Analysis of adaptive molecular mechanisms in response to low salinity in antennal gland of mud crab, Scylla paramamosain

2.1. Animals and sample collection

For the study long-term salinity adaptation, 12 adult S. paramamosain (100.0 ± 5.0 g, mean ± S.D.) were acquired for the collection of tissue samples and construct cDNA libraries. Crabs of the 5 ‰ group were long-term dwelling in the 5 ‰ sea (Cixi, China, 30°02′–30°24′N, 121°02–121°42′E) and crabs of the 23 ‰ group were long-term dwelling in the 23 ‰ sea (Xiamen, China, 24°23′–24°54′N, 117°53–118°26′E). At the beginning of the experiment, 6 crabs were selected as biological replicates in each experimental group. The crabs used for the study were reared from larvae/juveniles at a mud crab semi-intensive free-range farm.

The acute low salinity trial was carried out in indoor rectangular blue rectangular covered tanks (40 × 30 × 20 cm, L: W: H) in the Laboratory of Crustacean Genomics and Molecular Genetics, Ningbo University, Ningbo, China. 120 adult crabs used for the acute low salinity trial were obtained from a crab farm with a salinity of 23 ‰ in Xiamen, China. All the crab was stocked in 20 L tanks containing aerated seawater (salinity 23 ‰, pH 7.2) and acclimated to the laboratory culture condition for 14 days beforehand. During the acclimation, the brackish water was maintained at salinity 23 ‰, pH 7.2, temperature 24 ± 1 °C, and dissolved oxygen 6 ± 0.5 mg/L; the animals are fed twice a day with clam meat. When the experiment began, 60 crabs (sex ratio 1:1) were transferred from the salinity of 23 ‰–5 ‰; meanwhile, the other 60 crabs were maintained at the salinity of 23 ‰ as the control group. The acclimation medium of 5 ‰ salinities was maintained and changed every 24 h, using a salinity meter to measure. In 0 h (10 min), 3 h, 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h, 6 crabs were randomly selected for sampling at each time point as biological replicates. The survival rate was surveyed at the end of experiment.

Experimental design is to carry out RNAseq on long-term samples, and then carry out acute salinity stress experiment on 23 ‰ crabs in long-term experimental control group to detect physiological and biochemical indexes and mRNA expression. The antennal gland is located at the base of the eyestalk near the insertion of the second antenna, the superior aspect of the stomach, as 1 pair of oval sacs, opening at the base of the second antenna, consisting of a gland and a bladder. After the crab has been anesthetized on ice for 5 min and sacrificed by the destruction of the dorsal ganglia, it is kept on ice during the dissection procedure, the back is shelled, and after the gastric tissue has been removed, the position of the antennal glands is visualized and they are removed by forceps. For each group, the whole antennal gland of each crab was assigned for RNA extraction, protein extraction and microscopic studies, and hemolymph was drawn for osmolarity measurements. All animals were handled following the standard procedures of the Guide for the Use of Experimental Animals of Ningbo University.

2.2. The construction and analysis of transcriptomes

Total RNA (more than 2 μg) of the antennal glands of each crab was extracted following the method as described by Du et al. [53]. After enrichment and purification by the poly-T oligo-attached magnetic beads, the first-strand cDNA was synthesized by reverse transcriptase and random primers using the templates of artificially cleaved mRNA fragments. Next the second-strand cDNA was synthesized by DNA Polymerase I and RNase H. After purification and PCR amplification, the cDNA library was sequenced by the BGISEQ-500 sequencer (BGI, Shenzhen, China). The pair-end read length is 100.

The high-quality clean reads were generated by removing the adaptors, low-quality sequences (<Q20) and the sequences shorter than 50 bp of the RNA-Seq raw data using Solexa QA [54]. The differential gene expression analysis was pairwise comparison by DESeq2 [55] after the alignment of clean RNA-Seq reads onto the genome of S. paramamosain (GWHALOH00000000) using the HISAT2 package [56]. A gene with an adjusted P-value less than 0.05 and a fold change greater than 2 was defined as a differentially expressed gene. Gene annotation was conducted using the database of NR [57], GO [58], and KEGG [59]. Thereafter, GO enrichment and KEGG enrichment of the differentially expressed transcripts were analyzed using the R ClusterProfiler 4.0 with a P value of 0.05.

2.3. Real-time quantitative polymerase chain reaction (RT-qPCR)

Twelve DEGs in the transcriptome were selected for verification using qRT-qPCR to confirm the dependability of the transcriptome results. The same samples were used for both BGISEQ-500 sequencing and qRT-PCR validation. Total RNA of the antennal glands was extracted using the TRIzol reagent (Invitrogen, Carlsbad, US). The quantity and quality of RNA were determined by the NanoDrop 2000 micro-volume spectrophotometry (Thermo Fisher Scientific, Waltham, US) and the agarose gel electrophoresis, respectively. The cDNA was synthesized using the template of 2 μg extracted RNA by the PrimerScript™ RT reagent kit with gDNA Eraser (Perfect Real Time) (Takara Biotechnology, Kusatsu, Japan). RT-qPCR was carried out in the ABI 7500 real-time PCR system (Applied Biosystems, Foster City, US) following the program of 95 °C for 30 s, 32 cycles of 94 °C for 5 s, 55 °C for 30 s, and 72 °C for 10 s. All samples were run in triplicate, and each assay was repeated three times. For the standard curves of each gene, the amplification efficiencies were 95–105 % and the coefficient (R²) were 0.98 < R². The relative gene expression was calculated using the 2^−ΔΔCq method as described by Schmittgen and Livak [60], where the β-actin of S. paramamosain was used as the reference gene.

2.4. The measurement of osmolality and ion concentration

For each crab, 2 mL hemolymph was collected at the base of the third walking limb using a heparinized (100 IU/mL) syringe. The cells in hemolymph were removed by centrifugation (1000 g, 10 min, 4 °C). The hemolymph was firstly prepared using a T10B homogenizer (IKA, Staufen, Germany) following the method of Yao et al. [49]. The supernatant was collected after the hemolymph was centrifuged at 12,000 rpm for 20 min at 4 °C. The osmolality was analyzed using a Fiske 210 osmometer (Advanced Instruments, Norwood, US). The concentrations of Na⁺, Cl⁻, K⁺, Ca²⁺, and Mg²⁺ were measured using the commercial reagent kits (Jiancheng Bioengineering Institute, Nanjing, China) and the following method described by Long et al. [61]. The absorbance wavelength was set to 620, 480, 440, 610, and 540 nm on a SpectraMax M2 microplate reader (Molecular Devices, California, US) for measuring Na⁺, Cl⁻, K⁺, Ca²⁺, and Mg²⁺, respectively.

2.5. Enzyme linked immunosorbent assay (ELISA)

The whole antennal glands (about 0.1 g) were homogenized in PBS (w: v = 1: 9) before being centrifuged (3000 rpm, 10 min, 4 °C). The enzyme activities of CA-g and NKA in the supernatant were analyzed using the commercial ELISA kits (Qiaodu Biomart, Shanghai, China) following the instruction of manufacturer. Briefly, 50 μL diluted sample and 100 μL HRP conjugates were mixed in an antibody pretreated microplate well. After incubated for 60 min at 37 °C, the plate was thoroughly washed five times by the wash solution. Later, 50 μL chromogen solution A and 50 μL chromogen solution B were added to each well and gently mixed. After incubated for 15 min at 37 °C in the dark, 50 μL stop solution was added and the optical density (O.D.) was measured at wavelength 450 nm using the SpectraMax M2 microplate reader (Molecular Devices, California, US).

2.6. Histology and transmission electron microscopy (TEM)

Three whole antennal glands were fixed in the 0.1 M phosphate buffer (Na₂HPO₄, NaH₂PO₄) containing 4 % paraformaldehyde for histology and in the 0.1 M PB containing 2.5 % glutaraldehyde (in 0.1 M phosphate buffer) for 4–6 h at 4 °C for TEM. The osmolality of the fixative solution was adjusted to 950 mOsm/kg to prevent cell shrinkage/swelling. To prepare histological slides, fixed tissue samples were firstly embedded into the paraffin block after a series of washing and ethanol dehydration. At the region of interest, 5-μm thick sections were transverse sliced using a KD-2268 microtome (Kedee, Jinhua, China) and then mounted on adhesive glass slides. After paraffin removal by xylene and a series of ethanol dehydration, the slide was H&E stained and sealed by nail polish around the coverslip. Histological pictures were taken using an OLYMPUS DP72 optical microscope (Olympus, Tokyo, Japan). For TEM, after rinsing with 0.1 M PB three times (15 min each), the tissues were fixed in 1 % OsO₄ overnight. After a series of washes and later ethanol dehydration, the tissue blocks were cleared by propylene oxide and embedded in Epon 812 resin. Ultrathin sections (50–70 nm thick) were cut with a diamond knife (DiATOME, Nidau, Switzerland) on a RMC PT-XL ultramicrotome (Boeckeler Instrument, Tucson, US) and then mounted onto 200-mesh copper grids. The air-dried grids were stained with 3 % uranyl acetate solution for 30 min and 6 % lead citrate for 5 min prior to the observation by a Hitachi H-7650 transmission electron microscope (Hitachi, Tokyo, Japan) operating at 80 KV. The samples were assayed in triplicate and the experiments were performed in triplicate. All samples were assayed in triplicate and the sections done in triplicate.

2.7. Statistical analysis

After checking for normality and homogeneity of variance, data were subjected to a one-way ANOVA. If a significant main effect was detected, differences among treatment means were compared using Duncan’s multiple range test. All data are presented as the means ± standard deviation (mean ± SD). The level of significance was set at p < 0.05. All analyses were performed using SPSS 18.0.0 for Windows.

3.1. The comparative analysis of transcriptomes

Six cDNA libraries (three for each group) of antennal glands were successfully constructed, containing an average of 83,718,424 ± 1,124,465 good quality clean reads (Q20: 98.74 ± 0.10 %). The GC content of clean reads was 45.19 ± 0.45 % and the mapping rate to the genome was 76.92 ± 3.53 % (Table 1). There were 2507 differentially expressed genes (DEGs) between the group of 23 ‰ (1202 up-regulated) and 5 ‰ (1305 up-regulated) (Fig. 1A). Based on DEGs, the transcriptomes of four individual samples were clustered together for each experimental group (Fig. 1B). It demonstrated the transcriptomic similarity between samples within the same group and the transcriptomic variance between two groups.

Based on the GO annotation, most DEGs were involved in the subcategories of “binding” (44.65 %) and “catalytic activity” (36.56 %) of the category “molecular function”, the subcategories of “cellular processes” (26.47 %) and “metabolic processes” (22.81 %) of the category “biological process”, and the subcategories of “cells” (17.09 %), “cell part” (17.01 %), “membrane” (16.34 %), “membrane part” (15.16 %) and “organelles” (12.48 %) of the category “cellular component” (Fig. S1A).

The top KEGG pathways enriched by DEGs were related to the processes of ion regulation (“proximal tubule bicarbonate reclamation” and “mineral absorption”), hormone production (“steroid hormone biosynthesis”, “salivary secretion”, “glycolysis/gluconeogenesis” and “bile secretion”), signal transduction (“phosphatidylinositol signaling system”, “cAMP signaling pathway”, and “adrenergic signaling in cardiomyocytes”), and metabolism (“pyrimidine metabolism”, “purine metabolism”, “protein digestion and absorption” and “glutathione metabolism”) (Fig. S1B).

3.2. The DEGs related to osmoregulation

Compared with the 23 ‰ group, the up-regulated genes in the 5 ‰ group consisted of Na⁺/K⁺-ATPase (NKA), V-type proton (H⁺)-ATPase (VPA), cytoplasmic carbonic anhydrase (CA-c), adenylate cyclase (AC), ATP-binding cassette subfamily C (CFTR), solute carrier family 25 (SLC25), malate dehydrogenase (MDH) and phosphoenolpyruvate carboxykinase (PEPCK), while the down-regulated genes were solute carrier family 1 (NBC1), sodium/hydrogen exchanger (NHE), glycosyl-phosphatidylinositol-linked carbonic anhydrase (CA-g), and succinate dehydrogenase (SDH) (Table 2; Fig. 2A). The results of qRT-PCR confirmed that NKA, VPA, CA-c, AC, PEPCK, SLC25, MDH, and CFTR were higher expressed in the 5 ‰ group than the 23 ‰ group, and the expression of NBC1, NHE, CA-g, and SDH in the 5 ‰ group were lower than those in the 23 ‰ group (Fig. 2B). The primers used for RT-qPCR are listed in Table 3.

3.3. The long-term adaptive responses to the low salinity

To adapt to the low salinity environment, the hemolymph osmolality of 5 ‰ group was lowered to 651.0 ± 166.3 mOsm/kg in comparison to 851.2 ± 33.8 mOsm/kg of the 23 ‰ group (Fig. S2A). As the major ionic components in the hemolymph, the concentration of Na⁺, Cl⁻, Ca²⁺, Mg²⁺ and K⁺ dropped from 359.4 ± 2.8 to 204.5 ± 4.7 mmol/L (Fig. S2B), 231.1 ± 3.2 to 194.5 ± 5.5 mmol/L (Fig. S2C), 10.3 ± 0.1 to 8.6 ± 0.5 mmol/L (Fig. S2E), 10.9 ± 0.3 to 8.2 ± 0.1 mmol/L (Fig. S2F), and 7.1 ± 0.3 to 3.4 ± 0.2 mmol/L (Fig. S2D), respectively. The decreasing concentration of the major hemolymph ions were consistent with the lowered hemolymph osmolality.

As the key enzymes for ion transport, the enzyme activities of CA-c (Fig. 3A) and NKA (Fig. 3B) in the antennal glands increased in the 5 ‰ group compared with the 23 ‰ group. This result was also consistent with the upregulated mRNA expression of CA-c and NKA in the 5 ‰ group (Table 2 and Fig. 2A).

The sectioning level is crossed by the anterior part and includes both the proximal tubular region and the distal tubular region. The distal tubular region of the antennal gland contains tubular cells and a large coelomosac. There was an intermittent separation of hemolymphatic spaces between the coelomosac and the labyrinth cells of the tubular-lining labyrinth cells. Hemolymph vessels are found in the proximal tubular region of the antennal gland and is the entrance for the coelomosac artery.

LBRs are cuboid cells with apical brush-boarder microvilli, basal membrane folding, and numerous mitochondria. In the antennal gland, the main cell type is the continuous simple cuboidal labyrinthine (LBR). Arrows in (Fig. 4A’, B’) identify the width of the labyrinth lumen. In this study, results showed that the width of the labyrinth lumen of 5 ‰ group was more dilated to 103.57 ± 2.54 μm in comparison to 71.17 ± 6.8 μm of the 23 ‰ group (Fig. 4E). In addition, there were more mitochondria (8 ± 2) in the LBRs of the 5 ‰ group (black arrows in Fig. 4C and D) than those shown in 23 ‰ group (30 ± 8) (Fig. 4F).

3.4. The short-term adaptive responses to the sudden decline of salinity

Our result showed most mortality caused by the sudden decline of salinity took place within 24 h (Fig. S3). For the survived crabs, the hemolymph osmolality dropped from 824.0 ± 13.6 to 545.8 ± 27.6 mOsm/kg during the first 12 h and did not change afterwards (Fig. S4A); meanwhile, the environmental osmolality appeared to increase despite not being in a significant way. Accordingly, the concentration of Na⁺, K⁺, Mg²⁺ and Cl⁻ in the hemolymph decreased from 340.2 ± 4.9 to 203.5 ± 5.6 mmol/L (Fig. S4B), 6.8 ± 0.1 to 3.3 ± 0.1 mmol/L (Fig. S4D), 10.8 ± 0.1 to 7.0 ± 0.5 mmol/L (Fig. S4F), and 236.2 ± 4.9 to 203.5 ± 5.3 mmol/L (Fig. S4C); simultaneously, the environmental Na⁺, K⁺, Mg²⁺ and Cl⁻ increased from 147.0 ± 7.0 to 216.0 ± 8.0 mmol/L (Fig. S4B), 2.0 ± 0.2 to 3.3 ± 0.3 mmol/L (Fig. S4D), 4.7 ± 0.4 to 7.3 ± 0.5 mmol/L (Fig. S4F), 147.0 ± 7.0 to 222.7 ± 6.1 mmol/L (Fig. S4C). The new equilibrium of ion concentration between hemolymph and environment was achieved within 12–24 h.

The situation of Ca²⁺ was a bit different from that of other ions. In the 5 ‰ group, the hemolymph Ca²⁺ decreased from 9.9 ± 0.1 to 5.9 ± 0.1 mmol/L during the first 24 h and did not change afterwards; meanwhile, the environmental Ca²⁺ increased from 4.7 ± 0.2 to 7.2 ± 0.3 mmol/L during the first 6 h. Unlike the cases of other ions, the environmental Ca²⁺ of the 23 ‰ group was not maintained at the initial level, it decreased from 8.0 ± 0.2 to 5.3 ± 0.3 mmol/L during the first 6 h and did not change afterwards (Fig. S4E).

During the short-term adaptation to low salinity, the enzyme of CA-c responded faster than NKA and the responsive period of CA-c was longer. The enzyme activity of CA rose immediately and reached peak after 48 h (Fig. 5A); in comparison, NKA enzyme activity increased 3 h after the salinity change while the level stabilized at 6 h, which was sooner than CA (Fig. 5B). The gene expression of CA-c (Fig. S5E) and NKA (Fig. S5B) showed a correspondingly changing pattern; nonetheless, a plummeted expression of CA-c was observed from 72 to 96 h (Fig. S5E).

Similar to NKA, the rising expression of CFTR (Fig. S5H), SLC25 (Fig. S5I) and PEPCK (Fig. S5J) occurred within 24 h and the gene expression maintained at the peak level afterwards. For VPA (Fig. S5D) and AC (Fig. S5G), there was a temporary drop of the gene expression between 24 h and 72 h. For NHE (Fig. S5C), MDH (Fig. S5K) and SDH (Fig. S5L), the gene expression peaked at 24 h and started to fall afterwards. Unlike other genes, the expression of NBC1 (Fig. S5A) and CA-g (Fig. S5F) began to decline since 0 h, rebound from 12 h to 24 h, then fell again.

4. Discussion

Our results showed that the pathways of ion regulation including “proximal tubule bicarbonate reclamation” and “mineral absorption” were activated in the antennal glands of S. paramamosain during the adaptation to low salinity. The genes for the ion transporters, e.g. NKA, CA-c, VPA and NHE, were up-regulated. The increase of enzyme activities of NKA and CA-c confirmed the activation of these two genes. NKA is a membrane-associated enzyme responsible for transporting K⁺ in and Na⁺ out of the cell [23]. NHE is also a membrane protein which transports Na⁺ in and H⁺ out [62]. VPA can transport H⁺ across the plasma membrane [37]. CA catalyzes the bidirectional conversion of H₂O and CO₂ to H⁺ and HCO₃⁻, where H⁺ is often provided for other ion transporters [43].

The upregulation of AC might be related to the activation of AC-cAMP-PKA-ion transporter signaling pathway [63], which has been suggested as a crucial signal transduction pathway regulated by the crustacean neuroendocrine system [ [[64], [65], [66]]]. It has been shown that cAMP mediated neuroendocrine factors play important roles in the crustacean osmoregulation [ [[67], [68], [69]]]. In crustaceans, the gills are primarily responsible for osmoregulation, which involves efficient ion transport, regulation of hemolymph, and changes in metabolism through increased O₂ consumption to regulate hemolymph ion concentration [16,17,38]. These results showed the osmoregulatory function of antennal glands depends on the ionic reabsorption via the activation of ion transporters (Fig. 6). The response mechanisms of the gill and antennal glands may differ, and osmoregulation homeostasis is regulated by the coordinated action of gill and antennal glands. However, the deficiency is that we did not find differences in the response of genes related to osmotic regulation during long-term and short-term low-salt adaptation, which may be because only a few selected genes were detected for long-term and short-term exposure.

Our study also found that the expression of genes associated with energy reallocation is orchestrated in the antennal gland in response to low salinity. SDH and MDH are critical enzymes in the TCA cycle [70]. PEPCK converts oxaloacetate (OAA) to phosphoenolpyruvate (PEP) during gluconeogenesis [ [[71], [72], [73]]]. SLC25 mediates the translocation of substances including amino acids, sugars, and inorganic ions across mitochondrial membrane [74]. The upregulation of MDH, SLC25 and PEPCK and the downregulation of SDH implied the enhancement of gluconeogenesis (Fig. 6). This result was consistent with the observation of increased mitochondria in the luminal podocytes of the antennal gland in response to low salinity. It was possibly related to the requirement of extra energy from non-carbohydrate resources as a way of metabolic reprogramming for the intensive osmoregulation during low salinity adaptation [ [[75], [76], [77]]].

Our results showed that the hemolymph osmolality and the hemolymph concentrations of Na⁺, Cl⁻, Ca²⁺, Mg²⁺ and K⁺ decreased as adapted to the low salinity, and eventually the internal osmolality equilibrates to a steady state with the external ambient osmolality. After a sudden drop in salinity, the hemolymph osmolality of S. paramamosain balance to the environmental osmolality within 12–24 h. O₂ consumption, increased energy requirements and accelerated metabolism due to changes in salinity lead to physiological dysfunction and reduced immune defense capacity, as most mortality was also observed during the same period. Another study also observed a lowered hemolymph osmolality of S. paramamosain reared in low salinity water [6]. In contrast, an increase in hemolymph osmolality was observed in the freshwater crab Eriocheir sinensis when accumulated in a high salinity environment [78]. As a result, crabs such as S. paramamosain and E. sinensis are also known to be strong osmoregulator, maintaining hemolymph osmolality very similar to that of the external environment. E. sinensis mainly live in fresh water while S. paramamosain and P. trituberculatus are estuaries or ocean dwelling [ [25,37,38]]. The change of salinity is one of the important factors that directly affect the occupation of crustaceans to the environment [52,61]. For crustaceans, osmotic balance in low or high salinity is achieved by transport mechanisms, osmotic and ionic regulation [43,62]. Successfully occupying environments of different salinity requires the ability to respond quickly to changes in salinity.

Based on our cross section of the antennal gland, this explains the enlarged labyrinthic lumen inside the antennal gland observed in this and other studies during low salinity adaptation [79,80]. Low osmolality external water is likely to penetrate high osmolality hemolymph and dilute the hemolymph. To compensate for the increased volume of hemolymph, excess water is excreted through the antennal glands as low osmolality urine.

In summary, our study has revealed the osmoregulatory function of the antennal glands of S. paramamosain via the findings of up-regulation of the ion-transporter gene, deactivation of the enzyme activity of the ion-transporter, and rebalancing of hemolymph osmolality as the crab adapts to low salinity. For low salinity adaptations, crabs increase excretion of urine and ionic reabsorption from urine in the antennal glands. Additionally, we found that crabs occupying an environment of changing salinity may require additional energy expenditure to carry out transport mechanisms and osmotic and ionic regulation. It is necessary to acknowledge the limitations of this work. The only few selected genes we used to experimentally validate were examined for both long- and short-term exposures, the qPCR results might not reflect the global responses and expression profile in mud crab. Future research with larger scale is needed.

By Nan Mo, Tianyi Feng, Dandan Zhu, Jiaxin Liu, Shucheng Shao, Rui Han, Wentao Lu, Pingping Zhan, Zhaoxia Cui

Reference: https://www.sciencedirect.com/science/article/pii/S2405844024015871

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