Study on Ferritin Gene Expression to Evaluate the Health of White Leg Shrimp (Litopenaeus vannamei) Postlarvae Due to Changes in Water Temperature, Salinity, and pH

Abstract

The growth and survival of marine organisms are influenced by environmental factors such as water temperature, salinity, and pH. Unsuitable environmental conditions may negatively impact marine organisms. The white leg shrimp (Litopenaeus vannamei), a euryhaline organism highly adapted to salinity, is a valuable species for aquaculture. This study examined the effects of water temperature, salinity, and pH on the health of postlarvae L. vannamei. Stress levels within the organisms were analyzed through the expression of a biomarker gene. Ferritin was selected as the biomarker gene for analysis. The experimental animal samples used were the hepatopancreas of L. vannamei postlarvae. The analysis was performed by qRT-PCR. The results showed that the adaptation of L. vannamei postlarvae to temperature was dependent on salinity. Under low-salinity conditions (5 psu), ferritin expression increased at 25 °C and 30 °C after 48 h of exposure; however, it decreased after 72 h of exposure. Under normal salinity conditions (27 psu), ferritin expression increased from 24 h to 72 h at water temperatures of 25 °C and 30 °C. These results indicate that low-salinity conditions may enable L. vannamei postlarvae to rapidly adapt to high temperatures. In conclusion, L. vannamei postlarvae adapt more efficiently to high temperatures under low-salinity conditions than that under high-salinity conditions. The results of this study could beneficially impact L. vannamei farming.

1. Introduction

In marine ecosystems, environmental factors such as water temperature, salinity, and pH affect physiological processes, such as reproduction, physiology, metabolism, growth, and regulation of osmotic pressure, as well as the habitat of marine animals [1,2,3,4]. Marine animals are sensitive to temperature change, and may be affected by seasonal or long-term climate change [5]. These changes in temperature affect reproduction, growth, physiology, metabolism, energy balance, and diseases in marine organisms [1,6,7,8,9]. Changes in temperature may also disrupt the homeostasis of marine animals, to which the animals may adapt by changing their habitat [10,11,12]. Increased water temperature may cause hypoxia through a decrease in oxygen solubility and weakening of the binding force of hemoglobin [13]. Even for species targeted for aquaculture, water temperature conditions are crucial for seeding production, cultivation, and early development [7,14,15,16]. Temperature stress may affect animal immunity and metabolism through the generation of intracellular reactive oxygen species (ROS), which may trigger oxidative stress [17].

Salinity is another crucial environmental factor that affects marine animals, impacting their ecological tolerance, stress levels, and distribution [18]. The standard salinity concentration of the ocean is 33–34 psu. To recover from the imbalance in homeostasis caused by stress, marine animals may attempt to regain homeostasis through metabolic activities such as osmosis control, control of body fluid concentration, and oxygen consumption. However, during extreme changes in salinity, adaptation may not be possible, which may lead to fatal consequences [19,20,21,22,23,24]. pH represents the hydrogen ion concentration in water and is an indicator of acidity or basicity; it varies according to various dissolved substances and the assimilation and respiration of organisms and, therefore, affects the physiological processes of organisms.

Dissolved oxygen (DO) determines optimal aquaculture stocking density and production, as oxygen affects the growth and reproduction of aquaculture animals by influencing food intake, metabolism, and rearing conditions. The concentration of oxygen in water is limited by its solubility in water, which decreases with increasing temperature and salt concentration. DO levels are related to the pH; therefore, oxygen levels in water affect organisms variably based on the pH. The stress caused by environmental change may lead to the production of reactive oxygen species (ROS) in organisms, thereby influencing intracellular stress and cell and tissue damage. It may also lead to cell proliferation, decreased metabolism, and even death. Genes related to antioxidant activity may be activated as a defense mechanism against ROS.

Ferritin is a protein present in the cytoplasm and mitochondria of organisms that plays a role in preparing cells for damage caused by excess iron and in replenishing iron if it is lacking [25,26,27,28]. This replenishing process regulates intracellular iron homeostasis and helps remove heavy metals when their concentration in the cell is high [29]. Iron is an essential nutrient for organisms; however, high concentrations may cause oxidative stress and increase the expression of the ferritin gene, accompanied by an increase in antioxidant enzyme activity. Accordingly, iron may cause oxidative stress and ferritin may be associated with antioxidant activity, which protects the cells from oxidative damage. Ferritin plays a crucial role in marine animals, which is similar to its role in terrestrial organisms. In marine animals, ferritin primarily functions as an iron storage protein, helping to regulate iron levels in the body [30]. Ferritin in L. vannamei is also known to play a role in enhancing immunity, physiological responses, and survival [31]. Overall, the function of ferritin in marine animals is essential for maintaining iron homeostasis, supporting vital physiological processes, and protecting against oxidative stress, ultimately contributing to their overall health and well-being in aquatic environments [32]. Ferritin has been implicated in the immune response of shrimp, particularly in defense against pathogens and oxidative stress [33]. In aquaculture, shrimp are exposed to various environmental stressors, including temperature fluctuations, salinity changes, and pollutants. In the presence of these stressors, ferritin may play a role in mitigating the effects of oxidative stress and preventing oxidative damage.

L. vannamei can adapt to high temperatures, is euryhalinous, and is able to survive in a salinity range of 1–40 psu, which enables its adaptation to various ecological conditions. In Korea, indigenous shrimp farming varieties include Penaeus japonicus and Fenneropenaeus chinensis; however, these species are available in limited amounts due to their vulnerability to white spot disease caused by the white spot syndrome virus. Accordingly, L. vannamei is becoming the major species for shrimp farming in Korea and its demand is also increasing in other countries due to its high stocking density and environmental adaptability, especially to variable levels of salinity. However, several studies have reported that the ability of L. vannamei to adapt to a various extreme environmental conditions can have a negative impact on the organism if the environmental conditions such as water temperature, salinity, and pH are not appropriate [34,35,36,37,38]. However, reports on the postlarvae, which have weak immune systems, are rare.

In this study, we aimed to investigate the effects of changes in water temperature, salinity, and pH on L. vannamei postlarvae through the expression patterns of the ferritin gene. The expression pattern of the ferritin gene is believed to be able to serve as a biomarker gene that can indirectly indicate the level of stress in the body of L. vannamei postlarvae. Also, we hope that these gene expression patterns will reveal the level of stress in the body and how this organism may overcome stress through environmental adaptation.

2. Materials and Methods

2.1. Preparation of Experimental Animals

The L. vannamei postlarvae used in this study were obtained from the breeding farm of the National University of Agriculture and Fisheries of Korea. Seawater sterilized by autoclave was used as the breeding water. A 5 L glass beaker was used as the breeding container. A total of 30 postlarvae (length: 3.0 ± 0.2 cm) per experimental group were assigned; they were incubated in a multiroom incubator.

2.2. Water Temperature, Salinity, and pH

Water temperature was set at 15, 20, 25, and 30 °C. Salinity was set to 5 or 27 psu in each water temperature. The pH was set at 6.5 or 7.5 within each water temperature and salinity concentration combination. The experimental period was 72 h. Five experimental animals were collected from each experimental group at different time points (6, 24, 48, and 72 h) for sampling. Experimental animals housed at a water temperature of 15 °C were used as the control group.

2.3. Total RNA Extraction

Hepatopancreatic tissues were used for total RNA extraction. The tissues were washed with a saline solution, then placed in liquid nitrogen, and triturated. Total RNA was extracted using the RNAiso Plus reagent (TaKaRa Bio, Otsu, Japan) according to the manufacturer’s instructions. The extracted total RNA was quantified using a spectrophotometer (NanoVue, GE Healthcare, Amersham, UK), and RNA quality was confirmed based on the standard A260/280 ratio of 1.8–2.0.

2.4. Reverse Transcription–Quantitative Polymerase Chain Reaction (RT-qPCR)

One microgram of total RNA extracted from hepatopancreatic tissue was used as the template. Reverse transcription (RT) was performed using an oligo(dT)18 (0.5 μg) primer and the AccuPower RT premix kit (Bioneer Co., Daejeon, Korea) according to the manufacturer’s instructions. RT was carried out in a 20 μL reaction mixture at 42 °C for 1 h to synthesize cDNA. For RT-qPCR analysis, 1 μL of cDNA was used. The primers used for ferritin gene expression analysis were as follows: F (5′-CAAGTCCGCCAGAACTAC-3′) and R (5′-TGGCAAATCCAGGTAGAG-3′). The nucleotide sequences of the β-actin was used as an internal standard using the following primers: F (5′-CCACGAGACCACCTACAAC-3′) and R (5′-AGGCGAGGGCAGTGATTTC-3′). Each primer was used at a concentration of 20 pmol for RT-qPCR. RT-qPCR was performed after adjusting the final volume of the reaction solution to 20 μL using the SFCgreen Fast qPCR Master Mix (2×) kit (SFCprobes Co., Chungju, Korea) according to the manufacturer’s instructions. The cycling conditions for RT-qPCR were 94 °C for 5 min, followed by 35 cycles of 20 s at 94 °C, 20 s at 55 °C, and 20 s at 72 °C. To confirm the specificity and purity of all PCR products, melt curve analysis was carried out after amplification, under the following conditions: 94 °C for 15 s and 55 °C for 20 s. Fluorescence data were acquired during each annealing phase. Gene expression was assessed using the 2^−ΔΔCT method as described by Livak [39].

2.5. Statistical Analysis

Significant differences between the control and the experimental groups were analyzed using Student’s t-test and only those with p < 0.05 were considered significant.

3. Results and Discussion

Published research indicates that L. vannamei is subject to stress upon being exposed to varying water salinity and temperature [34,35,36]. In this study, we investigated this possibility by comparing the effects of water temperature, salinity, and pH on the health of L. vannamei postlarvae.

The expression of the ferritin in response to changes in water temperature over time was compared and analyzed in low- (5 psu; pH 6.5, 7.5) and high-salinity conditions (27 psu; pH 6.5, 7.5). The highest expression of ferritin at a salinity of 5 psu and pH 6.5 was observed in shrimp reared at 30 °C. There was a gradual and significant increase in ferritin expression through the experimental period in shrimp reared at 20 °C. With water temperatures of 25 and 30 °C, ferritin expression was the highest after 48 h (compared to that at 15 °C) and significantly decreased after 72 h (Figure 1). With salinity concentrations of 5 psu and a pH of 7.5, the expression of ferritin increased with increasing water temperatures and was higher than that observed at 15 °C. Ferritin expression increased over time in shrimp reared at 20, 25, and 30 °C. When the water temperature was 25 or 30 °C, ferritin expression was highest after 48 h; there was a trend for ferritin expression to decrease after 72 h (Figure 2). Under low-salinity conditions (5 psu), ferritin expression increased as the water temperature increased. This increased ferritin expression with increase in rearing water temperature under low salinity suggests that L. vannamei postlarvae can adapt to increasing water temperatures. In addition, the expression of ferritin increased with the increase in rearing water temperature and over time with no significant difference at pH 6.5 or 7.5. Ferritin expression was the highest at 48 h at rearing water temperatures of 25 and 30 °C, with a decrease at 72 h.

At a salinity concentration of 27 psu and pH of 6.5, ferritin gene expression increased over time with elevated rearing temperatures (20, 25, and 30 °C) compared to that at 15 °C. The expression patterns of ferritin with respect to temperature and time exhibited a gradual increase at 20, 25 °C, and 30 °C, showing elevated expression levels at 24 and 72 h. At 72 h, the expression decreased compared to that at 48 h, although this was not significant (Figure 3). At a salinity of 27 psu and pH of 7.5, ferritin expression exhibited patterns similar to those at a salinity of 27 psu and pH of 6.5 with respect to rearing temperature and time. Ferritin expression was higher at temperatures over 15 °C. Ferritin gene expression at 20 and 30 °C showed a gradual increase with respect to temperature and time, reaching the highest expression at 48 h and showing a significant decrease at 72 h. Similarly, at 25 °C, a gradual increase was observed in ferritin expression up to 48 h when it reached its highest expression, followed by an insignificant decrease at 72 h (Figure 4). Under high-salinity conditions (27 psu), ferritin gene expression increased with increasing water temperature. Similar to the results obtained at low-salinity conditions, these results suggest that an increase in rearing temperature imposes physiological stress on white shrimp. Furthermore, the expression of the ferritin gene, influenced by increases in rearing temperature and time, exhibited a significant increase at pH 6.5, maintaining high expression levels at 48 and 72 h. This suggests a state of stress owing to temperature adaptation for 72 h. At a pH of 7.5, a decreasing tendency was observed for the expression after 72 h.

The results of comparing ferritin expression in the low-salinity (5 psu; pH 6.5 and 7.5) and high-salinity (27 psu; pH 6.5 and 7.5) experimental groups were as follows: In the low-salinity (5 psu; pH 6.5 and 7.5) experimental groups, the highest ferritin expression was observed after 48 h at water temperatures of 25 and 30 °C, and decreased after 72 h. This suggests that under low-salinity conditions, environmental adaptation of L. vannamei occurs after 48 h and stress begins to decrease. However, in the high-salinity experimental groups, ferritin expression increased from 24 h to 72 h at water temperatures of 25 and 30 °C. This suggests that under high-salinity conditions (27 psu; pH 6.5 and 7.5), environmental adaptation of L. vannamei postlarvae was not achieved and that they were in a state of stress.

In summary, we found that an increase in water temperature under both low- and high-salinity conditions induced physiological stress in L. vannamei postlarvae. However, the transition to a stable state after temperature adaptation varied depending on salinity. Under low-salinity conditions (5 psu; pH 6.5 and 7.5), the recovery period appeared to be faster than that under high-salinity conditions (27 psu; pH 6.5 and 7.5). This conclusion is supported by the significant and rapid decrease in the expression pattern of ferritin at 72 h under low-salinity conditions (5 psu; pH 6.5 and 7.5) compared to that under high-salinity conditions (27 psu; pH 6.5 and 7.5).

In addition, the expression of ferritin at both low-salinity (5 psu; pH 6.5 and 7.5) and high-salinity conditions (27 psu; pH 6.5 and 7.5) at 6 h was consistently higher at 20, 25, and 30 °C than that at 15 °C. Upon comparing the expression of the groups exposed to different temperatures (20, 25, and 30 °C), no significant differences were observed (Figure 5). The expression of ferritin at 6 h, influenced by changes in temperature, salinity, and pH, showed no significant differences among the temperature groups (20, 25, and 30 °C).

At 24 h, ferritin expression under low-salinity conditions (5 psu; pH 6.5 and 7.5) was significantly higher at 20, 25 and 30 °C than that at 15 °C. No significant differences were observed in ferritin expression when comparing the groups exposed to 5 psu (pH 6.5) and 5 psu (pH 7.5) at each temperature (20, 25, and 30 °C). Under high-salinity conditions (27 psu; pH 6.5 and 7.5), a significant increase was observed in ferritin expression with an increase in temperature compared to that observed in the 15 °C group. Particularly, 27 psu (pH 6.5 and 7.5) led to a significant increase in ferritin expression at both 25 and 30 °C compared to the low-salinity conditions (5 psu; pH 6.5 and 7.5). No significant differences were observed between the results of the groups exposed to 27 psu (pH 6.5) and 27 psu (pH 7.5) at any temperature (20, 25, and 30 °C) (Figure 6). At 24 h, ferritin expression, influenced by changes in rearing temperature, salinity, and pH, exhibited significantly higher expression under high-salinity conditions (27 psu; pH 6.5 and 7.5) at 25 and 30 °C. Given these results, it is likely that a salinity concentration of 27 psu (pH 6.5 and 7.5) induced potent stress in L. vannamei postlarvae during the initial 24 h of rearing at temperatures of 25 °C and 30 °C, whereas low-salinity conditions (5 psu; pH 6.5 and 7.5) had no significant impact during the first 24 h.

Ferritin expression at 48 h increased significantly with increased water temperatures (20, 25, and 30 °C) compared to that at 15 °C for both the low-salinity (5 psu; pH 6.5 and 7.5) and high-salinity (27 psu; pH 6.5 and 7.5) conditions. In particular, low-salinity conditions (5 psu; pH 6.5 and 7.5) did not cause any significant changes in ferritin expression with increased temperatures up to 24 h of rearing; however, a gradual increase in ferritin expression was observed from 48 h onward. Similarly, high salinity (27 psu; pH 6.5 and 7.5) led to a progressive increase in ferritin gene expression with an increase in water temperature; ferritin expression levels were also elevated compared to those at 24 h. Comparing ferritin gene expression in response to increasing water temperature in the low-salinity (5 psu, pH 6.5, 7.5) and high-salinity (27 psu, pH 6.5, 7.5) groups, the high-salinity (27 psu, pH 7.5) group exhibited relatively higher expression than the other experimental groups (Figure 7). The characteristics of ferritin gene expression in response to changes in rearing temperature, salinity, and pH at 48 h revealed a significant increase in gene expression with an increase in water temperature under low-salinity conditions (5 psu; pH 6.5 and 7.5). Therefore, it is likely that a salinity concentration of 5 psu (pH 6.5 and 7.5) induces stress in L. vannamei postlarvae after 48 h of rearing at temperatures of 25 and 30 °C. Additionally, a high salinity (27 psu; pH 6.5 and 7.5) resulted in increased ferritin expression compared to that at 24 h.

At 72 h, the expression of ferritin under low-salinity conditions (5 psu; pH 6.5 and 7.5) sharply decreased at both 25 and 30 °C compared to at 48 h. Contrarily, the high-salinity conditions (27 psu; pH 6.5 and 7.5) showed a trend for increasing ferritin expression with increasing temperature. At 25 and 30 °C, high-salinity conditions (27 psu; pH 6.5 and 7.5) led to a significant increase in ferritin expression compared to low-salinity conditions (5 psu; pH 6.5 and 7.5; Figure 8). Ferritin expression abruptly decreased in response to changes in rearing temperature, salinity, and pH at 72 h with a rise in water temperature under low salinity (5 psu; pH 6.5 and 7.5). Therefore, it is possible that a salinity of 5 psu (pH 6.5 and 7.5) alleviates internal stress through temperature adaptation at rearing temperatures of 25 and 30 °C after 72 h. Conversely, high-salinity conditions (27 psu, pH 6.5, 7.5) maintained elevated levels of ferritin expression from 24 h to 72 h. These results suggest that in high-salinity conditions (27 psu; pH 6.5 and 7.5), shrimp still experience the stress of adaptation at 72 h, particularly at rearing temperatures of 20 and 25 °C.

The results of the study summarized the expression patterns of ferritin over time under low- (5 psu; pH 6.5 and 7.5) and high-salinity conditions (27 psu; pH 6.5 and 7.5) across different rearing temperatures, using the expression at 15 °C as a reference. At 20 °C, the expression patterns under both low- (5 psu; pH 6.5 and 7.5) and high-salinity (27 psu; pH 6.5 and 7.5) conditions showed an increasing trend over time (Figure 9). At 25 °C, the expression pattern of ferritin under low-salinity conditions (5 psu, pH 6.5, 7.5) showed no significant changes up to 24 h of rearing; however, expression had increased significantly at 48 h and then decreased significantly after 72 h. Under high-salinity conditions (27 psu; pH 6.5 and 7.5), ferritin expression gradually increased over time and remained significantly increased through 72 h of rearing. Comparing ferritin expression at 25 and 20 °C, all treatment groups had increased expression (Figure 10). At a rearing temperature of 30 °C, the expression patterns of ferritin in all treatment groups were similar to those at 25 °C. Under low-salinity conditions (5 psu; pH 6.5 and 7.5), no significant changes were observed in ferritin expression up to 24 h of rearing; however, expression significantly increased at 48 h and significantly and rapidly decreased at 72 h. Under high-salinity conditions (27 psu; pH 6.5 and 7.5), ferritin expression gradually increased from 24 h to 48 h. At 72 h, ferritin expression in the 27 psu (pH 6.5) group was similar to that at 48 h, whereas in the 27 psu (pH 7.5) group, ferritin expression at 72 h was lower than that at 48 h (Figure 11). These results suggest that salinity and rearing temperature are highly sensitive factors affecting the adaptation of L. vannamei postlarvae. At a temperature of approximately 20 °C, the impact of salinity on the occurrence of internal stress in L. vannamei postlarvae can be considered to be low. In summary, the optimal conditions for L. vannamei postlarvae rearing observed in this study were a rearing temperature of 25 or 30 °C at a low-salinity concentration (5 psu).

Cultured organisms are significantly influenced by environmental parameters. In the case of L. vannamei cultivation, the crucial parameters include water temperature and salinity, which affect cultivation. The optimal water temperature for L. vannamei cultivation has been shown to be 26–33 °C, considering aspects of growth and survival [40]. Research suggests that as the shrimp size increases, low water temperatures are preferable. For postlarvae, temperatures above 30 °C are recommended, while for adults, 27 °C is considered suitable; temperatures higher than this adversely affect growth [41,42]. Additionally, the metabolic rate and growth of L. vannamei decrease at rearing temperatures lower than 23 °C [41]. The acceptable range of salinity for L. vannamei is broad, ranging from 0.5 to 45 psu [43]. L. vannamei is a typical euryhaline crustacean that inhabits different locations throughout its life cycle. The larvae develop in the ocean, whereas postlarvae, juveniles, and adults inhabit estuaries and brackish waters [40]. Therefore, lower salinity is more suitable for L. vannamei postlarvae cultivation, lower than that of typical seawater. However, some reports suggest that shrimp growth rates are higher at 25 psu than at low-salinity conditions [44,45]. Low salinity is considered beneficial for habitats, whereas high salinity is considered better for growth. Failure to maintain optimal water temperature and salinity may lead to environmental stress, resulting in weakened immune responses and reduced productivity [40]. For L. vannamei cultivation, the recommended pH range for rearing water is 6.4–9.1; alkalinity is associated with low deformity rates [46]. In this study, the optimal conditions for L. vannamei postlarvae, considering water temperature, salinity, and pH, were characterized by a short and mild stress adaptation period at rearing temperatures of 25 and 30 °C under low salinity (5 psu). These conditions are suitable for survival, growth, and immunity of postlarvae of L. vannamei. The results of this research may be important for the production and cultivation of L. vannamei postlarvae.

4. Conclusions

If the environmental factors (temperature, salinity, and pH) of a marine organism’s habitat are not appropriate, their health can be adversely affected due to stress. This poor health can have adverse effects on reproduction, survival, and growth. When living organisms are under stress, ROS are typically produced. These ROS attack cells and deteriorate the health of the body. ROS are generated under prolonged stress, including oxidative stress, causing adverse effects on the immune and endocrine systems. Ferritin serves as a marker of antioxidant stress and can be used as an indicator of oxidative stress. We analyzed ferritin gene expression to determine the health status of L. vannamai postlarvae. Gene expression analysis is an efficient method for rapid and accurate evaluation of the health status of biological entities. From this perspective, the analysis of health status based on ferritin expression, considering environmental factors, such as water temperature, salinity, and pH, could be valuable. In this study, the appropriate water temperature and salinity for rearing L. vannamei postlarvae were presented through gene expression patterns. It was confirmed that low salinity (5 psu) under high temperature conditions for growth of L. vannamei postlarvae was positive. In the future, it is necessary to identify the conditions of appropriate environmental factors for L. vannamei postlarvae through research on the expression of various genes. In this study, analysis of ferritin expression suggested that low salinity (5 psu) and water temperatures of 25 and 30 °C are the most suitable environmental conditions for L. vannamei postlarvae.