Skip to main content

Transcriptome analysis of different life-history stages and screening of male-biased genes in Daphnia sinensis

Abstract

Background

In the life history of Daphnia, the reproductive mode of parthenogenesis and sexual reproduction alternate in aquatic ecosystem, which are often affected by environmental and genetic factors. Recently, the sex-biased genes are of great significance for clarifying the origin and evolution of reproductive transformation and the molecular regulation mechanism of sex determination in Daphnia. Although some genes on reproductive transition of Daphnia had been researched, molecular mechanism on the maintenance of sexually dimorphic phenotypes of Daphnia are still not well known, including differentially expressed genes in different life-history stages.

Results

In this study, four life-history stages of Daphnia sinensis, juvenile female (JF), parthenogenetic female (PF), sexual female (SF) and male (M), were performed for transcriptome, and male-biased genes were screened. A total of 110437 transcripts were obtained and assembled into 22996 unigenes. In the four life-history stages (JF, PF, SF and M), the number of unique unigenes is respectively 2863, 445, 437 and 586, and the number of common unigenes is 9708. The differentially expressed genes (DEGs) between male and other three female stages (M vs JF, M vs PF and M vs SF) were 4570, 4358 and 2855, respectively. GO gene enrichment analysis showed that the up-regulated genes in male were mainly enriched in hydrolase activity and peptidase activity. Thirty-six genes in male were significantly higher expression than in the three female stages, including one Doublesex (Dsx) gene, one laminin gene, five trypsin genes and one serine protease genes, and one chitin synthase gene and two chitinase genes.

Conclusions

Our results showed that thirty-six candidate genes may be as the male-biased genes involving in the maintenance of sexually dimorphic phenotypes. This work will provide a reference for further exploring the functional genes related to sex differentiation in Daphnia species. Moreover, according to previous investigations, we thought that the expression level of functional genes may be related to the life-history stages of organisms, and may be also affected by different Daphnia species.

Peer Review reports

Background

The phenotypes of male and female individuals for one species usually show difference greatly [1]. This difference is often driven by genes on the sex chromosome, which showed a particularly strong sex bias expression tendency [2, 3]. Sex-biased genes of male and female fundamentally lead to the difference between male and female phenotypes [4]. Therefore, the sex-biased genes are great significance to clarify the origin and evolution of reproductive transformation and molecular regulation mechanism of sex differentiation.

In the life history of Daphnia, parthenogenesis and sexual reproduction often alternate in aquatic ecosystem, which is affected by environmental (e.g. food, predation, photoperiod) and genetic (e.g. genotype) factors together [5]. Under suitable environmental conditions, they only produce female offspring by parthenogenesis. However, when environmental conditions deteriorate (such as fish predation, food shortage and higher population density), Daphnia species will transfer from parthenogenesis to sexual reproduction, producing male and sexual female, and then mate and fertilize, forming resting eggs [6,7,8,9]. Resting eggs can survive in lake sediments for several decades [10], and then hatch and form new populations in suitable conditions. Some investigations have indicated that Daphnia is an ideal model organism in studying ecology, environmental toxicology and evolutionary biology [11, 12].

The sex maintenance and the switch of Daphnia species have affected by environmental changes, making Daphnia an interesting comparative system for the study of sex-biased and reproductive genes. Some study of Daphnia pulex revealed that 50% of assayed transcripts show some degree of sex-bias [1]. Among them, female-biased transcription is enriched for translation, metabolic and regulatory genes associated with life-history. Colbourne et al. (2011) showed the detailed genomes information of D. pulex, the amount of genomic and transcriptomic resources for the genus has markedly increased, which greatly promoted our understanding of sex-biased genes [13]. Zhang et al. (2016) constructed the genetic data sets of the genes expressed in a sexual female and a parthenongentic female of Daphnia similoides. The study showed the gene may have a crucial role in reproductive switching of D. similoides. Male-biased expression is enriched for cuticle and protease function [14]. Huylmans et al. (2016) and Molinier et al. (2018) analyzed the sex-biased genes between the female and male of Daphnia galeata and Daphnia magna, respectively [15, 16]. Moreover, the female-biased genes of Daphnia sinensis might contribute to maintaining rapid production of parthenogenetic females, and nutrient uptake for the growth of neonates [17]. Therefore, the sex-biased genes (including male-biased genes) might play a pivotal role in steering the life-history and reproduction processes in Daphnia species.

Although some male-biased genes in other species of Daphnia have been reported, it is still worth exploring whether these genes perform the same function in D. sinensis. D. sinensis is a typical Daphnia species that is widely distributed in inland fresh waters, particularly in eutrophic waters. Our goals are to compare the transcriptome of D. sinensis at four life history stages (i.e. juvenile female, parthenogenetic female, sexual female and male), and to explore the role of male-biased genes in male production and male phenotype maintenance. Our study will provide some necessary fundamental data for further research of sex-biased genes of Daphnia in future.

Methods

D. sinensis and Tetradesmus obliquus culture

D. sinensis employed in the experiment were collected from Lake Chaohu in Anhui Province, China. Parthenogenetic females were cultivated under a 12 h light /12 h dark photoperiod at 25 ± 1℃ with a light intensity of 2500 lx, and fed with 40 mg L−1 of T. obliquus (wet weight). T. obliquus was obtained from the Freshwater Algae Culture Collection at Institute of Hydrobiology, Chinese Academy of Sciences, and cultured in BG-11 medium at 25 ± 1℃ with a 12 h light/12 h dark photoperiod.

Juvenile female (JF), parthenogenetic female (PF), sexual female (SF) and male (M) collection

In one experiment, 20 D. sinensis mothers were employed. Each mother was respectively cultured in a 50 ml beaker with 40 ml culture medium. The culture medium was replaced every two days. The culture medium was the filtered and aerated tap water. Offspring (birth time < 12 h) produced by D. sinensis mother were collected as juvenile female samples (JF), and each juvenile female sample was about 500 individuals. Juvenile female (JF) is female of D. sinensis within 12 h of birth. These neonates produced by the mother were removed in time from beakers during the experiment.

In another experiment, 60 D. sinensis mothers were employed. Each 10 mothers were placed in a 250 ml beaker with 200 ml culture medium, 6 replicates were conducted. The culture medium was replaced every two days. After 2–3 weeks, sexual females and males of D. sinensis could be observed when population density became higher. The parthenogenetic females (PF), sexual females (SF) and males (M) were selected under the microscope. Parthenogenetic female (PF) is female of D. sinensis carrying eggs for the first time; Sexual female (SF) is female of D. sinensis carrying resting eggs; Male is male of D. sinensis within 12 h of birth. About 50 parthenogenetic females and 50 sexual females were respectively collected as PF sample and SF sample. Male juveniles were collected and cultured in a 100 mL beaker with 80 mL culture medium for two weeks. Each male sample (M) contained about 60 male individuals.

JF, PF, SF and M samples were put into EP tubes, respectively. These samples were immediately frozen in liquid nitrogen and stored at -80℃. In this study, two replicates of JF, PF, SF and M samples were collected for transcriptome sequencing, respectively.

RNA isolation and cDNA library construction

Total RNA was respectively extracted from samples at four life-history stages of D. sinensis (juvenile females, parthenogenetic females, sexual females and males) using TRIzol reagent. RNA degradation and contamination was monitored using 1% agarose gels. RNA purity was checked using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). The RIN (RNA integrity number) value range of quality test is 5.8 to 6.6. RNA concentration was measured using Qubit® RNA Assay Kit in Qubit® 2.0 Flurometer (Life Technologies, CA, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA).

A total of 1.5 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) according to manufacturer’s recommendations, and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV reverse transcriptase (RNase H). Second strand cDNA synthesis was subsequently performed using DNA polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure was ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 250 ~ 300 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). 3 µl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min, followed by 5 min at 95 °C before PCR. Then, PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified with AMPure XP system and library quality was assessed on the Agilent Bioanalyzer 2100 system [14].

Clustering and sequencing

The clustering of the index-coded samples was performed with a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina). After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform and paired-end reads were generated.

De novo assembly of short reads and gene annotation

Raw data (raw reads) were firstly processed through in-house perl scripts. Clean data (clean reads) for the JF, PF, SF and M samples were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. Simultaneously, Q20, Q30, GC-content and sequence duplication level of the clean data were calculated. All the downstream analyses were based on clean data with high quality. Transcriptome assembly was accomplished based on the left.fq and right.fq using Trinity [18] with min_kmer_cov set to 2 by default, and all other parameters set default. The resulting sequences were named unigenes. The unigenes were annotated by BLASTx searching in NCBI non-redundant (Nr), Swiss-Prot, KEGG, PFAM and KOG and mapped to NCBI Nt database by BLASTn. Functional annotation by Gene Ontology (GO) terms was analyzed by using Blast2GO (http://www.balst2go.org/) software [19]. GO functional classification for unigenes was analyzed using WEGO software [20]. The similarity searches of unigenes were performed using the NCBI-BLAST network server (http://blast.ncbi.nlm.nih.gov/).

Differential expression genes and GO enrichment analysis

Differential expression analysis between two life-history stages (JF vs PF, PF vs SF and M vs PF) of D. sinensis was performed using the DEGseq 2 package. P-value was adjusted using Q-value [21]. Q-value < 0.05 and |log2 (fold change)|> 1 were set as the threshold for significantly differential expression [14]. GO enrichment analysis of DEGs was implemented by the GOseq packages based on Wallenius’ non-central hyper-geometric distribution [22].

Validation of DEGs using Real Time-PCR

Total RNA was extracted using TRIzol reagent (TaKaRa, Dalian, China). The ultramicro-spectrophotometer (MD2000D, Biofuture, UK) was used to assess sample purity and RNA concentration. RNA was reversely transcribed by PrimeScript™ RT reagent Kit (TaKaRa, Dalian, China).

RT-qPCR was performed with 76 genes, which were selected from top 30 up-regulation DEGs in M vs JF, M vs PF and M vs SF. The qPCR primers were designed using Beacon Designer 7.9 (PREMIER Biosoft International, Palo Alto, CA, USA) and listed in Table S3. We tested the amplication efficiency per primer before qPCR validation. DsimGAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as the reference gene and also listed in Table S3. The qPCR was performed in a LightCycler® 96 (Roche Diagnostics Gmbh, Basel, Switzerland) using a mixture of 5.0 μL AceQ qPCR SYBR Green Master Mix (Vazyme, China), 0.2 μL of each primer, 1.0 μL of sample cDNA and 3.6 μL of RNase Free dH2O. The amplification step was executed using a degeneration step at 95℃ for 10 min, followed by 40 cycles of 95℃ for 15 s and 60℃ for 60 s. The melting curve was employed to detect a single primer-specific peak, using 93℃ for 30 s and 60℃ for 45 s. All reactions were run in triplicate. The relative quantification results were analyzed using the Ct method (\({2}^{{-\Delta \Delta \mathrm{C}}_{\mathrm{T}}}\)) [23].

Statistical analysis

Statistical analysis was executed by SPSS 20.0 software. Significant differences of relative mRNA expression level between two life-history stages (M vs JF, M vs PF and M vs SF) were analyzed using multiple comparisons Turkey (HSD). All data were shown as mean ± SEM in this study.

Results

Transcriptome sequencing, assembly and annotation

Among transcriptome sequencing of four life-history stages (JF-juvenile female, PF-parthenogenetic female, SF-sexual female and M-male) of D. sinensis, 110437 transcripts were obtained, with a total length of 321269424 bp and an average length of 2909 bp. Moreover, 22996 unigenes were obtained, with a total length of 44512763 bp, an average length of 1936 bp and a N50 length of 4265 bp (Table S1). Compared with several common databases through BLASTx program, the most unigenes annotated to Nr database (13512, accounting for 58.75% of the total unigenes), followed by PFAM (10659, accounting for 46.35%) and GO (10659, accounting for 46.35%) (Table S2).

Homology analysis

The homologous sequences of D. sinensis unigenes were matched in Nr database. The relative species with higher homologous sequences were D. magna (70.5%), followed by D. pulex (10.0%), Tetrahymena thermophila (1.9%), Pseudocohnilembus persalinus (1.1%), Ichthyophthirius multifiliis (0.9%) and other (15.6%) (Fig. 1).

Fig. 1
figure 1

Percentage of homologous hits of D. sinensis unigenes to other species

Differentially expressed genes

The number of specific unigenes in JF, PF, SF and M life-history stages were 2863, 445, 437 and 586, respectively. There were 9708 common unigenes in four life-history stages (Fig. 2). The differentially expression genes (DEGs) between the two stages were determined by comparing the genes obtained in male with genes in the three female stages. In differentially expressed genes, the number of up-regulated genes and down-regulated genes were 2230 and 2340 in M vs JF, 2425 and 1933 in M vs PF, and 1473 and 1382 in M vs SF, respectively (Fig. 3).

Fig. 2
figure 2

Venn diagram of the number of unigenes with RPKM > 0.3 in four life-history stages (JF, PF, SF and M; RPKM: reads per kilo bases per million mapped)

Fig. 3
figure 3

Volcano plot of differentially expressed genes in M vs JF, M vs PF and M vs SF

Gene ontology annotation

To analyze the functions of these DEGs, we conducted 4670 DEGs in M vs JF, 4358 DEGs in M vs PF and 2855 DEGs in M vs SF by using the GO enrichment system (Q-value < 0.05). In M vs JF, up-regulated genes were mainly concentrated in protein metabolic process (506) and hydrolase activity (493), and the down-regulated genes were mainly concentrated in macromolecule biosynthetic process (429), cellular macromolecule biosynthetic process (426), cellular nitrogen compound biosynthetic process (421) and gene expression (418). In M vs PF, the up-regulated genes were mainly concentrated in hydrolase activity (503), and the down-regulated genes were mainly concentrated in nucleic acid binding (370) and gene expression (342). In M vs SF, the up-regulated genes were mainly concentrated in hydrolase activity (132), peptidase activity of acting on L-amino acid peptides (128) and proteolysis (119), and the down-regulated genes were mainly concentrated in nuclear acid binding (233) (Fig. 4).

Fig. 4
figure 4

GO enrichment analysis of differentially expressed genes in M vs JF, M vs PF and M vs SF. (red represents biological process, green represents cellular component, blue represents molecular function)

Male-biased candidate genes

In order to screen candidate genes related to male-biased genes of D. sinensis, a total of 76 genes were respectively obtained from the top 30 up-regulated DEGs in M vs JF, M vs PF and M vs SF. qPCR analysis showed that the relative expression levels of 36 genes in male (M) were significantly higher than those in the three female stages (JF, PF and SF) (P < 0.05), suggesting that these genes may participate in the male sex maintenance of D. sinensis (Fig. 5, Table 1). Among them, there are 11 known genes, including one Doublesex gene (Cluster-5789.12340), one laminin gene (Cluster-5789.8159), one chitin synthase gene (Cluster-5789.11830), two chitinase genes (Cluster-5789.5191 and Cluster-5789.7417), five trypsin genes (Cluster-5789.9553, Cluster-5789.3677, Cluster-5789.9554, Cluster-5789.11655 and Cluster-5789.7668) and one serine protease gene (Cluster-5789.2039). The other 25 genes were uncharacterized (Table 1).

Fig. 5
figure 5

qPCR results of differentially expressed genes related to male-biased genes of D. sinensis

Table 1 Thirty-six differentially expressed genes related to male-biased genes in D. sinensis

In addition, some previous known genes (Dsx1, Tra, antp and DMRT93B) related to male-biased genes in other Daphnia species appeared also in the differentially expressed genes of D. sinensis (Fig. 6). The expression levels of Dsx1 in male (M) was significantly (P < 0.05) higher than that in the other three stages (JF, PF and SF), and the expression level of antp in male (M) was significantly (P < 0.05) higher than those in both PF and SF whereas it was significantly lower than that in JF (the abbreviation reference Table S4).

Fig. 6
figure 6

qPCR results of some published genes related to male-biased genes in D. sinensis.(Dsx1 [13], antp [12] and Tra [14] may be related to male-biased genes in other Daphnia species)

Discussion

Usually, under worse conditions (such as fish predation, unsuited temperature and photoperiod), Daphnia transforms from parthenogenesis to sexual reproduction, producing male and sexual female, which mate and form resting eggs [24,25,26]. Colbourne et al. (2011) reported the detailed genomes information of D. pulex, the amount of genomic and transcriptomic database greatly promoted our understanding of sex-biased genes for Daphnia. The related study indicated that male-biased expression is enriched for cuticle and protease function. Moreover, male-biased genes seem to evolve faster than females-biased genes [1, 13]. Based on database, some studies have shown that several genes (e.g. Dsx, antp, Tra, DMRT93B) could play important roles in the male-biased genes of Daphnia [4, 27,28,29]. Doublesex (Dsx) gene is an important sex regulatory gene, and has been widely studied in Cladocera [30]. Kato et al. (2011) found that knock-out Dsx1 in male embryos of D. magna will lead to the production of female characteristics including ovarian maturation, while ectopic expression of Dsx1 in female embryos will lead to the life-history of male-like phenotype, and thought that Dsx1 is a key regulator of male phenotype in D. magna [4]. In D. carinata, Dsx1 and Dsx2 may be also involved in the sex differentiation [31]. Dsx contains two conserved domains: one is Dsx/Mab-3 (DM) domain at the N-terminal, and the other is oligomeric domain at the C-terminal [32]. DM-domain plays an important role in the sex maintenance of vertebrates [33, 34]. In this study, the expression of Doublesex2 (Cluster-5789.12340) in male was significantly (P < 0.05) higher than those in the three females (Juvenile female, parthenogenetic female and sexual female), suggesting that Doublesex2 as male-biased candidate genes may play an important role for male sex maintenance in D. sinensis. In D. magna, DMRT93B is contributed to the differentiation or maintenance of the testis [27]. However, DMRT93B did not express in the four life-history stages of D. sinensis in this study. The reason for this phenomenon may be that the expression of the same gene is different in different species or different developmental stages.

For other arthropods, the formations of differential morphology of legs and antennae were regulated by the antp gene [35, 36]. Our results also showed the expression level of antp gene in male was significantly (P < 0.05) higher than those in females (parthenogenetic female and sexual female). This suggested that antp may be responsible for male sex maintenance in D. sinensis. Schwarzenberger and Von Elert (2016) observed also that the antp expression level in the first antennae of D. magna male adults was significantly higher than that in the first antennae of female adults, and thought that antp may be involved in the molecular pathway of inducement to male phenotype of Daphnia [28]. Moreover, the expression of Tra in Daphnia carinata male was significantly higher than those in both parthenogenetic female and sexual female [31], which was also thought to be responsible for male sex maintenance of Daphnia. In this study, however, the expression level of Tra in D. sinensis male was significantly lower than that in parthenogenetic female, indicating that Tra may not play an important role in male-biased genes of D. sinensis. We speculated that the Tra gene maybe play different function in D. sinensis compared with D. carinata, or some analogous genes with Tra may coexist in D. sinensis.

Laminin (Ln) is a component of the basement membrane of male and female gonads in the frog Rana rugosa [37]. The synthesis of basement membrane is essential for sex differentiation of embryonic mammalian gonads [38]. In this study, the relative expression level of laminin subunit gamma-3 (Cluster-5789.8159) gene in male was significantly higher than those in the three females. The expression of laminin alpha 1 (LAMA1) in male and female bovine embryos showed sexual dimorphism [39]. Those results implied that laminin subunit gamma-3 gene may affect male sex differentiation through promoting the life-history of male gonads in D. sinensis. In addition, laminin alpha 5 chain is an early molecular marker of sexual differentiation in rat, which may be regulated by the sperm -determining factors [40].

Chitin is the second abundant polysaccharide in nature, and it is the main component of fungal cell wall and exoskeleton of arthropod. Chitin is synthesized by chitin synthase and degraded by chitinase (Cht) to maintain the sustainable growth and life-history of organisms [41]. The chitin content in some male insects is significantly higher than that in female insects [42]. In this study, the expression levels of chitin synthase (Cluster-5789.11830) and chitinase (Cluster-5789.5191, Cluster-5789.7417) genes in D. sinensis male showed all significantly higher than those in the three females. Same with Phenacoccus solenopsis, the expression levels of Cht 4 and Cht 4–1 in males were significantly higher than those in females [43]. Moreover, the Cht 4 gene in Nilaparvata lugens was only highly expressed in reproductive organs of adult male [44]. Those results suggested that the chitin synthase and chitinase genes play important roles for male sex maintenance in D. sinensis. In summary, 36 candicate genes were screened to be responsible for male sex maintenance of D. sinensis.

Conclusions

In this study, transcriptome sequences of the four life-history stages (JF-juvenile female, PF-parthenogenetic female, SF-sexual female and M-male) in D. sinensis were investigated, and candidate genes related to male-biased genes were screened (M vs JF, M vs PF and M vs SF). The number of specific unigenes in the four life-history stages (JF, PF, SF and M) were respectively 2863, 445, 437 and 586, with a common unigenes of 9708. Based on DEGs, the number of up-regulated genes and down-regulated genes were 2230 and 2340 in M vs JF, 2425 and 1933 in M vs PF, and 1473 and 1382 in M vs SF, respectively.

The 36 candidate genes related to male-biased genes of D. sinensis were obtained through screening from the top 30 up-regulated differentially expressed genes in M vs JF, M vs PF and M vs SF (P < 0.05). Among these genes, there are 11 known genes, such as Doublesex gene which was involved in sex differentiation of other Daphnia species, laminin gene which possibly related to the life-history of male gonads, and two chitinase genes which showed sexually dimorphic expression in D. sinensis. In addition, Dsx, Tra and antp genes related to male sex maintenance were also found in differentially expressed genes of four life-history stages in D. sinensis. The screening of the candidate genes will provide a reference for the identification of functional genes in Daphnia species and the molecular regulation mechanism of sex maintenance in Cladocera. Meanwhile, some results (e.g. DMRT93B) in D. sinensis were inconsistent with previous investigations, suggesting that the expression level of functional genes may be related to the life-history stage of organisms, and may be also affected by different Daphnia species. However, it was very difficult to find the differences of sex-biased genes among different Daphnia species because of the differences in methodology, number of biological replicates, aspects of data analysis and etc. On the other hand, studies on the sex-biased genes (especially male-biased genes) in Daphnia species are lack. Therefore, our results will provide some necessary fundamental data for further research of male-biased genes of Daphnia in future.

Availability of data and materials

The raw RNA-Seq data used in this study have been deposited in the Nation Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database under the accession number GSE197943. The web link is “https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE197943”.

References

  1. Ellegren H, Parsch J. The evolution of sex-biased genes and sex-biased gene expression. Nat Rev Genet. 2007;8(9):689–98.

    CAS  Article  Google Scholar 

  2. Bergero R, Charlesworth D. The evolution of restricted recombination in sex chromosomes. Trends Ecol Evol. 2009;24(2):94–102.

    Article  Google Scholar 

  3. Grath S, Parsch J. Sex-Biased Gene Expression. Annu Rev Genet. 2016;50(1):29–44.

    CAS  Article  Google Scholar 

  4. Kato Y, Kobayashi K, Watanabe H, Iguchi T. Environmental sex determination in the branchiopod crustacean Daphnia magna: deep conservation of a Doublesex gene in the sex-determining pathway. PLoS Genet. 2011;7:1–12.

    Article  Google Scholar 

  5. Deng HW. Environmental and genetic control of sexual reproduction in Daphnia. Heredity. 1996;76:449–58.

    Article  Google Scholar 

  6. Carvalho GR, Hughes RN. The effect of food availability, female culture-density and photoperiod on ephippia production in Daphnia magna Straus (Crustacea: Cladocera). Freshwater Biol. 1983;13:37–46.

    Article  Google Scholar 

  7. Hobaek A, Larsson P. Sex determination in Daphnia magna. Ecology. 1990;71:2255–68.

    Article  Google Scholar 

  8. Rojas NET, Marins MA, Rocha O. The effect of abiotic factors on the hatching of Moina micrura Kürz, 1874 (Crustacea: Cladocera) ephippial eggs. Braz J Biol. 2001;61(3):371–6.

    CAS  Article  Google Scholar 

  9. Deng DG, Zhang S, Li YY, Meng XL, Yang W, Li Y, Li XX. Effects of Microcystis aeruginosa on population dynamics and sexual reproduction in two Daphnia species. J Plankton Res. 2010;32(10):1385–92.

    Article  Google Scholar 

  10. Möst M, Oexle S, Marková S, Aidukaite D, Baumgartner L, Stich HB, Wessels M, Martin-Creuzburg D, Spaak P. Population genetic dynamics of an invasion reconstructed from the sediment egg bank. Mol Ecol. 2015;24:4074–93.

    Article  Google Scholar 

  11. Lampert W, Kinne O. Daphnia: development of a model organism in ecology and evolution. Oldendorf/Luhe: International Ecology Institute; 2011. p. 1–275.

    Google Scholar 

  12. Miner BE, De Meester L, Pfrender ME, Lampert W, Hairston NG. Linking genes to communities and ecosystems: Daphnia as an ecogenomic model. P Roy Soc B-Biol Sci. 2012;279:1873–82.

    Google Scholar 

  13. Colbourne JK, Pfrender ME, Gilbert D, Thomas WK, Tucker A, Oakley TH, Tokishita S, Aerts A, Arnold GJ, Basu MK, Bauer DJ, Cáceres CE, Carmel L, Casola C, Choi JH, Detter JC, Dong Q, Dusheyko S, Eads BD, Fröhlich T, Geiler-Samerotte KA, Gerlach D, Hatcher P, Jogdeo S, Krijgsveld J, Kriventseva EV, Kültz D, Laforsch C, Lindquist E, Lopez J, Manak JR, Muller J, Pangilinan J, Patwardhan RP, Pitluck S, Pritham EJ, Rechtsteiner A, Rho M, Rogozin IB, Sakarya O, Salamov A, Schaack S, Shapiro H, Shiga Y, Skalitzky C, Smith Z, Souvorov A, Sung W, Tang Z, Tsuchiya D, Tu H, Vos H, Wang M, Wolf YI, Yamagata H, Yamada T, Ye Y, Shaw JR, Andrews J, Crease TJ, Tang H, Lucas SM, Robertson HM, Bork P, Koonin EV, Zdobnov EM, Grigoriev IV, Lynch M, Boore JL. The ecoresponsive genome of Daphnia pulex. Science. 2011;331(6017):555–61.

    CAS  Article  Google Scholar 

  14. Zhang YN, Zhu XY, Wang WP, Wang Y, Wang L, Xu XX, Zhang K, Deng DG. Reproductive switching analysis of Daphnia similoides between sexual female and parthenogenetic female by transcriptome comparison. Sci Rep. 2016;27(6):34241.

    Article  Google Scholar 

  15. Huylmans AK, LópezEzquerra A, Parsch J, Cordellier M. De Novo Transcriptome Assembly and Sex-Biased Gene Expression in the Cyclical Parthenogenetic Daphnia galeata. Genome Biol Evol. 2016;8(10):3120–39.

  16. Molinier C, Reisser CMO, Fields P, Ségard A, Galimov Y, Haag CR. Identification of General Patterns of Sex-Biased Expression in Daphnia, a Genus with Environmental Sex Determination. G3 (Bethesda). 2018;8(5):1523–33.

  17. Jia J, Dong C, Han M, Ma S, Chen W, Dou J, Feng C, Liu X. Multi-omics perspective on studying reproductive biology in Daphnia sinensis. Genomics. 2022;114(2):110309.

    CAS  Article  Google Scholar 

  18. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E, Hacohen N, Gnirke A, Rhind N, di Palma F, Birren BW, Nusbaum C, Lindblad-Toh K, Friedman N, Regev A. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. 2011;29(7):644–52.

    CAS  Article  Google Scholar 

  19. Conesa A, Götz S, García-Gómez JM, Terol J, Talón M, Robles M. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics. 2005;21:3674–6.

    CAS  Article  Google Scholar 

  20. Ye J, Fang L, Zheng H, Zhang Y, Chen J, Zhang Z, Wang J, Li S, Li R, Bolund L, Wang J. WEGO: a web tool for plotting GO annotations. Nucleic Acids Res. 2006;34:293–7.

    Article  Google Scholar 

  21. Storey JD. The positive false discovery rate: a Bayesian interpretation and the q-value. Ann Stat. 2003;31(6):2013–35.

    Article  Google Scholar 

  22. Young MD, Wakefield MJ, Smyth GK, Oshlack A. Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol. 2010;11(2):R14.

    Article  Google Scholar 

  23. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-CT method. Methods. 2001;25(4):402–8.

    CAS  Article  Google Scholar 

  24. Camp AA, Haeba MH, LeBlanc GA. Complementary roles of photoperiod and temperature in environmental sex determination in Daphnia spp. J Exp Biol. 2019;222:1–24.

    Google Scholar 

  25. Eads BD, Andrews J, Colbourne JK. Ecological genomics in Daphnia: stress responses and environmental sex determination. Heredity. 2008;100(2):184–90.

    CAS  Article  Google Scholar 

  26. Gust KA, Kennedy AJ, Laird JG, Wilbanks MS, Barker ND, Guan X, Melby NL, Burgoon LD, Kjelland ME, Swannack TM. Different as night and day: behavioural and life history responses to varied photoperiods in Daphnia magna. Mol Ecol. 2019;28(19):4422–38.

    CAS  Article  Google Scholar 

  27. Kato Y, Kobayashi K, Oda S, Colbourn JK, Tatarazako N, Watanabe H, Iguchi T. Molecular cloning and sexually dimoraphic expression of DM-domain genes in Daphnia magna. Genomics. 2008;91:94–101.

    CAS  Article  Google Scholar 

  28. Schwarzenberger A, Von Elert E. What makes a man a man? prenatal antennapedia expression is involved in the formation of the male phenotype in Daphnia. Dev Genes Evol. 2016;226(1):47–51.

    CAS  Article  Google Scholar 

  29. Chen P, Xu SL, Zhou W, Guo XG, Wang CL, Wang DL, Zhao YL. Cloning and expression analysis of a transformer gene in Daphnia pulex during different reproduction stages. Anim Reprod Sci. 2014;146(3–4):227–37.

    CAS  Article  Google Scholar 

  30. Burtis KC, Baker BS. Drosophila doublesex gene controls somatic sexual differentiation by producing alternatively spliced mRNAs encoding related sex-specific polypeptides. Cell. 1989;56(6):997–1010.

    CAS  Article  Google Scholar 

  31. Kong L, Lv WW, Huang YH, Liu ZQ, Yang Y, Zhao YL. Cloning, expression and localization of the Daphnia carinata transformer gene DcarTra during different reproductive stages. Gene. 2015;566(2):248–56.

    CAS  Article  Google Scholar 

  32. Bayrer JR, Zhang W, Weiss MA. Dimerization of doublesex is mediated by a cryptic ubiquitin-associated domain fold: implications for sex-specific gene regulation. J Biol Chem. 2005;280(38):3289–96.

    Article  Google Scholar 

  33. Raymond CS, Shamu CE, Shen MM, Seifert KJ, Hirsch B, Hodgkin J, Zarkower D. Evidence for evolutionary conservation of sex-determining genes. Nature. 1998;391(6668):691–5.

    CAS  Article  Google Scholar 

  34. Matsuda M, Nagahama Y, Shinomiya A, Sato T, Matsuda C, Kobayashi T, Morrey CE, Shibata N, Asakawa S, Shimizu N, Hori H, Hamaguchi S, Sakaizumi M. DMY is a Y-specific DM-domain gene required for male development in the medaka fish. Nature. 2002;417(6888):559–663.

    CAS  Article  Google Scholar 

  35. Struhl G. A homoeotic mutation transforming leg to antenna in Drosophila. Nature. 1981;292(5824):635–8.

    CAS  Article  Google Scholar 

  36. Khadjeh S, Turetzek N, Pechmann M, Schwager EE, Wimmer EA, Damen WG, Prpic NM. Divergent role of the Hox gene Antennapedia in spiders is responsible for the convergent evolution of abdominal limb repression. P Natl Acad Sci USA. 2012;109(13):4921–6.

    CAS  Article  Google Scholar 

  37. Saotome K, Isomura T, Seki T, Nakamura Y, Nakamura M. Structural changes in gonadal basement membranes during sex differentiation in the frog Rana rugosa. J Exp Zool Part A. 2010;313(6):369–80.

    Article  Google Scholar 

  38. Pelliniemi LJ, Fröjdman K, Sundström J, Pöllänen P, Kuopio T. Cellular and molecular changes during sex differentiation of embryonic mammalian gonads. J Exp Zool. 1998;281(5):482–93.

    CAS  Article  Google Scholar 

  39. Bermejo-Alvarez P, Rizos D, Lonergan P, Gutierrez-Adan A. Transcriptional sexual dimorphism in elongating bovine embryos: implications for XCI and sex determination genes. Reproduction. 2011;141(6):801–8.

    CAS  Article  Google Scholar 

  40. Fröjdman K, Miner JH, Sanes JR, Pelliniemi LJ, Virtanen I. Sex-specific localization of laminin alpha 5 chain in the differentiating rat testis and ovary. Differentiation. 1999;64(3):151–9.

    Article  Google Scholar 

  41. Khoushab F, Yamabhai M. Chitin research revisited. Mar Drugs. 2010;8(7):1988–2012.

    CAS  Article  Google Scholar 

  42. Kulma M, Kouřimská L, Plachý V, Božik M, Adámková A, Vrabec V. Effect of sex on the nutritional value of house cricket, Acheta domestica L. Food Chem. 2019;272:267–72.

  43. Omar MAA, Ao Y, Li M, He K, Xu L, Tong H, Jiang M, Li F. The functional difference of eight chitinase genes between male and female of the cotton mealybug, Phenacoccus solenopsis. Insect Mol Biol. 2019;28(4):550–67.

  44. Xi Y, Pan PL, Ye YX, Yu B, Xu HJ, Zhang CX. Chitinase-like gene family in the brown planthopper, Nilaparvata lugens. Insect Mol Biol. 2015;24(1):29–40.

    CAS  Article  Google Scholar 

Download references

Acknowledgements

The authors thank the entire member of the Laboratory of invertebrate physiology and ecology, who took care of Daphnia sinensis culturing and collecting.

Funding

This work was supported by National Natural Science Foundation of China (No. 31370470, 31870451 and 32001155).

Author information

Authors and Affiliations

Authors

Contributions

All authors conceived the main ideas and participated in shaping this research project. Daogui Deng is the project leader. Ziyan Wang conducted experiments and wrote the manuscript. Feiyun Zhang assisted experiments and analyzed the data. Ziyan Wang, Wenping Wang and Daogui Deng reviewed and revised the manuscript. All authors have read and approved the final manuscript.

Corresponding authors

Correspondence to Wenping Wang or Daogui Deng.

Ethics declarations

Ethics approval and consent to participate

Field collection of Daphnia was carried out under the permission of the Ministry of Environment, and the field studies did not involve endangered or protected species.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1:

Table S1. Assembly analysis of transcriptome from four life-history stages of Daphnia sinensis. Table S2. Summary statistics on functional annotation of unigenes in Daphnia sinensis tanscriptome. Table S3. Primers for qPCR genes. Table S4. The abbreviation in this study.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. 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 in a credit line to the data.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Wang, Z., Zhang, F., Jin, Q. et al. Transcriptome analysis of different life-history stages and screening of male-biased genes in Daphnia sinensis. BMC Genomics 23, 589 (2022). https://doi.org/10.1186/s12864-022-08824-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12864-022-08824-x

Keywords

  • Daphnia
  • Life-history stages
  • Transcriptome
  • Male-biased genes
  • Differentially expressed genes