3.1. Lecithin

Plant-based lecithin can be extracted from sources, such as soybean, sunflower, rapeseed, walnut, chia seed, camelina seed, and corn, whereas animal-based lecithin is sourced from egg yolk, krill, fish roe, and fish products, among others (Table 2). Among these, soy lecithin (SL), EL, and KO are the most utilized sources of PL in crustacean diets, each possessing distinct FA profiles [39]. Despite the availability of animal-derived lecithin, plant-derived lecithin, particularly SL, is generally favored in crustacean diets due to its greater availability [38]. This section will provide a detailed discussion of these major sources of PL, which are commonly used in aquaculture.

3.2. Advantages and Disadvantages of Different PL Sources

SL is the most economically viable and widely available PL source for large-scale crustacean feed production, despite concerns over genetically modified organisms (GMOs) and lower purity [64, 65]. EL offers high-quality PL but is limited by high extraction costs and inconsistent supply [33, 38]. Krill oil lecithin (KOL) provides higher bioavailability and omega-3 but faces sustainability challenges due to limited harvests and ecological concerns [28, 66]. Among these sources, SL remains more feasible for long-term application in aquaculture systems. Table 3 presents a comparative assessment of major PL sources, naming SL, EL, and KOL according to their nutritional properties, economic feasibility, and sustainability within crustacean aquaculture.

5.1. Effects of PL on Crustacean Growth

The global seafood market is projected to reach $139 billion by 2027, up from $113 billion in 2020 [74]. Crustaceans, notably shrimp and prawns, account for ~20% of global consumption [75]. Therefore, optimizing growth and survival during the rearing phase (culture period) is critical for advancing crustacean aquaculture [76]. However, crustaceans cannot fully synthesize all essential nutrients, constraining their production [16, 77]. Thus, they require supplementation for a balanced diet during their early life stages, particularly during the culture period.

PLs are among the most essential dietary components of the crustacean diet, playing a unique role in enhancing various physiological processes like growth and development [78]. Dietary PL, such as lecithin and KO, are important sources of essential nutrients, including inositol, choline, vitamins, phosphorous, EFA, and energy, which are key requirements in many physiological functions [79]. These components contribute to vital cell functions like the integrity of cell membranes, and boosting metabolic functions like digestion, absorption, and consumption of nutrients, lipids in particular [79]. Additionally, dietary PL, particularly from marine sources are rich in EPA and DHA, which act as growth promoters by fulfilling the high demands of rapid tissue formation [80]. Dayal et al. [18] demonstrated that a high percentage of PL sources, such as SL, results in improved lipid accumulation and energy availability, thereby boosting the circulation and transfer of lipids from the hepatopancreas to hemolymph and other tissues, which cumulatively enhances crustacean growth.

In a mitochondrial study on P. trituberculatus, the mitochondrial electron transport chain complex activity assay revealed higher mitochondrial complex II activity and mitochondrial membrane potentials in the hepatopancreas of the KO-fed crab [81]. Moreover, the benefits of PL extend to the improvement of wider growth parameters of crustaceans, including WG, final body weight (FBW), SR, SGR, MF, and condition factor (CF) (Table 5). However, some studies have observed potential negative effects on certain growth parameters with high PL inclusion. The following sections in this review explore these findings in detail, considering parameters like SGR, WG, FCR, SR, CF, FBW MF, FE, protein efficiency ratio (PER), hepatosomatic index (HSI), and relative growth rate (RGR) (Table 5).

There is growing evidence that PL-enriched diets have significant potential to enhance various growth parameters in crustaceans, particularly FBW, HSI, WG, RGR, SGR, and PER. Among the summarized data, FBW and WG exhibited the most consistent positive responses across multiple studies (10). Positive effects on SGR, SR, and MF were also observed in several studies (four to eight). However, parameters like PER, FCR, and RGR showed limited evidence of improvement, with only one to three studies reporting the gains. It is noteworthy that some parameters, including FCR, SR, and MF can be negatively or neutrally affected by PL inclusion depending on the specific inclusion rate and crustacean species (Table 5).

The impact of PL inclusion rates ranging from 0.99% to 6.12% was investigated on the growth of several crustacean species, including P. trituberculatus, S. paramamosain, E. sinensis, L. vannamei, P. clarkii, and P. monodon [5, 11, 13, 18, 19, 43–47]. In a study by Sun et al. [43], significant increases in FBW and MF for P. trituberculatus were found, rising from 3.69 to 44.48 g and 2.76% to 3.29%, respectively. However, another study on P. trituberculatus showed minimal improvement in FCR, with the lowest value (1.28) observed at a 4% inclusion rate of SL [43]. Correspondingly, a higher SR of 92.86% was observed in S. paramamosain due to 3% EL inclusion but lower gains of 86.90% at an SL inclusion rate of 2% in the same species were observed [43, 53]. From these studies, S. paramamosain achieved the highest WG (3267.41%) at a 3% PL inclusion, while E. sinensis (3.59% SGR) and P. trituberculatus (4.03% HSI) benefited most from 2% to 3% PL inclusion [19, 45, 47]. Collectively, these studies identified optimal PL inclusion rates for maximizing WG, SGR, and HSI [19, 46].

5.2. Mechanism of PL Effects on Crustacean Growth

The optimized inclusion levels of PL in crustacean diets result in significantly improved growth performance and SRs across various species (Table 5). Figure 3 illustrates the role of PL on crustacean growth parameters, demonstrating that PL enrichment primarily positively impacts SGR, WG, FBW, and RGR. The increase in these parameters due to dietary PL (EL and KO) is attributed to the elevated levels of n-3 FA, particularly EPA and DHA. Notably, EPA and DHA readily get absorbed and utilized by crustaceans [58], unlike linolenic (n-3) and linoleic acids (n-6 Lc-PUFA), which cannot easily be converted into EFA that is crucial for growth and molting [80]. Moreover, terrestrial PL sources like SL, enhance the growth conditions in crustaceans by increasing the surface area of lipid droplets in the aquafeed, resulting in higher digestibility of feed [83]. Positive impacts of PL diet on crustacean MF have been reported in many crustaceans, such as E. sinensis and P. trituberculatus (Table 5).

Aside from these growth parameters, PL-enriched diets can also improve SR, which is the number of individuals surviving the production [84]. The increased SR with PL inclusion was potentially linked to enhanced antioxidant activity in crustaceans, reflecting the complex interaction between PL-enriched feed and antioxidants present in crustaceans [11]. In another study, feeding trials on P. trituberculatus reported higher levels of HSI due to PL-enriched feeds [13, 85]. The hepatopancreas, a crustacean organ for the storage of organic and inorganic compounds, is evaluated using HSI to assess relative energy reserves [86]. As illustrated in Figure 3, increased HSI with PL inclusion occurs because of higher lipid absorption, synthesis, and greater hepatic lipid deposition [23].

Additional important growth parameters considered in feeding trials to assess the impact of PL-enriched diets include FCR, PER, and FE. Positive impacts of these parameters have been recorded in various crustacean species, often depending on the specific inclusion rate [11, 13, 18, 43, 85]. FCR, an indicator of FE, refers to the amount of feed needed to grow 1 kg (live weight) of aquatic organisms. Thus, lower FCR values signify better feed utilization, allowing organisms to grow in a less polluted waterbody [87]. Furthermore, PER, a measure of protein quality, is expressed as the ratio of body WG to protein consumed [88]. The positive impact of PL-enriched diets on these growth parameters can be associated with the increased availability of energy for growth. This is facilitated by enhanced lipid transportation and mobilization from the hepatopancreas to the hemolymph and other tissues [89].

However, studies have shown that at higher PL inclusion rates, some parameters like SR, MF, CF, and HSI may be reduced or experience no significant improvement [5, 19, 44, 45, 53]. For example, no significant effect was observed in SR and CF in L. vannamei fed with PL-enriched diets containing 0.05% to 0.1% cholesterol, yet feeding the same diets to P. clarkii resulted in decreased MF levels [5, 44]. Other studies investigating S. paramamosain and E. sinensis found decreases in MF and HSI at increasing PL inclusion rates, highlighting the importance of optimizing feed formulations with PL to ensure optimal growth performance [23, 53]. Collectively, these studies demonstrated that lower HSI values at higher PL inclusion rates in E. sinensis were attributed to the suppression of lipid uptake gene expression and a reduction in excess lipid synthesis. This aligns with observations from earlier studies on P. trituberculatus and Larimichthys croceain, which reported that excessive dietary lipid levels can lead to increased abnormal lipids deposition, resulting in metabolic disturbances and inflammation in organisms, ultimately inhibiting growth [48, 90].

6.1. Effect of PL on Lipid Metabolism

Dietary PL supplies aquatic organisms with essential nutrients required for growth and development, particularly because of the inability of some species and juvenile crustaceans to biosynthesize it to meet their body requirements [16]. Primarily, the lipid-containing phosphorus plays a key role in enhancing lipid metabolism. PL emulsifies lipids, enhancing the bioavailability of crucial nutrients like EFA, phosphorous, choline, and inositol for vital biological functions [91]. Furthermore, dietary PL facilitates lipid transport from the gut epithelium to the hemolymph and various tissues, enabling the deposition of FA, cholesterol, and other lipophilic nutrients in crustaceans [92]. Dietary PL also serves as precursors for a wide range of biologically active lipid mediators critical to metabolism and physiological functions. These mediators, including eicosanoids, diacylglycerol (DAG), inositol phosphates (IP), and platelet-activating factors (PAFs), contribute to energy generation for normal physiological functions [93].

To further illustrate the influence of PL on lipid metabolism, studies employing molecular analysis of lipid uptake and synthesis genes in E. sinensis, such as fapt4, fatp6, fabp9, fabp10, fas, elovl6, and Δ9fad, support the role of PL-enriched diet in promoting lipid absorption and synthesis in the hepatopancreas, indicating improved overall metabolism [94]. Notably, a study on S. paramamosain revealed decreasing levels of srebp-1, fas, Δ6 fad, Δ9 fad, elovl4, and elovl6 with increasing PL inclusion rate where the highest fabp level was noted with 1% PL [82]. Indeed, extensive research has illuminated the metabolic benefits of PL-enriched diets for crustaceans. While most studies observed positive impacts of PL, the effect on specific metabolic parameters varies significantly with crustacean species, PL inclusion rate, and source. Therefore, the next section will examine the impact of PL on a range of key parameters, including TG, PL transfer protein (PLTP), low-density lipoprotein (LDL), high-density lipoprotein (HDL), total cholesterol content (T-CHO), very low-density lipoprotein (VLDL), phosphocholine cytidine transferase (PCT), carnitine palmitoyltransferase-1 (CPT-1), phospholipase A2 (PLA2), Acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) as presented in Table 6.

Table 6 summarizes the findings from 10 feeding trials investigating the impacts of PL-enriched diets on crustacean metabolism. These studies explored the effects of varying inclusion rates (0% to 6.12%) of PL derived from various sources (SL, EL, and KO). Notably, PL-enriched diets demonstrated the potential to improve specific metabolic parameters. For example, nine studies reported a significant boost to T-CHO levels, while elevated TG levels were observed in seven studies. Furthermore, three studies indicated an increase in VLDL content, whereas increases in metabolic enzymes like PCT, PLA2, and CPT-1 were reported in one study. In contrast, higher PL inclusion rates did not consistently yield positive effects as demonstrated in two studies that reported a negative impact on TG, HDL, and LDL, and one study that observed lower levels of PLTP, FAS, and ACC.

The effects of PL enrichment on individual crustacean species, as detailed in Table 6, reveal substantial variations in lipid metabolism. For instance, P. trituberculatus exhibited a notable increase in hepatopancreas T-CHO, reaching a maximum of 13.20 μg/mg when fed a diet containing 2% SL [47]. Similarly, S. paramamosain showed elevated TG levels (5.89 mmol/gprot) with 4% EL inclusion, but conversely, a lower inclusion rate (0.5%) resulted in decreased TG (2.80 mmol/gprot) [22, 57]. Significant increases in HDL and VLDL were also reported in E. sinensis due to PL-enriched diets [23]. This study found an increase in HDL to a maximum of 4.2 mmol/L with a 2.5% SL diet, while VLDL peaked at 2 mmol/L with 2.5% KO. Moreover, L. vannamei showed increased LDL levels to a maximum of 1.1 mmol/L with 4% KO, and a minimum of 0.85 mmol/L at 0% KO inclusion [24]. The variation in the responses to PL enrichment can be attributed to differences in lipid metabolic enzymes across species and inclusion rates as illustrated in several studies. Increased CPT-1 activity was reported in C. quadricarinatus with 3% SL [26], a response that was mirrored in P. trituberculatus fed with 3.63% SL, where PCT and PLA2 increased to a maximum of 105.18 and 47.37 U/mgprot, respectively [13]. However, some studies reported negative and contradictory results. For instance, while 1% SL resulted in the highest TG levels (3.10 mg/dl) in P. trituberculatus, 2% SL yielded the lowest (1.84 mg/dl) [47]. These variations in effects must be considered when developing species-specific and context-dependent PL inclusion strategies for crustacean aquaculture.

6.2. Mechanism of PL Effects in Crustacean Lipid Metabolism

Dietary PLs are essential for various cellular functions, including signaling, transportation, and regulation of membrane-bound enzyme activity [96]. In crustaceans, the hepatopancreas serves as the central regulatory organ for PL metabolism and storage (Figure 4), which dictates how dietary PL supplementation influences lipid distribution and metabolism [98]. In particular, supplementation with PL induces an increase in hepatopancreas lipids and a decrease in muscle lipids, implying that PLs aid in lipid deposition in the hepatopancreas while enhancing lipid catabolism in the muscle. Therefore, PL supplementation in crustacean diets can significantly affect various lipid metabolic factors, including HDL, LDL, VLDL, TG, T-CHO, and lipid metabolism enzymes (ACC, CPT-1, PCT, PLA-2, PLTP, FAS) (Table 6).

Lipoproteins (HDL, LDL, and VLDL) function as cholesterol transporters in the body, of which HDL exhibits a high protein-to-lipid ratio and is considered beneficial due to its inverse association with the risk of heart diseases [99]. Conversely, LDL, which primarily transports cholesterol, is associated with cardiovascular risks. VLDL plays a vital role in transporting TG from the hepatopancreas to the perihepatic tissues. This function is supported by supplemented PL, which maintains proper VLDL assembly and secretion into the serum [23, 45]. Similarly, VLDL is also regarded as a “bad” cholesterol due to its association with cardiovascular disease risks [100]. An increased or decreased HDL/LDL ratio reflects whether cholesterol in the serum is taken up by the hepatopancreas for metabolic activities or released from the hepatopancreas to peripheral tissues [95].

To illustrate the diverse effects of PL supplementation on crustacean lipid metabolism, feeding trials on E. sinensis demonstrated an increase in HDL, with the highest HDL (4.2 mmol/L) in the hepatopancreas reported at 2.5% SL inclusion rate. Conversely, a rapid decrease was observed in S. paramamosain fed a 1.5% SL diet, yielding the lowest HDL level (0.01 mmol/L) [22, 23]. Additionally, the highest VLDL level was reported in E. sinensis with 5% SL supplementation [94]. Despite these findings with SL, a recent study by [24] suggested that EL and KO supplementation may be more effective in promoting TG transport from the hepatopancreas to the ovary, potentially due to enhanced lipoprotein formation.

To further elucidate the mechanism of PL in crustacean lipid metabolism, dietary PL has been proposed to promote the absorption of cholesterol, TG, and n-3 FA from the gastrointestinal (GI) tract to the hepatopancreas. Subsequently, the absorbed lipids are delivered to various tissues through the hemolymph, resulting in elevated TG levels [6, 101]. Recently, [57] observed the highest TG accumulation (5.89 mmol/gprot) in S. paramamosain fed with an EL-supplemented diet. This increment was attributable to improved lipid emulsification by PL, facilitating the transportation process from the hepatopancreas to the hemolymph. Additionally, enhanced emulsification may have promoted lipid deposition and energy utilization within crustacean tissues [47].

Figure 4 summarizes the central role of crustacean hepatopancreas in bridging between the synthesis and secretion of digestive enzymes, lipid metabolism, nutrient absorption, and transportation. For instance, FAS, the primary limiting enzyme for lipid synthesis, predominantly found in the hepatopancreas and ovaries [102], exhibits a dose-dependent response to PL inclusion. Within an optimal range, PL supplementation enhances FAS activity, while excessive PL can lead to inhibition [13]. Similarly, PCT catalyzes a key regulated step in the cytidine diphosphate (CDP)-choline pathway, controlling the rate of PC and PC-related lipid production [103]. Furthermore, CPT-1, a mitochondrial membrane protein, regulates the rate-limiting step in FA oxidation, facilitating the transport of long-chain FA from the cytoplasm into mitochondria for oxidation [104]. Additionally, PLA2 enzymes play a regulatory role in the eicosanoid pathway by releasing free arachidonic acid from membrane PL, whereas ACC plays a crucial role in both the synthesis and oxidation of FAs, providing energy for cellular functions [105, 106]. Therefore, considering these enzymatic roles within the context of the central function of hepatopancreas, it becomes evident that lipid deposition in crustaceans is ultimately a product of the delicate balance between internal lipid synthesis and catabolism, both of which are significantly modulated by dietary PL [107].

7.1. Effect of PL on the Nutritional Profile of Crustaceans

Lipids are essential constituents of crustacean diets, playing multiple functions, including a source of energy source, supply of EFA, carrier for fat-soluble vitamins, formation of structural components for biomembranes, and serving as precursors for vital biological compounds like hormones and enzymes [108]. Notably, crustaceans cannot synthesize n-3 and omega-6 FA, and therefore they must be acquired from the diet. Specifically, alpha-linolenic acid (ALA) and linoleic acid (LA) serve as precursors for n-3 and n-6 PUFA, respectively [109].

Given the importance of lipids, numerous studies have investigated the effects of PL-enriched diets on crustacean nutrition (Table 7). These studies reveal significant changes in various FA components, including saturated fatty acid (SFA), MUFA, PUFA, EPA, and DHA in response to varying PL inclusion rates. However, some nutritional parameters were observed to decrease with PL-enriched diets. Therefore, this review aims to elucidate the effects of (saturated fatty acid) SFA, MUFA, n-3 PUFA, n-6 PUFA, Lc-PUFA, EPA, and DHA on crustacean nutrition. From these studies, we can observe widespread increases in DHA levels with increasing PL inclusion rates. However, PUFA levels showed a contrasting pattern, with decreases appearing in five studies. Furthermore, SFA levels exhibited mixed results, increasing in six studies and decreasing in two. Similarly, EPA levels increased in five but decreased in three. MUFA levels displayed the most inconsistent responses, with four studies showing increases and four showing decreases (Table 7).

The influence of PL inclusion on nutritional parameters demonstrates a species- and dose-dependent response. For instance, E. sinensis, P. trituberculatus, and S. paramamosain showed positive responses for most nutritional parameters at increasing PL-inclusion rates, except in MUFA and PUFA levels [19, 43, 45, 47, 53, 57]. For instance, S. paramamosain exhibited 46% SFA of total FA with a PL diet constituting a 4% level. This was significantly higher compared with the 31.71% SFA reported with a lower 4% PL inclusion rate for the same species [53]. Interestingly, S. paramamosain exhibited the highest PUFA level (21.04% of total FA) with 4% PL inclusion but a higher 6% PL inclusion level reversed this trend, resulting in decreased PUFA levels in P. clarkii (31.66%) [63, 97]. Finally, the DHA levels in S. paramamosain consistently increased with PL incorporation, while the EPA level decreased with a 3% inclusion rate and increased at a 2% PL-inclusion rate [53].

7.2. Mechanism of PL Effects on the Nutritional Profile of Crustaceans

Dietary inclusion of PL at optimal levels has demonstrated positive effects on various nutritional parameters in crustacean species (Figure 5) [110]. Crustaceans, like most marine fish, have a limited capacity to convert linolenic and linoleic acids to n-3 and n-6 Lc-PUFA, making dietary Lc-PUFA essential [80]. However, insufficient or excessive Lc-PUFA often results in a negative impact on crustaceans, including poor survival, stunted growth, and prolonged intermolt phases [111]. Thus, maintaining functional dietary EFA levels is critical for growth, development, and survival [112]. Dietary PL enhances Lc-PUFA absorption efficiency and could potentially improve lipid transport in swimming crabs [113].

DHA and EPA are critical for optimal crustacean growth, molting, and development. On the one hand, DHA is an essential constituent of biomembrane structure, particularly forming a key component of polar lipids in neural tissues (brain and eye) [80]. Consequently, there is an increased demand for DHA during periods of rapid growth to support developing tissues. On the other hand, EPA plays a significant role in the production of eicosanoids, bioactive regulatory compounds, and can contribute to DHA synthesis in some species with the necessary enzymes [114]. However, an imbalanced dietary DHA/EPA ratio can hinder neural development and immunity, potentially resulting in slowed growth or poor SRs of marine animal larvae. Moreover, high levels of EPA might also be detrimental to aquatic organisms [115]. Therefore, establishing optimal DHA/EPA ratios in diets is crucial for the healthy growth and development of crustaceans [80].

8.1. Effect of PL on the Immunity of Crustaceans

Crustaceans rely solely on an integrated innate immune system, combining cellular and humoral defenses [116]. Owing to this, their immunity is vulnerable to stressors like pathogens, environmental changes, physiological issues, and molting [117–120]. Molting, however, can enhance immunity by upregulating antimicrobial peptides, as demonstrated in recently molted mud crabs [120].

Dietary PLs have been investigated for their potential to modulate the crustacean immune system. Table 8 summarizes 12 studies examining the effects of various sources of PL (SL, EL, and KO) at different inclusion rates (0%–6.12%) on various immune parameters across different crustacean species. Overall, the data suggests that dietary PL can enhance the immune system and the expression of immune-related genes in different crustaceans. However, the effects of PL supplementation are not uniform. While many studies report a positive correlation between moderate to high PL inclusion and increased levels of parameters, like SOD, GSH-Px/G-Px, ProPO, total-antioxidant capacity (T-AOC), and LZM [11, 13, 16, 17, 20–22, 43–45, 53], others observe decreased levels of specific immune parameters at high PL inclusion.

For instance, significantly higher SOD activity (an increase from 317.81 to 418.96 unit/mL) was observed in L. vannamei with a 4% PL inclusion [11]. Similarly, CAT activity was also observed to increase in E. sinensis with a 2.5% KO inclusion rate (7 U/mgprot) [23]. On the contrary, variable CAT responses were reported in P. clarkii with increasing SL inclusion where a 6% SL inclusion resulted in 1.18 U/mL while an SL-free diet produced a 2.08 U/mL CAT activity [44]. Furthermore, parameters like MDA exhibited species-specific responses, exhibiting more downregulation than upregulation. For example, MDA levels decreased in seven studies with higher PL diets but increased in three [5, 17, 20, 23, 43, 45, 53] (Table 8).

Moreover, a few other parameters, such as T-AOC, LZM, alkaline phosphatase (AKP), acid phosphatase (ACP), and IMD mRNA, displayed occasional decreases in some studies, likely influenced by factors like species, PL source, and inclusion rate [11, 44]. These studies showed that the level of GSH-Px, T-AOC, and GSH increased in P. trituberculatus, exhibiting 328.14 U/mgprot (4% SL), 4.38 U/mgprot due (4.95% SL) and 251.34 μmol/gprot (3.63% SL), respectively [13, 43]. Another immune parameter, LZM, showed a positive response (123.53 U/mL) in P. clarkii due to a 2% SL inclusion rate [44].

8.2. Mechanism of PL Effect Crustacean Immunity

Figure 6 illustrates the diverse pathways through which dietary PL influence crustacean immunity, primarily by targeting humoral components. These components form the main line of defense against oxidative stress, with enzymes, such as SOD, CAT, T-AOC, GST, GSH, and GSH-Px playing key roles [122]. The enzymes remove hazardous toxins from the body and scavenge reactive oxygen species (ROS), protecting against oxidative stress and cell damage [123]. For instance, the ROS scavenging property of T-AOC, CAT, SOD, GSH, and GSH-Px acts in the first-line antioxidant defense mechanism. GST, on the other hand, modulates the immune response and mechanism and reduces cell damage caused by ROS [124]. Furthermore, GST activity can be used as a biomarker to assess exposure to organic pollutants in aquatic organisms [125]. Studies on crustacean species, including C. quadricarinatus, Penaeus vannamei, S. paramamosain, E. sinensis, L. vannamei, and P. trituberculatus have demonstrated the positive impacts of dietary PL supplementation on these six previously mentioned parameters [5, 11, 13, 20–23, 43–45, 53]. However, studies on P. clarkii exhibited a decrease in T-AOC content with increasing PL doses [44]. This variation in the antioxidant immune response of crustaceans due to a high PL diet is not well known but it can be possible due to the complex interaction between PL feeds and antioxidants [11]. It is also possible that the observed improvements in immune parameters might be due to the presence of high levels of linolenic acid and linoleic acid in the PL supplements [126].

MDA serves as another crucial immune-related antioxidant enzyme and biomarker in crustaceans which plays a role in free radical-induced lipid peroxidation, a process that can damage cells through oxidative stress [13, 127]. MDA levels fluctuate depending on the crustacean species and the amount of PL included in their diet. For instance, species like P. trituberculatus, S. paramamosain, E. sinensis, L. vannamei, and C. quadricarinatus showed decreased MDA levels with increasing PL intake, whereas others like S. paramamosain, P. clarkii, and P. trituberculatus exhibited the opposite trend [5, 17, 20, 22, 23, 43–45, 53]. Intriguingly, some studies on P. trituberculatus displayed both increased and decreased MDA levels, depending on the PL inclusion rate (6.12% vs. 4% SL, respectively) [13, 43]. Therefore, decreased MDA levels may indicate relief from oxidative damage, while increased levels could reflect lipid peroxidation and oxidative damage induced by high dietary lipid content [19, 128].

LZM, another key ubiquitous enzyme in crustacean immunity, lyses peptidoglycan cell walls of bacteria, particularly gram-positive ones, enhancing the nonspecific immune response [129]. PL-enriched diet can significantly increase LZM activity in crustaceans as demonstrated in P. clarkii, and L. vannamei fed with high PL doses [5, 44]. Furthermore, PL enriched diet also exerts a distinct effect on the expression of immune-related genes, including toll-like receptor mRNA, IMD mRNA, and LZM mRNA [11]. Variations in mRNA expression levels of proPO activating enzyme, serine protease, beta-glucan binding protein, and hemocyanin gene have also been reported in response to PL diets [21]. These effects are likely linked to higher levels of EPA, and DHA in the PL supplements, which are known to promote immune gene expression [130]. Additionally, the amino acid composition of PL-enriched feeds may contribute to enhanced immune gene expression like toll-like receptor mRNA, IMD mRNA, and LZM mRNA [131].

Moreover, ACP and AKP activities are also crucial parameters in crustacean immune defense mechanisms. ACP aids in digesting invading pathogens, while AKP facilitates detoxification, improved digestion, and nutrient absorption [132]. Both enzymes catalyze the hydrolysis of various phosphate groups, further improving the immune capacity of aquatic organisms [133, 134]. However, a study on P. clarkii fed with 6% SL reported lower activity levels of AKP and ACP [44], highlighting the need for further research to understand the specific factors influencing the impact of PL inclusion levels on different enzymes.

9.1. Effect of PL Enriched Diet on the Reproduction of Crustaceans

Vitellogenesis, the lipoprotein-based yolk formation process, is crucial for ovarian maturation in crustaceans [135]. Cholesterol, a key component of lipoproteins is derived from dietary PL, particularly PC and PE [20]. Its level in the hemolymph decreases gradually following yolk maturation, suggesting its role in enhancing secondary vitellogenesis and ovarian development in female shrimp [16]. It is important to note that, environmental factors like salinity, temperature, and photoperiod also play significant roles in crustacean reproduction [136]. For example, studies on crab Scylla olivacea and shrimp Neocaridina davidi demonstrated how temperature and salinity variations impact ovarian development [137, 138]. Similarly, the effect of photoperiod variation on the reproductive performance of Callinectes sapidus has also been reported [139].

While feeding trials often show the positive influences of PL-enriched diets on female reproductive performance, the specific mechanisms of crustacean reproduction remain under investigation. To elucidate these effects and their relevance to sustainable crustacean farming, eight studies examining the impact of PL-enriched diets on crustacean reproduction are summarized in Table 9.

The studies presented in Table 9 demonstrate the role of various PL sources (SL, EL, and KO) in crustacean diets at inclusion levels ranging from 0% to 8%. Overall, PL incorporation led to improved female reproductive performance, as evidenced by higher GSI in five studies, increased ovarian quality in four studies, and elevated levels of VG/VTG gene expression and reproductive hormones (E2, PROG) in three studies. Additionally, MF levels and VGR gene expression also showed increasing levels in two and one studies, respectively. However, some parameters were negatively affected (decreased) on PL inclusion, suggesting species-specificity and dependance on PL source and inclusion rates. For instance, levels of GSI and some hormones (LH, E2) decreased in one study, while gonadotropin-inhibiting hormone (GIH) and molting inhibiting hormone (MIH) were lower in two studies (Table 9).

The studies revealed that high to moderate PL inclusion rates (2%–6%) resulted in improved reproductive parameters. GSI increased in C. quadricarinatus, P. clarkii, L. vannamei, E. sinensis, and P. trituberculatus at these levels [12, 13, 20, 23, 95]. Notably, E. sinensis displayed a significant increase (7%–9% GSI) with 2.5% KO supplementation [23]. Furthermore, high to moderate PL-inclusion rates improved ovarian histology, ovarian TG, (reproductive hormone concentration) RHC, FA composition of the ovary, and overall ovarian morphology in C. quadricarinatus P. clarkii, L. vannamei, and E. sinensis [20, 23, 95]. Moreover, genes associated with female crustacean reproduction, vitellogenesis (VGR, VTG/VG, and FABP), also responded positively to PL enrichment. E. sinensis, L. vannamei, and P. trituberculatus exhibited increased expression of these genes with 2.5%–6% dietary PL [23, 25, 95].

9.2. Mechanism of PL in Crustacean Reproductive Organ Development

The GSI, which refers to the ratio of the gonad weight to body weight, is a reliable parameter for estimating the reproductive condition of female crustaceans [140, 141]. Figure 7 depicts the impact of PL supplementation on crustacean reproduction. It shows that PL-enriched feed can enhance the GSI, improve ovarian features, enhance lipid content, and explicitly regulate reproductive hormones including PROG, E2, and luteinizing hormone (LH). Studies show increased GSI in C. quadricarinatus, P. clarkii, L. vannamei, E. sinensis, L. vannamei, and P. trituberculatus with 2%–6% PL inclusion rate [13, 20, 23, 24, 95]. From these studies, the increase in GSI can be attributed to the elevated EPA and DHA deposition in the hepatopancreas in the PL-fed crustaceans [20]. However, limitations of GSI as an indicator of reproductive cycle performance in crustaceans have been recognized, as it offers limited insights into internal ovarian changes, particularly maturation [142]. Therefore, to gain a comprehensive understanding of gonadal development in response to PL-enriched diets, researchers have employed additional histological and biochemical parameters, including ovarian histology, ovarian TG, RHC, ovarian FA composition, and ovarian morphology [20, 23–25, 95]. These studies consistently demonstrated a positive impact on all parameters. For example, 2% KO supplementation in C. quadricarinatus supported the production of larger mature oocytes and elevated ovarian TG levels (0.5 mmol/gprot) [20].

Regarding ovarian development and morphology, P. clarkii exhibited a more compact and advanced structure with 6% SL supplementation [95]. In E. sinensis, increasing PL level positively impacted FA composition, resulting in elevated total Lc-PUFA and n-3 FA, likely because of higher deposition of lipids in the ovarian muscle [23]. This is significant as lipids are crucial in yolk biosynthesis and oocyte development, thereby promoting gonadal development [16].

Furthermore, higher levels of RHC were observed in C. quadricarinatus, E. chinensis, and P. trituberculatus, while elevated Vg gene expression was reported in E. chinensis, P. trituberculatus, and L. vannamei with increased PL intake [12, 13, 23, 24, 57]. This upregulation was attributed to the neuroendocrine regulatory network governing crustacean reproduction [46]. E2, a key sex steroid, is reported to exert a physiological influence on female crustaceans reproductive performance [143]. E2, along with MF, acts as a precursor for VG synthesis in the liver and oocyte development [24, 144]. As presented in this review, dietary PL provides energy for lipid mobilization and conversion to steroids, ultimately increasing VG levels [23].

Notably, a cellular study on L. vannamei identified distinct metabolic pathways for glycerophospholipid, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, alpha-linolenic acid, arachidonic acid, and glycerolipid for various PL-enriched feed. However, sphingolipid metabolism was observed exclusively in KO-fed shrimp, suggesting a higher energy providing capacity of KO during ovary development stages through enhanced lipid utilization [24].

10.1. PL on Gut Microbiome in Crustaceans

The gut microbiome plays a crucial role in crustacean health, influencing nutrient absorption, immune response, homeostatic balance, and overall productivity [145, 146]. Specific microbial communities within the crustacean gut serve as a first line of defense against pathogens and contribute to essential physiological functions [146–148]. These communities vary depending on rearing stage, diet, and health status, with Firmicutes, Proteobacteria, and Bacteroidetes being the dominant phyla [149–153]. For instance, the dominant phyla differ between E. sinensis (Proteobacteria and Tenericutes), S. paramamosain (Firmicutes, Proteobacteria, Bacteroidota, and Campilobacterota), and Penaeus sp. (Proteobacteria) [154–156].

Bioinformatics tools have been developed to predict variation in microbial diversity across samples [157]. One prominent tool, operational taxonomic units (OTUs) utilizes sequence data to define microbial communities across different taxonomic levels (phylum, class, order, family, genus, and species) [158]. OTUs are employed alpha diversity, which represents the average diversity of microbial species within a specific habitat. Commonly used alpha diversity indices are Simpson, Shannon, Abundance-based Coverage Estimator (ACE), and Chao1. The Simpson and Shannon indices measure species richness based on the degree of dominance by individual species. Conversely, the ACE index estimates the sample completeness or coverage, indicating how well a sampling effort captures the entire microbial population. Chao1 index requires abundance data from individual samples to determine species richness [159, 160].

PL-enriched diets can influence gut microbiome composition and diversity. However, these effects have received less attention compared to growth, reproduction, immunity, or nutrient metabolism. Feeding trials have investigated gut microbiome composition and diversity using parameters, such as richness index indicators (Shannon, Simpson, ACE, and Chao1) and dominant phyla. The impact of different PL sources (SL, EL, and KO) on crustacean gut microbiota is summarized in Table 10. Richness indices (Shannon, ACE, Chao1) increased with increasing dietary PL levels in three studies, whereas the Simpson index exhibited variable responses, showing increases in two studies and a decrease in one (Table 10). Commonly observed phyla included Proteobacteria, Firmicutes, Actinobacteria, Tenericutes, and Bacteroidetes, with prevalent genera, such as Aeromonas, Vibrio, Candidatus, Bacilloplasma, Citrobacter, Flavobacterium, and Shewanella [5, 26, 44].

The observed variations in gut microbiota depend on PL inclusion rate and specific crustacean species. For example, C. quadricarinatus exhibited a significant increase in the Shannon index (3.0) with 2% SL inclusion, while ACE and Chao1 indices reached their maximum values (~510) with 4% KO supplementation and minimum values (350) with 0% inclusion. Moreover, the highest Simpson index was recorded in P. clarkii (0.16) and L. vannamei (0.8) because of the 2% SL and 4% KO inclusion, respectively. Contrary, the Simpson index decreased in C. quadricarinatus (0.11) with 1% SL inclusion compared to 0.32 in PL-free diet. These findings suggest that PL can enhance gut microbiome richness and diversity in crustaceans, potentially contributing to improved health [161].

10.2. Mechanism of PL Enriched Diet on Crustacean Gut Microbiota

The mode of action of PL in the crustacean gut microbiome, as illustrated in Figure 8, has been investigated using alpha diversity indices, with studies reporting positive influence of PL-enriched diets. For example, in C. quadricarinatus both ACE and Chao1 indices were highest with 3% SL inclusion, while the Shannon index peaked at a 2% SL level, though notably, the Simpson index decreased with increasing SL inclusion [26]. Similar positive effects of PL enrichment on alpha diversity indices were also observed using higher dietary PL in L. vannamei (4% KO and 6%) and P. clarkii (2% SL) [5, 95].

In a study on L. vannamei, 2005 OTUs were identified, among which 163 OTUs were common between the control and PL-fed groups, representing 8.13% of the total. Among the major types of PL-enriched feed (KO, EL, and SL), KO supplementation led to a higher abundance of Fusibacter, a bacterium linked to antioxidant activity and immune function. This indicates that food PL can induce microbial diversity through the regulation of iron metabolism, which could boost immune resilience in female L. vannamei [5]. In another study on P. clarkii, a total of 30 phyla, 60 classes, 118 orders, 232 families, and 476 genera were isolated. Specifically, the relative abundance of Tenericutes was significantly different at varying levels of PL, while the lowest Firmicutes/Bacteroidetes ratio was observed with 6% PL supplementation. Furthermore, increasing dietary PL levels reduced the abundance of Shewanella but significantly increased that of Citrobacter [44].

The phylum Proteobacteria can be both pathogenic (reducing the growth performance of crustaceans) and beneficial, playing roles in biogeochemical processes and carbon, nitrogen, and sulfur cycling [162, 163]. Although it occurs as a benign microbe to the gut microbiome, any malfunctioning in the microbiome community can lead to its rapid proliferation, endangering shrimp production [154, 164]. As such, the effect of PL-levels on Proteobacteria abundance, exhibiting the species-specific nature, can have variable impacts. For instance, while higher PL levels have been shown to reduce Proteobacteria abundance in C. quadricarinatus and L. vannamei, they have been observed to increase it in P. clarkii [5, 26, 44].

Firmicutes and Bacteroidetes play a crucial role in crustacean health, contributing to gut immune homeostasis by promoting both immune responses and fermentation processes, which generate energy for physiological functions [5, 162, 165, 166]. Similarly, the Actinobacteria phylum is considered beneficial due to its role in enhancing crustacean immune function. The phylum is linked to various immune parameters like T-AOC, total nitric oxide synthase, HSP70, Trx, Lys, proPO, Muc-1, Muc-2, and Muc-5AC [166]. Consistent with these benefits, studies have shown the positive effects of PL-enriched diets on these bacteria phyla in C. quadricarinatus, L. vannamei, and P. clarkii [5, 26, 44].

The mechanism of PL influence extends to direct nutrient provision for gut microbes, potentially altering their composition and activity. For example, increased PL can enhance the activity of microbiome genes involved in lipid and energy metabolism, leading to greater interbacterial interactions. These interactions are reflected in observed changes like a reduced abundance of Proteobacteria alongside increases in Firmicutes, Actinobacteria, Tenericutes, and Bacteroidetes. Such shifts in bacterial populations suggest complex ecological niche processes and bacterial symbiosis influenced by dietary PL [167].

11.1. Effects of PL on Osmoregulation in Crustacean

Osmoregulation is the process by which organisms maintain a balance of dissolved substances (solutes) within and outside their cells (in the interstitial fluid) for proper function [168]. Aquatic crustaceans, through osmoregulation, can tolerate a wide range of salinities in their environment. This is achieved by controlling the osmotic pressure of their hemolymph through the regulation of hemolymph osmolytes [169]. The enzyme Na+/K+-ATPase (NKA) plays a crucial role in maintaining this osmotic pressure [170]. NKA is a member of the P-type ATPase family and is located in the cell membrane of various tissues and cells [171]. The NKA pump aids in preserving membrane potential and osmotic balance within cells [170].

NKA activity facilitates the movement of potassium (K⁺) and sodium (Na⁺) ions across the cell membrane against their concentration gradients. It maintains a higher concentration of potassium intracellularly and a higher concentration of sodium extracellularly. Several studies suggest that suitable dietary PL levels could play a significant role in maintaining NKA activity and, consequently, the osmoregulation process in aquatic organisms [172–174]. However, research on specific PL requirements for osmoregulation in crustacean aquaculture is limited. This highlights a gap in our knowledge regarding the impact of PL on NKA activity and osmoregulation.

Furthermore, existing research on the effect of PL on NKA activity presents contradictory findings. For example, a study on S. paramamosain observed a decreasing trend in the NKA mRNA expression with increasing dietary PL levels, with the lowest expression at 4% PL and the highest in the PL-free diet [19]. Conversely, another study on S. paramamosain reported a significant increase in NKA activity with increasing PL inclusion, with 4% EL yielding the highest activity and 0.1% the lowest [57]. These findings emphasize the need for further research to understand the complex relationship between PL and NKA activity in crustaceans. Changes in environmental salinity can trigger modifications in NKA activity, allowing crustaceans to adapt to varying osmotic conditions and survive in diverse aquatic environments [175]. Therefore, future studies must rigorously control environmental salinity as a potential confounding factor when evaluating the impact of PL impact on NKA activity and osmoregulation.