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Effects of stocking density on the
growth performance, mitophagy,
endocytosis and metabolism of
Cherax quadricarinatus in
integrated rice–crayfish farming
systems
Yin Dong
1†
, Rui Jia
1
,
2†
, Yiran Hou
2
, Weixu Diao
1
, Bing Li
1
,
2
* and
Jian Zhu
1
,
2
*
1
Wuxi Fisheries College, Nanjing Agricultural University, Wuxi, China,
2
Key Laboratory of Integrated
Rice-Fish Farming Ecology, Ministry of Agriculture and Rural Affairs, Freshwater Fisheries Research
Center, Chinese Academy of Fishery Sciences, Wuxi, China
Red claw crayfish (Cherax quadricarinatus) is an economic freshwater shrimp with
great commercial potential. However, the suitable stocking density of C.
quadricarinatus is still unclear in integrated rice–crayfish farming system. Thus,
this study aimed to investigate the effects of stocking density on growth
performance, mitophagy, endocytosis and metabolism of C. quadricarinatus.
The C. quadricarinatus was reared at low density (LD, 35.73 g/m
2
), middle
density (MD, 71.46 g/m
2
) and high density (HD, 107.19 g/m
2
)inanintegrated
rice–crayfish farming system. After 90 days of farming, the growth performance
of C. quadricarinatus significantly decreased in the MD and HD groups relative to
that in the LD group. The HD treatment caused oxidative stress and lipid
peroxidation at the end of the experiment in hepatopancreas. Transcriptome
analysis showed that there were 1,531 DEGs (differently expressed genes)
between the LD group and HD group, including 1,028 upregulated genes and
503 downregulated genes. KEGG (Kyoto Encyclopedia of Genes and Genomes)
enrichment analysis indicated that the DEGs were significantly enriched in
endocytosis and mitophagy pathways. Meanwhile, four lipid metabolism
pathways, including biosynthesis of unsaturated fatty acids, fatty acid
biosynthesis, glycerolipid metabolism and glycerophospholipid metabolism,
exhibited an upregulated tendency in the HD group. In conclusion, our data
showed that when the stocking density reached up to 207.15 g/m
2
in HD
group, the growth performance of C. quadricarinatus was significantly inhibited
in this system. Meanwhile, the data indicated that C. quadricarinatus may respond
to the stressful condition via activating antioxidant defense system, endocytosis,
mitophagy and metabolism-related pathways in hepatopancreas.
KEYWORDS
Cherax quadricarinatus, stocking density, transcriptome analysis, growth
performance, oxidative stress, metabolism
OPEN ACCESS
EDITED BY
Youji Wang,
Shanghai Ocean University, China
REVIEWED BY
Xianyong Bu,
Ocean University of China, China
Haibo Yu,
Northwest A&F University, China
*CORRESPONDENCE
Bing Li,
lib@ffrc.cn
Jian Zhu,
zhuj@ffrc.cn
†
These authors have contributed equally
to this work
SPECIALTY SECTION
This article was submitted to Aquatic
Physiology,
a section of the journal
Frontiers in Physiology
RECEIVED 09 September 2022
ACCEPTED 16 November 2022
PUBLISHED 28 November 2022
CITATION
Dong Y, Jia R, Hou Y, Diao W, Li B and
Zhu J (2022), Effects of stocking density
on the growth performance, mitophagy,
endocytosis and metabolism of Cherax
quadricarinatus in integrated
rice–crayfish farming systems.
Front. Physiol. 13:1040712.
doi: 10.3389/fphys.2022.1040712
COPYRIGHT
© 2022 Dong, Jia, Hou, Diao, Li and Zhu.
This is an open-access article
distributed under the terms of the
Creative Commons Attribution License
(CC BY). The use, distribution or
reproduction in other forums is
permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original
publication in this journal is cited, in
accordance with accepted academic
practice. No use, distribution or
reproduction is permitted which does
not comply with these terms.
Frontiers in Physiology frontiersin.org01
TYPE Original Research
PUBLISHED 28 November 2022
DOI 10.3389/fphys.2022.1040712
Introduction
In aquaculture practice, semi-intensive and intensive aquaculture
modes are commonly selected by farmerstoobtainhighereconomic
benefits, where the farmed animals maintain a high stocking density
(Roy et al., 2022). As one of the critical factors, stocking density
influences the water quality, growth, survival, behavior, and health of
farmed aquatic animals (Ani et al., 2022;Hossain et al., 2022). Studies
on stocking density have been conducted in many aquatic species,
such as turbot (Scophthalmus maximus)(Liu B et al., 2019), rainbow
trout (Oncorhynchus mykiss)(Zahedi et al., 2019), Pacificwhite
shrimp (Litopenaeus vannamei)(Deng et al., 2019)andSiberian
sturgeon (Acipenser baerii)(Aidos et al., 2018). A variety of adverse
effects are caused in aquatic animals when they are stocked in a high
density. It has been reported that the growth performance and
survival rate of aquatic animals are decreased under a high
stocking density due to the intensification of interspecific
competition and/or the deterioration of water quality (Trenzado
et al., 2006;Yuan et al., 2018). High stocking density, as a stressor,
may disturb the balance of homeostasis in farmed aquatic animals,
which further negatively impacts on immune function and
physiological status, even increases the susceptibility to diseases
(Lin et al., 2015;Ellison et al., 2018;Liu B et al., 2019;Ellison
et al., 2020). An increasing stocking density induces oxidative stress
leading to high catalase (CAT) activity and malonaldehyde (MDA)
content in channel catfish (Ictalurus punctatus)(Refaey et al., 2022).
In addition, some studies demonstrated that high stocking density
could decrease digestive enzyme activity (Liu et al., 2018)andchange
body composition (Aidos et al., 2018). Hence, an appropriate
stocking density is vital in aquaculture, which may take into
consideration both welfare of animal and economic benefits of
farming.
Transcriptomic analysis is considered as a reliable tool and has
been widely used to assess the molecular mechanism of abnormal
physiological change caused by adverse stimulus in aquatic animals.
Under high stocking density, transcriptomic analysis reveals adverse
alterations in muscle quality and immune function of grass carp
(Ctenopharyngodon idellus)(Zhao et al., 2019). According to
transcriptomic analysis, Ellison et al. (2018) demonstrated that
rearing density significantly impacts susceptibility of Nile tilapia
(Oreochromis niloticus)totheoomyceteSaprolegnia parasitica.In
large yellow croaker (Larimichthys crocea), transcriptomic analysis
showed that short crowding stress can induce an immune response,
but long-term high stocking density may suppress the immunity
(Sun et al., 2017). It should be noted that transcriptomic analysis can
provide more molecular function information, which contributes to
understanding the mechanism of rearing density in aquatic animals.
In China, there are multiple aquaculture models including
pond farming, lake/reservoir farming, indoor recirculating
aquaculture and integrated rice-aquatic animals farming.
Among these, integrated rice-aquatic animals farming has
expanded rapidly in the last 10 years, and the farming area is
2.56 million hectares and aquatic production reaches up to
3.25 million tons in 2020 (National Fesheries Technology
Extension Center, 2021). It has been considered as a
sustainable strategy that improves the utilization of land and
water resources, provides food for human, and alleviates
environmental pollution resulted from agricultural production
(Bashir et al., 2020). In the rice-aquatic animals co-culture
system, the excrement of aquatics animals as fertilizer can be
utilized by rice to meet the growth requirement for nitrogen and
phosphorus, while weeds, insects and plankton can be eaten by
aquatic animals as food, which reduces the input of commercial
diet (Vromant et al., 2001;Tsuruta et al., 2010). It has been
reported that rice-aquatic animals co-culture system has higher
ecosystem service value compared with rice monoculture (Liu
et al., 2020). In farming practice, various aquatic animals, such as
common carp (Cyprinus carpio)(Liu et al., 2022), Chinese mitten
crab (Eriocheir sinensis)(Jiang et al., 2021), red swamp crayfish
(Procambarus clarkia)(Si et al., 2018) and Chinese soft-shelled
turtle (Trionyx sinensis)(Wu et al., 2021), have been co-cultured
with rice. It is worth noting that existing research majorly focuses
on the assessment of ecological and economic values, microbial
diversity and soil nutrition in the integrated rice-aquatic animals
farming system (Si et al., 2018;Liu et al., 2020), but the suitable
stocking density of aquatic animals and the effects of stoking
density on physiological function are rarely evaluated.
C. quadricarinatus, also known as red claw crayfish, has
considerable potential for commercial culture due to high
growth rate and well tolerance to stressful conditions
(Naranjo-Páramo et al., 2004). There are two farming
models for C. quadricarinatus including pond monoculture
and rice-crayfish co-culture in China. The suitable stocking
density of C. quadricarinatus in the pond monoculture has been
reported, and the effect of high stocking density on growth
performance was evaluated (Pinto and Rouse, 1996;Naranjo-
Páramo et al., 2021). However, in rice-crayfish co-culture
system, the suitable stocking density has not been reported.
In addition, it is unclear that how high density affects the
physiological, biochemical and molecular variations of C.
quadricarinatus.Therefore,inthisstudy,wesetdifferent
rearing densities in the integrated rice-crayfish system, and
then compared the growth performance and biochemical
parameters among different stocking densities after 90 days
farming. Further, we evaluated the underlying mechanism of
stress induced by high stocking density in C. quadricarinatus
via transcriptomic analysis.
Materials and methods
Animals, experimental design and
sampling
C. quadricarinatus used in the study were purchased from
Zhejiang Freshwater Fisheries Research Institute (Huzhou,
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Dong et al. 10.3389/fphys.2022.1040712
China). The experiment was carried out at the farm of Freshwater
Fisheries Research Center (Jingjiang, China). The integrated rice-
crayfish farming system consists of a rice field (360 m
2
) and a
canal refuge (0.8 m in depth, 40 m
2
)(Supplementary Figure S1).
In the system, rice seeding (Nangeng 5,055) was transplanted in
the middle of June 2021, and the rice was harvested in early
November. The management of rice fields was based on local
agricultural practice experience. Basic fertilizer (16% nitrogen,
8% phosphorus and 16% potassium) was applied before rice
transplantation, and no fertilizer was used after C.
quadricarinatus farming.
In the rice-crayfish system, we reared three densities of C.
quadricarinatus:lowdensity(LD,35.73g/m
2
), middle density
(MD, 71.46 g/m
2
)andhighdensity(HD,107.19g/m
2
). Each
density included three repetitions. The average initial weight was
14.29 ± 1.05 g/crayfish. The experiment lasted 90 days from 22 July
2021. During the experiment, the crayfish were fed on a commercial
feed (crude protein ≥30%, crude fat ≥3%, crude fiber ≤8%, crude
ash ≤18%, total phosphorus ≥1%, and calcium 1%–3.5%) once every
day. The daily feed ration was adjusted according to crayfish weight
(1%–2% of weight). During the trial period, the water quality
maintains within a reasonable range, such as 0.19–0.48 mg/L
total ammonia nitrogen, 0.01–0.04 mg/L nitrite, 23.4–32.1°C
temperature and 3.21–5.08 mg/L dissolved oxygen. The amount
of fed diet and mortality of crayfish were recorded.
To assess the growth performance, the body weight was
measured every 30 days via randomly catching 20% of the
crayfish in each group. After 90 days of farming,
45 individuals in each group were sampled at random for
biochemical and transcriptome analyses. In biochemical
analysis, the hepatopancreas of five individuals was pooled
into a sample, while 15 individuals’hepatopancreas were
mixed into a sample to sequence transcriptome. All samples
were stored in liquid nitrogen temporarily, and then stored for a
long time under −80°C. The use of crayfish in this study was
approved by the Freshwater Fisheries Research Centre (FFRC),
Wuxi, China. All experiment operations were performed
according to the requirement of animal welfare.
Growth performance
The growth performance was assessed by calculating specific
growth rate (SGR, % day
−1
), weight gain ratio (WGR, %) and
survival rate (SR, %) under three different densities. The
calculation formulas are presented as follows:
SGR 100 × (lnW2−lnW1)n
WGR 100 × (W2−W1)W1
SR 100 × f inal number initial number
Where, W
1
and W
2
are initial weight (g) and final weight (g), n is
the days of the feeding trial.
Determination of oxidative stress
parameters
Oxidative stress parameters including total antioxidant
capacity (T-AOC), malondialdehyde (MDA), superoxide
dismutase (SOD), catalase (CAT), glutathione (GSH) and
glutathione peroxidase (GPx) were determined in
hepatopancreas. The determination of all parameters was
conducted according to the method described by the
manufacturers. In addition, total protein (TP) was detected to
calculate the levels of oxidative stress parameters. The
commercial kits for TP, T-AOC, SOD and GSH were
provided by Nanjing Jiancheng Bioengineering Institute
(Nanjing, China). The kits of CAT and MDA were purchased
by Beyotime Biotechnology (Shanghai, China). The kit of GPx
was ordered from Grace Biotechnology Co., Ltd (Suzhou, China).
RNA extraction, cDNA library
construction, and transcriptome
sequencing
The hepatopancreas from the LD and HD groups was used to
transcriptome analysis. Total RNA of hepatopancreas was
isolated using TRIzol reagent (Invitrogen, Carlsbad, CA,
United States). RNA concentration and integrality was
measured using Qubit2.0 Fluorometer (Life Technologies, CA,
United States) and Agilent 2,100 Bioanalyzer, respectively
(Agilent Technologies, CA, United States). The RNA was used
to synthesize cDNA and then construct DNA libraries by PCR
amplification according to a standard procedure. The libraries
were sequenced using Illumina Novaseq6000 (Gene Denovo
Biotechnology Co., Guangzhou, China). The raw data of
transcriptome sequences have been submitted to NCBI
Sequence Read Archive (SRA) database (NO. PRJNA884003).
To ensure data quality, low-quality reads in the raw data were
filtered by fastp (version 0.18.0) (Chen et al., 2018). The value of
Q20 (the base quality score ≥20), Q30 (the base quality
score ≥30), and GC (GC content in clean reads) of clean
reads were counted. The filtered reads were assembled by
Trinity (version 2.8.4) (Grabherr et al., 2011), and the
integrity of assemble was evaluated by Benchmarking
universal Single-Copy Orthologs (BUSCO). Assembled
unigenes were annotated via nr (Non-Redundant Protein
Sequence Database), SwissProt, KEGG (Kyoto Encyclopedia of
Genes and Genomes) and COG/KOG (Clusters of Orthologous
Groups of proteins) databases. The unigene expression was
calculated and normalized to RPKM (Reads Per Kilobase of
transcript per Million mapped reads) by RSEM (Li and
Dewey, 2011). Principal component analysis (PCA) was used
to evaluate the relationship of samples.
The differently expressed genes (DEGs) was selected using
DESeq2 (version 1.20.0) (Love et al., 2014) with the threshold
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Dong et al. 10.3389/fphys.2022.1040712
value: false discovery rate (FDR) <0.05 and fold change (FC) ≥2.
All DEGs were annotated to GO (Gene Ontology) and KEGG
databases to conduct enrichment analysis. Furthermore, gene set
enrichment analysis (GSEA) was used to assess the differences of
genes in important KEGG pathways (Subramanian et al., 2005).
Validation of Differently expressed genes
by quantitative real-time PCR
To verify the reliability of RNA-seq, the key genes related to
endocytosis and mitophagy in hepatopancreas were selected and
detected via quantitative real-time PCR (qPCR). Total RNA of
hepatopancreas from the LD and HD groups was isolated using
TRIzol reagent (Takara Biomedical Technology Co., Ltd, Beijing,
China) according to the manufacturer’s instructions. The isolated
RNA was used to synthesize cDNA using a commercial kit
(Takara, RR047A). The TB Green Premix EX Taq ™kit
(Takara, RR820A) was used to amplify sequence of target
gene. The β-actin was selected as a reference gene, and the
relative expression of target gene was calculated by the 2
−ΔΔCq
method (Livak and Schmittgen, 2001). The specific primers are
shown in Supplementary Table S1.
Statistical analysis
SPSS (version 25.0) software was used to conduct statistical
analysis in this study. All values were presented as mean ± SE
(standard error). The data of growth performance and
antioxidant parameters were analyzed by one-way ANOVA
with LSD post-hoc test, and the relative levels of mRNA
between the LD group and the HD group were analyzed by
t-test. The normal distribution and homogeneity of the variances
was assessed by Shapiro-Wilk test and Levene test, respectively.
Pearson test was used for correlation analysis between the qPCR
data and RNA-seq data. Differences were considered to be
significant if the p-value <0.05.
Results
Growth performance parameters
The variation of body weight, SR, SGR and WGR of C.
quadricarinatus are presented in Figure 1. The average body
weight of C. quadricarinatus exhibited an upward trend in
different groups as the farming time increased. After 90 days
of farming, the average body weight was lower in the MD and HD
groups than that in the LD group (p<0.05). Meanwhile, the SGR
and WGR significantly decreased in the MD and HD groups
relative to that in the LD group (p<0.05). However, the SR
showed similar change among different groups (p>0.05).
Antioxidant parameters
At the end of the trial, significant differences in antioxidant
ability of C. quadricarinatus were observed among different
stocking densities (Figure 2). The activities of SOD, CAT and
GPx and the contents of MDA and GSH were higher in the HD
group than those in the LD group (p<0.05) after 90 days of
farming, whereas the CAT activity was lower in the HD group
than that in the LD group (p<0.05). In addition, the levels of
GSH and Gpx were also significantly increased in the MD group
compared with the LD group (p<0.05; Figures 2C,D).
Transcriptome sequencing and analysis of
differently expressed genes
In order to better understand the adverse effects of high stocking
density, we analyzed the genes expression profile in hepatopancreas
via RNA-seq. After filtering of raw reads, 41,402,944 (99.64%)—
47,175,044 (99.66%) clean reads were obtained, which were
assembled into unigenes for further analysis. The results of base
quality score are as follow: the values of Q20, Q30, and GC were
97.75%–98.05%, 93.58%–94.37% and 44.71%–48.25%, respectively.
The mapped ratio was greater than 92.54% (Table 1).
The result of PCA showed that the LD group and HD group
were distinctly separated, and samples had higher correlation
within group (Figure 3A). In hepatopancreas, a total of
1,531 DEGs were identified between the LD group and HD
group, including 1,028 upregulated genes and 503 downregulated
genes (Figures 3B,C).
GO enrichment analysis of differently
expressed genes
All DEGs were subjected to GO enrichment analysis to identify
significantly enriched biological process, molecular function and
cellular component (Figure 4). In the biological process category,
the DEGs were enriched mainly in organonitrogen compound
catabolic process (p.adjust = 0.003), proteolysis involved in cellular
protein catabolic process (p.adjust = 0.032), modification-dependent
protein catabolic process (p.adjust = 0.040), modification-dependent
macromolecule catabolic process (p.adjust = 0.040) and cellular
protein catabolic process (p.adjust = 0.040) (Figure 4B). In the
molecular function category, binding and catalytic activity
involved the most DEGs (Figure 4A), and lipid transporter
activity (p.adjust = 0.090) and cobalt ion transmembrane
transporter activity (p.adjust = 0.090) were the top two GO terms
(Figure 4C). In the cellular component category, the DEGs mainly
participated in cell, cell part and organelle (Figure 4A), and RNA
polymerase III transcription factor complex (p.adjust = 0.002) and
transcription factor TFIIIC complex (p.adjust = 0.003) were the top
two GO terms (Figure 4D).
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KEGG enrichment analysis of differently
expressed genes
To understand biological function and key signaling pathways,
the DEGs were enriched in KEGG database (Figure 5). The result
indicated that annotated DEGs were mapped to 124 specificKEGG
pathways, and metabolism was the most abundant KEGG A class
(Figure 5A). The top 10 enriched KEGG pathways were presented in
Figure 5B. It was worth nothing that the DEGs principally enriched in
immune function (e.g., endocytosis, mitophagy-animal, autophagy-
other eukaryotes and phagosome) and metabolism function (e.g.,
taurine and hypotaurine metabolism and biosynthesis of unsaturated
fatty acids). In addition, the FoxO signaling pathway and TGF-bata
signaling pathway were also affected by different stocking densities.
Change of mitophagy pathway
At the end of the trial, 13 genes in mitophagy pathway were
significantly changed, including 12 upregulated genes and
1 downregulated gene (p<0.05; Figure 6A). The qPCR data
showed that 7 key genes regulated mitophagy pathway, including
casein kinase II subunit alpha (csnk2a), sequestosome-1 (p62), Bcl-2
nineteen kilodalton interacting protein 3 (bnip3), autophagy-related
gene 9A (atg9), TBC1 domain family member 15-like isoform X2
(tbc1d15), microtubule-associated proteins 1A/1B light chain 3A
(lc3a) and FUN14 domain-containing protein 1 (fundc1)were
also significantly upregulated in the HD group (p<0.05;
Figure 6B), which was significantly consistent with the RNA-seq
data (r = 0.872, p=0.010;Figure 6C).
Change of endocytosis pathway
After 90 days of farming, the endocytosis pathway in the
hepatopancreas of C. quadricarinatus was significantly changed.
KEGG enrichment analysis showed that a total of 32 DEGs were
enriched in the endocytosis pathway, including 30 upregulated
genes and 2 downregulated genes (Figure 7A). Further, the
mRNA levels of 11 key genes involved in the endocytosis
FIGURE 1
Mean body weight (A), specific growth rate (B), weight gain ratio (C) and survival rate (D) of C. quadricarinatus under different densities in an
integrated rice–crayfish farming system. Values are presented as means ± SE. Means with different superscripts denote significant differences (p<
0.05). LD, low stocking density; MD, middle stocking density; HD, high stocking density.
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pathway, including heat shock protein 70 (hsp70), clathrin light
chain (clta), ras-related protein Rab-5C (rab5c), rab7a,rab11a,
suppressor protein of bem1/bed5 double mutants (vps4), sorting
nexin-12 (snx12), actin-related protein 2/3 complex subunit 4
(arpc4), adaptor protein 2 complex subunit mu (ap-2), dynamin
superfamily protein (dnm) and ras-like GTP-binding protein
(roh1), were also upregulated in the HD group compared with
the LD group (p<0.05; Figure 7B). Meanwhile, correlation
analysis indicated that the RNA-seq data were significantly
consistent with the qPCR data (r = 0.852, p= 0.001; Figure 7C).
FIGURE 2
Antioxidative parameters in hepatopancreas of C. quadricarinatus under different densities in an integrated rice–shrimp farming system after
90 days. Values are presented as means ± SE (n=9).(A) SOD; (B) CAT; (C) GSH; (D) GPx, (E) T-AOC; (F) MDA. Means with different superscripts
denote significant differences (p<0.05). LD, low stocking density; MD, middle stocking density; HD, high stocking density.
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Change of lipid metabolism pathways
The result of GSEA indicated that the lipid metabolism
function was significantly influenced by stocking density
(Figure 8). Under high stocking density, four lipid
metabolism-related pathways, including biosynthesis of
unsaturated fatty acids (KO01040), fatty acid biosynthesis
(KO00061), glycerolipid metabolism (KO00561) and
glycerophospholipid metabolism (KO00564) were more likely
to be upregulated.
FIGURE 3
Differently expressed genes (DEGs) in the hepatopancreas of C. quadricarinatus between the LD and HD group. (A) The correlation of samples
between the LD and HD groups. (B) The number of DEGs in the hepatopancreas between the LD and HD groups. (C) Volcano plot of DEGs between
the LD and HD groups.
TABLE 1 Valid data used in transcriptome analysis.
Samples Raw reads Clean reads Q20 (%) Q30 (%) GC (%) Total maped
(%)
LD-1 46,379,574 46,172,628 (99.55%) 97.91 94.08 48.08 92.96
LD-2 42,423,984 42,184,242 (99.43%) 97.83 93.91 48.25 92.80
LD-3 44,891,590 44,647,100 (99.46%) 98.05 94.37 47.70 93.35
HD-1 47,334,822 47,175,044 (99.66%) 97.97 94.08 44.73 92.82
HD-2 41,551,364 41,402,944 (99.64%) 97.75 93.58 44.71 92.54
HD-3 44,255,976 44,104,972 (99.66%) 97.94 94.03 44.79 92.91
Q20 and Q30, the base quality score (Q score) was no less than 20 and 30, respectively, in clean reads. GC, GC, content in clean reads; LD, low density group; HD, high density group.
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Discussion
Effect of stocking density on growth
performance
Growth performance was vital parameter for assessing effects
of stocking density on farmed fish or shrimp. Under excessive
stocking density, growth performance of aquatic animal is
inhibited, which may reduce economic benefits of farming. In
this study, C. quadricarinatus in the HD group exhibited a lower
growth performance than that in the LD group as evidenced by
decreased body weight, SGR and WGR. The growth performance
was inhibited when the stocking density reached to 207.15 g/m
2
in the rice-crayfish system after 90 days of farming. The similar
results have also reported in other shrimp species. For example,
the growth rate of white shrimp (Penaeus vannamei) reared at
high density (180 shrimp/m
2
) was significantly lower than that at
low density (90 shrimp/m
2
) at the 30th week (Araneda et al.,
2008). The black tiger shrimp (Penaeus monodon) under low
density (400 shrimp/m
3
) had a higher average daily weight gain,
SGR, and final biomass than that at middle density (450 shrimp/
m
3
) and high density (500 shrimp/m
3
), while the FCR (food
conversion ratio) was lower at low density after 127 days
(AftabUddin et al., 2020). There are many reasons for the
poor growth performance at high density, such as the
availability of food, water quality, habitable space and
physiological status (Riar et al., 2021). A study on P. monodon
has demonstrated that negative effects of stocking density on
growth performance may be attributed to deterioration of water
quality (Nga et al., 2005). Irwin et al. (1999) suggested that the
turbot reared at high density had lower growth rate due to the
increase of social interactions. In this work, the water quality
parameters were similar in the three groups and met the C.
quadricarinatus farming standard (Pan et al., 2017) because of
the purification of rice fields. Thus, the inhibition of growth
performance was more likely due to the interspecific
competition. In the integrated rice–crayfish farming system,
the shallow water and rice cropping limited the survival space,
FIGURE 4
GO enrichment analysis in the hepatopancreas of C. quadricarinatus under two stocking densities (LD-vs-HD). (A) GO enrichment terms of
DEGs in three ontologies. (B) The top 8 GO terms in the biological process. (C) Top 5 GO terms in the molecular function. (D) The top five GO terms in
the cellular components.
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which may lead to intensification of interspecific competition
under high stocking density. A considerable amount of energy
was consumed to cope with intensive social interaction, resulting
in the reduction of energy used for growth and metabolism
activity (Liu et al., 2016;Wang et al., 2022b).
Effect of stocking density on antioxidative
status
The antioxidant defense system is crucial to the stress
response mechanism (Jiang et al., 2014). It has been proven
that the activities of antioxidant enzymes can be regarded as
indicators of oxidative stress (Wang et al., 2013). Various
antioxidant enzymes and non-enzymatic antioxidants can
effectively remove excess reactive oxygen species (ROS) and
protect against oxidative damage (Zhang et al., 2022).
However, when the stress exceeds the tolerance, the
scavenging capacity of the antioxidant defense system is
decreased (Winston, 1991;Mathew et al., 2007). In
aquatic animals, high stocking density can cause oxidative
stress and change antioxidative status, which is related to
species and farming conditions (Liu et al., 2017;Dorothy
et al., 2021;Campa-Córdova et al., 2022). The antioxidant
parameters, such as SOD, CAT, GPx, GSH and T-AOC, were
inhibited by high stocking density in largemouth bass
(Micropterus salmoides)(Kommaly et al., 2021)andtiger
puffer (Takifugu rubripes)(Zhang et al., 2019), which may
cause an oxidative damage. However, the study on L.
vannamei reared in biofloc system showed that the
activities of antioxidant enzymes (SOD, CAT, and GPX)
were increased with increasing stocking density (Dorothy
et al., 2021). Similarly, the data in olive flounder
(Paralichthys olivaceus) indicated that high stocking
density significantly increased the expression and
activities of SOD and CAT (Choi et al., 2019). In this
study, the levels of SOD, GPx, GSH and T-AOC increased
in the HD group after 90 days farming, indicating
antioxidant defense system was activated to copy with the
adverse stimulation caused by high stocking density. In
addition, the activity of CAT in the HD group was
significantly lower than that in the LD group, indicating
chronic oxidative stress may depress the activity of CAT.
This was probably because the CAT consumption was
FIGURE 5
KEGG enrichment analysis in the hepatopancreas of C. quadricarinatus under two stocking densities (LD-vs-HD). (A) The result of DEGs
enrichment in the KEGG. (B) The top 10 enriched KEGG pathways.
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Dong et al. 10.3389/fphys.2022.1040712
FIGURE 6
Changes of mitophagy pathway in the hepatopancreas of C. quadricarinatus between the LD group and the HD group. (A) DEGs enriched in
mitophagy pathway. (B) Mitophagy-related gene expression measured by qPCR, values are presented as means ± SE (n=3)(C) The correlation
between the qPCR data and RNA-seq data.
FIGURE 7
Changes of endocytosis pathway in the hepatopancreas of C. quadricarinatus between the LD group and the HD group. (A) DEGs enriched in
endocytosis pathway. (B) Endocytosis-related gene expression measured by qPCR, values are presented as means ± SE (n=3).(C) The correlation
between the qPCR data and RNA-seq data.
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Dong et al. 10.3389/fphys.2022.1040712
greater than its synthesis under chronic oxidative stress
caused by high stocking density.
Under oxidative stress, the excess ROS attacks lipids,
inducing lipid peroxidation (Yin et al., 2011). MDA is a final
product of lipid peroxidation (Wang et al., 2022a). Hence, it is
generally recognized that the content of MDA can reflect the level
of oxidative damage in organisms (Han et al., 2022). It has been
reported that the content of MDA significantly increased with the
increasing stocking density in many aquatic animals, such as M.
salmoides (Jia et al., 2022), S. maximu (Jia et al., 2016), and blunt
snout bream (Megalobrama amblycephala)(Wang et al., 2018).
In this study, the significant increase of MDA content in the HD
group also proved that long-term high stocking density caused
oxidative stress.
Effect of stocking density on mitophagy
The liver is a highly dynamic metabolic organ and a major
site of protein synthesis, lipid metabolism and detoxification
(Qian et al., 2021). These metabolic processes require a high
amount of energy. Mitochondria is the primary energy-
generating organelle and its function readily deteriorates
under stress condition. To maintain mitochondrial quality,
FIGURE 8
Changes in lipid metabolism pathways identified using GSEA in the hepatopancreas of C. quadricarinatus under two stocking densities (LD-
vs-HD). (A) Biosynthesis of unsaturated fatty acids. (B) Fatty acid biosynthesis. (C) Glycerolipid metabolism. (D) Glycerophospholipid metabolism.
The |Normalized ES| >1, nominal p-value <0.05 and FDR <0.25 in each gene set were set as threshold values for statistical significance.
Frontiers in Physiology frontiersin.org11
Dong et al. 10.3389/fphys.2022.1040712
cells can invoke a mitochondria-specific form of degradative
process, named mitophagy, to remove damaged and
dysfunctional mitochondria (Saito et al., 2021). Mitophagy
shares some key regulatory proteins, such as LC3 and p62,
with macroautophagy, and it is also regulated by specific
proteins including PTEN-induced putative kinase 1 (PINK1),
Bnip3, and Fundc1 (Urbina-Varela et al., 2020). The mitophagy
is susceptible to external stress, such as hypoxia and nutrient
deprivation, which, in turn, significantly influences
mitochondrial function, cell survival and energy homeostasis
(Ke, 2020). It has been reported that mitophagy is triggered to
scavenge impaired mitochondria and reduce ROS level under
oxidative stress (Fan et al., 2019a). Thus, mitophagy is considered
as a protective response against oxidative damage induced by
adverse stimuli (Fan et al., 2019b;Garza-Lombo et al., 2020). In
aquatic animals, lots of exogenous stimuli, such as starvation,
hypoxia, and bacterial or viral infections, have been proven to
cause the activation of autophagy. For example, copper exposure
upregulated the mRNA levels of LC3a, PINK1, Parkin, and
induced mitophagy in L. crocea (Pan et al., 2020). Dietary
methionine deficiency led to induction of mitophagy via
PINK1/PARKIN axis in the liver of rainbow trout
(Oncorhynchus mykiss)(Seite et al., 2018). In this study, the
KEGG analysis showed that the mitophagy pathway was
activated under high stocking density. It is worth noting that
the key genes including lc3a, p62, bnip3, and fundc1 were
significantly upregulated in HD group. We speculated that
high stocking density is a stressor that could induce ROS
production and further trigger mitophagy. The activation of
mitophagy is an adaptive response which may alleviate
oxidative damage (Tian et al., 2022). Similar data were also
reported in previous study, where mitophagy was activated to
maintain cellular homeostasis in loach fin cells under oxidative
stress induced by doxycycline exposure (Shan et al., 2022).
Effect of stocking density on endocytosis
Endocytosis is an essential and highly dynamic biological process
that is responsible for the internalization of transmembrane receptor
ligand complexes, lipids and pathogens (Schroeder and McNiven,
2014).Itplaysanimportantroleinmaintaining cellular homeostasis
and interacting with environments (Mellman, 1996), which is mainly
manifested in controlling the composition of plasma membrane
(Doherty and McMahon, 2009), participating in immune regulation
(Lv et al., 2021), absorption of nutrients and other signal transduction
of physiological activities (Leborgne-Castel and Luu, 2009). It has
been reported that environmental stress-induced adaptive programs
can regulate bulk endocytic flux and alter endocytosis (Shin et al.,
2021). Increased endocytosis may strengthen cellular resilience via
elevating nutrient intake under stress condition (Sébastien, 2021). In
addition, the alteration of endocytosis during stress may counteract
deleterious effects through switching on or amplifying specific
cellular pathways (Lopez-Hernandez et al., 2020). Previous studies
also suggested that the significant change of endocytosis pathway
could be induced by adverse environmental conditions in aquatic
animals, such as nitrite (Yu et al., 2019), salinity (Qin et al., 2020),
copper exposure (Xing et al., 2021) and cadmium exposure (Zhang
et al., 2021). Vega et al. (2010) suggested the upregulated endocytosis
played an important role in the recovery from damage under stress
conditions. In the present study, we found that high stocking density
induced the upregulation of endocytosis in hepatopancreas of C.
quadricarinatus, which probably was a result of oxidative stress. The
increased endocytosis may be an adaptive response which enhanced
nutrients uptake to maintain the cellular homeostasis.
Effect of stocking density on metabolism
Dynamic change in metabolic function is a major mechanism
responded to external stress in aquatic animals. High stocking density
as a stressor has been confirmed to induce a change in amino acid
carbohydrate and triglyceride metabolism in liver of Patagonian
blennie (Eleginops maclovinus)(Oyarzún et al., 2020). Under a high
stocking density, glucose metabolic enzymes were activated, which
enhanced energy production to resist the environment stimuli in
abalone (Haliotis discus hannai)(Gao et al., 2018). A
transcriptomic analysis of M. salmoides indicated that high stocking
density caused abnormal lipid metabolism (Jia et al., 2022). In line with
previous studies, our data also showed that the metabolic function of
hepatopancreas, such as organonitrogen compound catabolic process,
proteolysis involved in cellular protein catabolic process, modification-
dependent protein catabolic process and cellular protein catabolic
process, was significantly influenced by high stocking density. We
suspected the changes of metabolic function may produce more energy
to adapt to the stress condition, meaning that less energy and matter
were used for growth under high stocking density.
It has been reported that hepatic lipid metabolism is highly
susceptible to adverse stress (Guo et al., 2022). In aquatic animals,
high stocking density-induced changes of lipid metabolism has been
widely reported. The lipid contents were reduced in liver of gilthead
seabream (Sparus aurata)andpiabanha(Brycon insignis)under
high stocking density, which may reflect a higher lipid utilization to
cope with the stress (Montero et al., 2001;Tolussi et al., 2010).
Multiple omics analysis showed that high density caused abnormal
lipid metabolism in grass carp (Ctenopharyngodon Idella) and lenok
(Brachymystax lenok)(Liu Y et al., 2019;He et al., 2021). Similarly,
our data exhibited altered lipid metabolism in hepatopancreas of C.
quadricarinatus under high stocking density. Meanwhile, the four
key pathways associated with lipid metabolism, including fatty acid
biosynthesis, biosynthesis of unsaturated fatty acids, glycerolipid
metabolism and glycerophospholipid metabolism, displayed an
upregulated tendency in the HD group, which may be an
adaptive response to stressful condition. The results were
consistent with previous studies. For example, activated
biosynthesis of unsaturated fatty acids can maintain the
Frontiers in Physiology frontiersin.org12
Dong et al. 10.3389/fphys.2022.1040712
membrane fluidity, regulate physiological state and provide energy
under starvation and hypoxia stress (Jiao et al., 2020;Ma et al., 2021);
Glycerolipid metabolism had an active effect on response to thermal
stress in the liver of S. maximus (Zhao et al., 2021); yeast
(Saccharomyces cerevisiae) maintained the membrane
homeostasis by activating glycerophospholipid metabolism (Xia
et al., 2019).
Conculsion
This study showed that C. quadricarinatus is suitable for
growing in integrated rice-crayfish farming system according to
the growth performance and survival rate. However, the growth
performance of C. quadricarinatus was significantly inhibited when
the stocking density reached up to 207.15 g/m
2
(the stocking density
of the HD group on 90th day). Meanwhile, the high stocking density
caused oxidative stress after 90 days of farming. In order to cope with
the adverse change of physiological state, the endocytosis, autophagy
and lipid metabolism pathways were activated in the hepatopancreas
of C. quadricarinatus, which may maintain cellular homeostasis,
strengthen cellular resilience and provide energy. In addition, the
activation of these pathways consumed a considerable amount of
energy, resulting in the reduction of energy used for growth activity,
which may be a potential mechanism to explain the inhibition of
growth under high stocking density. In summary, our study
provided a reference for optimizing the stocking density of C.
quadricarinatus in an integrated rice-crayfish farming system.
Data availability statement
The datasets presented in this study can be found in online
repositories. The name of the repository and accession number
can be found below: NCBI; PRJNA884003.
Ethics statement
The animal study was reviewed and approved by Freshwater
Fisheries Research Center, Chinese Academy of Fishery Sciences.
Author contributions
YD conceptualization, writing—original draft preparation, and
software; RJ methodology, investigation validation, formal analysis,
and resources; YH and WD data cu-ration, writing—review, and
editing; JZ and BL visualization, supervision, project administration,
and funding acquisition. All authors read and approved the final
version of the manuscript.
Funding
This study was supported by Central Public-interest
Scientific Institution Basal Research Fund, CAFS (2020TD60),
earmarked fund for CARS (CARS-45–20), and National Key
R&D Program of China (2019YFD0900305).
Conflicts of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the
authors and do not necessarily represent those of their
affiliated organizations, or those of the publisher, the
editors and the reviewers. Any product that may be
evaluated in this article, or claim that may be made by its
manufacturer, is not guaranteed or endorsed by the
publisher.
Supplementary material
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fphys.
2022.1040712/full#supplementary-material
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