​Molecular Mechanism of Extreme Hypoxia Tolerance Difference between Male and Female Adult Fish and Juvenile Fish of Acrossocheilus fasciatus by Transcriptomics

It is always worth thinking about the adaptability of different organisms to extreme hypoxia in nature. Different individuals have different responses to extreme hypoxia. Abundant researches showed that fish size has significant effect on hypoxia tolerance (Nilsson et al., 2010). Research discovered that small-sized Megalobrama amblycephala performed better in hypoxia tolerance than their fellows with larger sizes (Chen et al., 2017). Nonetheless, Sloman got an opposite result when studying Astronotus ocellatus (Sloman et al., 2006). As a result, the relationship between fish size and hypoxia tolerance has always been in the spotlight. The influence of gender on extreme hypoxia tolerance cannot be underestimated. Abundant researches showed that hypoxia-treated male rats show obvious heart damage and arrhythmias compared with females. However, there are few studies on the effects of gender and age on acute hypoxia adaptation in fish at the same time, especially in the molecular field, so it is necessary to continue intensive study.
 
Asphyxiation point and oxygen consumption rate are important evidences to represent fish metabolism, with asphyxiation point being a limit index of fish hypoxia tolerance (Jia-Er et al., 2008). Therefore, asphyxia point is a good experimental condition to study the difference of extreme hypoxia tolerance caused by different gender and age. Fish of different genders and sizes possessed different metabolic demand, resulting in diverse molecular mechanisms towards hypoxia response. Up to now, molecular mechanism related to hypoxia in A. fasciatus remains unknown. So further research on the molecular mechanism of extremely low oxygen is needed.
       
Acrossocheilus fasciatus (A. fasciatus) is termed as Cypriniformes, Cyprinidae, Acrossocheilinae, which is a small-sized fish in mountain streams, widely distributed in areas south of the Yangtze River. The requirement of water quality for A. fasciatus is too demanding that the drawback of hypoxia intolerance badly inhibits the promotion of breeding well-bred A. fasciatus. 

Acrossocheilus fasciatus (A. fasciatus) used in this experiment were obtained from artificial breeding populations from fishery research Department of Zhejiang Ocean University. The experimental site was carried out in the Fisheries Laboratory of Zhejiang Ocean University in 2021. Experimental subjects are juvenile A. fasciatus with body length of (3.8±0.23 cm) and body weight of (0.97±0.23 g) in the juvenile stage and genderually mature adult fish with female groups with body length of (13±1.17 cm) and bodyweight of (19.45±1.86 g). Male groups with body length of (12±1.05 cm) and body weight of (16.13±1.57 g).
       
Total 100 A. fasciatus in each of the three groups were bred in three separate culture ponds with biofilter circulation system. The domestication experiment lasted for 1 month in the biofilter circulation system under the condition of 24^oC and dissolved oxygen (DO) of 7.8 mg/L±0.2 mg/L (normoxia). During domestication, fish were fed twice per day with commercial fish feed. Daily inspection of water quality was performed and dead animals as well as particles were removed immediately.
 
Ethics statement
 
This study was conducted in accordance with the guidelines and approval of Institutional Animal Care and Use Committee at the Zhejiang Laboratory Animal Research Center and Zhejiang Ocean University. The approval number is 20210207.
 
Measurement of asphyxiation point
 
We have prepared 45 transparent glass bottles (500 mL) with completely sealed lids as breathing chambers for three sizes of fish. Before the experiment, the 45 transparent glass bottles were filled with water. The initial dissolved oxygen in the water was maintained at 7.8 mg/L±0.2 mg/L and the water temperature was maintained at 24 degrees. We randomly selected 15 items from each group of female adult fish, male adult fish and juvenile fish in the previous breeding ponds and a total of 45 fish was placed in these 45 experimental glass bottles independently and then sealed. When the fish was observed in a dying state, YSI Pro20 Oxygen Dissolver (USA) was used to record the DO of the water in the breathing chamber at that time. We defined the dissolved oxygen value at this time as the asphyxiation point of each fish. After recording the asphyxiation point, take out the fish in each breathing room, measure and record the weight and body length. The experimental data were analyzed by SPSS 20.0 software and each data was expressed as mean ± SD. Duncan test was used to compare the mean of each treatment and the difference was statistically significant (P<0.05).
 
Extreme hypoxia test
       
In the formal experiment, randomly selected 15 healthy A. fasciatus in each of the three groups were transported from culture ponds to three sealed glass containers (5L) filled with water, with the DO of 7.8 mg/L±0.2 mg/L, water temperature constant at 24 degrees as extreme hypoxia treatment groups. In each of the groups, the first 5 fish to reach death stage were divided into hypoxia sensitive group and the last 5 fish to reach death stage were divided into hypoxia tolerant group. DO in glass containers dropped with oxygen consumption by fish. Fish were immediately brought out for sampling when the first 5 fish and the last 5 fish of each group displayed death stage. Subsequently, 5 fish in each of the three groups in the nomoxia culture pond with constant DO of 7.8 mg/L±0.2 mg/L, as nomoxia control group, were brought out and bathed in eugenol (dilution 1:1000) for anaesthetic. After measuring body weight and length, fish were dissected to get their liver tissues, which were immediately frozened in liquid nitrogen and stored in -80^oC till RNA extraction.
 
Extraction of RNA, synthesis of cDNA and transcriptome sequencing
 
After gathering all of the samples, TRIzol^® reagent (Invitrogen) was used in accordance with the instruction. Total liver RNA of 9 groups were extracted and the 9 groups were hypoxia sensitive, hypoxia tolerance and nomoxia control groups of each of the three groups respectively Each group of RNA is a mixture of five individuals and labeled as F1 ( adult female fish group 1), F2 (female adult fish group 2), FC (Normoxic control group of adult female fish), M1 (adult male fish group 1), M2(adult male fish group 2), MC (Male adult normoxic control group), J1 (juvenile fish group 1), J2 (juvenile fish group 2) and JC (Normoxic control group) respectively. Extracted RNAs in each of the groups were the mixture of 5 individuals. NanoPhotometer® spectrophotometer (IMPLEN, California, U.S.) was utilized to check RNA purity and Qubit^®RNA analyzing kit in Qubit^®2.0 fluorophotometer was used to detect RNA concentration (Invitrogen, California, U.S.). According to the recommendation of manufacturer, NEBNext^®Ultra™RNA Library Prep Kit for Illumina^® (NEB, U.S,) was used to generate sequencing library. Later, RNA-seq library was sequenced by Illumina sequencing technology.
 
Differentially expressed genes and enrichment analysis
 
The threshold level to identify significant DEGSs was set as p value (Padj)<0.05 and |log2 fold change|>1.0. Those with p<0.05 were considered as significantly enriched. Unigene sequences was aligned with NR, Swiss-Prot, GO, COG, KOG, KEGG by BLAST software. After predicting the amino acid sequences of Unigene, HMMER software and Pfam database was used for alignment, obtaining annotations of Unigene.
 
Transcriptome data certification by transcriptome data deriving from qRT-PCR
 
Total RNA reversal was conducted under the guidance of construction. Sequences of related genes were searched based on the results of transcirptome sequencing. And 8 specific primers to be used in qRT-PCR were designed based on sequences in the coding region (Table 1). TB PrimeScript^™RT Kit (TaKaRa) was placed in the ABI QuantStudio 3 system for qRT-PCR analysis. Relative expression level of each gene was calculated by 2^-ΔΔCT method. Three replications were tested for each of the groups and all the samples in triplicate were detected on the same plate. The expression level of target gene was normalized into the expression of β-actin, presented as relative expression of target gene (relative mRNA).
 

Analysis of asphyxiation point results
 
Under the condition of water temperature of 24^oC, the average asphyxiation point of female adult was 0.56, the average asphyxiation point of male adult was 0.66 and the average asphyxiation point of juvenile was 0.858 (Fig 1). It is obvious that the asphyxiation point of adult fish is lower than that of young fish and the asphyxiation point of female fish is lower than that of male fish. Female adults showed the strongest tolerance to extreme hypoxia. Through regression analysis, we can get that the slope of fitting curve of body weight is larger (Fig 2), The regression equation can be expressed as y=0.880-0.013a-0.002b (y stands for asphyxia point, a stands for body weight, b stands for body height), which indicates that body weight has more influence on asphyxia point than body length.
 

 

       
The difference of hypoxia tolerance caused by different age and gender of organisms has always been the focus of researchers. A great deal of studies argued that size of fish had essential impact on hypoxia tolerant ability. In this research, the asphyxiation point of A. fasciatus is higher than hypoxia tolerant fresh water fish such as Pelteobagrus fulvidraco and Carassius aruatus (Wu et al., 2014). In the comparison of asphyxiation point in Acrossocheilus fasciatus (A. fasciatus) with different sizes, an decreasing tendency was observed with the increase of age and weight. The asphyxiation point of female adult fish was the lowest among the three specifications, showing the strongest extreme hypoxia tolerance.
 
Statistic of transciptome data and analysis of differentially expressed genes
 
Juvenile fish, male fish and female fish qualified for sequencing were selected and samples were processed under extreme anoxia stress and no stress (control) respectively. Then, transcriptomic sequencing was performed, gathering 80.16 Gb Clean Data. Detailed data of sequenced samples and their quality could be found in (Table 2). According to the Venn (Fig 3) and the volcano maps (Fig 4). It can be seen that the number of DEGSs of adult fish is much higher than that of juvenile. Further analysis showed that the number of DEGS in adult male and female significantly exceeded the number in juvenile. This phenomenon was also reported in other fish such as grass carp (Jin et al., 2017), yellow catfish, indicating that improving the number of gene expression was the main response to adapt to acute hypoxia environment.
 

 

 

       
Differentially expressed genes were further analyzed by GO functional enrichment analysis (Fig 7). Differentiallyexpressed genes across 6 comparison groups were mainly enriched in 3 GO terms (Cell Component: 17, Molecular Function: 14 and Biological Process: 23) and 54 subterms (Fig 5). In general, M1 vs MC clearly had more enriched than the other comparisons and the number of enriched genes was relatively distributed more evenly in each of the terms. The results indicated that under hypoxia stress, processes related to cellular metabolism and synthesis and catalysis were highly activated in the liver and gill of A. fasciatus. KEGG enrichment analysis was carried out on the signal pathway of differentially expressed genes (Fig 6). The results showed that the differentially expressed genes in six comparison groups were significantly enriched in Signal transduction, transport and Catabolism and other signaling pathways. The above results showed that acute hypoxia activated endocrine, protein, amino acid and Multiple biological processes such as fat and carbohydrate metabolism to regulate A. fasciatus adapt to extreme hypoxia stress (Qian et al., 2019). Hypoxia stress changed the endocrine system of A. fasciatus.
 

 

 

 
Screening for key genes under extreme hypoxia stress
 
In order to further explore the reasons why female fish of A. fasciatus are more tolerant to extreme hypoxia, top 10 genes with the greatest significant expression were screened (Table 3), In particular, differentially expressed genes were significantly enriched in the Ferroptosis, MAPK signaling pathway, etc. Under hypoxia, a series of responses takes place inside cells to accommodate hypoxia (Jiang et al., 1996). We found that the expression of ncoa4 and facl4 was significantly down regulated and the expression of jnk, gpx4 and jip-1 was significantly increased in three sizes of fish under extreme hypoxia. Specially, the increase and decrease of these genes were most obvious in female adult fish. Ferroptosis is an iron-dependent novel manner of programmed cell death, with the essence being intracellular lipid oxide metabolism disorder (Xie et al., 2016). Drastic environmental stress makes it easier for the occurance of ferroptosis (Speer et al., 2013). In this research, expression of genes related to ferroptosis, such as glutathione gene gcl and gss, transferrin, ferritin heavy chain, was up-regulated in the three groups, which was similar to the results in researches about other fish under stress condition. In this research, expression of NCOA4 was lower in adult groups compared to juvenile groups. The expression level in female groups was even more significantly reduced, suggesting that female groups were equipped with stronger anti-oxidative stress damage ability by down-regulating the expression of NCOA4 (Gryzik et al., 2001). Hypoxia stress response was achieved by the interaction of regulatory network based on these pathways and genes involved in the pathways as well as complicated molecular adaptation strategies.
 

 
qPCR validation
 
To verify transcriptome data, sequences of 8 genes (osbp6, bco1, mrc2, klf5, nr, cfh, ho, hif-1a) were randomly selectedaccording to the transcriptome data and qRT-PCR was used to detect their relative expression level (Fig 7). Results suggested that the tendency of up-regulation and down-regulation was in accordance with transcriptome data, certificating the reliability of results of the transcriptome data. It is noteworthy that heme oxygenase-1 (HO-1), as the initial enzyme and rate-limiting enzyme of heme degradation, has anti-inflammatory, antioxidant, anti-apoptotic and other physiological effects (Shi et al., 2018). The highest expression was observed in juvenile fish under extremely low oxygen stress, which indicated that the damage of juvenile fish was the most serious.  As a hypoxia induciblefactor, Hif1a has the highest expression in juvenile fish under hypoxia stress, which also shows that hypoxia stress has a more serious impact on the body of juvenile fish.

In a nutshell, by measuring asphyxiation point, understanding exuberant metabolic time span and hypoxia tolerance, it is helpful to better explain the reason why females have stronger hypoxia tolerance. Moreover, by comparative analysis of transcriptome data under hypoxia stress, crucial genes related to extreme hypoxia stress and enhancement of hypoxia tolerance were discovered. This research held a great importance in widening the knowledge of physiological mechanism of A. fasciatus under extreme aquatic conditions.