2.1 Adaptation in different environments

The success of decapods in freshwaters is related to their osmoregulation capacity, which is the osmotic (and ionic) homeostasis within a cell or organism relative to the ionic property of the surrounding environment. This process, termed osmoregulation involves a series of mechanisms where different epithelial tissues are utilized. Anisosmotic Extracellular Regulation (AER), the term used by various researchers including Florkin and their associates^16-19 (Figure 1), involves salt absorption or secretion of salt through epithelia such as gill and antennal glands aided by low cuticle permeabilities.^{5, 16, 20} Eventually, in freshwaters, high osmotic gradient is maintained, while in brackish waters, stable haemolymph osmolalities are kept.²⁰ Among freshwater animals, decapods are superior osmoregulators, maintaining 400–500 mOsm/kg H₂O of haemolymph osmolality despite water osmolalities of approximately ~15 mOsm/kg H₂O.¹⁸ The osmoregulation mechanism is assisted by decreased epithelial osmotic and ionic permeability, primarily by production of dilute urine and active branchial salt absorption.^{5, 18, 21, 22}

However, Freire et al.⁶ believe that in conditions of changing external salinity, such as in estuarine/brackishwater habitats, efficient cell volume regulation mechanisms (IIR = isosmotic intracellular regulation) proposed first by Florkin²³ are also essential and have a longer evolutionary significance than osmoregulatory (AER) mechanisms (Figure 1). Under such mechanisms, during the medium salinity reduction, the extracellular fluid (ECF) is diluted which induces the influx of water and the tissue cells swell. This is compensated by the process of regulatory volume decrease (RVD), in which the efflux of solutes (inorganic and organic) from the cells to the ECF occurs leading to water efflux. On the other hand, in high saline conditions, ECF osmolality increases which induces the efflux of water and ultimately leads to cell shrinkage. This is complemented by the regulatory volume increase (RVI) mechanism in which an influx of solutes (inorganic and organic) from ECF towards the cell and ultimately water inflow occurs, restoring cell volume^{24, 25} (Figure 1).

2.2 IIR mechanisms and evolutionary significance

The IIR mechanisms are also considered a prerequisite in the initial stages of development in low-saline waters.²¹ Over time, the extracellular mechanisms have evolved, leading to less pressure on the IIR strategies and decreased IIR capacities.^{21, 26} In crustaceans exhibiting varying degrees of osmoregulatory/osmoconforming behaviour, euryhalinity is the synergism of AER and IIR mechanisms.^{18, 23, 27} Thus, the osmoregulators (freshwater species) are in less demand of IIR mechanisms than euryhaline conformers who rely entirely on IIR mechanisms.^{17, 18} Nevertheless, freshwater species like Macrobrachium potiuna display the highest capacity of IIR and AER among other palaemonid shrimps of the genus Macrobrachium.^{10, 28} Whereas, the estuarine species, Palaemon pandaliformis although being a powerful osmoregulator AER displays a low capacity of IIR.¹⁰ It follows that both AER and IIR mechanisms work in tandem to maintain osmo-ionic homeostasis (or allostasis) to different degrees, depending on the species' niche and evolutionary history.^{5, 28-30}

2.3 Haemolymph's role and osmoregulation

In crustaceans, haemolymphs serves as an intermediary between cells/tissues and the ambient environment, significantly influencing IIR. The haemolymph osmolality is primarily determined by the inorganic ions usually sodium and chloride; however, free amino acids (FAA) synthesized or formed by the breakdown of haemolymph/muscle proteins are also believed to serve osmoeffectors in several crustacean species.^31-33 FAA like glycine, proline and alanine play a significant role in osmoregulation during exposure to different salinity environments as found in Penaeus stylirostris, P. japonicus, Paleomon elegans and crayfish Procambarus clarkii.^{31, 34-36} Salinity has a strong relationship with haemolymph osmolality and the latter has been found to decrease in several penaeids with a drop in salinity.³⁷ For instance, the Pacific white shrimp (L. vannamei) exhibited varied haemolymph osmolality depending on the stage and salinity, reflecting its adaptive mechanisms. Additionally, organic osmolytes like free amino acids significantly contribute to the RVD mechanism in decapods. Interestingly, hyper-/hypo-regulators like L. vannamei rely less on cell volume regulation than euryhaline osmoconformers.

2.4 Species-specific adaptations

The penaeid species exhibit hyper- and hypo-osmotic regulation with salinities below and above isosmotic values respectively, as demonstrated by numerous studies.^{38, 39} The isosmotic point of Penaeidae varies from 671.3 mOsm/kg (21.1 ppt) in the case of L. vannamei⁴⁰ to 824 mOsm/kg (26.8 ppt) in the case of L. setiferus.⁴¹ L. vannamei has been shown to maintain a narrow range of haemolymph osmolality in various salinity levels (1, 5, 7, 15 and 25 ppt).⁴⁰

On the other hand, the Palaemonid shrimps, originally of marine origin, have successfully invaded freshwaters via independent events.^{8, 42} The freshwater prawns and shrimps of the Macrobrachium genus hyperosmoregulate in freshwater or low salinities, and become hypoconformers at higher salinities (except M. equidens and M. olfersii).⁴³ Their larval development adaptations and migration principles emphasize their habitat preferences and survival strategies.^{5, 8, 44}

2.5 Energetic consideration and ion transport

The ability to actively transport (uptake) essential ions from dilute waters, especially freshwaters, requires more energy due to the existence of a large ionic concentration gradient.⁴⁵ The survival and growth in these water are predominantly determined by the energetically driven ion-motive ATPase enzymes, such as Na+/K+-ATPase and V-type H+-ATPase.^{46, 47} It suggests that the presence of salt makes it energetically cost-effective for certain species to complete the larval cycle in brackish waters. In such a medium the larvae maintain the hydro-mineral balance between the external environment and the haemolymph, which promotes its growth and survival. It also defines the migration principle towards upstream and downstream in most of the Macrobrachium species in search of brackish waters during the spawning period for hatching.⁸ Additionally, migration patterns in search of optimal salinity waters during spawning emphasize the importance of salinity for their life cycles.

Overall, decapods have demonstrated exceptional success in freshwater habitats because of their excellent osmoregulation abilities. To maintain ionic homeostasis, many epithelial tissues, including gill and antennal glands, are used in osmoregulation, which combines isosmotic intracellular regulation (IIR) and anisosmotic extracellular regulation (AER) mechanisms. The IIR mechanisms are more significant in euryhaline conformers that live in brackish environments, whereas AER mechanisms are more common in freshwater species. Yet, depending on the species' habitat and evolutionary history, both AER and IIR processes cooperate to maintain osmo-ionic homeostasis to varying degrees. The IIR is primarily affected by inorganic ions and free amino acids, with the haemolymph serving as an intermediary medium between the cells and the environment (Figure 1).

3.1 Na⁺/K⁺-ATPase

NKA was first discovered in the nerve cells of shore crab (Carcinus maenas) by Jens Christian Skou (1957) who described the influence of Na⁺ and K⁺ on the ATP hydrolytic activity for which he was awarded the Nobel prize in 1997. It is simply a sodium pump that plays an important role in the energy-demanding process like osmolyte transportation, acid–base regulation, nitrogenous waste removal and gas exchange in gills. The main role of this enzyme is to activate the ion exchange between the internal body and surrounding water to maintain the osmotic balance in both vertebrates and invertebrates.^{52, 53}

NKA enzyme belongs to P-type ATPase family of genes and structurally it has a catalytic α-subunit comprising about 1000 amino acids (binding sites for Na⁺and K⁺ and ATP) and an auxiliary β subunit (55–60 kDa) which regulates ion binding at the α subunit and acts as a chaperone.^55-57 The γ-subunit of enzyme in crustacean NKA was also found by Silva et al. (2012). Molecular cloning of α-subunit of NKA from three crustacean species viz, brine shrimp (Artemia franciscana), American lobster (Homarus americanus) and the blue crab (Callinectes sapidus) helped to decode its primary structure^58-60 and results revealed that the subunit is highly conserved across the species.

NKA is found in the membrane of epithelial cells in organs responsible for osmoregulation which pumps out 3 Na⁺ ions in blood\haemolymph and pumps in 2 K⁺ ions into the cell at the expense of one ATP molecule.^{61, 62} In numerous studies on both invertebrates and vertebrates, the expression of NKA is regulated during the change of salinities.^{61, 63-66} In low salinities, the expression of NKA transcript is high which leads to the assimilation of more sodium from the surrounding medium.⁶¹ When the animals were transferred from freshwater to higher salinity, the expression of NKA was increased.⁶⁷ Other studies supporting the facts are that the NKA enzyme has a higher affinity towards Na⁺ in the case of freshwater crustacea while this property is reversed in marine osmoconformers (reviewed by Lucu and Towle⁶¹). A study revealed that in the larval stages (zoea V and decapodid) at both 5 and 25 ppt salinities, the expression of NKA-α was higher in M. pantanalense, an inland species, compared to M. amazonicum, an estuarine species. However, this difference in NKA-α expression was not observed in the juvenile stage of these species.⁶⁸

Immunohistochemical analysis of the gill in some palaemonids (M. rosenbergii, M. olfersi, M. amazonicum and M. acanthurus) revealed that the NKA is mostly located on lamellar septum cells of gills.⁴⁷ In the giant prawn, M rosenbergii, all the individual gills were observed to have a similar NAK activity suggesting their joint importance in establishing the osmotic balance.⁶⁹ In general, the difference is only noticed in the crabs, making their gills unique in all invertebrates to respond in the osmoregulatory process.

The NKA activity in crustacea is influenced by physicochemical factors like the ionic composition of the medium, pH, temperature and some inhibitors. The pH value ranges from 7.0 to 7.7 for the optimum activity of the enzyme.^{70, 71} The optimum temperature for the maximum activity ranges from 37°C to 40°C in freshwater and brackishwater crustacea with some exceptions like in Callinectes sapidus and Orconectes limosus the values are 23°C and 27°C, respectively.^{70, 72, 73} The phospholipid bilayer's composition and fluidity are believed to modulate NAK activity. As far as inhibitors of NKA activity are concerned, orthovanadate an organic compound and ouabain have been found to completely inhibit this enzyme's function.⁷⁴

In sternal epithelial cells, it was found that the activity of the enzyme increases in the premolt and intramolt stages of crustacea. It suggests that the ATPase of the NKA helps in Na⁺ and K⁺ exchange during the mineralization process of the cuticle when ecdysis is happening.⁷⁵ The increased absorption of water during ecdysis dilutes the haemolymph ions and to protect the dilutions, the NKA is activated which re-establishes the concentrations of Na⁺, K⁺ and Cl⁻.⁷⁶

3.2 Carbonic anhydrase

CA is a Zn metalloenzyme that catalyses the rapid inter-conversion of CO₂ and HCO₃⁻.⁷⁷ The reaction is like CO₂ + H₂O⇌ H⁺ + HCO₃⁻, where H⁺ + HCO₃⁻ serves as counter-transporter for Na⁺/H⁺ (NH₄⁺) exchangers and Cl⁻/HCO₃⁻ cotransporter.⁷⁸ The CA plays a key role in the salt transport mechanism in the species at low salinities, especially in estuarine species.⁷⁹ In crustacea, the major role of this enzyme is to regulate the branchial ion transport.^{5, 30} Knockdown of CA in crustacea downregulated other important osmoregulatory genes suggesting its role in acid–base balance.⁸⁰ For the catalytic function of CA, there is a binding site for Zn, threonine-99 loop, the substrate binding pocket and the mechanism for proton shuttling.^81-83 The tissue distribution studies of CA in crustacea disclosed its presence in gills, hepatopancreas and muscle while low expression was detected in the heart, haemocytes and intestine brachial chamber.^{80, 84-88}

There are three isoforms of CA: glycosyl–phosphatidylinositol-linked carbonic anhydrase (CAg), which becomes active in high salinity conditions; cytoplasmic carbonic anhydrase (CAc), which functions in low salinity environments; and beta carbonic anhydrase (CAb), for which information is currently limited.⁸⁶ The available literature on the isoforms reveals that CAg is localized on the outer membrane of cells and accelerates the mobilization of haemolymph HCO₃ to molecular CO₂ and helps in the excretion of the latter through the gills.⁸⁹ Similarly, CAc is in the cytoplasm and helps in counter exchange H⁺ and HCO₃⁻ ions for the Na⁺/H⁺ and Cl⁻/HCO₃⁻ in gills.⁸⁹ CAc is the main isoform of CA that plays a role in the pH changes as well as in maintaining the systematic acid balance in crustaceans.⁸⁰ Ridge tail white prawn Exopaiaemon carinicauda, white shrimp L. vannamei and freshwater crayfish, Cherax quadricarinatus, exposed to changing pH and saline-alkaline stress showed upregulation of CAc and CAg mRNA expression levels in gills while CAb transcript level remained unchanged, suggesting the role of former two in coping with the stress conditions in crustacea.^{80, 86, 87}

In conclusion, CA plays a vital role in acid–base balance and osmoregulation, especially in high-saline environments. Besides this, it constitutes one of the primary components of osmotic regulation and is highly expressed in the organs that play a role in osmotic and ionic regulation. The function of CAb isoform in crustacea is unavailable so to elucidate its role in osmoregulation it becomes an interesting area of research.

3.3 Na⁺/K⁺/2Cl⁻ cotransporter

NKCC is a membrane transport protein that belongs to the cation-chloride cotransporter (CCC) family of proteins and helps in osmoregulation by transporting Na⁺, K⁺ and Cl⁻ across cell membranes simultaneously.⁹⁰ It sometimes acts as a symporter and maintains electroneutrality by transporting two positively charged ions in parallel with two negatively charged ions, that is, Na⁺ and K⁺ with 2 Cl⁻.⁸⁹ In vertebrates, NKCC manifests in two isoforms: NKCC1 which facilitates the trans-epithelial secretion of chloride ions and modulates the excitability of certain neurons. On the other hand, NKCC2 plays a crucial role in the renal system by contributing to the reabsorption of salt in the kidneys.^91-93 Basal expression reports of NKCC exhibited its presence in all the tissues with the highest in the posterior gills.⁹⁴

In weak hyper-osmoregulators, NKCC plays a crucial role in apical NaCl absorption. Neohelice granulata exposed to varying degrees of salt water (dilute or concentrated) resulted in an increase in the mRNA expression of NKCC.⁹² Most of the studies in crustacea determine that NKCC is upregulated when exposed to low salinity.^{92, 93, 95} Similarly, M. australiense, a freshwater prawn challenged with increased salinity resulted in an increase in the mRNA expression of NKCC.⁵⁴ Available reports confirmed that NKCC has an osmoregulatory function in M rosenbergii, M. australiense and M. koombooloomba.^{52-54, 96} The mRNA expression of NKCC was upregulated in the moult cycle, especially during the postmolt stage suggesting the involvement of this transcript in branchial ion uptake during ecdysis that correlates with the role of NKCC in the gills in osmoregulation. The expression of the NKCC gene in palaemonid shrimps is positively correlated with the over-regulation of Cl⁻ in the haemolymph, indicating a potential role of the NKCC symporter in salt uptake. Additionally, the synthesis of gill NKCC protein is associated with the hyper-regulation of haemolymph osmolality, further supporting this hypothesis.⁹⁷

In general, NKCC transcript is highly expressed in the gills of crustacea and plays a significant role in maintaining osmoregulation in high-saline environments.

3.4 Na⁺/H⁺-exchanger

NHE belongs to superfamily cation/proton antiporters (CPA) and is a member of the solute carrier (SLC) 9A family that transports Na⁺ in exchange for H⁺ (1:1 stoichiometry) during the regulation of ionic balance, pH homeostasis, cell volume regulation and other osmoregulatory processes.^98-100 Since its first isolation in the rat kidney, 9 isoforms (1–9) of the genes have been identified in higher animals reviewed by Donowitz et al.¹⁰¹ All the isoforms of NHE have a similar topological structure containing N- terminus 11–13 transmembrane domains for ion exchange and have a regulatory region on C-terminus. In invertebrates, the NHE has been studied in brush border cells of hepatopancreas and results reveal that a different type of electrogenic exchange protein is found which displays a transport stoichiometry of 2Na⁺/1H⁺.¹⁰² At physiological pHi (intracellular pH) in cells, NHE basal activity is very low but when pHi is low, the activity of NHE activity increases sharply for acidified pHi adjustment by promptly extruding protons in exchange for extracellular Na⁺.¹⁰³ In, P. vannamei, the knockdown of NHE resulted in reduced haemolymph pH when exposed to low-pH challenge, suggesting its role in maintaining the acid\base homeostasis in crustacea.¹⁰³ Supporting this data, in ridge tail white prawn, Exopalaemon carinicauda, the knockdown of CAc resulted in the downregulation of NHE and finally the mortality of the species reared under saline–alkaline stress.⁸⁰ A study revealed that in the larval stages (zoea V and decapodid) of M. pantanalense and M. amazonicum at both 5 and 25 ppt salinities, the expression of NHE3 was higher at 5 ppt.⁶⁸ In general, the role of NHE was deciphered by different approaches and the evidence supports that it maintains the extracellular (circulating) and intracellular pH regulation in crustacea.

3.5 V-type proton (H⁺)-ATPase

VAT or HAT are membrane-associated, highly conserved enzymes that acidify intracellular organelles and boost secondary ion transport across whole epithelia by proton delivery from the cytoplasm to the extracellular medium and in the plasma membrane, it pumps protons in many tissue types.¹⁰⁴ Initially, it was isolated from the intracellular vacuoles and later in the plasma membranes where it performs its leading role.¹⁰⁵ In M. amazonicum, the subcellular localization by immunofluorescence demonstrated its presence in the gills and in the branchiostegites.¹⁰⁶ VAT is formed of two domains V0 and V1 that, in turn, have many subunits, namely six subunits (a, d, c, c′, c″ and e) for V0 and eight (A–H) for V1 which are arranged differently.^{107, 108} In the V1 domain, A and B subunits have catalytic sites for ATP hydrolysis which is used to carry H⁺ across the membrane, whereas V1 has the function of proton translocation.^109-112 In freshwater crustacea, that are hyper-osmotic, VTA plays an important role in maintaining osmoregulation.^{95, 113}

The VAT mRNA expression studies in crustacea have been mostly performed by exposing them to varying pH and the transcript level was found to be upregulated when invertebrates were treated with low salinity or low pH.^{67, 114} A negative correlation between pH and VTA has been reported, in which at low pH VTA is upregulated and vice versa.¹¹⁴ Individuals transferred from the optimum to varying (higher and lower) salinities, resulting in the downregulation of VTA.⁹³ When M. amazonicum was exposed to a salinity level of 21%, there was a rapid increase in VAT mRNA expression within the first hour. However, this expression sharply declined to a negligible level after a period of 10 days.^{46, 115} So, VAT is the main enzyme that maintains osmoregulation and is implemented during pH and salinity changes in crustacea.

3.6 Other important genes

Besides the master genes involved in osmoregulation in crustacea, lots of minor genes also play a key role in maintaining homeostasis in changing environments. Alkaline phosphatase is involved in ion binding; precipitation of ions and metabolic process during osmoregulation.⁵³ Arginin kinase, which is involved in the production of ATP in invertebrates through hydrolysis, is doubled when some crustacea are transferred from high salinity to low salinity, suggesting its role in the osmoregulation. Aquaporin, a key player in the regulation of water channels, is a crucial component among genes responsible for osmoregulation. Its significance is particularly notable in the maintenance of homeostasis in freshwater environments.⁵⁴ Similarly, some other genes include Crustacean cardiovascular peptide (involved in body fluid regulation, stress tolerance), Bestrophin (maintains cellular water homeostasis), ATP-binding cassette 12 (ABC12, engaged in osmotic stress regulation), Calreticulin (Ca²⁺ homeostasis), Mitogen-activated protein kinase 38 and Mitochondrial carrier protein (osmotic stress response) that have been studied to play an active role in the osmoregulation.^{52, 53, 96} The minor genes involved in osmoregulation are discussed properly in a review by Rahi et al.⁵⁰

In general, the genomic basis for osmoregulation in crustacea particularly in decapods has been studied well and still, there is a scope to use powerful tools like sequencing technologies, transcriptomics and proteomics to elucidate the exact mechanism the genes play in maintaining the homeostasis. To enhance our understanding of the genomic mechanisms involved in osmoregulation among crustaceans, it is imperative to expand the scope of sequencing beyond model species and incorporate a broader range of non-model crustacean species.

4.1 Combined effect of the temperature and salinity on growth of P. vannamei

While the average water temperature in most of the shrimp farms varies from 15°C to 32°C, the best survival temperature for the growth of L. vannamei juveniles and adults is about 20–30°C and 27°C, respectively.^{119, 120} Kır et al.¹²¹ found that L. vannamei has a remarkable ability to survive in a broad range of temperatures, ranging from 7.2°C to 41.9°C (CT_min − CT_max: 7.2–41.9°C) and suggested a temperature range of 25–30°C for effective production. The exposure of shrimp to lower water temperatures induces oxidative stress, DNA damage, lipid peroxidation, changes in osmolality, arrests growth, or even causes death.^122-124 The reduction in water temperature was seen to lower the osmoregulatory capacity of P. stylirostris both at low and high salinity and the effect was more pronounced in sub-adults than juveniles.¹²⁵ During the cold stress (13°C for 24 h), L. vannamei exhibited significant alterations in 30 proteins (up- and down-regulated) studied.¹²⁶ The physiological response of L. vannamei to temperature fluctuation (28–13°C) in low-salinity water was studied by Wang et al.¹²⁷ It was found that the number and volume of β-cells in hepatopancreas tubules significantly increased. The lipids and protein in plasma responded more rapidly to temperature fluctuation, while the contents of glucose remained stable while lowering the temperature from 28°C to 13°C. It was concluded that the increase of β-cells facilitates gluconeogenesis to synthesize glucose from protein and lipid, by which shrimps maintain glucose demand under cold stress. The protein and lipid were observed to be the main energy source of L. vannamei during temperature fluctuation.¹²⁷ To understand further the mechanism of homeostasis during temperature fluctuation, Wang et al.¹²⁸ studied the intestinal transcriptome profiles of L. vannamei reared at 5 ppt. Metabolic inhibition was found as a strategy to cope with cold stress and lipid metabolism served as an important factor during temperature fluctuation. Also, cold stress caused histological damage to the shrimp intestine, which was self-repaired after the temperature was reversed. The sudden exposure of L. vannamei reared at 5 ppt and pH 8.0 to low temperature (from 23 ± 2°C to 12 ± 2°C) induced oxidative stress, DNA damage, lipid peroxidation and changes in osmolality within 3 h.¹²² The oxygen consumption in juveniles of L. vannamei was more stable at 25°C and 30°C for 13 and 25 ppt salinity in all size groups.¹²⁹ In L. vannamei subjected to continuous salinities, the isosmotic point increased from 717 to 823 mmol/kg as a function of temperature. The best growth was achieved under these testing conditions with the T–S combination of 32°C and 28 ppt.¹³⁰ Overall, it has been found that L. vannamei can self-regulate the changes in temperature to a certain extent. However, with a sudden increase in water temperature, there is a significant effect on survival, behavioural responses and immunological parameters.^{127, 131}

In crustaceans, there is an overproduction of reactive oxygen species (respiratory burst) and a sharp reduction in total haemocyte count at low temperatures.^{132, 133} In conclusion, water temperature plays a crucial role in the survival and growth of L. vannamei in shrimp farms. The ideal water temperature for juveniles and adults is around 20–30°C and 27°C, respectively. The study of homeostasis mechanisms during temperature fluctuation can lead to a better understanding of how to improve the shrimp farming industry's management practices.

4.2 Salinity and nutritional importance

Nutrients are the components or part of food/feed which provide nourishment and are required for the body's functions like maintenance, development, growth and reproduction. It can also be defined as the component of food, the excess or deficiency of which may produce a malfunction of the body. Nutrients play numerous roles and are essential for the shrimp, like any other animals, to stay alive, active and healthy to grow. These are the building blocks of all the tissues of the animals and are involved in all the chemical reactions that facilitate life processes. Dietary requirements can be established for protein and amino acids, lipids and fatty acids, carbohydrates, vitamins and minerals. The salinity gradients have a profound impact on the nutritional requirements of the penaeid shrimps. The comprehensive description of the nutritional requirements of (P. vannamei) till 2015 was illustrated in the review of Li et al.¹³⁴ In this section, the detailed compilation of most of the studies published after 2015 on various aspects of nutritional requirements or interventions at different salinity gradients or salinity stresses will be summarized (Table 2).

4.3 Effect of salinity on digestive physiology

The digestive physiology of certain economically significant crustaceans is influenced by the changing salinity levels in their surrounding culture water.¹⁷³ The deviation of the optimum salinity levels (20–25 ppt)¹³⁴ for shrimp rearing alters the normal physiological homeostasis leading to osmoregulatory-dependent pressure in cells, thus increasing the body energy demand (Figure 2).⁸⁸ Likewise, it also triggers the activity of substrate-specific digestive enzyme activities in the hepatopancreas of L. vannamei.¹³⁷ Previously, authors have demonstrated that salinity has a variable impact on hepatopancreatic digestive enzymes, osmoregulation, growth metrics, immune responses and survival of shrimp.^{139, 174-176}

Li et al.¹⁷⁵ conducted a study on P. vannamei, investigating the impact of different salinity levels (3, 17 and 32 ppt) on the activities of digestive enzymes. A significantly higher trypsin and amylase activity was observed at 3 ppt salinity followed by 32 ppt. However, lipase-specific activity did not vary significantly despite variations in salinity levels. The surge in digestive enzyme activities may be due to an increase in the number of hepatopancreatic luminal B cells (blasenzellen cells) at both extreme salinities. It facilitates the synthesis and secretion of digestive enzymes to compensate for extra energy demand through osmoregulation via gill. Similarly, a decrease in the abundance of luminal R cells (restzellen cells) at 3 ppt indicates the utilization of cell reserves to cope with low salinity conditions. In contrast to this, the activity of the digestive and antioxidant enzymes decreased at very high salinities, particularly above 50 ppt salinity¹⁷⁶ Alkaline proteases such as trypsin and chymotrypsin are the key digestive enzymes of protein metabolism in shrimp.¹³⁶ Apart from this, they also modulate the immune system by triggering prophenoloxidase defence system.^{177, 178} Synthesis and production of these enzymes depends on the mRNA and protein synthesis.¹⁷⁹ In a study, Gao et al.¹³⁹ assessed the impact of varying salinity levels (2, 10, 20 and 30 ppt) on metabolic-related gene expression of P. vannamei. The hepatopancreatic trypsin and chymotrypsin mRNA expression significantly downregulated with the decreasing salinity levels and the lowest mRNA expression was observed at 2 ppt salinity. The study concludes that the rate of synthesis of these digestive enzymes was inhibited at low salinity leading to stress, ultimately poor growth and immunodepression. Gao et al.¹⁷⁴ evaluated the effect of short-term (24 h) and long-term salinity stress (56 days, 2 ppt as low saline water and 30 ppt as natural seawater) on hepatopancreatic digestive-related genes of P. vannamei. Relative mRNA expressions of trypsin and chymotrypsin 1 and hepatopancreatic genes were unchanged in short-term salinity stress but decreased by 1.33- and 1.54-fold in long-term salinity stress at low salinity. The findings specify that these genes support short-term stress recovery in shrimp, but long-term salinity stress leads to immunodepression and poor digestive performance and growth of shrimp. However, mRNA expression of the chitinase gene increased 3.07-fold at short-term salinity stress but downregulated by 1.45-fold at long-term low salinity stress conditions. This observation indicates that the activity of this enzyme increased to digest more food to compensate for the hypo-osmotic stress-mediated metabolic energy demand via activation of the glycolytic pathway¹⁸⁰ and immuno-protective efforts of P. vannamei. But long-term hypo-osmotic stress leads to damage of the hepatopancreas that resulted in immunodepression, abnormalities in digestive physiology and growth retardation of shrimp. Gaxiola et al.¹⁷³ studied the factorial effects of salinity levels (15 and 40 ppt), dietary carbohydrate (2.3% and 42.3%) and moult cycle of L. vannamei and found that the specific activity of carbohydrases (α-amylase, hexokinase, α-glucosidase and hexokinase IV) were significantly altered in both the rearing salinity conditions. At low salinity, α-amylase activity was highest when fed with low and high levels of dietary carbohydrates (salinity and moult stages interaction) whereas highest α-glucosidase activity was found in low carbohydrate-fed group (salinity and dietary carbohydrate interaction). The findings are particularly associated with the hormonal regulation of growing stages over synthesis and production of digestive enzymes concomitant with the digestion of carbohydrates for metabolic energy needs at low salinity levels. However, hexokinase and hexokinase IV activity were found highest in high carbohydrate-fed groups (salinity and carbohydrate levels interaction). Elevated activities of hexokinase and hexokinase IV are linked with higher need of glycogen and energy for faster moulting processes and physiological adaptations for growth regulated by crustacean hyperglycemic hormone (CHH).^{181, 182} Chen et al.¹⁸³ described higher hepatopancreatic lipid metabolic enzyme activities viz., fatty acid synthase (FAS), hormone-sensitive lipase (HSL), Δ5 fatty acid desaturase (Δ5 FAD), Δ6 fatty acid desaturase (Δ6 FAD), adipose triacylglycerol lipase (ATGL) and elongase of very long chain fatty acids 6 (ELOVL 6) at high salinity (30 ppt) as compared to very low salinity (3 ppt). This function is mainly attributed to the higher degradation of fatty acids to produce triacylglycerols which are further transported to gill via lipoproteins to satiate the higher energy demand for salinity-dependent osmoregulatory stress. At low salinity, temperature also impairs the plasma protein metabolic enzymes and ALT (alanine aminotransferase) activities in P. vannamei. A decrease in temperature from 28°C to 13°C at low saline conditions (5 ppt) gradually decreases the plasma ALT activities and ruptured B cells were detected in the dilated lumen of hepatopancreas indicating severe damage of the organ as well as the reduced digestive capacity of shrimp L. vannamei.¹²⁷ However, the restoration of hepatopancreatic B cells occurred during the temperature reoccurrence process (13–28°C).

4.4 Salinity changes: immune response and impact on disease outcome

Beyond the baseline metabolic rates needed for sustaining life, additional energy is expended on activities such as locomotion, digesting food, building new tissues, reproducing and responding to stress or disease. Growth can be hindered or even reversed when these demands increase either due to heightened stress or disease. The energy required for osmoregulation is considered a part of the baseline maintenance component.¹⁸⁴ Different species or groups of animals exhibit various strategies for osmoregulation. Salinity stress negatively affects immune parameters in crustaceans and can increase the virulence of pathogens in hosts, leading to increased mortality. Rapid fluctuations in salinity levels can have detrimental effects on the osmoregulation, physiology and immune responses of decapod crustaceans.¹⁸⁵ Several studies have investigated the effects of salinity on the immune responses of different crustacean species (Figure 3). Luqing and Lingxu¹⁸⁶ observed a gradual decrease in the bacteriolytic and antibacterial activity of the haemocytes of Fenneropenaeus chinensis and L. vannamei in response to a rapid reduction in salinity from 30 to 15 ppt over a 10 h period. However, the phenoloxidase activity of these shrimps increased over the same period. Vargas-Albores et al.¹⁸⁷ demonstrated that prophenoloxidase activity increased directly with salinity in Farfantepenaeus californiensis reared at salinity levels between 28 and 44 ppt. Similarly, several other reports have indicated a decrease in haemocyte count and an increase in phenoloxidase activity in response to changes in salinity in Marsupenaeus japonicus¹⁸⁸ and Portunus trituberculatus.¹⁸⁹

The impact of salinity on haemocyte numbers in crustaceans has been studied by several researchers. Cheng and Chen¹⁹⁰ observed an increase in haemocyte numbers in M. rosenbergii when salinity levels changed from 0 to 5, 10, or 15 ppt over a period of 7 days. On the other hand, Perazzolo et al.¹⁹¹ observed that the total heamocyte count (THC) Farfantepenaeus paulensis gradually declined when salinity was decreased from 34 to 22 ppt or 13 ppt over 3 days, before stabilizing between 7 and 28 days. Le Moullac and Haffner¹³² observed that F. paulensis reared in 34 ppt salinity had a significantly higher THC than those reared in 22 and 13 ppt. Similarly, giant freshwater prawn M. rosenbergii reared in 15 ppt had a significantly higher THC compared to the prawns reared in freshwater.¹⁹⁰ From these findings, it can be inferred that salinity levels have a significant impact on haemocyte count in crustaceans. Overall, decreasing salinity levels may lead to a decline in haemocyte count, indicating potential suppression of immune response in crustaceans. Conversely, higher salinity levels may promote an increase in haemocyte count, suggesting enhanced immune response. These findings highlight the importance of salinity as a crucial environmental factor influencing the immune responses of crustaceans, which may have implications for their health, survival and overall immune defence against pathogens. The neuroendocrine-immunoregulatory network has also been found to play a principal role in adapting to salinity changes in crustaceans. Zhao et al.¹⁹² proposed that the network serves as the main centre for sensing stress and initiating an immune response in L. vannamei. Furthermore, several studies have reported that salinity stress can cause a decrease in haemocyte count and enzyme activity related to disease resistance, while an increase in sensitivity to pathogens in crustaceans.^{132, 187, 193} Lu-Qing et al.¹⁹⁴ have suggested that the haemocyte count of shrimp decreases under low salinity due to an increase in haemolymph volume. As a result, the total haemocyte count appears to decrease in relation to the haemolymph volume. This may explain why there is no significant change in the immune enzyme activity per unit protein in the haemolymph with decreasing salinity.

Overall, salinity stress can have significant effects on the immune responses of various crustacean species, including changes in haemocyte count, bacteriolytic and antibacterial activity and phenoloxidase activity. These findings suggest that the impact of salinity stress on crustacean immune responses is complex and may depend on various factors, including species, salinity levels and exposure duration.