Marine Biotechnology

https://doi.org/10.1007/s10126-022-10186-0

RESEARCH

Transcriptomes of Testes at Different Developmental Stages

in the Opsariichthys bidens Predict Key Genes for Testis Development
and Spermatogenesis
Jie Ding1,2 · Daojun Tang1 · Yibo Zhang1,2 · Xinming Gao1 · Chen Du1 · Weiliang Shen2 · Shan Jin1 · Junquan Zhu1

Received: 13 October 2022 / Accepted: 21 November 2022

© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022

Abstract
Testis development is a complex process involving multiple genes, and the molecular mechanisms underlying testis develop-
ment in Opsariichthys bidens remain unclear. We performed transcriptome sequencing analysis on a total of 12 samples of
testes from stages II, III, IV, and V of O. bidens and obtained a total of 79.52 Gb clean data, as well as 288,573 transcripts and
116,215 unigenes. Differential expression analysis showed that 22,857 differentially expressed genes (DEGs) were screened
in six comparison groups (III vs. II, IV vs. II, V vs. II, IV vs. III, V vs. III, and V vs. IV). Kyoto Encyclopedia of Genes and
Genomes enrichment analysis of DEGs showed that six comparison groups were significantly enriched for a total of 20 signifi-
cantly up- or down-regulated pathways, including six pathways related to signal transduction, three pathways related to energy
metabolism, five pathways related to disease, and two pathways related to ribosomes. Furthermore, our investigation revealed
that DEGs were enriched in several important functional pathways, such as Huntington’s disease signaling pathway, TGF-β
signaling pathway, and ribosome signaling pathway. Protein–protein interaction network analysis of DEGs identified 63 up-
regulated hub genes, including 9 kinesin genes and 2 cytoplasmic dynein genes, and 39 down-regulated hub genes, including
13 ribosomal protein genes. This result contributes to the knowledge of spermatogenesis and testis development in O. bidens.

Keywords  Opsariichthys bidens · Transcriptome · Testis development · Hub gene

Introduction testis development and spermatogenesis in teleosts is limited to

The testes are an important component of the reproductive
system of teleosts and are mainly responsible for the produc-
tion of male gametes, i.e., mature spermatozoa, through sper-
matogenesis (Schulz et al. 2010). However, our knowledge of
Jie Ding and Daojun Tang are the co-first authors.
fishes used for basic research or aquaculture (Hong et al. 2004;
Leal et al. 2009). During spermatogenesis, spermatogonia are
the original diploid population of male germ cells that gradu-
ally evolve into many highly differentiated spermatozoa through
mitosis, meiosis, and sperm formation (Coward et al. 2002).
Therefore, it is particularly important to characterize gene expres-
sion during testis development and spermatogenesis in animals

* Junquan Zhu Weiliang Shen

zhujunquan@nbu.edu.cn sweleon@163.com
Jie Ding Shan Jin
dj704728781@163.com jinshan@nbu.edu.cn
Daojun Tang 1
Key Laboratory of Applied Marine Biotechnology
tangdaojun@nbu.edu.cn
of Ministry of Education, College of Marine Sciences,
Yibo Zhang Ningbo University, Ningbo 315832, Zhejiang, China
zyb15058456761@163.com 2
Ningbo Academy of Oceanology and Fishery,
Xinming Gao Ningbo 315103, Zhejiang, China
nbugxm4851@163.com
Chen Du
1026954273@qq.com

13
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at the genomic level and to identify hub genes and signaling
pathways related to the development of testes and spermatozoa.
In recent years, with the rapid development of high-
throughput sequencing technologies, it has become possible
to explore the gene expression patterns of specific tissues at
particular developmental stages and their physiological func-
tions at the transcriptome level (Chen et al. 2017; Ding et al.
2020). For example, during spermatogenesis in Scophthal-
mus maximus, transcriptome dynamics can help screen key
pathways and genes regulating male gametes (Huang et al.
2020); at different stages of reproduction in Channa puncta-
tus, transcriptome analysis of the testes revealed genetic regu-
lation of spermatogenic and steroidogenic events (Roy et al.
2017); comparative transcriptome analysis of the differences
in expressed genes and signaling pathways in XY and YY
testes of Pelteobagrus fulvidraco (Wu et al. 2015), and tran-
scriptome sequencing revealed the traits of spermatogenesis
and testicular development in Larimichthys crocea (Luo et al.
2019). However, information on the testes at different devel-
opmental stages in teleosts is limited.
The Chinese hook snout carp (Opsariichthys bidens, Opsari-
ichthys, Cyprinidae, Cypriniformes) is a small fish living in moun-
tain streams and rivers and is unique to the freshwater waters of
East Asia. Owing to its tender and fresh meat, high nutritional
value, rapid growth, and strong reproduction, it has become a
new fishery specialty industry in many Chinese provinces. Con-
sequently, the development of industrial O. bidens breeding has
emerged as an important industry to help revitalize China’s coun-
tryside. To date, studies on the basic biology of O. bidens have
mostly focused on artificial reproduction and breeding (Sui et al.
2012), cytogenetics (Han et al. 2014), kinship geography (Berrebi
et al. 2006; Chen et al. 2018b), food analysis (Chen et al. 2020),
age growth (Zhang et al. 2017), parasitization (Rim et al. 1996),
and swimming capability (Cai et al. 2020). However, studies on
the molecular mechanisms of testis development and spermatozoa
in O. bidens have not been reported.
Consequently, in this study, transcripts and their expression
profiles were analyzed at the whole-genome level in the testes of
O. bidens at different developmental stages using transcriptome
sequencing (RNA-Seq). Subsequently, hub genes and signaling
pathways that may be involved in testis development and sper-
matogenesis in O. bidens were identified. This provides basic
data for further research on the reproductive development mecha-
nism of male O. bidens as well as an important theoretical basis
for the subsequent selection of O. bidens breeding.
Materials and Methods
Experimental Fish and Sample Collection
The fish were obtained from the breeding pond of the Ron-
gxin Aquaculture Cooperative, Shaoxing City, Zhejiang
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Province, China. Male O. bidens individuals were selected
every month from October 2018 to June 2019. The sam-
pling information is included in Supplementary Table S1.
The sampled fish was dissected after anesthesia with
50 mg/L MS-222 (3-aminobenzoic acid ethyl ester meth-
anesulfonate, Sigma, Saint Louis, MO, USA). Subse-
quently, the testes were excised and either fixed in Boone’s
solution for histological observation or preserved in liquid
nitrogen and stored at − 80 °C for RNA extraction. Next,
the fixed testes were embedded in paraffin, sectioned, and
stained with hematoxylin and eosin (HE). Images were
acquired using an inverted microscope (Zeiss).
RNA Extraction and Quality Testing
The testes samples of O. bidens were histologically observed
and identified based on the developmental period. Testes
identified as stages II, III, IV, and V were used for RNA
extraction. Stage I testes were not sequenced because the
sample size was too small for RNA extraction. Total RNA
was extracted from stages II, III, IV, and V testes using TRI-
zol (Invitrogen, Carlsbad, CA, USA). Three testes samples
were taken from each developmental stage and named II-1,
II-2, II-3, III-1, III-2, III-3, IV-1, IV-2, IV-3, V-1, V-2, and
V-3. The concentration of RNA, O ­ D260/280, and O
­ D260/230
of each sample was detected using the NanoPhotometer
(Thermo Fisher Scientific, Waltham, MA, USA), and RNA
integrity (RIN) was accurately measured using the Agilent
2100 BioAnalyzer (Agilent Technologies, Santa Clara,
CA, USA). RNA samples with a concentration ≥ 200 ng/
µL, ­OD260/280 between 1.8 and 2.2, O ­ D260/230 ≥ 2.0, and
RIN ≥ 7.0 were selected for subsequent library construction.
Library Construction and Illumina Sequencing
The selected samples were used for cDNA library construc-
tion using the NEBNext® UltraTM RNA Library Prep Kit
(Illumina, San Diego, CA, USA). After library construc-
tion and quality control, the libraries of different samples
were pooled according to the effective concentration and
target downstream data volume, and 12 cDNA libraries were
sequenced in both directions using the Illumina HiSeq 2500
platform, with a sequencing read length of 125 bp at one end
and a sequencing volume of 6 G for each sample.
Quality Control
The raw reads were filtered with adapters, reads containing
N (N means that the base information cannot be determined)
and low-quality reads (quality scores lower than 20) using
Fast QC software (Chen et al. 2018a) to obtain high-quality
Marine Biotechnology
clean reads. Subsequent data analysis was performed on
the basis of these clean reads. Furthermore, Q20, Q30, GC
content, and sequence repeat levels of the clean reads were
counted. All raw data were uploaded on NCBI (accession
numbers: SRR15969033–SRR15969044).
De Novo Assembly
As there is currently no reference genome for O. bidens, the
clean reads had to be spliced to obtain reference sequences
for subsequent data analysis. The clean reads were subse-
quently assembled using Trinity (–min_contig_length 300
–min_kmer_cov 3 –min_glue 3 and other default parameters)
(Grabherr et al. 2011), and further processed by TGICL (-l 40
-c 10 -v 25 -O “-repeat_stringency 0.95 -minmatch 35 -min-
score 35” and other default parameters) (Pertea et al. 2003)
to delete redundant sequences and produce unique transcripts
(unigenes). Transcript sequences obtained by Trinity splicing
were then used as reference sequences for subsequent analyses.
Finally, BUSCO (Simao et al. 2015) was used to evaluate the
splicing quality of the unigene and cluster. Additionally, the
accuracy and completeness of the splicing results were evalu-
ated based on the ratio and completeness of the comparison.
Gene Function Annotation
For annotation analysis, unigenes were BLASTed against
seven major gene/protein databases, i.e., the National Center
for Biotechnology Information (NCBI) non-redundant pro-
tein sequence (NR) database, NCBI non-redundant nucleo-
tide sequences (NT), SwissProt, protein family (PFAM),
Clusters of Orthologous Groups of Proteins (COG), Gene
Ontology (GO), and Kyoto Encyclopedia of Genes and
Genomes (KEGG) databases. The cutoff E-value was set at
1e-5, and only the top hit was used to annotate each unigene.
Gene Expression Level Analysis
The transcriptome obtained by Trinity splicing was used as the
reference sequence. The clean reads of each sample were com-
pared with the reference sequence using Bowtie2 (-q –phred64
–sensitive –dpad 5 –mp 1,1 – np 1 –score-min L,0,-0.1 -I 1
-X 1000 -k 200 and other default parameters) (Bo and Dewey
2011), and the number of reads of each gene was counted for
each sample. For the number of fragments to truly reflect the
transcript expression level, we used the fragments per kilobase
of exon per million fragments mapped (FPKM) value to quan-
tify the gene expression level (Zhao et al. 2021).
Differential expression analysis was then performed using
the DESeq2 package (Love et al. 2014), with P-value < 0.05
and |log2 (fold change)|≥ 1 as the threshold for significant
differential expression. Genes that met the criteria were
identified as differentially expressed Genes (DEGs).
GO and KEGG Analysis
GO functional and KEGG pathway enrichment analyses
were performed for all DEGs in each comparison using
GOseq (Young et al. 2010) and KOBAS (Xie et al. 2011),
respectively, with a q-value cutoff of 0.05. The DEGs asso-
ciated with the significantly enriched GO terms and KEGG
pathways were subjected to further analyses to identify genes
potentially involved in the regulation of testis development.
Protein–Protein Interaction (PPI)
All the DEGs obtained were compared to the STRING data-
base by BLASTx, and the protein interactions of the refer-
ence species were used to construct the interaction network
in the STRING protein interaction database online webpage
(http://​string-​db.​org). Cytoscape (Shannon et al. 2003) was
used for visualization and analysis. To further screen the core
regulatory genes, the CytoHubba plugin (Chin et al. 2014)
in Cytoscape software was used for the weight analysis of
the connectivity of the PPI network, and the eight weighting
algorithms in the plug-in (MCC, DMNC, MNC, degree, EPC,
EcCentricity, closeness, and radiality) were used to obtain the
intersection of the top 5% of genes as hub genes.
qRT‑PCR Validation of the DEGs
Based on the transcriptomic dataset, 15 unigenes (cep192, dync1
i1, kif11, kif18a, kif20a, kif22, kif23, kif4, nek2, plk4, rlp24, rpl17
, rps19, rps29, and rps7) related to various gene pathways were
selected and analyzed by real-time PCR (RT-PCR). The RNA to
be tested was first reverse-transcribed into cDNA using the Prime-
ScriptTM RT reagent Kit (Takara, Dalian, China) as a template for
real-time fluorescent quantitative PCR analysis, followed by quan-
titative PCR experiments on a Roche Light Cycler 480 II using
SYBR Green Master I (Roche, Basel, Switzerland). The thermal
cycling program was as follows: activation at 95 °C for 3 min, fol-
lowed by 40 cycles of 95 °C for 20 s, 60 °C for 20 s, and 72 °C for
20 s. Primers for the target genes were synthesized and provided
by BGI Genomics (Shenzhen, China), and β-actin was used as the
reference gene. The primer sequences used for qPCR are listed
in Supplementary Table S2. qRT-PCR was performed with three
biological replicates per group and three technical replicates, and
the results were analyzed using the ­2−ΔΔct method.
Results
Histological Structure of the Testes
Combining the characteristics of the development of the
O. bidens itself and referring to the developmental staging
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methods of Lin (Lin et al. 2021a) and Tang (Tang et al.
2020), the spermatozoa of the O. bidens can be divided into
six periods from I to VI. The spermatozoa of 3-month-old
fish are stage I; those of 4-month-old fish are stage II; those
of 6- to 9-month-old fish are stage III; those of 10-month-
old fish are stage IV; those of 11- to 12-month-old fish are
stage V, and those of 13- to 14-month-old fish are stage
VI. Histological examination of stage II–V testes of O.
bidens showed that the stage II testes consisted mainly of
spermatogonia and primary spermatocytes, with spermato-
gonia mainly distributed at the margins of the seminiferous
lobula and primary spermatocytes mainly distributed inside
the seminiferous lobula. Furthermore, the lobular cavity had
been formed (Fig. 1A, a). Stage III testes consisted mainly
of spermatogonia, primary spermatocytes, and secondary
spermatocytes; early spermatids were also present (Fig. 1B,
b). Spermatogonia, primary spermatocytes, secondary sper-
matocytes, and spermatids were composed of spermatogenic
cysts and were sequentially distributed in the seminifer-
ous lobula. The volume of spermatogenic cells decreased
sequentially during spermatogenesis. In addition to the pres-
ence of spermatogonia, primary spermatocytes, secondary
spermatocytes, and spermatids at different developmental
stages, sperms were also present in the lobular cavity of
stage IV testes; the number of spermatid cysts was increased
compared to that of stage III testes (Fig. 1C, c). Stage V
represents mature testis, with semen flowing out from the
belly of the fish when gently squeezed and the lobular cavity
being filled with sperm (Fig. 1D, d).
Sequence Analysis and Assembly
After sequencing on the Illumina HiSeq 2500 platform, a
total of 273,829,483 raw reads were obtained for the 12
cDNA libraries. After filtering out sequences containing
splice, ploy-N, and low-quality RNA, a total of 265,062,021
clean reads were obtained, with a total base count of
79.52  Gb, and the average number of bases per sample
reached 6.63 Gb (Table 1). Among them, more than 98.01%
Fig. 1  Histological features of
stages II–V testis. A, a: stage II
testis; B, b: stage III testis; C,
c: stage IV testis; D, d: stage V
testis. LL, lobular cavity; SG,
spermatogonia; PS, primary
spermatocyte; SS, secondary
spermatocyte; SL, seminifer-
ous lobula; ST, spermatid; SP,
sperm
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of the sequence mismatches were less than 1% (Q20), and
more than 94.16% of the sequence mismatches were less
than 1‰ (Q30).
The clean reads were assembled by splicing using Trin-
ity, resulting in 288,573 transcripts and 116,215 unigenes.
The length of the transcripts ranged from 301 to 24,939 bp,
with an average length of 1752 bp, 3201 bp for N50, and
677 bp for N90 (Fig. 2A), and the average length of the
unigenes was 1225 bp, 2279 bp for N50, and 461 bp for
N90 (Fig. 2B). We used BUSCO to evaluate the effect of the
assembly of the testes transcriptome of O. bidens, and the
results showed that the integrity of the transcripts reached
99.67% and the integrity of the unigenes reached 89.67%
(Fig. 2C), both of which indicated good results of the splic-
ing and assembly of this reference-free transcriptome.
Principal component analysis (PCA) of the transcriptome
samples showed a good correlation between the three paral-
lels for all stages (Fig. 2D). These results indicate that the
transcriptome sequencing results are of good quality to meet
the requirements for subsequent analyses.
Gene Function Annotation
To analyze the potential functions of the unigenes, 116,215
assembled sequences were BLASTx-searched against major
databases. All unigenes were matched to at least one of the
databases, and 9173 transcripts (7.89%) were successfully
matched in all seven databases (Fig. 3A). Among them, the
least number of transcripts was annotated to the KOG data-
base (9.19%), and most transcripts were annotated to the NT
database (79.74%). In addition, the species with the highest
similarity among the annotated sequences were Anabarilius
grahami (34.5%), followed by Sinocyclocheilus rhinocerous
(9.7%), Cyprinus carpio (9.3%), Sinocyclocheilus anshuien-
sis (7.9%), and Carassius auratus (7.2%), all belonging to
the Cypriniformes family fish (Fig. 3B).
Among the KOG annotation results (Fig. S1), “signal
transduction mechanisms” were the most annotated cat-
egory, indicating the importance of signal transduction in
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Table 1  Statistics of sequencing data for the 12 samples

Sample name Raw reads Clean reads Clean bases (Gb) Error (%) Q20 (%) Q30 (%) GC content (%)

II-1 21,952,612 21,409,666 6.42 0.02 98.15 94.53 46.32

II-2 23,213,761 22,662,151 6.80 0.03 98.01 94.16 47.71
II-3 21,541,089 20,686,048 6.21 0.02 98.27 94.76 47.19
III-1 23,107,708 22,431,321 6.73 0.02 98.14 94.44 45.85
III-2 23,122,270 22,420,659 6.73 0.02 98.22 94.59 46.31
III-3 22,205,524 21,578,116 6.47 0.02 98.08 94.38 46.07
IV-1 22,964,367 22,108,460 6.63 0.02 98.26 94.74 46.26
IV-2 22,873,251 22,141,408 6.64 0.02 98.20 94.56 46.76
IV-3 23,696,753 22,845,764 6.85 0.02 98.17 94.54 46.75
V-1 22,752,385 21,767,231 6.53 0.02 98.22 94.64 46.65
V-2 23,279,054 22,586,984 6.78 0.02 98.17 94.50 46.36
V-3 23,120,709 22,424,213 6.73 0.03 98.01 94.17 46.28
Total 273,829,483 265,062,021 79.52

the development of O. bidens testes. In the GO annota-
tion results, “cellular-process,” “metabolic process,” and
“regulation of biological process” are the most observed
subcategories regarding biological processes. For the cell
component, “cellular anatomical entity,” “intracellular,” and
“protein-containing complex” were the most prominent sub-
categories. With respect to molecular function, “binding,”
“catalytic activity,” and “transporter activity” were the sub-
categories with the highest proportions (Fig. 3C).
In the KEGG annotation, 17,420 unigenes were anno-
tated to 289 KEGG pathways in 34 categories (Fig. S2).
Among them, “Signal transduction” was the most repre-
sented category (2708), followed by “Immune system”
(1346), “Endocrine system” (1206), “Signaling molecules
and interaction” (1043), and “Transport and catabo-
lism” (1042). The top ten KEGG pathways are listed in
Table S3.

Fig. 2  Transcriptome sequenc-
ing and assembly data analysis.
A Length frequency distribution
of spliced transcripts of the tes-
tes transcriptome of O. bidens.
B Length frequency distribution
of spliced unigenes of the testes
transcriptome of O. bidens. C
Results of BUSCO evaluation
of spliced transcripts (differ-
ent colors represent different
types of spliced transcripts). D
PCA of samples from different
periods of testes development in
the O. bidens 

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Fig. 3  Unigene function annota-
tion results. A Histogram of
unigene annotation ratio in
seven major databases. B Taxo-
nomic map of species annotated
with unigene functions. C His-
togram of GO annotation results
for unigene
Differential Expression Analysis
In this experiment, transcriptome samples from all develop-
mental stages were compared two by two, and FDR < 0.05
and |log2 (fold change)|≥ 1 were used as the criteria to screen
DEGs. A total of 22,857 DEGs were screened in six com-
parison groups (III vs. II, IV vs. II, V vs. II, IV vs. III, V
vs. III, and V vs. IV), of which the number of up-regulated
genes was 276, 4665, 2457, 1179, 219, and 107 (Fig. 4A),
respectively. Furthermore, there were 857, 5990, 4226, 1711,
819, and 351 (Fig. 4B) down-regulated genes, respectively.
Most DEGs (10,655) were detected in the IV vs. II stages,
and the fewest DEGs (458) were detected in the V vs. IV
stages of testes development. This suggests that there are sig-
nificant spatial and temporal differences in gene expression
patterns and molecular mechanisms during various periods
of testicular development in O. bidens.
Following the strategy of comparing transcriptome
samples from two adjacent developmental periods (i.e., III
vs. II, IV vs. III, and V vs. IV), 4481 DEGs were obtained.
The number of DEGs showed a trend of increasing and
then decreasing, and the highest number of DEGs was
found in the stage IV vs. stage III comparison group. This
suggests that there is a large difference in transcriptome
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level in stage IV in comparison with other developmental
periods. Consequently, this may be a critical period for
spermatophore development in O. bidens. In addition, the
heat map of DEGs showed that the expression patterns of
stages II and III were similarly clustered into one category
(Fig. 4D), whereas stages IV and V were clustered into
another category. This indicates that there may be large
differences in gene expression during the development of
the testes of O. bidens from stage III to stage IV.
GO Enrichment Analysis of DEGs.
To better investigate the functions of these DEGs during tes-
tis development in O. bidens, we used the R package GOseq
to analyze all DEGs for GO enrichment. In the group com-
paring stage III vs. stage II, the GO terms that were signifi-
cantly enriched (Q < 0.05) were mostly related to microtubules
and microtubule movement, including microtubule-based
movement (GO:0007018), microtubule-based process
(GO:0007017), microtubule motor activity (GO:0003777),
microtubule (GO:0005874), dynein complex (GO:0030286),
microtubule cytoskeleton (GO:0015630), and microtubule-
associated complex (GO:0005875) (Fig. 5A). In the group
comparing stage IV vs. stage II and the group comparing
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Fig. 4  Venn diagram and heat
map of DEGs. A Venn diagram
of up-regulated DEGs. B Venn
diagram of down-regulated
DEGs. C Venn diagram of all
DEGs. D Cluster analysis of dif-
ferentially expressed genes for
all transcriptome samples
stage V vs. stage II, the GO terms that were significantly
enriched (Q < 0.05) were mostly associated with the binding
of macromolecules, including ATP binding (GO:0005524),
adenyl ribonucleotide binding (GO:0032559), nucleotide
binding (GO:0000166), carbohydrate derivative binding
(GO:0097367), and ribonucleotide binding (GO:0032553)
(Fig. 5B and D). In the groups comparing stage IV and stage III,
the significantly enriched GO terms were in ribosome-related
functional groups, such as ribonucleoprotein complex biogen-
esis (GO:0022613), ribosome biogenesis (GO:0042254), and
structural constituents of ribosomes (GO:0003735) (Fig. 5C).
In the groups comparing stages V and III as well as the group
comparing stage V vs. stage IV, there was no significant enrich-
ment (Q > 0.05) in the relevant functional groups.
KEGG Enrichment Analysis of DEGs
To identify the most important biochemical metabolic
pathways and signal transduction pathways involved in the
screened DEGs, we performed a KEGG pathway enrich-
ment analysis. A total of 20 pathways were significantly up
or downregulated in the five comparison groups (Fig. 6),
with the largest number of signaling pathways (14) signifi-
cantly enriched in the group comparing stages V vs. stage
IV. There were six pathways related to signaling, includ-
ing cytokine-cytokine receptor interaction (ko04060),
signaling pathways regulating pluripotency of stem cells
(ko04550), TGF-beta signaling pathway (ko04350), tight
junction (ko04530), adrenergic signaling in cardiomyocytes
(ko04261), and synaptic vesicle cycle (ko04721). Further-
more, there were three pathways related to energy metabo-
lism, including purine metabolism (ko00230), pyrimidine
metabolism (ko00240), and protein digestion and absorption
(ko04974). In addition, there were five pathways related to
diseases, including Huntington’s disease (ko05016), alcohol-
ism (ko05034), prion diseases (ko05020), systemic lupus
erythematosus (ko05322), and viral myocarditis (ko05416).
There were two pathways related to ribosomes, including
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Fig. 5  GO enrichment analysis of DEGs in the testes of O. bidens at different developmental stages. A III vs. II, B IV vs. II, C IV vs. III, and D
V vs. II. BP, biological process; CC, cellular component; MF, molecular function
ribosome (ko03010) and ribosome biogenesis in eukaryotes
(ko03008), as well as some pathways related to cell cycle
and cancer (Table S4).
Trend Analysis of DEGs
We performed a trend analysis of all DEGs in the testes at
the four developmental stages using STEM. All DEGs were
clustered into 20 modules, based on different expression
trends or profiles. Modules 0, 1, 2, 7, 12, 17, 18, and 19 were
significantly enriched (P < 0.05; Fig. 7). KEGG enrichment
analysis of these significantly enriched modules showed that
ribosome biogenesis in eukaryotes; oxidative phosphoryla-
tion; one carbon pool by folate, valine, leucine, and isoleu-
cine degradation; Parkinson’s disease; amino acid biosyn-
thesis; and carbon fixation in photosynthetic organisms were
enriched in module 1, and systemic lupus erythematosus and
alcoholism pathways were enriched in module 19 (Fig. S3).
PPI Analysis of DEGs
After constructing PPI networks for up- and down-regu-
lated DEGs separately and performing visual analysis, we
obtained a PPI network of up-regulated genes containing
1906 nodes and 14,579 edges (Fig. S4A) and a PPI network
of down-regulated genes containing 1931 nodes and 25,693
edges (Fig. S4B). To identify hub genes, the intersection of
the top 5% of genes obtained by eight weighting algorithms
in the plug-in CytoHubba was used for the weighting analy-
sis of the connectivity of the PPI network. The PPI network
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analysis of up-regulated genes identified a total of 63 hub
genes (Table S5; Fig. 8A). These hub genes were function-
ally annotated, and most of them were found to be associated
with mitotic and meiotic processes. Among them, 11 genes
were related to motor proteins, including nine members of
the kinesin family, namely kif18a, kif22, kif2c, kif23, kif11,
kif2a, kif4, kif14, and kif20a, and two members of the cyto-
plasmic dynein family, namely dync1i1 and dync1i2. PPI
network analysis of the down-regulated genes screened a
total of 39 hub genes (Table S6; Fig. 8B), most of which
were associated with ribosome and ribosome functions,
namely, rpl11, rps7, rlp24, rpl10a, rpl12, rplp0, rpS19, rpl9,
rpl36a, rpl2, rp13a, rpl17, and rps29.
Data Validation by Quantitative Real‑Time PCR
To verify the reliability of the transcriptome data, a fluores-
cence real-time quantitative method was used to calculate
the expression levels of the 15 DEGs (Fig. 9A, B, C, D,
E, F, G, H, I, J, K, L, M, N, and O). Correlation analysis
revealed a significant linear correlation between the two
results (R2 = 0.8989, P < 0.01), indicating that the transcrip-
tome data were highly reliable (Fig. 9P).
Discussion
O. bidens is a widely distributed stream-dwelling fish,
which reproduces rapidly and is fast-growing. Furthermore,
it is popular with consumers because of its tasty meat and
Marine Biotechnology
nutritional value. With breakthroughs in artificial breeding
technology, production has increased significantly for O.
bidens (Johansson et al. 2006). Despite its importance in
aquaculture, genetic research on this species is still in its
infancy, particularly studies related to sexual development
(Tang et al. 2020; Lin et al. 2021a; Jiang et al. 2021). High-
throughput sequencing has been widely used in many stud-
ies for gene expression profiling, identification of genetic
mutations, and species diversity analysis (Ding et al. 2020;
Zhao et al. 2016; Lin 2015). In this study, we assembled a
comprehensive and high-quality reference transcriptome for
O. bidens based on RNA-seq data. RNA extracted from the
testes at different developmental stages of O. bidens was
used to construct a library, which ensured the comprehen-
siveness of the resulting transcriptome assembly. Ultimately,
116,215 unigenes were obtained, and subsequent quality
assessment confirmed the accuracy and completeness of
the unigenes. This is the first reference transcriptome for O.
bidens and will be valuable for molecular and genetic stud-
ies of O. bidens and other related species of the subfamily
Danioninae, including gene cloning and molecular marker
development (Perdices et al. 2005; Johansson et al. 2006).
Signaling Pathways Associated with Testes
Development
KEGG enrichment analysis of DEGs showed that more than
20 signaling pathways were significantly enriched, indicat-
ing that testis development in O. bidens is a complex process
that requires the joint participation and regulation of mul-
tiple signaling pathways. Among them, many DEGs were
enriched in signaling pathways such as Huntington’s disease,
ribosome, ribosome biogenesis in eukaryotes, and the TGF-
beta signaling pathway.
The Huntington’s disease signaling pathway was the
most significantly up-regulated signaling pathway in the
group comparing stage III vs. stage II and was frequently
observed in other species when studying testis development
(Jia et al. 2018). Huntington’s disease is a neurodegenera-
tive disease, the main cause of which is a mutation in the
Huntington gene on chromosome 4 of the patient, which
produces a mutated protein that gradually gathered together
in cells and forms large molecular clusters accumulating in
the brain, affecting the function of nerve cells (Difiglia et al.
1997; Mangiarini et al. 1996). In a study of the functions of
genes in this pathway, many genes were found to be associ-
ated with testis development or spermatogenesis, such as
the Htt gene. This gene plays a role in spermatogenesis by
regulating processes such as translation and DNA packaging
in the mouse testis. This is observed in cases where mice
exhibit small testes and display azoospermia after knockout
(Yan et al. 2016). Huntington-interacting protein 1 (Hip-1)
is important for the regulation of cytokinesis and is required
for the differentiation, proliferation, and survival of mouse
spermatogonia (Rao et al. 2001). In the present study, we
found that many genes of this pathway were expressed at sig-
nificantly high levels in stage III of testis development, such
as dnai1, dnai2, dnal4, creb5, and other genes (Table S4).
Dnai1 and Dnai2 are members of the dynein intermedi-
ate chain family. Their mutations can lead to abnormali-
ties in respiratory ciliary function and ultrastructure, which
is an important mechanism of primary ciliary dyskinesia
(Escudier et al. 2009). In addition, mutations in DNAI1 in
male Drosophila have been associated with sterility caused
by motile spermatozoa (Fatima 2011). Stage III testes in
O. bidens are the spermatocyte division phase, where the
number of spermatocytes is significantly increased. The
high expression of dnai1 and dnai2 may be associated with
the formation of flagella and the migration of major orga-
nelles such as centrioles and mitochondria during spermatid
formation.
The TGF-β signaling pathway plays an important regu-
latory role in the growth, development, and differentia-
tion of reproductive organs and cells, including the prolif-
eration of spermatogonia, meiosis of spermatocytes, and
spermatogenesis (Kubiczkova et al. 2012). TGF-β family
members play key roles in reproductive regulation. Activin
type I receptor genes ALK2 and ALK4 of the TGF-β super-
family play important roles in promoting male germ cell
differentiation and entering the process of mitotic stasis.
Inhibition of the ALK2 and ALK4 genes in mice causes
male-specific gene (Nanog) expression to be significantly
suppressed (Miles et al. 2013). In the present study, both
genes were significantly overexpressed in mature testes
(stage V), indicating their important regulatory role in the
testis development of O. bidens. It has been shown that
ALK4 interrupts the entry of male germ cells into meiosis
via the TGFβ signaling pathway (Miles et al. 2013) and that
the high expression of ALK4 in stage V (sperm matura-
tion) may be an attempt to conserve energy by inhibiting
the meiotic process and devoting more energy to energy-
consuming processes such as material transport during
spermatogenesis. The mediator Smad1 plays a key role in
transmitting TGF-β signals from cell surface receptors to
the nucleus, and Smad1 gene expression in rat testes tends
to increase gradually with age (Hu et al. 2003). Knock-
down of the Smad1 gene in male mice causes sterility and
metastatic testicular tumors (Pangas et al. 2008). Smad1
has been also shown to be an important regulator of prolif-
eration and protein synthesis in human support cells (Guo
et al. 2015; Hai et al. 2015). Support cells play a key role
in regulating spermatogenesis and testicular development
by providing structural and nutritional support; therefore,
the high expression of smad1 in stage V may be related to
support cells providing morphological maintenance for the
expansion of the seminiferous lobules. Additionally, the
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◂Fig. 6  An overview of the KEGG pathways significantly enriched in
DEGs in the A III vs. II, B IV vs. II, C V vs. II, D IV vs. III, E V vs.
III, and F V vs. IV group. The vertical axis represents the pathway
categories, and the horizontal axis shows the enrichment factor. The
point size shows the number of DEGs among the pathway. The point
color shows different Q values as indicated on the right
transcriptomes of other fish species, such as Danio rerio
(Crespo et al. 2016; Santos et al. 2007), Clarias fuscus (Lin
et al. 2021b), Salmo salar (Kleppe et al. 2020), and Tricho-
podus pectoralis (Boonanuntanasarn et  al. 2020), have
shown a significant enrichment of DEGs in this pathway,
suggesting the importance of this pathway in the develop-
ment of testes in fish.
Both the ribosome signaling pathway and the ribosome bio-
genesis in the eukaryotes signaling pathway were significantly
enriched in DEGs in the late testes development stage of O.
bidens. This correlates to data on the testis transcriptome of
other fish, such as Silurus asotus (Shen et al. 2020), Etheostoma
caeruleum (Bahamonde et al. 2016), and Danio rerio (Zheng
et al. 2019), suggesting that the normal function of ribosomes
is important for spermatogenesis. Knockdown of the RpL36
gene in Drosophila melanogaster spermathecae results in a
significant reduction or even absence of mature spermatozoa
in the seminal vesicles (Fang et al. 2021). In the present study,
rpl36 was also significantly overexpressed in stage V testes of
O. bidens, suggesting that rpl36 may be important for sperm
capacitation after sexual maturation. It is worth noting that many
Fig. 7  Gene expression trends
significantly enriched in differ-
ent stages. The X-axis shows
stages, and the Y-axis presents
the normalized expression data
of various genes. The upper
left and lower left of the figure
represent the ID and P values of
the module, respectively. Mod-
ules with color are significantly
enriched trends, and modules
without color are non-signifi-
cantly enriched trends
genes in this pathway, such as rpl10a, rpl39, rps13, smd3, and
erpl22, are highly expressed in the early stage of testis develop-
ment. Studies have shown that rpl10a and rpl39 are involved in
gonadal differentiation in Oncorhynchus mykiss as well as the
proliferation and differentiation of spermatogonia and support
cells (Makkapan et al. 2014). Furthermore, erpl22 has a spe-
cial translational effect on germ cell differentiation (Mageeney
and Ware 2019), while smd3 controls the homeostasis of germ
stem cells (Yu et al. 2019), and rps13 is involved in regulating
the proliferation and differentiation of germ stem cells (Wang
et al. 2020). The high expression of these genes in the primordial
gonads indicates that this pathway is involved in the regulation
of not only late testes development but also primordial gonad
differentiation.
Testis development is a complex process involving a
number of signaling pathways and genes. Although the
interactions between genes in signaling pathways have been
extensively studied, the interactions between DEGs and the
regulatory role of signaling pathways obtained from the tran-
scriptomic data of O. bidens require further investigation.
Important Candidate Genes in Testes Development
The processes of testis development and spermatogenesis are
usually regulated by hub genes, and PPI analysis of DEGs
can help identify hub genes. In this study, 63 hub genes were
identified among the up-regulated DEGs, most of which
were associated with mitotic and meiotic processes, such as
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Fig. 8  Visualization of the
candidate hub genes from the
up-regulated genes (A) and
down-regulated genes (B). Node
size represents the importance
of a node. The edge denotes the
interaction strength

kinesin family genes kif2a, kif2c, kif4, kif11, kif14, kif18a,
kif20a, kif22, and kif23, and the kinesin family genes dync1i1
and dync1i2 (Fig. 10A). The kinesin superfamily comprises a
group of molecular motor proteins that transport various pro-
tein complexes, biomolecules, and other substances essential
to cells and play an important role in spermatogenesis. For
example, kif2c is a mitotic centromere-related protein that is
mainly involved in chromosome separation, formation of the
bipolar spindle apparatus, microtubule depolymerization, cell
cycle regulation, and mitosis. It not only plays an important
role in spindle apparatus assembly but also ensures that chro-
mosomes are properly separated during mitosis (Rizk et al.
2009). Other family members such as kif2a and kif2c play
similar roles and are important for the correct assembly of
the spindle apparatus (Manning et al. 2007), while kif4 and
kif22 have been shown to be important for spindle apparatus

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Fig. 9  Validation of the DEGs by qRT-PCR. The Y-axis on the left
represents the relative gene expression level measured by qRT-PCR,
while the Y-axis on the right represents te log2 fold change of the
assembly and chromosome positioning during mid-meiotic
chromosomes in Xenopus laevis. This is in addition to their
involvement in important processes such as the maintenance
of normal chromosome structure and DNA damage and repair
during interphase cytokinesis (Mazumdar et al. 2004; Funabiki
and Murray 2000). Another family member, kif11, plays a role
in spindle formation during mitosis and meiosis in early germ
cells, is highly expressed in pre-meiotic spermatogonia and
spermatocytes, and is thought to be a marker for spermatogen-
esis (Hara-Yokoyama et al. 2019). The mitotic kinesin kif14
plays an important role in the cell cycle and cell migration by
affecting spindle formation and separation of chromosomes
and cytoplasm (Hentrich and Surrey 2010), while kif18a is a
microtubule depolymerization motor protein gene that con-
trols mitotic chromosome alignment by inhibiting chromosome
oscillation during mid-mitosis in spermatogonia (Tanenbaum
et al. 2011). Furthermore, kif20a is thought to be an important
kinesin responsible for sperm cell adhesion localization and
gene measured by RNA-Seq for the same stages using the same RNA
samples. All qRT-PCR data are shown as mean ± standard error; n = 3
sperm transport in spermatogenic epithelial cells and is also
involved in mitosis, organelles, and cell membrane transport
(Yang et al. 2021), while kif23 has been shown to be a com-
ponent of intercellular bridges during spermatogenesis as well
as a component of male and female embryonic intercellular
bridges (Greenbaum et al. 2009). These important kinesins
are involved in spermatogenic processes, including the mitotic
proliferation of spermatogonia and meiosis of spermatocytes,
as well as acrosome genesis, nuclear formation, flagellum for-
mation, and sperm transport, which are important for studying
testes development and spermatogenesis in O. bidens (Ma et al.
2017). Cytoplasmic dynein 1 intermediate chain 1 (dync1i1)
and dync1i2 are important cargo-binding subunits of cyto-
plasmic kinases that play an important role in cargo binding
and determine the specificity of transported cargo (Kuta et al.
2010; Li et al. 2021). In addition, they can bind microtubulin
to regulate dynein movement. This suggests that they may play
an important role in spermatogenesis in O. bidens.
13

Among the down-regulated DEGs, a total of 39 hub genes
were identified, most of which were associated with ribosomal
proteins, such as rpl11, rps7, rlp24, rpl10a, rpl12, rplp0, rpf2,
rps19, rpl9, rpl36a, rpl2, rp13a, rpl17, and rps29 (Fig. 10B).
Ribosomal proteins are the translation machinery for cellular
protein biosynthesis and play an important role in various
physiological activities during biological growth and develop-
ment. For example, ribosomal protein L24 (rpl24), a protein
that makes up the 60S subunit of the ribosome, has also been
reported to play an important role in spermatogenesis and
spermatozoa development in Marsupenaeus japonicus (Zhang
et al. 2007). The rps7 gene has been shown to interact with
the sex-determining region of the Y chromosome to induce
testis development (Sato et al. 2011). Furthermore, rps29 is
stably expressed in the testes of mice and rats at different
developmental stages and can be used as an internal reference
gene (Gong et al. 2014; Svingen et al. 2015). In addition,
there is growing evidence that ribosomal protein genes have
extra-ribosomal functions, in addition to their involvement in
protein synthesis. The rps19 gene encodes one of the small
subunit proteins that make up ribosome 40S, and its defect is
thought to cause congenital pure red blood cell aplastic ane-
mia (Sieff and Lodish 2007). Zebrafish with rps19 deficiency
have stunted growth, defective erythroid differentiation, hypo-
proliferative anemia, and increased apoptosis (Danilova et al.
2008). Phosphorylation of rpl12 affects the translation of
specific mRNAs during mitosis (Imami et al. 2018), while
rpl11 is associated with and inhibits the transcriptional activ-
ity of peroxisome proliferator-activated receptor-alpha (Gray
et al. 2006). Furthermore, rplp0 can mediate phospholipase
Fig. 10  Heat map analysis of
hub genes. A Kinesin family
genes. B Ribosomal protein-
related genes. Genes shown in
red are upregulated, and those
shown in green are downregu-
lated, relative to the control
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A and acyltransferase 4 (PLAAT4)-induced cell cycle arrest
and apoptosis (Wang et al. 2019). The presence of other func-
tions of ribosomal proteins, in addition to their involvement
in protein biosynthesis, such as transcriptional regulation or
apoptosis, is also a key question that deserves further explora-
tion to help better understand the molecular mechanisms that
control important physiological processes, such as spermato-
zoa development in fish.
In addition, we have identified a number of interesting hub
genes. FGFR1, a tyrosine-protein kinase, acts as a cell-surface
receptor for fibroblast growth factors and plays an essential role
in the regulation of embryonic development, cell proliferation,
differentiation, and migration (Eswarakumar et al. 2005; Turner
and Grose 2010). However, FGFR1 has not been explored in
studies related to testis development in other carp fishes and has
only been found to be highly expressed in the testis in Mega-
lobrama amblycephala (Zhang et al. 2015). There is little evi-
dence for their involvement in the regulation of sperm function,
and their high expression in the O. bidens may suggest their role
as important regulators in testis development, which warrants
further investigation. EMG1 is a methyltransferase that methyl-
ates pseudouridine at position 1248 of 18S rRNA (Wurm et al.
2010). Several studies have shown that DNA methylation medi-
ates the control of splicing and stability of 3′-untranslated region
(UTR) mRNA in spermatocytes and sperm cells (Chen et al.
2022). Thus, DNA methylation may be required for the meiotic
and haploid stages of spermatogenesis in the O. bidens. This has
been found in other carp species such as zebrafish and Hypoph-
thalmichthys nobilis where DNA methylation is associated with
spermatogenesis (Fu et al. 2021; Stromqvist et al. 2010).
Marine Biotechnology
Conclusion
The transcriptome analysis of the testis at different
developmental periods of O. bidens yielded DEGs and
revealed many candidate genes and signaling pathways
that may be involved in testis development and sper-
matogenesis, providing important candidate genes for
the study of testis differentiation in O. bidens. This has
yielded valuable information for subsequent study of tes-
tis development and for further study of the reproductive
biology of O. bidens.
Supplementary Information  The online version contains supplemen-
tary material available at https://​doi.​org/​10.​1007/​s10126-​022-​10186-0
Author Contribution  JQZ conceived and supervised the study. JD and
DJT designed and managed the experiments and wrote the manuscript.
YBZ performed the analysis and designed the charts and tables. YBZ,
JD, CD, WLS, and JQZ conducted the experiments. All authors have
read and approved the manuscript.
Funding  This research was funded by the Ningbo Science and Tech-
nology Plan Projects (2019C10057), the Science and Technology
Planning Programs of Zhejiang Province (2020C02014, 2016C32062),
the National Natural Science Foundation of China (NSFC)-Zhejiang
Joint Fund for the Integration of Industrialisation and Informatisation
(U1809212) and the Collaborative Innovation Center for Zhejiang
Marine High-efficiency and Healthy Aquaculture, and was sponsored
by the K. C. Wong Magna Fund in Ningbo University.
Data Availability  The data used to support to the findings of this study
are available from the corresponding author upon reasonable request.
Declarations  Difiglia M, Sapp E, Chase KO, Davies SW, Bates GP, Vonsattel JP,
Competing interests  The authors declare no competing interests.
Ethics Approval  The principles and procedures of the sampling meth-
ods were in strict accordance with the requirements of the Governing
Regulation for the Use of Experimental Animals in Zhejiang Province
(Zhejiang Provincial Government Order No. 263, released in 17 August
2009, effective from 1 October 2010) and approved by the Animal Care
and Use Committee of Ningbo University.
Conflict of Interest  The authors declare no competing interests.
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