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