Hirudo nipponia, a blood-sucking leech native to East Asia, possesses a rich repertoire of active ingredients in its saliva, showcasing significant medical potential due to its anticoagulant, anti-inflammatory, and antibacterial effects against human diseases. Despite previous studies on the transcriptomic and proteomic characteristics of leech saliva, which have identified medicinal compounds, our knowledge of tissue-specific transcriptomes and their spatial expression patterns remains incomplete. In this study, we conducted an extensive transcriptomic profiling of the salivary gland tissue in H. nipponia based on de novo assemblies of tissue-specific transcriptomes from the salivary gland, teeth, and general head region. Through gene ontology (GO) analysis and hierarchical clustering, we discovered a novel set of anti-coagulant factors—i.e., Hni-Antistasin, Hni-Ghilanten, Hni-Bdellin, Hni-Hirudin—as well as a previously unrecognized immune-related gene, Hni-GLIPR1 and uncharacterized salivary gland specific transcripts. By employing in situ hybridization, we provided the first visualization of gene expression sites within the salivary gland of H. nipponia. Our findings expand on our understanding of transcripts specifically expressed in the salivary gland of blood-sucking leeches, offering valuable resources for the exploration of previously unidentified substances with medicinal applications.

Background

Slit and Robo are evolutionarily conserved ligand and receptor proteins, respectively, but the number of slit and robo gene paralogs varies across recent bilaterian genomes. Previous studies indicate that this ligand-receptor complex is involved in axon guidance. Given the lack of data regarding Slit/Robo in the Lophotrochozoa compared to Ecdysozoa and Deuterostomia, the present study aims to identify and characterize the expression of Slit/Robo orthologs in leech development.

Results

We identified one slit (Hau-slit), and two robo genes (Hau-robo1 and Hau-robo2), and characterized their expression spatiotemporally during the development of the glossiphoniid leech Helobdella austinensis. Throughout segmentation and organogenesis, Hau-slit and Hau-robo1 are broadly expressed in complex and roughly complementary patterns in the ventral and dorsal midline, nerve ganglia, foregut, visceral mesoderm and/or endoderm of the crop, rectum and reproductive organs. Before yolk exhaustion, Hau-robo1 is also expressed where the pigmented eye spots will later develop, and Hau-slit is expressed in the area between these future eye spots. In contrast, Hau-robo2 expression is extremely limited, appearing first in the developing pigmented eye spots, and later in the three additional pairs of cryptic eye spots in head region that never develop pigment. Comparing the expression of robo orthologs between H. austinensis and another glossiphoniid leech, Alboglossiphonia lata allows to that robo1 and robo2 operate combinatorially to differentially specify pigmented and cryptic eyespots within the glossiphoniid leeches.

Conclusions

Our results support a conserved role in neurogenesis, midline formation and eye spot development for Slit/Robo in the Lophotrochozoa, and provide relevant data for evo-devo studies related to nervous system evolution.

We estimated the genome size of a relict longhorn beetle, Callipogon relictus Semenov (Cerambycidae: Prioninae)—the Korean natural monument no. 218 and a Class I endangered species—using a combination of flow cytometry and k-mer analysis. The two independent methods enabled accurate estimation of the genome size in Cerambycidae for the first time. The genome size of C. relictus was 1.8 ± 0.2 Gb, representing one of the largest cerambycid genomes studied to date. An accurate estimation of genome size of a critically endangered longhorned beetle is a major milestone in our understanding and characterization of the C. relictus genome. Ultimately, the findings provide useful insight into insect genomics and genome size evolution, particularly among beetles.

 Stag beetles (family Lucanidae Latreille, 1804) are one of the earliest branching lineages of scarab beetles that are characterized by the striking development of the male mandibles. Despite stag beetles' popularity among traditional taxonomists and amateur collectors, there has been almost no study of lucanid relationships and evolution. Entomologists, including Jeannel (1942), have long recognized resemblance between the austral stag beetles of the tribes Chiasognathini, Colophonini, Lamprimini, Pholidotini, Rhyssonotini, and Streptocerini, but this hypothesis of their close relationship across the continents has never been tested. To gain further insight into lucanid phylogeny and biogeography, we reconstructed the first molecular phylogeny of world stag beetles using DNA sequences from mitochondrial 16S rDNA, nuclear 18S and 28S rDNA, and the nuclear protein-coding (NPC) gene wingless for 93 lucanid species representing all extant subfamilies and 24 out of the 27 tribes, together with 14 representative samples of other early branching scarabaeoid families and two staphyliniform beetle families as outgroups. Both Bayesian inference (BI) and maximum likelihood inference (MLI) strongly supported the monophyly of Lucanidae sensu lato that includes Diphyllostomatidae. Within Lucanidae sensu stricto, the subfamilies Lucaninae and Lampriminae appeared monophyletic under both methods of phylogenetic inferences; however, Aesalinae and Syndesinae were found to be polyphyletic. A time-calibrated phylogeny based on five fossil data estimated the origin of crown group Lucanidae as circa 160 million years ago (MYA). Divergence between the Neotropical and Australasian groups of the Chiasognathini was estimated to be circa 47MYA, with the South African Colophonini branching off from the ancient Chiasognathini lineage around 87MYA. Another Gondwanan relationship was recovered between the Australasian Eucarteria and the Neotropical Casignetus, which diverged circa 58MYA. Lastly, as Jeannel's hypothesis predicted, divergence within Lampriminae between the Australasian Lamprima and the Neotropical Streptocerus was estimated to be circa 37MYA. The split of these lineages were generally concordant with the pattern of continental break-up of the super-continent Gondwana, and our biogeographic reconstructions based on the dispersal-extinction-cladogenesis model (DEC) corroborate our view that the divergences in these austral lineages were caused by vicariance events following the Gondwanan break-up. In addition, the phylogenetic position and geographic origin of the Hawaiian genus Apterocyclus was revealed for the first time. Overall, our results provide the framework toward studying lucanid relationships and divergence time estimates, which allowed for more accurate biogeographic explanations and discussions on ancestral lucanids and the evolutionary origin of the enlarged male mandibles.

This paper provides a review of the family Lucanidae in Korea, which consists of 17 species belonging to 9 genera. One new species, Dorcus tenuihirsutus sp. nov., is described and the following taxonomical changes are proposed: Aegus laevicollis Saunders, 1854 is newly identified as Aegus laevicollis subnitidus Waterhouse, 1873; Macrodorcas striatipennis Motschulsky, 1861 is removed from the Korean fauna because this record is thought to have been based on the misinterpretation of locality and misidentification; Neolucanus saundersii Parry, 1864 is removed from the Korean fauna because this record is thought to have been based on misidentification.