Hirudo nipponia (Annelida, Clitellata, and Hirudiniformes) is widely distributed in many regions of the globe (Liu et al., 2018). Physiologically, leeches are narrow, slightly flattened, cylindrical, and small-sized creatures used as raw material in Chinese traditional medicine with diverse functioning such as anticoagulation, exorcizing blood stasis, and promoting menstruation (Saglam, 2019). They are hermaphroditic where the male part matures first and then mates heterozygously, and oviparously (Ceylan, 2020). After hatching, leech seedlings moult once every 10 days, leeches grow for one age after one molt, 30–90 days is the optimum growth period for leeches, and it takes approximately 80 days to grow from seedling to adult size (Manav et al., 2019). However, H. nipponia has a prolonged maturation cycle with low reproduction ability as the H. nipponia begins to lay eggs in the second year. The average number of spawning cocoons of H. nipponia was the lowest compared with Whitmania pigra Whitman and Hirudinaria Poecilobdella manillensis (Ceylan, 2020).

H. nipponia as a traditional Chinese medicine contains hirudin which is the strongest thrombin natural inhibitor found so far (Straub et al., 2011; Liu et al., 2019) and it has unique properties of hypolipidemic, anti-tumor, anti-fibrotic, and anti-inflammatory. Furthermore, it has widely been used clinically to treat a variety of diseases such as hypertension, hyperlipidemia, cerebral infarction and malignancies (Sig et al., 2017; Pickrell et al., 2020; Wang et al., 2021). In addition, leeches have the ability to self-repair damage to the central nervous system and restore function, and are often used as model organisms in the field of neurobiology (Tasiemski and Salzet, 2017; Meiser et al., 2019; Heath-Heckman et al., 2021). In recent years, the number of wild leeches has gradually been decreased due to environmental destruction and increased hunting which results in high market demand but low supply. Thus, H. nipponia breeding at the farm level with selective traits is emerging everywhere all around the world (Chen et al., 2018; Yu et al., 2021).

Many studies have focused on the role of effective components in leech and their cultivation (Iwama et al., 2019; Wang et al., 2021). Recent advancements in high-throughput technologies provide an opportunity to dig out in-depth and comprehensive high quality leech genomes. Previously Liu et al. (2018) conducted a comparative transcriptomic study on three leech species including rod-shaped bull leech, broad-bodied golden leech and the cave mountain leech, and reported differentially expressed genes related to the olfactory transduction pathway among them. Moreover, another transcriptome-based study of the H. nipponia salivary gland identified a large number of anticoagulants, anti-inflammatory, antibacterial and anti-tumour proteins (Lu et al., 2018). But very little is reported related to the omics of H. nipponia, which is the major obstacle in the development and application of H. nipponia gene resources effectively. In this study, RNA-seq was used to sequence the transcriptome of H. nipponia at different developmental stages including larvae (L), young (Y), and adult (A) to provide a scientific basis for later research on the breeding, growth, and development of H. nipponia and related functional genes as well as the lncRNAs which are important for chromosomal modification, transcriptional activation, and interference.

In this study, we reported transcriptomic analyses from three different developmental stages (L, Y, and A) of H. nipponia, thus significantly expanding the genetic resources available for the leech. Our study also provides a new theoretical basis for studying the molecular genetics-based physiological mechanisms involved in the development of H. nipponia which ultimately benefits to improve the aquaculture privilege of H. nipponia. Based on growth characteristics, the leeches have categorized into three growth or developmental stages such as the larva that have not sucked blood for about 10 days after hatching, young worm included those that have sucked blood three times after hatching for about 2 months, and the adults sucked blood more than six times after 2 years of hatching and are sexually mature. Totally, 1,348 DEGs and 223 DelncRNAs were found in three growth periods, among them, the highest number of differential genes were identified in larval and adult leeches, while the lowest number of differential genes were figured out in young and adult leeches, which may be related to the fact that 3-month-old young leeches are similar in size to adult leeches (Tan et al., 2002; Lu et al., 2018). Furthermore, the GO and KEGG enrichment analyses showed that differential genes expression patterns were mainly associated with protein hydrolysis, enzyme activity, metabolism, and cell growth and development. As leeches grow rapidly during the young worm period, requiring large amounts of food intake and energy through synthesis, digestion, and metabolism of food, our data show that genes associated with digestion and metabolism were highly expressed in the young worm stage. In addition, genes encoding enzymes with protease activity (metalloproteases and thiol proteases) were found upregulated in young worms, consistent with studies showing that the non-reproductive stage (often referred to as the feeding stage) exhibits more aggressive feeding behavior than the adult stage (Zhao et al., 2021).

lncRNAs are long-stranded non-coding RNAs that are greater than 200 nt in length and do not have a coding role in living organisms. Although lncRNAs do not encode proteins but they have a crucial role in life activities. It has been demonstrated that lncRNAs are involved in X chromosome inactivation, chromosomal remodeling, transcriptional activation and interference as well as growth and development, metabolism and differentiation, and tumor production. Compared to other species, the study on leech lncRNAs is not well documented. In this study, during three developmental periods (L, Y, and A) a total of 223 differentially expressed lncRNA molecules were identified. Further analysis of these lncRNA molecules indicated that these lncRNA molecules are likely to play an important role in the growth and development of H. nipponia. Our study data provide new insights into the regulatory mechanisms of lncRNAs in the growth and development of H. nipponia.

Typically, actin, Ef1a, and GAPDH are used as internal reference genes for RT-qPCR testing, however we selected UBQ as internal reference gene. This is because β-actin or GAPDH are differentially expressed genes according to the results of our RNA-seq sequencing datat therefore we cannot use β-actin or GAPDH as internal reference genes for qRT-PCR analysis. Since there is lack of literature on the selection of internal reference genes in H. nipponia, we looked to other species and found that UBQ could be used as a regular internal reference gene (Robledo et al., 2014; Peña et al., 2010) and was not a differentially expressed gene in our RNA-seq data, so it was selected as an internal reference gene.

Previous studies have shown that the degree of muscular growth has changed significantly at different growth stages along with the underlying mechanisms regulating muscle growth might differ (Li et al., 2021). Our results indicated that the genes attributing muscular development were highly expressed and showed a tendency of upregulation as the leech body develops. The muscle-building block protein of annelids, paramyosin (MYSP), plays a key function in the muscular tension contraction process (Sonobe et al., 2016; Silva et al., 2020). In addition to performing basic contractile functions, proto-myosin (TPM) is the actin-binding protein in muscle filaments is also involved in many vital activities in the body, such as cytoplasmic division and signal transduction. It can bind helically to actin filaments and regulate interactions with myosin (Zhang et al., 2018; Jurewicz et al., 2020; Pavadai et al., 2020). Actin is an important member of the actin family that controls almost all forms of cell and organelle motility which also regulates growth, development, bioregulation, and repair (Hammer et al., 2019; Kadzik et al., 2020; Liu et al., 2020). Myosins consist of the myosin heavy chain (MYH) and the myosin light chain. The MYH family is one of the key members in the regulation of muscle development and affects muscle production and repair after injury, mainly through the regulation of cell proliferation and differentiation (Pan et al., 2016; Squire, 2019; Hill et al., 2021).

Leeches are typically hermaphroditic, with the male gonads maturing first than female gonads. Compared to other species of leeches, H. nipponia has low reproductive performance. Though analyzing the DEGs, we screened the genes or proteins associated with gonadal development or oogenesis. For example, SPA17, IQCG, ACTB, 5-HT, and the HOX gene family. The IQCG is also involved in spermatogenesis and is a key regulator of cilia/flagellar motility and is associated with sperm motility (Harris et al., 2014; Gonzalez-Gutierrez et al., 2021). Additionally, the studies have also reported that β-actin is potentially involved in sperm maturation, sperm excretion, and the ability to maintain sperm motility. Early studies on mammalian (guinea pig) sperm showed that actin was involved in sperm motility and random severance of actin filaments inhibited the flagella motility (Yenia, et al., 2007). Moreover, comparative proteomic study on normal and weak spermatozoa revealed that reduced expression of β-actin results in feeble spermatozoa (Martinez-Heredia et al., 2008). Similarly, reports on ovarian development have shown that 5-HT significantly play an important biological role in gonadal maturation and serial egg production (Kondo et al., 2017). The HOX gene is a family of regulatory molecules encoding highly conserved transcription factors whose expression is regulated by sex steroids, which are significantly involved in reproductive tract development, endometrial cycle growth, and embryo implantation (Du and Taylor, 2015; Cohen et al., 2020).

Furthermore, the leech is a fascinating model organism for researching brain regeneration mechanisms because of its capacity to regenerate the central nervous system (CNS) throughout its life cycle (Meriaux et al., 2011; Lu et al., 2018). Second, the leech’s CNS neurons are constantly expanding, implying that the process through which the leech develops and promotes synaptic connectivity may never be turned off. In our study, genes associated with neural development such as TTLL5, FLNA, PCDHGA10, SLC5A8, and NKX2-8 were expressed in all three developmental stages but were highly expressed in the larval stage. These genes are thought to have a major role in the formation of certain synaptic connections as well as the maintenance of neuronal energy status and function. They are regulated in neurons which re-establish axonal and synaptic connections. In addition to hirustasin, other anticoagulant genes including ANTA, ADAMTS, THST, and DECO were also expressed in the leech, and they were highly expressed in young leeches. Hirustasin and THST inhibited trypsin, chymotrypsin, histone G and tissue kinase-releasing enzyme, but not coagulation factor Xa activity (Kwak et al., 2019). In contrast, ANTA is a potent inhibitor of Factor Xa. Additionally, DECO inhibits the interaction of fibrinogen with platelet receptors expressed, on the glycoprotein IIb-IIIa complex. It prevents blood clotting when feeding and/or storing ingested blood (El-Obeid and Al-Badr, 1982). Adult leeches are frequently used as medicine in production applications and their anticoagulant features make it worthwhile to utilize leeches in medicine, like H. nipponia, which is commonly used for the treatment of various ailments (Koeppen et al., 2019; Singh et al., 2020) Our findings indicated that the anticoagulant gene is highly expressed in young leech suggesting the possibility that young leeches are more effective in medicine while further experimental verification of the exact function is needed.

In this study, the transcriptome of H. nipponia was systematically analyzed in three different developmental stages. Trait and histological observations revealed that adult leeches had significantly higher body length and body weight and a significantly higher number of myenteric tissues than larval and young leeches. Transcriptome analysis revealed that the number of DEGs was highest in L vs. A; DEGs enrichment analysis was mainly related to metabolic pathways; genes related to muscle development, genes related to gonadal development or oviposition regulation and genes related to neural development were significantly period-specific, indicating that these changes were associated with leech growth and development. In addition, we found that hirudin and anticoagulation-related genes were highly expressed in the young stage.