The Genome of Medicinal Leech (Whitmania pigra) and comparative genomic study for Exploration of Bioactive Ingredients

doi:10.21203/rs.3.rs-31354/v2

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Research article

The Genome of Medicinal Leech (Whitmania pigra) and comparative genomic study for Exploration of Bioactive Ingredients

https://doi.org/10.21203/rs.3.rs-31354/v2

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published 24 Jan, 2022

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Background

Leeches are classic annelids that have a huge diversity and closely related to people, especially medicinal leeches. Medicinal leeches have been widely utilized in medicine based on the pharmacological activities of their bioactive ingredients. Comparative genomic study of these leeches enables us to understand the difference among medicinal leeches and other leeches and facilitates the discovery of bioactive ingredients.

Results

In this study, we reported the genome of Whitmania pigra and compared it with Hirudo medicinalis and Helobdella robusta. The assembled genome size of W. pigra is 177 Mbp, close to the estimated genome. Approximately about 23% of the genome was repetitive. A total of 26,743 protein-coding genes were subsequently predicted. W. pigra have 12346 (46%) and 10295 (38%) orthologous genes with H. medicinalis and H. robusta, respectively. About 20% and 24% genes in W. pigra showed syntenic arrangement with H. medicinalis and H. robusta, respectively, revealed by gene synteny analysis. Furthermore, W. pigra, H. medicinalis and H. robusta expanded different gene families enriched in different biological processes. By inspecting genome distribution and gene structure of hirudin, we identified a new hirudin gene g17108 (hirudin_2) with different cysteine pattern. Finally, we systematically explored and compared the active substances in the genomes of three leeches. The results showed that W. pigra and H. medicinalis exceed H. robusta in both kinds and gene number of active molecules.

Conclusions

This study reported the genome of W. pigra and compared it with other two leeches, which provides an important genome resource and new insight into the exploration and development of bioactive molecules of medicinal leeches.

Epigenetics & Genomics

Whitmania pigra

Genome

Bioactive Ingredients

Helobdella robusta

Hirudo medicinalis

Leeches are segmented parasitic or predatory worms that belong to the phylum Annelida and the subclass Hirudinea with ability to extend or contract their bodies[1-3]. Most leeches live in freshwater environments, while some species can be found in terrestrial and marine environments. The best-known leeches, such as European medicinal leech Hirudo medicinalis are hematophagous, feeding on vertebrate blood and invertebrate hemolymph[4-6]. H. medicinalis attaches to the host by means of its two suckers and bites through the skin of its victim. Most leech species, however, are predatory, feeding primarily by swallowing other invertebrates. Almost 700 species of leeches are currently recognized, of which some 100 are marine species, 90 terrestrial and the remainder freshwater taxa.

Although a huge diversity and close relationship to people, we know little about the genome of leeches. In 2013, one leech H. robusta was sequenced to study bilaterian evolution [7]. H. robusta is a freshwater leech in the family glossiphoniidae, and a type of annelid with anterior and posterior suckers that are used for locomotion and feeding on blood. Its early development has been studied extensively. For another important family hirudinidae, the genome of H. medicinalis has just been published during the review period of this study[8, 9]. The family hirudinidae includes medicinal leeches which have been widely utilized in medical procedures for thousands of years. Because of their important bioactive ingredients, medicinal leeches, such as H. medicinalis and related species, have engendered great interest from pharmaceutical companies.

Comparative study of these available genomes of leeches facilitates the discovery of bioactive ingredients. In this study, we reported the genome of another medicinal leech W. pigra in the family hirudinidae and compared it with other two leeches (Figure 1A). W. pigra, an Asian freshwater leech, is non-blood feeding, despite the placement of this genus within the family hirudinidae [10]. The family hirudinidae also includes H. medicinalis and several other blood feeding species. W. pigra is considered macrophagous, suggesting that it commonly swallows or takes bites out of prey sources[11-13]. W. pigra is recorded in the current Chinese Pharmacopoeia as the source of leeches, and the most commonly available from Chinese commercial leech market[14]. We first analyzed the genome of W. pigra and conducted gene synteny analysis among the three leeches H. robusta, W. pigra, and H. medicinalis. Then we analyzed the expansion and contraction of gene family among seven related species (H. robusta, Lottia gigantea, Capitella teleta, Schmidtea mediterranea, Schistosoma mansoni, W. pigra, H. medicinalis). The sequence diversity, genome distribution and gene structure of hirudin were also studied. At last, we explored nine kinds of bioactive compounds in the genomes of the three leeches. This study pointed out the differences in the genome of the three leeches W. pigra, H. medicinalis and H. robusta, and provided insight into the exploration and development of the bioactive molecules of medicinal leeches.

Summary of genome assembly and annotation for W. pigra

Using a whole-genome shotgun strategy with the Illumina HiSeq™ 2000 platform, we sequenced the genome of W. pigra from Wuhan, the provincial capital Hubei, China. The de novo assembly of a 146 Gbp high-quality sequences from 2 paired-end and 3 mate-pair libraries provided 100-fold coverage with a total assembly length of 177 Mbp (Table 1), which approximates the genome size estimated by 23 K-mer distribution (Figure S1). The scaffold N50 is 728 Kbp. 3495 scaffolds are with length >2 Kbp. Repeat content comprised 23% of the W. pigra genome, which is 10% lower than that of the H. robusta [7]. The W. pigra shares a similar profile of GC content (35%) with H. robusta (33%). A total of 26,743 protein-coding genes were predicted in W. pigra. W. pigra and H. Robusta showed similar gene model features in a whole. However, W. pigra has shorter intron length and longer protein length compared with H. robusta (Table 1). A total of 17123 protein-coding genes were annotated in all three common databases Uniprot, TrEMBL and interPro (Figure 1B). We identified 12346 and 10295 orthologous genes between W. pigra and H. medicinalis, and between W. pigra and H. robusta, respectively, using reciprocal best blast hits (RBHs) method (Figure 1C). There are a large proportion of genes (14398 and 16449) in W. pigra not assigned as orthologous genes.

Table 1. Summary for genome sequencing, assembly and annotation

	*H. robusta* (Ref [7])	*W. pigra* (this study)	*H. medicinalis* (Ref [8])
Size of genome assembly	228 Mbp	177 Mbp	187M
Num. of Scaffolds	1,993	10,050	14,042
Num. of Scaffolds (> 2Kbp)	1124	3495	105
Scaffold N50	3,060 Kbp	728 Kbp	97 Kbp
Total reads	3,176,156	118,388,619	62,184,084
Reads mapping to genome (%)	2,839,951 (89%)	112,480,685 (95%)	NA
Sequencing coverage depth	7.92X	100X	73X
Repetitive content (%)	33	23	NA
GC (%)	33	35	41
Num. of predicted genes	23,400	26,743	14,596
Protein length (aa)	376	438	464
Mean exon length	203 bp	205 bp	224
Mean intron length	526 bp	391 bp	716
Mean number of exons per gene	6.1	6.4	8

GC, fraction of guanine plus cytosine nucleobases; Scaffold N50, the length such that half of the assembled sequence is in scaffolds longer than this length.

Gene synteny among the genomes of W. pigra, H. medicinalis and H. robusta

The above result showed that W. pigra only has 46.2% (12346) orthologous genes in H. medicinalis, and 38.4% (10295) orthologous genes in H. robusta. To further compare the genome similarity among the three leeches, we performed a careful analysis of syntenic blocks between W. pigra and H. medicinalis, and between W. pigra and H. robusta using MCScanX[15]. As small scaffolds are not useful for gene synteny analysis, we only considered the scaffold with more than 30 genes. Finally, we identified 21 scaffolds in H. medicinalis had syntenic blocks matched to the 13 scaffolds in W. pigra. In contrast, there are 33 scaffolds in H. robusta matched to the 21 scaffolds in W. pigra (Figure 2). Overall, the genome of W. pigra has a good collinearity relationship with other two genomes. We further examined the synthetic blocks in the larger scaffolds Wh8, wh9, wh17, and wh22. We found that H. medicinalis tends to have larger synthetic blocks matched to the scaffolds of W. pigra than H. robusta. It suggests that compared to H. robusta, W. pigra has a more similar genome structure with H. medicinalis.

The expansion and contraction of gene family in the W. pigra genome

After analysis of gene synteny, we further analyzed the expansion and contraction of gene family among the seven species: H. robusta, Lottia gigantea, Capitella teleta, Schmidtea mediterranea, Schistosoma mansoni, W. pigra, H. medicinalis. We compared the predicted proteomes of seven species, yielding a total of 13563 orthologous gene families that comprised 108245 genes. We found 1488, 832 and 1266 gene families expanded in W. pigra, H. medicinalis and H. robusta, respectively (Figure 1D). Of these families, there are 63, 1 and 59 families that are evolving rapidly (P<0.05) in W. pigra, H. medicinalis and H. robusta, respectively (Figure 1E). These rapidly evolving families are species-specific and little overlap between the two species (Figure 1E). To reveal the molecular function and structural domain of these rapidly evolving families, we performed enrichment analyses by gene ontology terms and interPro domains. The enrichment results showed a clear difference among the three leeches. For W. pigra, the expanded families are enriched in the following functions: protein histidine kinase activity, O−acyltransferase activity, thiamine pyrophosphate binding, carbohydrate binding, proteolysis and so on. For H. robusta, the expanded families are mainly enriched in the functions such as sodium channel activity, sodium ion transport, zinc ion binding, and RNA−DNA hybrid ribonuclease activity. For H. medicinalis, only two functions endopeptidase inhibitor activity and extracellular region are enriched (Figure 3A). In contrast, for the contracted families, there are little go terms enriched in W. pigra and H. robusta, but more go terms enriched in H. medicinalis. For example, iron ion binding, heme binding, proteolysis, and sodium channel activity functions are enriched by the contracted family in H. medicinalis (Figure 3C). Corresponding to these functions, specific protein domains are enriched in different leeches. These results imply the three species may take different adaptive strategies. And the different functions and domains are potentially related to environmental adaptation and bioactive peptides properties of the three leeches.

Phylogenetic analysis and sequence alignment of the hirudin gene family

As the most well-studied natural anticoagulant from leeches, hirudin has served as a standard for designing natural coagulation inhibitors[16]. Hirudin may be useful in the therapy of thrombosis because of its specific antithrombin effects[17]. We identified two hirudin genes g14352 and g17108 (Figure 4A) in W. pigra in this study. We named g14352 and g17108 as hirudin_1, hirudin_2, respectively (Figure 5). For comparison, we also identified three hirudin genes g9136, g9138, and g9139 in H. medicinalis. These five hirudin genes and 38 hirudin-like sequences from protein database UniProt were used to clarify the phylogenetic relationships of these hirudin genes (Figure 4A). They are clustered into three clades (named Groups 1, 2 and 3) (Figure 4A). Three groups are highly supported with bootstrap value >95. The sequences (Group 3) from W. pigra do not cluster with the other hirudin genes. Groups 1, 2 and 3 follow different cysteine patterns CX(7)CX(1)CX(5)CX(5)CX(10)C, CX(7)CX(1)CX(5)CX(5)CX(8)C and CX(8)CX(1)CX(5)CX(5)CX(10)C, respectively (Figure 4B). The pattern of group 1 is the typical cysteine pattern of the hirudin. In contrast, gene g17108 (hirudin_2) of W. pigra shows the third cysteine pattern, which inserts an extra amino acid between the first and second cysteines. The gene g17108 (hirudin_2) is a new kind of hirudin, which has not been reported before.

Genome-wide distribution, gene structure and transcript levels of hirudin genes

Although there are a lot of studies about hirudin, the genome-wide distribution and gene structure of hirudin have not been reported. By sequence searching, we found that g14352 (hirudin_1) and g17108 (hirudin_2) are located at different scaffolds 5072 and 278, respectively (Figure 5A). The left and right sides of g17108 (hirudin_2) are surrounded by multiple genes (24 and 69 genes, respectively). We can infer that the two genes are separated by great distances (>210 Kbp). It suggested a lot of genome rearrangement events happened after gene duplication of hirudin genes. Furthermore, gene structures of the two hirudin genes are also different. g14352 (hirudin_1) only has three exons. In contrast, g17108 (hirudin_2) has four exons, which encode a signal peptide and a longer tail. Therefore, protein hirudin_2 has a longer sequence than hirudin_1 (Figure 4B and 5B).

Exploration of bioactive ingredients in the W. pigra genome

There are more than 20 bioactive substances identified from leeches, such as Antistasin, hirustasin, ghilantens, hirudin [2, 6, 18, 19]. These molecules have analgesic, anti-inflammatory, anticoagulant, platelet inhibitory, thrombin regulatory functions, and so on. W. pigra, H. medicinalis and H. robusta are belong to the family hirudinidae and glossiphoniidae, respectively. The detailed distribution of these bioactive substances in different leech species is still unknown. It is essential to identify and compare these active molecules in different leeches. Using the genome data, we systematic explored and compared five classes of active substances in W. pigra, H. medicinalis and H. robusta (Table 2). All 9 common active molecules were found in W. pigra. It is noteworthy that hirustasin, hirudin and destabilase I genes are absent in the H. robusta. There are far more gene copies for the active molecules in W. pigra than in H. robusta (57 vs 24). W. pigra exceeds H. robusta in both kinds and gene number of active molecules. The gene copy of bioactive ingredients of W. pigra also exceeds that of H. medicinalis.

Table 2. The exploration of five class of active substances in W. pigra, H. medicinalis and H. robusta

Modes of action	Bioactive molecules	Gene copy (H. robusta)	Gene copy (W. pigra)	Gene copy (*H. medicinalis*)
Analgesic and anti-inflammatory effect	Antistasin	6	9	5
	Hirustasin	0	18	7
	Ghilanten	9	10	7
Extracellular matrix degradation	Hyaluronidase	1	4	5
Inhibition of platelet function	Saratin	1	4	1
Anticoagulant effect	hirudin	0	2	3
	Factor Xa inhibitor	2	1	1
	Therostasin	5	4	2
	Destabilase I ^a	0	5	2
Antimicrobial effect	Destabilase I ^a	0	5	2
Total		24	57	33

^a Destabilase I, involved in anticoagulant effect and antimicrobial effect.

Gene expression analysis of W. pigra

We make full use of the available RNA-seq data to analyze the gene expression in W. pigra. We divided all genes of W. pigra into four parts (No expression, Low, Medium, High). The expression of these genes is shown in Figure 5C. These genes are involved in different molecular functions (Figure 5D). No expressed genes are enriched in the functions of ATPase activity, oxidoreductase activity, DNA−binding transcription factor activity, transposase activity and so on. Low expressed genes are enriched in the functions of ion transport, G protein−coupled receptor activity, carbohydrate binding, microtubule motor activity and so on. Medium expressed genes are enriched in the functions of nucleus, zinc ion binding, Rho guanyl−nucleotide exchange factor activity, protein dephosphorylation and so on. High expressed genes are enriched in the functions of translation, ribosome, serine−type endopeptidase inhibitor activity, enzyme inhibitor activity and so on. Finally, we examined the gene expression of bioactive peptide (Figure 5E). All kinds of bioactive peptides were expressed. Of these peptides, antistasin, therostasin, and hirudin have higher expression, while factor Xa inhibitor and ghilanten have lower expression. The result implies that these bioactive peptides may play different roles in the survival of W. pigra.

"Medicinal leech" represents the leeches in the family hirudinidae of the order hirudinida. Medicinal leeches have been widely utilized in medical procedures for thousands of years and were approved by the US Food and Drug Administration in June, 2004 as a medical device due to their mechanically relieving venous congestion and delivering anti-coagulants[20, 21]. W. pigra is the most commonly available from Chinese commercial leech market. Although its importance in medicine and the significance of medicinal leeches in biological research, there is no genome data available for any species in the family hirudinidae until 2020. The genome of H. robusta has been sequenced to study bilaterian evolution in 2013. H. robusta is a leech of the family glossiphoniidae, which is very far from the family hirudinidae [22]. The genome of H. medicinalis in the family hirudinidae was just published during the review period of this study[8, 9]. In this study, we reported the genome of W. pigra, another medicinal leech in the family hirudinidae. We characterized the genome by analysis of gene synteny, gene family and the genome distribution of bioactive molecules and comparing it with H. robusta and H. medicinalis.

The results of the expansion and contraction of gene family revealed very clear different patterns among W. pigra, H. medicinalis and H. robusta. This suggests that the three leeches used different survival strategies to adapt to living environment. These results also suggest that although W. pigra and H. medicinalis both are medical leeches, they displayed different patterns of expanded and contracted families. Therefore, the features of one leech cannot simply be applied to another leech.

In the respect of active substances, we found a huge difference between W. pigra and H. robusta after systematic comparison of five classes of active substances. Hirudin, hirustasin, and destabilase I genes are absent in H. robusta. In contrast, all 9 common active molecules were found in W. pigra. There are two hirudin genes in W. pigra. Furthermore, two hirudin genes display different cysteine patterns in the protein sequence. The gene g17108 (hirudin_2) is a new kind of hirudin, which has not been reported before. The alignment of all available hirudin sequence shows a diversity of hirudin, which provides insight into the development of a new hirudin with more potent activity. Significantly, although W. pigra and H. medicinalis are both medicinal leeches, the gene copy of bioactive ingredients of W. pigra far exceeds that of H. medicinalis.

In summary, the genome of another medicinal leech (W. pigra) was reported in this study. The genomes of three leeches, W. pigra, H. medicinalis and H. robusta, show many differences in the respects of orthologous genes, gene synteny and gene family. Furthermore, W. pigra exceeds H. robusta in both kind and gene number of active substances, such as hirudin, hirustasin, and destabilase I genes. This study pointed out the differences in the genome of two medicinal leeches, W. pigra and H. medicinalis and provided insight into the exploration and development of bioactive molecules of medicinal leeches.

Sample preparation and genome and RNA-seq sequencing

Samples of W. pigra were collected from East Lake in Wuhan, the provincial capital Hubei, China. Animal care and handling were conducted in accordance with the stipulations of Ethics Committee of Kunming University. Genomic DNA was extracted from the whole body of W. pigra after cleaned off their gastric tracts and the blood. Two short paired-end (300 and 500 bp) and three mate-end (5, 8, and 10 Kbp, respectively) sequencing libraries were constructed with the standard protocol provided by Illumina (San Diego, United States), and then sequenced on an Illumina HiSeq™ 2000 platform. Low-quality and duplicated reads were filtered out through fastp (v0.20.0) software[23].

For transcriptome-based gene prediction, RNA was extracted from the whole body of W. pigra after cleaned off their gastric tracts and the blood. Prepared tissues (approximately 200 mg) were preserved in liquid nitrogen for RNA extraction. Total RNAs were purified with RNA Easy Kit (QIANGEN, German) according to the manufacturers' instructions. RNA yields and the quality were measured by agarose gel electrophoresis and spectrophotometer (Thermo, USA). Equal amounts of total RNA (20 μg, 50.8 μg/ml) purified from each tissue were separately stored and mRNA was isolated with Oligo-dT Purist Kit (TaKaRa, Japan) according to the standard protocol. All the libraries were prepared using the Illumina TruSeq RNA sample preparation kit (San Diego, United States) then were sequenced by Illumina HiSeq™ 2000 platform at Biomarker Technologies Co. Ltd. of Beijing, China.

Estimation of genome sizes and genome assembly

Genome sizes were estimated using JELLYFISH [24] and GenomeScope [25] with an optimal k-mer size (K-mer=23). Genome sizes were calculated from the following equation: Genome size = 23-mer_number / 23-mer_depth, where 23-mer_number is the total number of each unique 23-mer and 23-mer depth is the highest frequency that occurred. Consequently, the estimated genome size of W. pigra was ~ 162Mbp. By taking the estimated genome size as a reference, total sequence data accounted for ~100-fold coverage. The clean reads were used for de novo assembly by Platanus (v1.2.4)[26] with default parameters. Subsequently, intra-scaffold gaps were filled using the reads of short-insert libraries by gap_close command. The final assembled genome size was ~ 177 Mbp. The summary for assembly results are list in Table 1. Only scaffolds with lengths longer than 500 bp were used in further analyses.

Genome annotation

Homolog and de novo strategies were both applied to identify the repetitive sequence in the W. pigra genome. Software LTRfinder (v1.07)[27] and RepeatModeler (v1.0.11, http://www.repeatmasker.org/RepeatModeler) were used for ab initio prediction. The results obtained from these tools were combined to form a new repetitive sequence database. This database was then merged with Repbase [28, 29]. Repetitive sequences in the W. pigra genome were identified by homolog searching with the final merged database by RepeatMasker (v1.332)[30]. We identified 40 Mbp repetitive sequences, which accounted for 23% of the W. pigra assembled genome (Table 1). Protein coding genes were predicted using GeneMark-ES (v4.3.8) and AUGUSTUS (v3.3.0) implemented in the BRAKER2 pipeline[31, 32] using RNA-seq alignments as evidence. The RNA-seq bam files generated by HISAT2 [33, 34] were combined and fed into BRAKER. A total of 26,743 protein-coding genes were generated for the W. pigra genome.

All protein sequences from the BRAKER2 results were aligned to TrEMBL and UniProt [35] databases using BlastP at E-value ≤1e^-5. Gene functions were also annotated using the InterProScan software[36-38] by searching publically available databases including Pfam[39, 40], PRINTS[41], ProDom[42] and SMART[43]. In summary, approximately 95% (25,496/26,743) of the genes were supported by at least one related function assignments from the public databases (TrEMBL, UniProt, and InterPro).

Comparative genomic analysis

To define gene families that descended from a single gene in the last common ancestor, we downloaded the protein-coding genes of H. robusta, Lottia gigantea, Capitella teleta, Schmidtea mediterranea, Schistosoma mansoni from NCBI. The protein-coding genes of H. medicinalis were downloaded from http://download.ripcm.com/hirudo_genome. The protein-coding gene of W. pigra were derived from BRAKER2. All proteins of the seven species were processed with OrthoFinder-Diamond (v1.1.10) to provide information about orthologous gene families. OrthoFinder is robust to incomplete models, differing gene lengths, and larger phylogenetic distances [44]. Gene families (orthogroups) in OrthoFinder are defined as homologous genes descended from a single gene from the last common ancestor of the species examined. It is assumed that a parental gene of each orthogroup was present in the common ancestor of the seven species investigated. We applied the likelihood model implemented in the software package CAFE (v4.1)[45] to identify the expanded and contracted gene family along each branch of the phylogenetic tree. The phylogenetic tree was constructed in the process of defining gene families.

Phylogenetic analysis of gene family

Protein sequences in the gene family were aligned using Clustal W [46] with fine adjustment by hand. Then the aligned sequences were used for phylogenetic analysis using MEGA X [47]. The evolutionary history was inferred by using the Maximum Likelihood method and JTT matrix-based model [48]. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The default parameters were used for sequence alignment, phylogenetic analysis.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

The whole genome sequence data reported in this paper has been deposited in the Genome Warehouse in National Genomics Data Center [49], Beijing Institute of Genomics (BIG), Chinese Academy of Sciences, under accession number GWHABJR00000000 that is publicly accessible at https://bigd.big.ac.cn/gwh.

Competing interests

The authors declare no competing interests.

Funding

This work was supported by grants from the National Natural Science Foundation of China (31360516 and 31401142), the Joint Special Project of Universities in Yunnan (2017FH001-004), Yunnan Provincial Training Programs of Youth Leader in Academic and Technical Reserve Talent (2018HB101), Yunnan Provincial Ten Thousand People Plan, the start-up fund of Kunming university of science and technology (KKZ3201927005), and Yunnan Fundamental Research Projects (2019FB050).

Authors' contributions

ZCL and SXD conceived the study and designed experiments. LT, SXD and DJK performed the experiments. SXD, XT and ZCL analyzed the data. XRT, XXB, YS, and YQZ contributed reagents/materials/analysis tools. ZCL, LT and SXD wrote and revised the paper.

Acknowledgements

We thank our colleagues, Drs. Gong-Hua Li and Wen-Xing Li for helpful comments on the manuscript.

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The Genome of Medicinal Leech (Whitmania pigra) and comparative genomic study for Exploration of Bioactive Ingredients

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