The Gobiidae is the largest family of marine fishes, which consists of more than 200 genera and nearly 2,000 species, and is characterized by wide distribution, high diversity, and strong adaptability (Tassell, 2011). Acanthogobius ommaturus is a large, demersal, and fast-growing annual fish in the family of Gobiidae, which is widely distributed in coastal waters or brackish waters of the Northwest Pacific Ocean surrounding China, Korea, Japan, and Indonesia (Wu and Zhong, 2008). Individuals of this species grow rapidly, and have a nearly linear growth curve in the first 6 months during the year of its life cycle, and then the growth rate slows down. The standard length and body weight will continue to increase for spawning by the next April, after which the parents die (Wang et al., 2011). Studies have shown that annual fish species usually have rapid growth and sexual maturation to maximize reproduction (Lanés et al., 2014; Lanés et al., 2016). Annual fish, whether male or female, need to invest a large amount of energy for reproduction (Berois et al., 2015; Godoy et al., 2019). The body weight of A. ommaturus can reach more than 450 g in 1 year (Chen, 1978), which is completely rare in annual fish (Jakub et al., 2021). This suggests that the species has a strong growth potential and energy storage capacity. In addition, as a demersal fish inhabiting coastal region and estuaries, A. ommaturus often faces environmental changes, such as light, temperature, salinity, and other factors. To cope with complex and changing living environments, A. ommaturus needs to store energy and allocate energy reasonably. However, the energy metabolism or storage of A. ommaturus, especially in molecular level, has not been well studied.

Lipids with their constituent fatty acids (FAs) and proteins are the major organic constituents in fish, while carbohydrates are much less abundant in fish. Actually, the protein content is much less than the lipid content, which reflects that lipids are usually the major source of metabolism energy in fish for growth, reproduction, and movement (Tocher, 2003). Notably, FAs are an important composition of lipids, participating in a wide variety of metabolic pathways (Watkins et al., 2007). NADPH (nicotinamide adenine dinucleotide phosphate) produced by the FA oxidation can provide metabolism energy in the form of ATP through the oxidation phosphorylation process (FrOyland, 2015). The capelin, herring, and salmonids prove that FAs are the preferred source of metabolic energy (Henderson et al., 1984a; Henderson et al., 1984b; Henderson and Almatar, 1989; Tocher, 2003). Lipid homeostasis is the balance between lipid uptake, storage, biosynthesis, transportation, metabolism, and catabolism (Tocher, 2003), which plays an important role in maintaining normal life activities of fish. The lipids are only stored in the liver in the majority of gobiids (Akiyoshi and Inoue, 2004; Cuevas et al., 2016; Louiz et al., 2018), and gobies can maintain a lifelong high level of fat storage without pathological changes caused by abnormal lipid metabolism, suggesting that there may be a special mechanism to maintain lipid homeostasis in these fishes. Hence, elucidating the lipid metabolism of A. ommaturus is of great value for understanding the evolution of energy metabolism in teleost and the pathogenesis of abnormal lipid metabolism.

A high-quality, complete, and contiguous genome is essential to analyze the biological characteristics of ecology and evolution (Ravi and Venkatesh, 2018; Bian et al., 2019). Examples are sex-determination mechanisms (Cai et al., 2021), loss of pelvic fin (Lin et al., 2016), loss of adaptive immunity (Star et al., 2011), and genome compaction (Aparicio et al., 2002). Although many fish genome data were previously released at NCBI, most of them were assembled based on short reads with limited contiguity and quality. Compared to second-generation sequencing technologies, third-generation sequencing technologies, such as PacBio (Pacific Biosciences, Menlo Park, CA, USA) and Nanopore (ONT, Oxford, UK), can produce long reads and avoid many gaps (Ge et al., 2019). These long-read assembly approaches can improve contiguity and span repetitive regions, which provides a path forward for genome assembly at a high level (Gordon et al., 2016). Additionally, the high-throughput chromosome conformation capture (Hi-C)-assisted genome assembly technique has been used to assemble chromosome-level genome for many fishes, such as Epinephelus lanceolatus (Zhou et al., 2019), Oplegnathus fasciatus (Xiao et al., 2019), Lota lota (Han et al., 2021), Micropterus salmoides (Sun C. et al., 2021), and Mugilogobius chulae (Cai et al., 2021).

The high-quality complete genomes of fish have proliferated in recent years. Gobies are an important group in the ecosystem as the bait of many fish, and are one of the most diverse families of vertebrates on earth (Tassell, 2011). Unfortunately, little genomic information on gobies is available, and only a few goby genomes reach chromosomal level. In this study, the chromosome-level genome assembly of A. ommaturus was constructed by combining Illumina short reads, Pacbio long reads, and Hi-C sequencing data, and its phylogenic relationships with other fishes were elucidated by comparative genome analysis. Based on genome data, we aimed to identify the lipid metabolism and candidate aging genes. This work may provide important resources to study the mapping of traits with economic importance, evolutionary position, and it provides an accurate reference sequence for related species.

In the present study, the whole genome of A. ommaturus was generated by PacBio sequencing combined with the Hi-C approach, yielding high-quality genome annotations and a chromosome-level genome for this economically important species. A total of 33 million Hi-C raw reads were finally clustered into 22 chromosomes, which is consistent with a previous karyotype study of A. ommaturus (Wang and Zhao, 1993). In addition, we sorted out the chromosome number of some fishes in Gobiidae based on literature, the NCBI database, and the Fish Karyome database. The chromosome number of Gobiidae fish varies from 22 to 38 ( Supplementary Table 20 ). The chromosome number of A. ommaturus is the same as its relative species, Acanthogobius flavimanus. The genome size of 921.49 Mb of A. ommaturus was in the middle of the genome size of its relative species (M. chulae: 1.002 Gb, Neogobius melanostomus: 1.00 Gb, B. pectinirostris: 955.75 Mb, Rhinogobius similis: 890.10 Mb). We identified 18,752 protein-coding genes in A. ommaturus, which was similar to those in Scartelaos histophorus (18,156) (You et al., 2014) and Lepisosteus oculatus (18,328) (Braasch et al., 2016), but lower than those in M. chulae (20,531) (Cai et al., 2021), B. pectinirostris (20,798) (You et al., 2014), and D. rerio (26,260) (Howe et al., 2013). The annotation data of relative species in databases were less, which may affect the homology prediction results. Moreover, the gene copy number of this species may decrease during evolution. A previous study has shown that the functions of genes are redundant after gene and genome duplications during evolution. Therefore, a single-copy gene is sufficient to perform its function, while the sub-copies of gene may accumulate harmful mutations and loss (Meyer and Schartl, 1999). Notably, the functionality and number of genes have to be further confirmed, and the current finding could only be the first preliminary step towards identifying key genes.

We discovered that the effective population size of A. ommaturus experienced a bottleneck event from 200 to 100 Ka, which may be related to the Guxiang Glacial Stage (ShangZhe et al., 2007). Glacial stage can affect atmospheric circulation, monsoon strength, animal and plant changes, soil development, and sea level rise and fall, resulting in a decline in population size. The population size of A. ommaturus gradually increased from 70 to 30 Ka, which may have something to do with several warm periods during this time and the influence of the Kuroshio (Hu et al., 2021).

Annual fish often demand great energy for rapid growth and sexual maturity in the juvenile stage. A. ommaturus feeds on a variety of shrimps in the juvenile stage (Sun et al., 1996), which may be associated with energy requirements. Previous studies have shown that A. ommaturus enhanced energy metabolism to increase their ability to survive in salinity changes (Sun et al., 2020), and the energy-related pathways were significantly enriched in response to temperature changes (Sun Z. et al., 2021). To facilitate rapid energy mobilization, A. ommaturus may have evolved a regulatory mechanism. In the A. ommaturus genome, acsbg2, znf638, and the ldlr gene family were expanded compared to 14 other fishes, and there were eight copies of the acsbg2 gene. Nine genes (acsl6, acsf3, hrasls, stard3, fads2, fabp6, pparγ, acat1, and apoe) with essential roles in lipid metabolism were identified as PSGs.

Lipids play important roles in numerous biological processes, such as inflammation response, biofilms, reproduction, and energy source and storage (Lopes-Marques et al., 2018). FAs are important components of many lipids, including structural lipids, storage molecules, and signaling molecules. By now, FA catabolism is the main energy source of many fish (Tocher, 2003). They can be degraded for energy production to serve many essential functions in living organisms (Watkins et al., 2007). Notably, any anabolic and catabolic process of FA participation has a critical initial step, the “activation” of FAs. The activation reaction is catalyzed by ACS to produce a thioester with CoA (Watkins, 1997). The ACS is essential to lipid metabolism ( Figure 4C ). The acsbg2 gene has been demonstrated to convert FAs to active Acyl-CoA using C18:1 and C18:2 as substrates (Pei et al., 2006). acsls typically activate C16:0 and C18:1 (Watkins, 1997). Huang et al. (2014) have researched the FAs in the muscle of A. ommaturus, and the results showed that the FAs were mainly C22:6 (~24.77%), C16:0 (~20.62%), C18:1 (~17.31%), C20:5 (~14.78%), C18:0 (~7.74%), and C18:2 (~3.84%) (Huang et al., 2014). The total content of these six FAs is up to 90%, which shows that the acsbg2 and acsl6 genes play an important role in A. ommaturus FA metabolism and synthesis.

The most important simple lipid is cholesterol (Tocher, 2003). ldlr is a kind of lipoprotein-carrying cholesterol, which plays a role for essential energy production, cell membrane, and cholesterol homeostasis (Brown and Goldstein, 1986; Go and Mani, 2012). ldlr gene variants with impaired function result in early-onset atherosclerosis known as familial hypercholesterolemia (FH) (Go and Mani, 2012). The ldlr gene knockout zebrafish showed moderate hypercholesterolemia when fed a normal diet (Liu et al., 2017). The ldlr gene family including lrp1, lrp2, lrp4, and lrp6 were expanded in A. ommaturus. lrp1 is involved in FA uptake, and lrp6 is involved in the synthesis of TG and FAs, which both suggested that this species has a strong ability to regulate lipid homeostasis. In addition, znf638 is a key regulator of adipogenic differentiation (Meruvu et al., 2011). Pparγ and apoe are also essential to lipid metabolism (Ren et al., 2002; Huang and Mahley, 2014). The expansion of lipid metabolism genes in A. ommaturus may help it to adapt to the complex environment, for instance, by allocating energy properly to respond to changes in external environmental factors.

Aging is broadly defined as the time-dependent irreversible physiological decline that affects most organisms, which has been demonstrated into nine hallmarks. These hallmarks are as follows: loss of proteostasis, telomere attrition, stem cell exhaustion, epigenetic alterations, mitochondrial dysfunction, cellular senescence, genomic instability, deregulated nutrient sensing, and altered intercellular communication (López-Otín et al., 2013). A. ommaturus that has a life span of 1 year may be a good model for aging studies. Analysis of positive selection in the genome of A. ommaturus identified 20 genes associated with aging, such as ercc6, hspd1, igf1, polg, taf1, and tert. These genes can be mainly divided into two categories based on the characteristics of aging.

(I) Genomic instability. The accumulation of various types of genetic damage throughout lifetime is the common feature of aging (Moskalev et al., 2013). Cells may undergo a series of phenotypic changes, from cell cycle arrest, cellular senescence, to malignant transformation when DNA damage reaches a certain level (Erol, 2011). Progeria aging syndromes are often the result of increasing DNA damage accumulation (Burtner and Kennedy, 2010). ercc6, known as CSB, is involved in DNA unwinding and DNA repair (Licht et al., 2003). A previous study showed that ercc6 and xpa mutant mice die before weaning and display some premature aging phenotypes, such as attenuated growth, retinal degeneration, kyphosis, progressive neurological dysfunction, and cachexia (Van et al., 2007). Cockayne syndrome (CS) is a rare progeroid syndrome caused by mutations of ercc6 (CSB) genes (Karikkineth et al., 2017), which has characteristics of normal aging including vision and hearing loss, early-onset neurodegeneration, and impaired mitophagy in early life, ultimately leading to premature death (Lee et al., 2019). Thus, the ercc6 (FDR = 0.032) under positive selection may be a candidate aging gene in A. ommaturus.

(II) Telomere attrition. Telomerase consists of the protein component tert and the RNA component terc, which prolong telomeres after replication to maintain the telomere length (Harel et al., 2015). Telomeres are considered as a good biomarker in age research (Boonekamp et al., 2013), because they shorten during vertebrate aging, including Nothobranchius furzeri (Hartmann et al., 2009; Artandi and DePinho, 2010). The syndromes of telomere shortening, such as dyskeratosis, were caused by mutations in tert or other genes in the telomere-protecting complex (Armanios, 2009). The clinical presentations of dyskeratosis congenita are characterized by premature aging, such as pulmonary fibrosis, marrow failure (Armanios, 2009), reduced fertility (Bessler et al., 2010), and several cancers (Alter et al., 2009). A previous study produced tert-deficient fish by CRISPR/Cas9 technology, and the results showed that tert-deficient fish exhibit the loss of telomerase function, reduced fertility, and defects in highly proliferative tissues, and have genetic ability (Harel et al., 2015). Thus, mutations in tert could lead to a series of premature aging in fish. In this study, the variants of F to S in the telomerase RNA binding domain might have consequences in lifespan regulation. We speculated that tert under positive selection may be associated with the short lifespan of A. ommaturus. In addition, there are other candidate genes in A. ommaturus PSGs within the hallmarks of aging pathways (López-Otín et al., 2013), including mitochondrial dysfunction (polg and ppargc1a), deregulated nutrient sensing (igf1), loss of proteostasis (hsdpd1), and cellular senescence (taf1). In summary, A. ommaturus is an annual fish with a high-quality genome in the chromosomal level, which will be valuable for the identification of conserved genes important for lifespan, and the candidate genes related to aging can provide insight into the evolutionary power of lifespan strategies.

In this study, we presented a high-quality chromosome-level genome assembly of A. ommaturus. The final size of the A. ommaturus genome assembly was 921.49 Mb, with a contig N50 size of 15.70 Mb. The assembled sequences were clustered into 22 chromosomes by using Hi-C data. A total of 18,752 protein-coding genes were predicted. Phylogenetic analysis showed that A. ommaturus is closely related to P. magnuspinnatus and B. pectinirostris; the divergence time was appropriately 31.9 MYA. Several genes related to lipid metabolism such as acsbg2, znf638, and the ldlr gene family were expanded in comparative genomic analyses. Several candidate genes involved in aging were identified in positive selection of A. ommaturus. The high-quality genome assembly and annotation information supplied important genomic data to further investigate the evolution of A. ommaturus with other species, and it will be important for conservation applications and determining the location of important traits in this fish.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: https://www.ncbi.nlm.nih.gov/, PRJNA795319; https://ngdc.cncb.ac.cn/, GWHBHRM00000000.

The animal study was reviewed and approved by the Ocean University of China Academic Committee.

NS conceived and managed the project. YP and ZS collected the sequencing samples. YP and LZ performed the experiment and analysis. YP wrote the manuscript, and TG, NS, and LZ revised the manuscript. All authors reviewed and approved the final manuscript. All authors contributed to the article and approved the submitted version.

This work was supported by the National Key R&D Program of China (2018YFD0900905) and the National Natural Science Foundation of China (No.U20A2087).

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.

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