Transcriptional response of immune-related genes in Litopenaeus vannamei cultured in recirculating aquaculture systems with elevated CO 2

- This short-term study evaluated the effect of non-lethal high CO 2 concentration on the transcriptional response of immune-related genes of Pacific white shrimp ( Litopenaeus vannamei ) cultured in recirculating aquaculture systems (RAS). Two experimental groups were created: high CO 2 (47.67±2.04 mg L −1 ) and low CO 2 (2.0±1.93 mg L −1 ). Shrimp of 8.85±1.20 g were placed randomly at a density equivalent to 100 individuals m −3 and were monitored at 6, 12, 18, and 24 h. The transcriptional response of immune-related genes was analyzed by qPCR. Gene expression of hemocyanin, prophenoloxidase, and heat shock protein 60 was downregulated at 24 h, suggesting affectations on oxygen transportation, melanization, and protein functioning of L. vannamei under high CO 2 concentrations. Also, gene up-regulation of lipopolysaccharide- and β-glucan-binding protein and cytosolic manganese superoxide dismutase can impair the bacterial recognition and antioxidant defense of shrimp exposed to high CO 2 concentrations. These results suggest that concentration at about 47 mg L −1 of CO 2 can significantly influence the transcriptional response modulation of immune-related genes.


Introduction
Worldwide, crustacean farming reached a production of 9,386,500 tons in 2018; Pacific white shrimp (Litopenaeus vannamei) is the most representative and widely cultivated species, contributing 53% of total shrimp production (FAO, 2020). Due to aquaculture exponential growth and the adaptability of L. vannamei to intensive farming, recirculating aquaculture systems (RAS) has become an eco-sustainable alternative to traditional systems used for shrimp farming (Chen et al., 2019). Higher stocking densities in RAS require high feeding rates, thus increasing organic matter decomposition and CO 2 concentrations (Good et al., 2010). Therefore, high carbon dioxide (CO 2 ) levels are a characteristic of these culture systems (Khan et al., 2018).

Material and Methods
The research was conducted in Ciudad Obregon, Sonora, Mexico (27°29'03.6" N, 109°56'4.2" W), and animal use was conducted with ethical standards and approved by the institutional Ethics and Biosafety Committee (2020-04).
For the present study, two RAS with six circular tanks (0.9 × 1.10 m) each and with a capacity of 700 L were used. One RAS with six tanks was used to receive the additional CO 2 through a diffuser from a pressurized CO 2 -gas bottle until achieving dissolved concentrations of 47.67±2.04 mg L −1 for the high treatment. The remaining six tanks did not receive CO 2 (control treatment), so the levels were 2.0±1.93 mg L −1 CO 2 . One hundred and eighty shrimp (8.85±1.20 g) were randomly distributed in the 12 tanks at a density of 15 individuals per tank with a working volume of 150 L, equivalent to 100 shrimp m −3 density. The RAS remained with aeration, without water changes, and feeding was suspended during the test time (24 h). Afterwards, one shrimp per replicate (six per treatment) was collected at different times (6, 12, 18, and 24 h) to obtain 400 µL of shrimp hemolymph according to that described by Martinez-Porchas et al. (2020). The hemolymph was centrifuged at 3,500 rpm for 10 min at 4 ℃, the plasma was discarded, and the cell pellet was resuspended in TRIzol for RNA extraction and frozen at −70 ℃ until analysis. Total RNA was extracted with the TRIzol reagent. Concentration and purity of RNA were analyzed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific), and an A260: A280 ratio between 1.8 and 2.2 was ensured. Total RNA was treated with RNA-free DNase (Promega ® ). The cDNA was synthesized using the ImProm-II™ Reverse Transcription System (Promega ® ) with oligo d(T)20 (T4OLIGO), using 500 ng of total RNA. The cDNA was diluted with 80 μL of ultrapure water, and 5 μL were used as template for the quantitative real time PCR (qPCR) reaction.
Transcriptional response was analyzed based on five immune-related genes and β-actin as reference gene (Table 1) (Zhang et al., 2013). The qPCR amplifications were performed in final reaction volumes of 15 μL following the instructions of MyTaq DNA polymerase (Bioline™) with 0.2 μM of each primer (T4OLIGO), 0.0125 μM of EvaGreen ® 20X (Biotium), and 5 μL of cDNA. The qPCR was performed on a StepOne Real Time PCR System (Thermo Fisher Scientific). Conditions for qPCR were initial denaturation for at 95 °C for 10 min, followed by 40 denaturation cycles at 95 °C for 15 s, and annealing/ extension at 60 °C for 1 min. An analysis of the dissociation curve (60-95 °C at a temperature transition rate of 0.5 °C s −1 ) was performed for each pair of primers. The levels of gene-relative expressions Transcriptional response of immune-related genes in Litopenaeus vannamei cultured in recirculating... Hernández et al. 3 were calculated according to the 2 −ΔΔCT equation (Livak and Schmittgen, 2001). Data from relative gene expression were transformed with Log10 + 1 to achieve normal distribution according to that described by Rodriguez-Anaya et al. (2020).
All data are presented as mean±SE. Data collected at 6, 12, 18, and 24 h were evaluated by one-way analysis of variance. If any significance was observed, Tukey's test was performed for a comparison of the means. Statistical analysis was performed with Statgraphics Centurion XVI. Significance was set at 95% probability levels.
Variables were analyzed according to the following mathematical model: in which Y ij = observed variable, μ = overall mean, β i = effect of CO 2 level, and ε ij = random error associated to each observation.

Results
Carbon dioxide treatments did not affect (P>0.05) salinity, temperature, and dissolved oxygen, while pH was affected by high CO 2 level ( Table 2).
Significant differences compared with low CO 2 treatment: *P<0.05.

Discussion
In RAS, the optimal CO 2 range is 5 to 10 mg L −1 , but high densities can produce concentrations above 20 mg L −1 (Furtado et al., 2017). Although CO 2 concentrations between 20 and 60 mg L −1 are not lethal, the pH hemolymph decreases, causing negative effects on shrimp metabolism (Furtado et al., 2016;Furtado et al., 2017), including transcriptional response of genes related to shrimp immunity (Zhou et al., 2010;Johnson et al., 2015). During this study, a significant decrease in water pH was observed in the high CO 2 treatment compared with the control treatment. Therefore, we hypothesized that a high CO 2 concentration between non-lethal levels could influence expression of genes related to oxygen transportation and hemolytic activity, melanization, pathogen recognition, antioxidant defense, and stress response of L. vannamei cultured in RAS.
Data are presented as mean±SE, n = 6 each group. Low CO 2 treatment is represented by black bar and high CO 2 is represented by white bar.
Significant differences compared with low CO 2 treatment: *P<0.05, **P<0.01.  Transcriptional response of immune-related genes in Litopenaeus vannamei cultured in recirculating... Hernández et al. 6 Shrimp, as all organisms, regulate their physiological activity by modulating the transcriptional response of their genes for homeostasis maintenance (Fierro-Coronado et al., 2019). The hemocyanin function is related to oxygen transportation and non-specific innate immune defense (Zhang et al., 2009;Li et al., 2017). A 24-h short-term study with shrimp under hypercapnic hypoxia reported a decrease in hemocyanin gene expression due to a global reduction in shrimp protein synthesis (Johnson et al., 2015).
Our results showed an upregulation of Hc gene expression at 6 h, but the gene expression significantly decreased over time suggesting an effect on oxygen transportation. Prophenoloxidase participates in melanization, and its activation promotes phagocytosis, encapsulation, and nodule formation for the protection against invading pathogenic microorganisms (Vazquez et al., 2009). Two long-term studies (1 to 14 days) with brine shrimp (Artemia sinica) evidenced an increase in proPO gene expression during the seventh day post water acidification (Zheng et al., 2015;Chang et al., 2016). Although ours was a short-time study, in which proPO gene expression increased at 6 and 12 h but decreased at 24 h, we agree that non-lethal high CO 2 can affect pathogen recognition via proPO-activating system.
The specific defense mechanisms against bacteria, fungi, and viruses are activated by pattern recognition proteins such as lipopolysaccharide-and β-glucan-binding protein, which help in bacterial agglutination and removal by phagocytosis (Aguirre-Guzman et al., 2009). Gene expression of Gram-negative bacteria-binding protein, a pattern recognition protein similar to LGBP, was enhanced when brine shrimp was exposed to high CO 2 concentration (Zheng et al., 2015). In this study, the transcriptional response of LGBP gene significantly increased during the trial.
These results suggest that genes encoding for pattern recognition proteins are biologically responsive to water acidification; therefore, the bacterial recognition and removal could be affected by acidification stress.
Superoxide dismutase (SOD) is the main antioxidant defense pathway in response to oxidative stress caused by reactive oxygen species (ROS) (Campa-Córdova et al., 2002). Pacific oyster (Crassostrea gigas) under high CO 2 showed varied SOD gene expression, down-and up-regulation . Our data indicated that cytMnSOD gene expression decreased at 12 and 18 h but increased at 24 h, suggesting that water acidification can impair ROS metabolism, causing damage to proteins, lipids, and DNA by oxidative stress.
Heat shock proteins (HSP) play an important role in protecting organisms from almost any sudden change in the cellular environment that induces protein damage (Li, 2017), and their expression can take more time (>4 h) to reach the high expression levels under stress factors (Dennis III et al., 2015). Green crab (Carcinus maenas) under high CO 2 concentrations during more than seven days altered its HSP gene expression (Fehsenfeld et al., 2011). The transcriptional response data of the HSP60 gene indicated a downregulation during this study, but the bioassay duration could be a factor for not reaching the maximum expression levels. However, the protein function can be detrimental under high CO 2 concentration.
Our study demonstrated how non-lethal high CO 2 level influenced the transcriptional response of immune-related genes of L. vannamei. The gene expression modulation by water acidification promotes metabolic suppression, reduced protein synthesis and respiratory stress, and reduced metabolic scope, causing pathogen invasion, disease transmission, and host susceptibility (Chang et al., 2016;Wang et al., 2016). On the other hand, the energy destined to transcriptional response modulation can reduce shrimp muscular growth and productive performance (Silveira et al., 2018). Therefore, further long-term studies are necessary to determine how non-lethal high CO 2 concentrations influence growth, tissue histology, nutrient absorption, and physiologic response of shrimp cultured in RAS.

Conclusions
The transcriptional response of immune-related genes of L. vannamei cultured in recirculating aquaculture systems was affected by high CO 2.