Lehmann et al., 2016; Pruisscher et al., 2022).
Here we use a time course analysis across the initiation, maintenance, and termination stages of diapause, following time points used in a previous RNA-seq study (Pruisscher et al., 2022). Our goal is to identify candidate miRNAs that may be important in regulating the progression of diapause, allowing future studies to query their role in among population variation in related phenotypes. First, we identify the miRNA genes and their location in the genome. Second, we then look at expression changes through diapause, cluster these changes into distinct patterns, and compare them with mRNA results. Finally, we identify interesting miRNA candidate genes based upon differential expression across multiple tissues during a critical diapause transition, along with some previous candidates from the diapause literature