Recent progress in the development of genomic techniques, including next generation sequencers, has greatly facilitated transcriptome analysis of ecologically important animals to reveal variations in mRNA expression patterns among closely related species and ecotypes within species [1–3]. Divergence in mRNA expression patterns is known to contribute to phenotypic evolution [4, 5], although amino acid alterations in proteins are also important . While a great deal is known about variation in mRNA expression profiles, information regarding naturally occurring variation in the expression patterns of small RNAs is limited, except for a few cases in plants [7, 8] and cichlids .
Non-coding small RNAs, ranging from 20 to 30 nucleotides in length, mediate the regulation of gene expression [10–13]. The members of one class of small RNAs, microRNAs (miRNAs), are typically 20–24 nucleotides long and are highly conserved across diverse taxa [11, 12]. miRNA post-transcriptionally regulates messenger RNA (mRNA). A miRNA interacts with ten to hundreds of target mRNAs to induce degradation or suppress translation . Another function of miRNA is epigenetic modification of genomic DNA: miRNAs interact with target DNAs to alter the chromatin state and suppress mRNA transcription . miRNAs comprise more than 1% of animal genes [15, 16], suggesting that they play important roles in many biological processes. Recent functional studies in laboratory model animals such as mice, flies, and nematodes have demonstrated that miRNAs are important for regulating development, growth, pathogen resistance, and neural functions [11, 12, 17–19].
Another class of small RNAs is the Piwi-interacting RNAs (piRNAs), which are typically 24–32 nucleotides long and interact with Piwi proteins to suppress the expression of transposons and other functional genes [13, 20]. piRNAs often possess uridine at the 5’-end (5′U) [13, 20]. piRNAs are expressed from intergenic repetitive elements, active transposons, and piRNA clusters. Importantly, piRNAs may contribute to hybrid dysgenesis [21, 22]. For example, some Drosophila strains contain transposons as well as piRNAs that inhibit transposon activity, whereas other strains lack both transposons and inhibitory piRNAs. Because piRNAs are maternally transmitted, hybrid progeny resulting from a cross between a mother lacking both transposons and piRNAs and a father possessing both will inherit the transposons, but not the inhibitory piRNAs. This abnormal activity of transposons in the germ line is likely to result in sterility [21, 22]. Thus, maternally transmitted piRNAs can explain why hybrid abnormalities are observed in only one direction of the inter-strain crosses. piRNAs are expressed not only in the gonads, but also in the brain, and they may be involved in the regulation of neuronal functions [23–25]. Compared with miRNAs, piRNAs are less well conserved across taxa. Yet another class of small RNAs, endogenous small interfering RNAs (endo-siRNAs), are usually 21 nucleotides and have been found in some taxa, including nematodes , flies [27–29], and mammals [30, 31], but it has not been well characterized in other animals.
Evolutionary genetic studies examining small RNAs are important for several reasons. First, genome-wide allele-specific mRNA expression analyses have revealed that both cis- and trans-regulatory changes contribute to differential expression of mRNAs among closely related species [32–34]. Small RNAs can act as trans-regulatory factors, which contribute to differential mRNA expression . Additionally, cis-regulatory changes may include mutations at the target sites of small RNAs ; for example, SNPs and insertion-deletion polymorphisms were identified within miRNA-binding sites of 3’-untranslated regions [37, 38]. Variations in small RNA transcriptomes and sequences were found to be associated with phenotypic variation in humans and laboratory animals. For example, miRNA and miRNA target site polymorphisms and mutations have been found in humans and are associated with disease susceptibility [39–42]. Polymorphism in a miRNA target site is associated with variation of muscularity in pigs . Second, small RNAs regulate translation of mRNAs. Therefore, transcriptome studies of mRNA alone can overlook the divergence in the total outcome of gene expression among species. Third, piRNAs may contribute to hybrid abnormalities (see above), but generalities regarding the roles of piRNA in different types of hybrid abnormalities remain unclear.
In the present study, we compared brain small RNA transcriptomes between incipient species of the threespine stickleback (Gasterosteus aculeatus). The threespine stickleback is a good model for linking ecological and genetic studies of adaptive evolution and speciation [44–52]. The threespine stickleback has undergone tremendous diversification over the past few million years [44, 45, 49]. Evolutionary diversification within the stickleback species complex led to a speciation continuum, which ranges from populations with interspecific phenotypic polymorphism to strong divergence with near-complete reproductive isolation [44, 53]. Recent genetic studies have revealed that differences in the expression of genes involved in morphological development [54, 55], physiology [56, 57], and immune function  may underlie adaptive divergence among populations or species. Sex bias of the mRNA transcriptome has also been investigated, and genes located on sex chromosomes were found to be female-biased, likely owing to Y-chromosome degeneration and lack of dosage compensation . However, transcriptome analysis of small RNAs has not yet been conducted in any stickleback system.
This study focused on Japanese threespine stickleback species pairs, including a Pacific Ocean form and a Japan Sea form. These sticklebacks diverged during a period of geographical isolation between the Sea of Japan and the Pacific Ocean approximately 1.5–2 million years ago [60, 61]. Currently, they are sympatric in eastern Hokkaido, but they are reproductively isolated with a very low level of hybridization [60–62]. In the Japan Sea form, a chromosomal fusion occurred between linkage group (LG) 9 and the ancestral Y chromosome (LG 19), resulting in the evolution of the X1X2Y multiple sex chromosome system . In contrast, the Pacific Ocean form has a simple XY sex chromosome system . Previously, we found that the Pacific Ocean and Japan Sea forms diverge in male courtship behaviors and female mate choice behaviors, contributing to behavioral isolation between these two forms [60, 62, 63]. Furthermore, we found that divergence in the intensity of courtship behaviour, which is important for mate choice, mapped to a neo-sex chromosome (LG 9).
To better understand the genetic mechanisms affecting behavioral differences between this Japanese stickleback species pair, it is essential to understand divergence in small RNA transcriptomes of the brain. Both miRNAs and piRNAs play important roles in a diverse array of neuronal functions such as neuronal differentiation, neural stem cell renewal, neuronal outgrowth, and dendritic spine morphogenesis [23, 24, 64]. Furthermore, variation in miRNA expression patterns in the brain may contribute to behavioral differences among laboratory mouse strains . Additionally, in the Japanese stickleback system, courtship dysfunction may exist in hybrids because a substantial number of hybrids did not perform any courtship behavior in a previous experiment (Supplementary data in ). Therefore, it is necessary to examine whether small RNAs, especially piRNAs, which may be regulating transposon activity in the brain, affect hybrid behavior. Here, we characterized the divergence in small RNA transcriptomes in the brain between the species pairs of Japanese threespine stickleback.