Tissue expression profiles unveil the gene interaction of hepatopancreas, eyestalk, and ovary in the precocious female Chinese mitten crab, Eriocheir sinensis
- 122 Downloads
Sexual precocity is a common biological phenomenon in animal species. A large number of precocity individuals were identified in Chinese mitten crab Eriocheir sinensis, which caused huge economic loss annually. However, the underlying genetic basis of precocity in E. sinensis remains unclear to date.
In this study, morphological and histological observation and comparative transcriptome analysis were conducted among different stages of precocious one-year-old and normal two-year-old sexually mature E. sinensis. The expression profiles of the ovary, hepatopancreas, and eyestalk tissues were presented and compared. Genes associated with lipid metabolic process, lipid transport, vitelline membrane formation, vitelline synthesis, and neuropeptide hormone-related genes were upregulated in the ovary, hepatopancreas, and eyestalk of precocious E. sinensis. Our results indicated that the eyestalk was involved in the neuroendocrine system providing neuropeptide hormones that may induce vitellogenesis in the hepatopancreas and further stimulate ovary development. The hepatopancreas is a site for energy storage and vitellogenin synthesis, and it may assist oogenesis through lipid transport in precocious E. sinensis.
We provided not only an effective and convenient phenotype measurement method for the identification of potential precocious E. sinensis detection but also valuable genetic resources and novel insights into the molecular mechanism of precocity in E. sinensis. The genetic basis of precocity in E. sinensis is an integrated gene regulatory network of eyestalk, hepatopancreas, and ovary tissues.
KeywordsTranscriptome Ovary development Genetic network
Differentially expressed genes
RNA integrity number
Transcripts per million transcripts
Sexual precocity, which refers to the early maturity of the reproductive system (gonad) during puberty, is a natural phenomenon in most animal species, even humans [1, 2, 3]. This phenomenon can cause growth and development retardation, increased illness rate, and other associated physiological defects [1, 4]. Sexual precocity is a complex physiological process induced by extrinsic environmental factors and intrinsic genetic factors [2, 5]. The early development of gonads is considered a molecular response to environmental factors, such as hormones, nutrition, temperature, and disease . However, the genetic mechanism underlying sexual precocity remains unclear to date.
In vertebrate, gonad development is regulated by the hypothalamus-pituitary-gonad axis (HPG). However, in invertebrate, the regulation of reproductive system is vague [6, 7]. The Chinese mitten crab Eriocheir sinensis is an economic crustacean widely cultured in China that suffers from severe precocious problems . Huge economic losses in the E. sinensis aquaculture industry are caused by substantial proportions of precocious E. sinensis individuals every year [2, 9]. Environmental factors such as temperature, salinity, light, and stocking density induce sexual precocity in E. sinensis, however, the intrinsic molecular response to the stimulation of environmental factors is largely unknown in E. sinensis [10, 11, 12].
The X-organ-sinus gland complex neuroendocrine system in eyestalk functions similarly to the HPG axis in crustaceans [7, 13, 14]. Gonad inhibiting hormone (GIH), molt-inhibiting hormone (MIH), crustacean hyperglycaemic hormone (CHH), and neuropeptide F (NPF) genes/neuropeptides expressed and synthesized in eyestalk play essential roles in regulating the gonad development of E. sinensis . Meanwhile, the hepatopancreas is an essential organ for energy metabolism, providing essential energy source for the gonad development of E. sinensis . Studies have also indicated exogenous vitellogenin is synthesized in the hepatopancreas and transferred to the ovary during vitellogenesis process in E. sinensis . However, how the environmental factors stimulate and activate the early gonad development and the specific biological function of eyestalk and hepatopancreas in regulating gonad development are largely unknown in precocious E. sinensis.
In this study, potential precocious female E. sinensis individuals were collected based on phenotypic characters (ratio of abdominal sternite length) and then confirmed by histological observation. Comparative transcriptome analysis was conducted on the eyestalk, hepatopancreas, and ovary tissues of precocious female E. sinensis from different ovary developmental stages to 1) provide a convenient way to identify precocious E. sinensis in aquaculture, 2) reveal the tissue-expression profiles in precocious female E. sinensis in chronological order, 3) identify potential candidate genes/pathways involved in early ovary development, and 4) provide novel insights into the genetic network of eyestalk and hepatopancreas in regulating ovary development in E. sinensis.
Animal sampling and ethics
This study was approved by the Institutional Animal Care and Use Committee (IACUS) of Shanghai Ocean University (Shanghai, China). Sampling procedures complied with the guideline of IACUS on the care and use of animals for scientific purposes. All the E. sinensis individuals in this study were collected from the Aquatic Animal Germplasm Station of Shanghai Ocean University (Shanghai, China) and were anesthetized on ice before sampling. In this study, potential precocious female E. sinensis individuals were firstly collected according to the ratio of abdominal sternite length (B5-B5’/C5-C5’) and then confirmed by histological observation (Fig. 1).
Measurement of phenotypic characters and histological observation
For the collected E. sinensis individuals, phenotypic characters such as body weight, ovary weight, and hepatopancreas weight were measured, and abdominal sternite length was recorded as previously described . The hepatopancreas index (HSI), gonad index (GI), and ratio of abdominal sternite length were calculated using the following formulas:
Hepatopancreas index = (wet hepatopancreas weight/wet body weight) × 100%,
Gonad index = (Wet gonad weight/wet body weight) × 100%.
Rate of abdominal sternite length = (B5-B5’)/(C5-C5’) (Fig. 1a). Ovary tissues from potential precocious E. sinensis individuals were fixed using Bouin’s fixative (Sangon Biotech, China) at room temperature for 24 h. Then, ovary tissue-slices were prepared and stained with hematoxylin-eosin (HE). The tissue-slices were observed under a DM500 microscope system (LEIKA, Germany) and Image Analysis Software Toup View.
RNA isolation and transcriptome sequencing
According to the histological observation results, precocious E. sinensis individuals with ovary developmental stages in major growth stage I, major growth stage II, and sexually mature stage were collected with three biological replicates in each group. Meanwhile, three normally developed two-year-old female E. sinensis individuals (sexually mature stage) were also sampled as the control group. The eyestalk, hepatopancreas, and ovary tissues were quickly collected and stored in liquid nitrogen before RNA extraction. Total RNA was extracted from each collected E. sinensis individual with RNAiso Reagent (Takara, China) according to the manufacturer’s instructions. RNA integrity and quantity were examined using agarose gel electrophoresis and an Agilent 2100 Bioanalyzer (Agilent, Shanghai, China), respectively. A total of 5 μg RNA with an RNA integrity number (RIN) exceeding 8.0 was used for RNA-seq library construction using the Truseq™ RNA sample Prep Kit for Illumina (Illumina, USA). These indexed libraries were sequenced on an Illumina Hiseq™4000, with 150 bp pair-end reads produced.
Differential expression and enrichment analysis
After sequencing, raw sequencing reads were first trimmed using Trimmomatic software . Then, clean reads were mapped to our previously assembled reference transcriptome assembly (NCBI TSA accession number: GGQO00000000) using Bowtie 1.0.0 . Gene abundance, the TPM (transcripts per million transcripts) value was measured using the RSEM 1.3.0 software . The resulting data matrix with expression value (TPM) for all the samples was generated and used as input data for differential expression analysis. Then, differentially expressed genes (DEGs) were identified by DESeq2 software using P < 0.001 for the false discovery rate (FDR) and a fold change > 22 . After normalizing the DEG TPM values using log2 and mean centered, cluster analysis was performed using the hierarchical cluster method based on the euclidean distance using heatmap module in R. GO and KEGG enrichment analysis of the DEGs was conducted using DAVID annotation software with P value < 0.05 . Pearson correlation was calculated and plotted by corrplot package in R.
Quantitative real-time PCR (qRT-PCR) was carried out to validate the DEGs identified in this study. Eight DEGs in the ovary, hepatopancreas, and eyestalk were chosen for qRT-PCR assays. PCR primers were designed according to our previous reference transcriptome assembly (Additional file 1: Table S1). In this study, three reference genes ubiquitin conjugating enzyme (Ube), beta-actin (β-actin), and ribosomal S27 fusion protein (S27) were selected to normalize the gene expression level. qRT-PCR was conducted using SYBR Green Premix Ex Taq (Takara, China) in a QIAxcel real-time PCR system (Qiagen, German). A standard curve was first generated to assess amplification accuracy, and primers with an amplification efficiency between 95 and 105%, and Pearson correlation (R2) > 0.98 were chosen for following qRT-PCR experiments. Three biological and three technical replicates were chosen for each selected DEG. The relative expression was estimated using the 2–ΔΔCt method with normally developed, sexually mature E. sinensis individuals as a calibration control . Relative expression results were presented as the fold-change relative to normally developed, sexually mature E. sinensis individuals. Statistical significance (P < 0.05) was determined using one-way ANOVA tests under SPSS 25.0.
Phenotypic character measurement and ovary histological observation
Information on phenotypic characters and histological observation of precocious E.sinensis
Ratio of abdominal sternite length
No. of individuals
Average hepatopancreas index
Average gonad index
Ovary developmental stage
5.62 ± 0.72 g
10.45 ± 1.56%
13.72 ± 1.23 g
7.16 ± 1.52%
0.16 ± 0.09%
Major growth stage I
19.47 ± 4.01 g
6.03 ± 1.70%
0.34 ± 0.10%
Major growth stage II
40.90 ± 1.68 g
5.40 ± 0.98%
7.59 ± 1.07%
Sexually Mature stage
Tissue gene expression profiles in precocious E. sinensis
A total of 806 DEGs were identified among different groups of precocious hepatopancreas. Two clusters were defined based on the hierarchical clustering results. GO and KEGG enrichment indicated that genes such as sodium/bile acid cotransporter (SLC10A1), sodium-dependent nutrient amino acid transporter 1 (NAAT1), vitelline membrane outer layer protein 1 (VMO1), vitellogenin (VG), beta-hexosaminidase subunit beta (HEXB), hemolymph juvenile hormone binding protein (JHBP), glucosylceramidase (GBA), and arylsulfatase A (ARSA) in cluster 1 were enriched in lipid transport (GO:0006869), vitelline membrane formation (GO:0030704), response to estrogen (GO:0043627) and sphingolipid metabolism pathway (mmu00600) and were highly expressed in the sexually mature precocious group (Fig. 2c,d Cluster 1, Additional file 2: Table S2). Genes such as glycerol-3-phosphate acyltransferase 3 (GPAT3), bile salt-activated lipase (CEL), lysosomal acid lipase (LIPA), and pancreatic triacylglycerol lipase (PNLIP) in cluster 2 were associated with lipid metabolic process (GO:0006629) and fat digestion and absorption pathway (mmu04975) (Fig. 2c,d Cluster 2, Additional file 2: Table S2).
After the comparison of normal two-year-old sexually mature hepatopancreas with sexually mature precocious hepatopancreas, 372 DEGs were identified. Genes up-regulated in completely sexually mature precocious hepatopancreas, such as VMO1 and VG were enriched in vitellogenin synthesis, vitelline membrane formation, and lipid transport biological process (Additional file 3: Table S3, Fig. 3).
A total of 1081 DEGs were identified among different groups of precocious eyestalks. Two clusters were defined based on the hierarchical clustering results revealing different expression patterns. GO and KEGG enrichment analysis indicated genes such as neuropeptide F (NPF), MIH, CHH, vasotocin-neurophysin VT 1 (VT1), glycoprotein hormone beta-5 (GPHB5), and corticotropin-releasing factor-binding protein (CRHBP) in cluster 1 were highly expressed in sexually mature precocious eyestalk and were enriched in neuropeptide signaling pathway (GO:0007218) (Fig. 2e,f Cluster1, Additional file 2: Table S2). Genes such as cuticle protein CP498, cuticle protein AM1159, chitotriosidase-1 (CHIT1), probable chitinase 2 (CHT2), ATP-binding cassette sub-family G member 5 (ABCG5), and ATP-binding cassette sub-family G member 8 (ABCG8) in cluster 2 were involved in chitin catabolic process, retinoid metabolic process (GO:0001523), sterol transport (GO:0015918), and amino sugar and nucleotide sugar metabolism pathway (hsa00520) (Fig. 2e,f Cluster 2, Additional file 2: Table S2).
A total of 449 DEGs were identified between normal two-year-old sexually mature eyestalk and sexually mature precocious eyestalk. Up-regulated genes such as VT1, NPF, prohormone-3 (PROH3), helicostatins, and pro-neuropeptide Y (NPY) in completely sexually mature precocious eyestalk were associated with neuropeptide hormone activity and neuropeptide signaling pathway (Additional file 3: Table S3, Fig. 3).
DEGs related to neuropeptide hormone activity and lipid transport
Gene expression values from a total of 15 DEGs (GO:0005184, neuropeptide hormone activity) and 12 DEGs (GO:0006869, lipid transport) from the studied individuals were extracted. Out of the 15 DEGs related to neuropeptide hormone activity, 12 were identified in eyestalk tissue, and most of the DEGs (MIH, GPHB5, NPA, CHH, VT1, NPF, RPCH, PDH1, CCAP, NPY) were upregulated in precocious eyestalk than in the normal sexually mature eyestalk. In addition, 11 out of the 12 DEGs related to lipid transport were identified in the hepatopancreas tissue, and most of the DEGs (SLC10A1, apolipophorin, VG, LDLR-A, and SLC10A2) were upregulated in the precocious hepatopancreas than in the normal sexually mature hepatopancreas (Additional file 4: Table S4).
Sexual precocity is a complex biological process involving many genes/pathways in specific organs to induce early gonad development [1, 5]. In this study, we utilized the ratio of abdomen sternite length to discriminate ovary developmental stages and believed it is a convenient method for the early detection of potential precocious E. sinensis. E. sinensis individuals with the ratio of abdomen sternite length above 0.70 during the first year are potential precocious E. sinensis that should be abandoned in the aquaculture.
After comparison of the expression profiles of the ovary between two-year-old and one-year-old sexually mature E. sinensis, only 11 DEGs were identified and tissue histological observation showed the normal function of precocious ovary. This result indicated that precocious E. sinensis individuals are functional and capable of spawning . However, significantly different gene expression profiles were identified in the hepatopancreas and eyestalk between normal and precocious E. sinensis, indicating the important function of the hepatopancreas and eyestalk in regulating ovary development.
The hepatopancreas is an essential organ for the energy storage and metabolism of crustaceans, providing the required energy for growth and development [16, 26]. It is also a site for the synthesis and metabolism of certain steroid hormones required by crustaceans during vitellogenesis [17, 27]. In this study, DEGs associated with lipid metabolic process were upregulated in major growth stages I and II during vitellogenesis in precocious E. sinensis (Fig. 2d Cluster 2), indicating the initiation of ovary development depends on the lipid metabolism in the hepatopancreas, which may provide energy and steroid hormones for early ovary development [28, 29].
Previous studies also pointed out that nutriments such as sugar/lipid are absorbed and accumulated in the hepatopancreas; and excessive nutrition is transferred to the gonads continuously, thereby inducing early gonad development . Interestingly, DEGs associated with lipid transport were also upregulated in precocious E. sinensis in both hepatopancreas and ovary tissues (Fig. 2b Cluster 2, Fig. 3). These up-regulated lipid transport related genes may provide the genetic basis for the lipid transfer from the hepatopancreas to the ovary [29, 30, 31]. The hepatopancreas index measured in this study decreased with the increased gonad index, suggesting that the hepatopancreas may transfer the required energy/lipid to the ovary (Fig. 1). Meanwhile, vitellogenin (endogenous and exogenous) is the key factor component in vitellogenesis in E. sinensis. Endogenous VG is synthesized by oocyte, while exogenous VG is synthesized in the hepatopancreas and transferred to the ovary . In this study, VG gene expression in the hepatopancreas positively correlated with oogenesis, and lipid transport related DEGs in the ovary indicating excessive VG expression in the hepatopancreas may stimulate ovary development in precocious E. sinensis. VMO1, which is associated with vitelline membrane formation, was also up-regulated in precocious E. sinensis, indicating that hepatopancreas may also participates in vitelline membrane formation process. Our results confirmed the essential roles of the hepatopancreas in regulating ovary development, energy storage, and steroid hormone synthesis for oogenesis. Vitellogenin synthesis and vitelline membrane formation for vitellogenesis were fulfilled in the hepatopancreas. The intrinsic genetic factors of sexual precocity in E. sinensis at some content was caused by abnormal expression of the above-mentioned candidate DEGs in hepatopancreas.
The X-organ-sinus gland complex system in the eyestalk is an important neuroendocrine system in crustaceans . Previous study indicated that regulating the neuroendocrine system of shrimp and crab improve the growth rate and the maturity time, and that eyestalk ablation stimulates gonad development and ovulation . Consistent with previous hypotheses, the present study identified more neuropeptide hormones such as NPF, NPY, and prohormone-3 as up-regulated DEGs in the precocious eyestalk in this study. This study indicates the essential regulatory mechanism of the eyestalk in ovary development. NPF and NPY belonging to the NPY family are neuropeptide hormones that accelerate ovarian maturation in female Schistocerca gregaria , and regulate visceromotor functions during egg laying . It is well known that CHH and MIH genes inhibit periodic molting in E. sinensis, and E.sinensis stop molting and initiate their gonad development after the last reproductive molting during their life [36, 37]. Therefore, extremely highly expressed CHH and MIH in the eyestalk may inhibit the molting and induce precocity in advance. Interestingly, DEGs in the eyestalk such as NPY, RPCH, helicostatins, GHBP5, NPF, ORCKA, and CCAP were positively correlated with VG genes expression in the hepatopancreas. This result indicates these neuropeptide hormone genes may target hepatopancreas and induce VG expression. However, further functional experiments need to be conducted to confirm the hypothesis (Fig. 4b). All the upregulated genes in the precocious eyestalk indicated that the neuropeptide hormone synthesized in the eyestalk may stimulate ovary development similar to the HPG axis. Our results proved that the eyestalk is indeed an essential organ for gonad development and possibly regulates vitellogenesis in precocious E. sinensis. However, the interplay of the eyestalk, hepatopancreas, and ovary requires further functional research.
Sexual precocity is a serious situation in the aquaculture of E. sinensis, and scientific researchers and farmers struggled to find solutions and elucidate the genetic mechanism of sexual precocity. In this study, few expression differences were identified between the precocious and normal sexually mature ovary, and early ovary development may be affected by abnormally developed eyestalk and hepatopancreas. Several stimulation factors, such as high temperature, salt, stock density, and nutrition, may induce the metabolic disorder of genes associated with neuropeptide, and steroid hormones, leading to the abundant expression and accumulation of VG in the hepatopancreas and further initiating the ovary development. However, comprehensive functional studies should be conducted to elucidate the genetic mechanism underlying sexual precocity, especially the regulatory mechanism for eyestalk and hepatopancreas. Our study provides valuable genetic resources for the research of sexual precocity in E. sinensis in the future. Meanwhile, the effective convenient phenotype measurement method and related candidate DEGs identified in this study provide guidance for the detection of precocious E. sinensis in aquaculture.
We would like to appreciate Mr. Yada Shen for helping collect samples.
This work was funded by Agriculture Research System of Shanghai, China (Grant No. 201804), Shanghai Agriculture Applied Technology Development Program, China (Grant No. G2017-02-08-00-10-F00076), Shanghai Science and Technology Committee Programs, China (No.16391905300; No. 13DZ2251800), and Leading Agricultural Talents in Shanghai Project, China (Grant No. D-8004-16-0217). The funders had no role in study design, data collection and analysis, decision to publish, or the manuscript preparation.
Availability of data and materials
Sequencing reads are available at NCBI SRA database (SRR7777398, SRR7777399, SRR7777400, SRR7777401, SRR7777402, SRR7777403, SRR7777404, SRR7777405, SRR7777406, SRR7777407, SRR7777408, SRR7777409).
CHW and JW designed the study; XWC, XH, WCY, SH collected material and performed the experiments; JW and XWC performed data analyses; JW and XWC wrote the first draft of the manuscript; CHW revised the manuscript. All authors have read the final and approved the final version of the manuscript.
Ethics approval and consent to participate
Compliance with ethical standards. All the animals used in this study were approved by the Institutional Animal Care and Use Committee (IACUS) of Shanghai Ocean University (Shanghai, China). Sampling procedures complied with the guideline of IACUS on the care and use of animals for scientific purposes.
Consent for publication
The authors declare no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
- 3.Takada L, Barbero MMD, Oliveira HN, de Camargo GMF, Fernandes Júnior GA, Aspilcueta-Borquis RR, Souza FRP, Boligon AA, Melo TP, Regatieri IC, et al. Genomic association for sexual precocity in beef heifers using pre-selection of genes and haplotype reconstruction. PLoS One. 2018;13(1):e0190197.CrossRefGoogle Scholar
- 8.Wang W, Wang C, Ma X. Ecological aquaculture of Chinese mitten crab (in Chinese). 2nd ed. Beijing: China Agriculture Press; 2013.Google Scholar
- 12.Wu X, Wang Z, Cheng YX, Zeng C, Yang X, Lu J. Effects of dietary phospholipids and highly unsaturated fatty acids on the precocity, survival, growth and hepatic lipid composition of juvenile Chinese mitten crab, Eriocheir sinensis (H. Milne-Edwards). Aquac Res. 2011;42(3):457–68.CrossRefGoogle Scholar
- 15.Ci Y. Preliminary research on the molecular mechanism of precocious Chinese mitten crab, Eriocheir sinensis (in Chinese). Master: Shanghai Ocean University; 2015.Google Scholar
- 17.Wang W, Wu X, Liu Z, Zheng H, Cheng Y. Insights into hepatopancreatic functions for nutrition metabolism and ovarian development in the crab Portunus trituberculatus: gene discovery in the comparative transcriptome of different hepatopancreas stages. PLoS One. 2014;9(1):e84921.CrossRefGoogle Scholar
- 19.Li C, Wang C, Li S. Discriminant analysis on preconciousness of Chinese mitten crab, Eriocheir sinensis (in Chinese). Fisheries Science & Technology Information. 1998;2:25–8.Google Scholar
- 24.Huang da W, Sherman BT Fau - Lempicki RA, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc2009;4: 44–57.Google Scholar
- 28.Du N, Lai W, Chen P, Cheng Y, Nan C. Studies on vitellogenesis of Eriocheir sinensis (in Chinese). Acta Zool Sin. 1999;45(1):88–92.Google Scholar
- 29.Wang C, Li S, Li C, Zhao J. Observation and analysis on appearing difference of precocious crab of Yangtze population and Liaohe population cultured in ponds (in Chinese). Journal of Lake Sciences. 2001;1:57–62.Google Scholar
- 30.Teng W, Cheng Y, Wu X, Yang X, Bian W, Lu Q, Wang W. A comparative study on biological index changes concerned with gonad development between two population of the Chinese mitten crab (Eriocheir sinensis): Rhine and Yangtze. J Shanghai Fish Univ. 2008;1:65–71.Google Scholar
- 31.Li S, Wang C, Zhao N. Studies on gonad development rule of lake stocked mitten crab of Yangtze population (in Chinese). Acta Hydrobiologica Sinica. 2001;4:350–7.Google Scholar
- 33.Wang X, Zhang Z, Wang Y, Jia X, Li P. Research progress on regulation mechanism of ovary development in shrimps and crabs. Biotechnol Bull. 2013;7:29–35.Google Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.