Introduction
Most species have developed endogenously driven circadian rhythms in physiology and behavior that are attuned to changes in the daily 12 h light/12 h dark cycle (Hussain and Pan, 2015; Schilperoort et al., 2020). In other words, the photoperiod profoundly influences the circadian rhythm of biochemical, physiological, and behavioral processes in almost living organisms (Wright et al., 2013; Dannerfjord et al., 2021). Epidemiological studies and substantial experimental evidence have repeatedly reported that changes in the light/dark cycles have been closely associated with various metabolic disorders (Rutter et al., 2002; Maury et al., 2010). In aquatic animals, the light/dark cycle acts as an important biological factor that influences the entire life cycle from embryonic development to sexual maturation (Downing and Litvak, 2001; Migaud et al., 2010; Arambam et al., 2020). In recent years, growing evidence has shown that the effects of the light/dark cycle on various species are diverse, some have a natural preference for the dark environment while some have an improved physiological state under high-light intensity (Serra et al., 1999; Ruchin, 2006; Li et al., 2021b; Wei et al., 2021). Therefore, it is a promising research direction for further studies.
The circadian nature of physiology and behavior is regulated by a circadian clock that is located in the suprachiasmatic nucleus (SCN) of the hypothalamus (Dannerfjord et al., 2021). Apart from the SCN, the tissues are capable of generating multiple circadian biological rhythms (Hastings et al., 2018; Pett et al., 2018). Earlier studies have demonstrated the mechanism of dark-light-adaptation changes in crustaceans compound eyes (Migaud et al., 2010). Nevertheless, our understanding regarding the influential mechanism underlying the photoperiods on other tissue is unknown in crustaceans and needs to be investigated further. Currently, the relationship between the light/dark cycle and intestinal microbiota has aroused widespread concern (Peyric et al., 2013; Fortes-Silva et al., 2016; Kim et al., 2019). Furthermore, different light/dark cycles could affect growth, digestibility, and physiological metabolism in fish (Leiner and MacKenzie, 2001; Li et al., 2021b). The crustacean hepatopancreas is an important organ constituting the main site for nutrient digestion, absorption, and metabolism (Li et al., 2021a). Therefore, the hepatopancreas and intestine can be the potential targets for studying the responsive mechanism of shrimp in response to different light/dark cycles.
The Pacific white shrimp Litopenaeus vannamei is considered a paramount aquaculture species, accounting for more than 70% of the total global shrimp production worldwide (Jiao et al., 2020). L. vannamei starts benthic life soon after the mysis stage or the early stage of postlarvae. Currently, the effects of different light/dark cycles on larval survival and growth performance have been studied in L. vannamei and Macrobrachium rosenbergii (Sanudin et al., 2014; Wei et al., 2021). Until present, evidence regarding the regulation of physiological metabolic process under different light/dark cycles is limited in L. vannamei. Thus, in this study, we mainly investigated the effects of different light/dark cycles (12 h light/12 h dark, 0 h light/24 h dark) on the hepatopancreas metabolism and intestinal microbiota in L. vannamei using multiomics techniques. Moreover, body color was also measured. This study might provide an important theoretical basis and reference for further understanding the influence of light/dark cycles on the body color, hepatopancreas metabolism, and intestinal microbiota in shrimp.
Discussion
The change in somatic color in crustacea has received a great deal of attention as a conspicuous and quantifiable phenomenon related to physiological and ecological factors. Crustacean showed an ability to change coloration in response to photoperiod, which may play roles including photoprotection and enhancing camouflage in a unique marine environment (Russell and Dierssen, 2018). In our study, one interesting finding was that the body color of L. vannamei became darker after dark treatment for an 8-week feeding trail, which may be related to the decreased gene expression of crustacyanin subunit C (from transcriptome data). Although the molecular mechanism of the crustacean body color change is not clear, several studies have indicated that the regulation of body color can be associated with gene expression of crustacyanin (Zhao et al., 2021). Astaxanthin, one of the carotenoids found in nature, appears to be the main pigment responsible for color in crustaceans, accounting for approximately 65–98% of all the carotenoids found in shrimp species (Wade et al., 2005; Ertl et al., 2013). The stability of the highly reactive astaxanthin pigment is provided by crustacyanin, a protein that binds to the pigment to form a carotenoprotein complex, which stabilizes the pigment as well as the tertiary and quaternary structures of proteins (Wade et al., 2009). Crustacyanin subunit A and C expression levels were lower in dark shrimp than that in light shrimp (Ertl et al., 2013). In addition, it has been reported that L. vannamei infected with Vibrio parahaemolyticus, V. fluvialis, V. mimicus, V. alginolyticus, and Vibrio sp. found to take less feed and body color tended to be darker (Mastan, 2015). Later, the intestinal microbiota analysis confirmed that the relative number of Vibrio has been increased after the dark treatment. Taken together, we suspected that dark treatment decreased the gene expression of crustacyanin subunit C and increased intestinal Vibrio number and, therefore, changed body color in L. vannamei. However, currently, the molecular mechanism remains unclear, which allows us to dig deeper into the possible mechanisms in the future.
Serum enzymes such as AST and ALT are generally used in assessing liver function (Senior, 2012). We found that dark treatment elevated serum levels of AST and ALT, which are usually considered as a sign of hepatocellular damage (Panteghini et al., 1983). The transcriptomics data further revealed the influence of different photoperiods (12 h light/12 h dark, 0 h light/24 h dark) on the metabolism in L. vannamei. We observed that DEGs mostly involved in nutrient digestion, absorption, and transport were significantly suppressed after dark treatment for the 8-week feeding trail, which might partially explain the changed serum levels of ALB, LDL, and glucose. In detail, digestive enzymes (i.e., chitinase and chitin deacetylase 9-like protein), nutrition transport regulation (i.e., glucose transporters, ion/metal transporters, putative sodium-dependent multivitamin transporter, transient receptor potential channel pyrexia, solute carrier family 22 members, and selenium-binding protein 1-A), and lipoprotein binding (i.e., lipoprotein receptor 2A, SEC14-like protein 2, and osbpl1a) genes were downregulated. Similarly, Sanudin et al. (2014) reported that the ingestion rate of frozen Artemia nauplii under dark conditions (24 h dark) was lower than that under light conditions (24 h light) in the smallest shrimp (total length of 0.5 cm) (Sanudin et al., 2014).
Most organisms have evolved an internal circadian clock that drives circadian rhythms in metabolism, physiology, and behavior (Ko and Takahashi, 2006). The circadian clocks use a 24 h light-dark cycle as the environmental signal (zeitgeber) to establish endogenous circadian timing systems that synchronize biological functions (Roenneberg et al., 2013). In our study, we observed that circadian clock genes including cryptochrome, aryl hydrocarbon receptor nuclear translocator-like protein 1 (BMAL1), period circadian protein homolog 1, and circadian locomotor output cycles kaput protein were downregulated under constant darkness treatment in the hepatopancreas of L. vannamei. In similar to our data, continuous darkness decreased the mRNA levels of clock genes cryptochrome 1 (Cry1) and period 2 (Per2) in the eyestalk of L. amboinensis (Choi et al., 2019). In mammals, darkness exposure for 8 weeks decreased the mRNA levels of BMAL1 in the brain and muscle and decreased the mRNA levels of Per1 and Per2 in the liver (Li et al., 2020).
Molecular insights into the mechanisms of circadian rhythms have provided clues that posttranslational modifications control the circadian clock function (Gallego and Virshup, 2007). Posttranslational modifications regulate the localization, degradation, and activity of these clock genes by methylation, phosphorylation, acetylation, sumoylation, ubiquitination, etc. We observed that darkness exposure downregulated genes, which were associated with protein ubiquitination (i.e., ubiquitin conjugation factor E4 B), acetylation (i.e., KAT8 regulatory NSL complex subunit 3 and NAD-dependent protein deacylase Sirt4), and methylation (i.e., thiopurine S-methyltransferase-like). Ubiquitin conjugation factor E4 B functions as an E4 ligase that mediates the assembly of polyubiquitin chains on substrates ubiquitinated by another E3 ubiquitin ligase (Hatakeyama et al., 2001). NAD-dependent protein deacylase SIRT4 is a lysine deacetylase and acts as a crucial player in maintaining insulin secretion and glucose homeostasis (Anderson et al., 2017). As part of the NSL complex, KAT8 regulatory NSL complex subunit 3 is involved in the acetylation of nucleosomal histone H4 on several lysine residues and may be involved in the regulation of transcription (Cai et al., 2010). Thiopurine S-methyltransferase-like drives methylation by directing thiopurines [6-mercaptopurine (6MP)] toward methylated entities [6-methylmercaptopurine (6MMP)] and, thus, decreases levels of active 6-thioguanine nucleotides (Urbancic et al., 2019).
A marked oscillation in hormone exhibited daily rhythms under light/dark cycles (Bayarri et al., 2004; Oliveira et al., 2013). Merging evidence suggests that disturbance of the circadian rhythm of the body increases endocrine disorder risk (Gamble et al., 2014; Zimmet et al., 2019). We found that constant darkness exposure exhibited decreased several gene expressions, which was involved in hormone regulation, including insulin-like receptor, C-type allatostatin, iodotyrosine dehalogenase 1, pituitary homeobox x-like, and thyroid hormone receptor beta-A-like in the hepatopancreas of L. vannamei. In invertebrates, the role of the insulin pathways includes not only glucose homeostasis but also the regulation of a variety of fundamental processes, such as growth, aging, and reproduction (Claeys et al., 2002). As the pivotal protein in any insulin family-based signaling pathway, the insulin-like receptor is responsible for mediating the signal carried by insulin-like peptides (ILPs) from the intercellular to the intracellular environment (Sharabi et al., 2016). A similar result was reported that constant darkness exposure exhibited decreased BMAL1 expression and inhibition of the NAMPT/NAD + /SIRT1 pathway, further suppressed GLUT4 expression, and lead to insulin resistance in the liver cells (Li et al., 2020). C-type allatostatin is named because of its potent inhibitory effect on juvenile hormone biosynthesis by the corpora allata in insects and also modulates the immune response in the hepatopancreas of mud crab Scylla paramamosain (Xu et al., 2020). The thyroid hormone receptors are members of the steroid receptor superfamily that mediate the action of thyroid hormone signaling in numerous tissues to regulate important physiological and developmental processes (Anyetei-Anum et al., 2018). Iodotyrosine dehalogenase 1 is a transmembrane protein involved in the encoding iodotyrosine deiodinase, and it controls the reuse of iodide for thyroid hormone synthesis (Gnidehou et al., 2004). Pituitary homeobox plays an important role in regulating the transcription of enzymes involved in steroidogenesis (Hiroi et al., 2003). Our results provide the first evidence that constant dark treatment could influence hormone regulation in the hepatopancreas of L. vannamei.
The involvement of the light-dark cycle as an important regulator of immune functions has been extensively described in mammals, but there is a paucity of information on the influence of this biological phenomenon in aquatic animals. Limited studies have reported that the innate immune system had a circadian rhythm based on the light-dark cycle in fish (Esteban et al., 2006; Ceballos-Francisco et al., 2020). Serum-mediated bacterial killing activities and enzyme activities associated with immune defense (i.e., lysozyme, alkaline phosphatase, and myeloperoxidase) reached the highest during the light phase in rainbow trout (Oncorhynchus mykiss) (Lazado et al., 2018). High-light intensity (16D:8L and 0D:24L dark/light cycles) increased lymphocyte count and improved the overall physiological state of carp (Cyprinus carpio L.) yearlings, while zero illumination had an inverse effect (Ruchin, 2006). We found that dark treatment significantly suppressed immune-related gene expression in the hepatopancreas of L. vannamei, including beta-1,3-glucan binding protein, C-type lectin, antilipopolysaccharide factor-like protein, and NF-kappa-B inhibitor delta. Beta-1,3-glucan binding protein might act as a pathogenic recognition protein to activate shrimp immune defense against invasive pathogens by the agglutination, binding, and enhancing encapsulation and phenoloxidase activity of the hemocytes (Phupet et al., 2018). C-type lectin could inhibit the proliferation of hemolymph microbiota by maintaining the expression of antimicrobial peptides in shrimp (Wang et al., 2014). An antilipopolysaccharide factor is a small protein that has been verified responsive to different pathogen challenges and with broad-spectrum antimicrobial activities (Li et al., 2015). NF-kappa-B inhibitor delta functions in the regulation of inflammatory responses and regulates the expression of IL-2, IL-6, and other cytokines through regulation of the NF-kappa-B activity (Arnold et al., 2012). These results indicated that constant dark treatment impaired the immune function in the hepatopancreas of L. vannamei.
Intestinal microbiota modulates host physiological processes and plays an important role in promoting and maintaining the health of the host. Dark treatment significantly increased the relative abundance of genus Ruegeria, Vibrio, Actibacter, Roseovarius, Ilumatobacter, and Kriegella at the intestine of L. vannamei. It is worth noting that dark treatment promoted the proliferation of pathogenic bacteria. Vibrio spp. is the most typical and well-known pathogen causing vibriosis infections in aquatic animals (Arunkumar et al., 2020). As a member of the Roseobacter clade, Roseovarius crassostreae is a pathogenic bacteria of Roseovarius Oyster Disease and has resulted in mass mortalities of cultured Crassostrea virginica in the northeastern United States (Maloy et al., 2007). Furthermore, microbiota KEGG functional analysis demonstrated that dark treatment increased the abundance of pathways in Vibrio cholerae infection, Staphylococcus aureus infection, and biofilm formation of Vibrio cholerae. Besides, the relative abundance of Ruegeria was increased after the dark treatment at the intestine of L. vannamei. Ruegeria is a globally distributed gram-negative marine bacterium, which could produce the antibacterial compound tropodithietic acid and biofilm formation against several marine pathogens (Garcia et al., 2014). Ilumatobacter genome includes two type I polyketide synthases. The type I polyketide synthases can catalyze the synthesis of polyketides, which are highly effective antibiotics (Zhao et al., 2019). The above findings suggested that increased Ruegeria and Ilumatobacter might help combat pathogenic bacteria. Moreover, the dark treatment significantly decreased the relative abundance of Formosa, Demequina, and Lutimonas. Demequina can help its host absorb and utilize carbohydrates by producing α-amylase, xylanase, and cellulase (Al-Naamani et al., 2015). Lutimonas is a strictly aerobic heterotrophic nitrifying bacterium for the degradation of ammonia (Fu et al., 2009). To date, less is known about the physiological function of Actibacter, Kriegella, and Formosa, which needs to be further clarified in the future.