Leeches are well-known annelids due to their obligate blood-feeding habits. Some leech species secrete various biologically active substances which have important medical and pharmaceutical value in antithrombotic treatments. In this study, we provided a high-quality genome of the Asian buffalo leech (Hirudinaria manillensis), based on which we performed a systematic identification of potential antithrombotic genes and their corresponding proteins. Combining automatic and manual prediction, we identified 21 antithrombotic gene families including fourteen coagulation inhibitors, three platelet aggregation inhibitors, three fibrinolysis enhancers, and one tissue penetration enhancer. A total of 72 antithrombotic genes, including two pseudogenes, were identified, including most of their corresponding proteins forming three or more disulfide bonds. Three protein families (LDTI, antistasin, and granulin) had internal tandem repeats containing 6, 10, and 12 conserved cysteines, respectively. We also measured the anticoagulant activities of the five identified hirudins (hirudin_Hman1 ~ hirudin_Hman5). The results showed that three (hirudin_Hman1, hirudin_Hman2, and hirudin_Hman5), but not the remaining two, exhibited anticoagulant activities. Our study provides the most comprehensive collection of antithrombotic biomacromolecules from a leech to date. These results will greatly facilitate the research and application of leech derivatives for medical and pharmaceutical purposes in the treatment of thrombotic diseases.

Despite being a non-hematophagous leech, Whitmania pigra is widely used in traditional Chinese medicine for the treatment of antithrombotic diseases. In this study, we provide a high quality genome of W. pigra and based on which, we performed a systematic identification of the potential antithrombotic genes and their corresponding proteins. We identified twenty antithrombotic gene families including thirteen coagulation inhibitors, three platelet aggregation inhibitors, three fibrinolysis enhancers, and one tissue penetration enhancer. Unexpectedly, a total of 79 antithrombotic genes were identified, more than a typical blood-feeding Hirudinaria manillensis, which had only 72 antithrombotic genes. In addition, combining with the RNA-seq data of W. pigra and H. manillensis, we calculated the expression levels of antithrombotic genes of the two species. Five and four gene families had significantly higher and lower expression levels in W. pigra than in H. manillensis, respectively. These results showed that the number and expression level of antithrombotic genes of a non-hematophagous leech are not always less than those of a hematophagous leech. Our study provides the most comprehensive collection of antithrombotic biomacromolecules from a non-hematophagous leech to date and will significantly enhance the investigation and utilization of leech derivatives in thrombosis therapy research and pharmaceutical applications.

Vitellogenin (Vg), a storage protein, has been significantly studied for its egg yolk precursor role in oviparous animals. Recent studies found that vitellogenin and its Vg‐like homologs were fundamentally involved in many other biological processes in social insects such as female caste differences and oxidative stress resilience. In this study, we conducted the first large‐scale molecular evolutionary analyses of vitellogenin coding genes (Vg) and Vg‐like genes of bumble bees, a primitively eusocial insect belonging to the genus Bombus. We obtained sequences for each of the four genes (Vg, Vg‐like‐A, Vg‐like‐B, and Vg‐like‐C) from 27 bumble bee genomes (nine were newly sequenced in this study), and sequences from the two closest clades of Bombus, including five Apis species and five Tetragonula species. Our molecular evolutionary analyses show that in bumble bee, the conventional Vg experienced strong positive selection, while the Vg‐like genes showed overall relaxation of purifying selection. In Apis and Tetragonula; however, all four genes were found under purifying selection. Furthermore, the conventional Vg showed signs of strong positive selection in most subgenera in Bombus, apart from the obligate parasitic subgenus Psithyrus which has no caste differentiation. Together, these results indicate that the conventional Vg, a key pleiotropic gene in social insects, is the most rapidly evolving copy, potentially due to its multiple known social functions for both worker and queen castes. This study shows that concerted evolution and purifying selection shaped the evolution of the Vg gene family following their ancient gene duplication and may be the leading forces behind the evolution of new potential protein function enabling functional social pleiotropy.

The complete mitochondrial genome of Linoclostis gonatias (Lepidoptera: Xyloryctidae) is 15,528 bp in length, containing 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNAs, and a putative control region. Except for cox1 starts with CGA, all other PCGs use the typical ATN codons. Most of the PCGs end with the complete stop codon TAA, whereas cox2 terminates with the incomplete stop codon T. The BI analysis was performed using a dataset matrix containing 13 PCGs concatenated from the mitogenomes of Gelechioidea species. The monophyly of Xyloryctidae was highly supported. In addition, Oecophoridae was inferred as the sister group of Xyloryctidae.

The avian family Ardeidae comprises long-legged freshwater and coastal birds. There has been considerable disagreement concerning the intrafamilial relationships of Ardeidae. Mitochondrial cytochrome c oxidase subunit I (COI) was used as a marker for the identification and phylogenetic analysis of avian species. In the present study, we analyzed the COI barcodes of 32 species from 17 genera belonging to the family Ardeidae. Each bird species possessed a barcode distinct from that of other bird species except for Egretta thula and E. garzetta, which shared one barcoding sequence. Kimura two-parameter distances were calculated between barcodes. The average genetic distance between species was 34-fold higher than the average genetic distance within species. Neighbor-joining and maximum likelihood methods were used to construct phylogenetic trees. Most species could be discriminated by their distinct clades in the phylogenetic tree. Both methods of phylogenetic reconstruction suggested that Zebrilus, Tigrisoma, and Cochlearius were an offshoot of the primitive herons. COI gene analysis suggested that the other herons could be divided into two clades: Botaurinae and Ardeinae. Our results support the Great Egret and Intermediate Egret being in separate genera, Casmerodius and Mesophoyx, respectively.