Lipids, represented by various classes and subclasses, perform various biological functions in living organisms. In aquatic animals, acylglycerols, particularly triglycerides, are the major source of energy for physical activities and cellular functions (Sargent et al., 1993; Tocher et al., 2008; Rey et al., 2018; Xie et al., 2020). Most energy in the form of ATP is produced from fatty acids associated with triglycerides through the beta-oxidation process (Berg et al., 2002a). In addition to energy provision, lipids play essential roles in cellular structure (Alberts et al., 2002). Phospholipids and sterols are key components of cell membrane structure, which is essential for the osmoregulation mechanism of marine animals (Chapelle et al., 1982; Sargent et al., 1993; Huang et al., 2019); moreover, phospholipids and sterols are also precursors of cellular signaling molecules and hormones that regulate growth, molting, the immune system and reproduction (Tocher et al., 2008).

The core of lipid molecules is esterified with diverse fatty acid constituents, which vary in chain length and saturation. Among the varieties of fatty acid types, n-3 and n-6 long-chain polyunsaturated fatty acids (LC-PUFAs) are essential for growth and health of marine animals, in particular at the larval stage (Glencross, 2009). They are an energy source and precursors of eicosanoids, which are local hormones that function in signaling and regulation of several biological mechanisms including immune response, growth and development (Bhathena, 2006; Wimuttisuk et al., 2013; Engelking, 2015; Stillwell, 2016). However, these fatty acids must be obtained from the diet because of the low activities of fatty acid elongation and desaturation in marine animals (Kanazawa et al., 1979; Glencross, 2009). Deficiency of LC-PUFAs causes abnormal development and growth of several marine animals (Watanabe, 1993; Harel et al., 2002; Samocha et al., 2010). Several studies have been carried out to supplement LC-PUFAs in the diet for marine animals (Ishizaki et al., 1998; Boglino et al., 2012; Lund et al., 2012; Watanabe et al., 2016; Visudtiphole et al., 2018).

Thraustochytrids are single-cell protists, which are abundant producers of LC-PUFAs including docosahexaenoic acid (DHA, 22:6 n-3), eicosapentaenoic acid (EPA, 20:5 n-3), docosapentaenoic acid (DPA, 22:5 n-3), and arachidonic acid (ARA, 20:4 n-6). They have been applied to aquafeed as an alternative LC-PUFA source to fish oil (Lewis et al., 1999; Unagul et al., 2017). In larviculture, they are used to enrich LC-PUFAs in live Artemia or rotifer, which are fed to marine animal larvae to promote growth and health (Leger et al., 1986; Barclay and Zeller, 1996; Harel et al., 2002). The effect of thraustochytrid enrichment on shrimp larviculture has been studied at the early post-larval stage (PL1), the earliest that can ingest enriched Artemia (Visudtiphole et al., 2018). However, supplementation of dietary LC-PUFAs for shrimp larvae at earlier developmental stages was not studied. Therefore, in this study, a thraustochytrid, Aurantiochytrium limacinum BCC52274 (AL), was applied to Penaeus monodon larvae from mysis 1 (M1) to PL2 to supplement DHA in the diet. To do so, a common feed for the larvae, Thalasiosira weissflogii (TW), which is deficient in LC-PUFAs, was replaced with AL at graded ratios and the effects of the supplementation on larval growth and lipidomics were then evaluated.

Lipidomics is the holistic study of lipid molecules found in biological organisms. With the complexity of lipid molecules, analytical tools in combination with lipid structure database are required to elucidate lipid molecules from unknown samples in both qualitative and quantitative analyses (Cajka and Fiehn, 2014). Lipidomic analysis using liquid chromatography-high resolution mass spectrometry (LC-HRMS) has been employed to understand the physiological processes of marine species including blue mussels and Pacific white shrimp (Huang et al., 2019; Laudicella et al., 2020). The technique has also been applied to study the diet effect on aquatic animals—for example, different lipid sources and fish meal replacement in swimming crab and Pacific white shrimp, respectively (Xie et al., 2020; Yuan et al., 2021). Therefore, as well as growth evaluation, lipidomic analysis using LC-HRMS was carried out to examine the effects of the AL supplement on lipid profiles in shrimp. Moreover, since AL was found to contain distinctly high amounts of not only LC-PUFAs (DHA and DPA) but also a saturated fatty acid (16:0), the acquired lipidomic data were further analyzed to understand how the two different types of fatty acids were differentially accumulated in lipid classes and subclasses of the shrimp. The obtained results substantiate different biological roles of the two types of fatty acids in shrimp larvae.

Previously, dietary supplementation of AL through Artemia enrichment had been studied to promote growth performance of P. vannamei post-larvae from PL1 to PL19 (Visudtiphole et al., 2018). In the present study, AL supplementation was conducted in P. monodon larvae from M1 to PL2 by direct suspension of the AL cells into the larviculture. This approach is more efficient than the enrichment technique because all fatty acids in AL are taken in directly by the shrimp and not retroconverted by the enriched Artemia (Navarro et al., 1999; Han et al., 2001; Visudtiphole et al., 2018). The supplementation resulted in accumulation of the key fatty acids present in AL i.e., 16:0, DPA, and DHA, in PL2 shrimp. No effect on the growth of larvae was observed during the supplementation period; however, the effect on growth was exhibited later in the post-supplementation period from PL2 to PL12. Average body weight and biomass were increased in PL12 shrimp that had been fed earlier with higher amounts of AL. Dietary supplementation using other high-n-3 LC-PUFA substances, such as fish oil, was also shown to enhance growth performance of shrimp at the PL stage (Leger et al., 1985; Immanuel et al., 2001). Taken together with the previous results in P. vannamei PL and from the other studies, dietary n-3 LC-PUFAs may be more important for growth of shrimp during PL than the larval stage. This result could be ascribed for two reasons. First, the supplementation period during the mysis stage might not be long enough for difference in growth to be exhibited. Second, development of penaeid mysis is secondary lecithotrophy, in which the larvae partially rely on the nutrients from maternal reserves in eggs and also from the food (Mourente et al., 1995; Palacios et al., 2001). The supplemented LC-PUFAs were thus accumulated during the mysis stage and subsequently used for growth during the later PL stage, when AL was no longer supplied. This is also supported by the indifferent levels of most LC-PUFAs in PL12 shrimp.

The fatty acid profile of the aquatic animal normally reflects that of the ingested diet (Glencross, 2009; Kumar et al., 2018a). Likewise, fatty acid profiles of the PL shrimp in this study reflected those of the supplied diet, both at PL2 in which marked differences in fatty acid profiles were observed among groups with different feeding regimens, and later at PL12 when fatty acid profiles became more similar after all groups were later fed with the same diet. In contrast to the fatty acid profile, lipid class and subclass profiles of the PL shrimp and feed showed weaker correspondence. This could be accounted for digestion of macro-lipid molecules in the digestive tract and subsequent modification of lipid molecules in the animal. Macro-lipid molecules, especially triacylglycerols, have to be digested to smaller-sized molecules before being absorbed through the intestine and subsequently repacked and modified to various lipid class products (Senior, 1964; Iqbal and Hussain, 2009).

Interestingly, increased ingestion of TG present in AL was associated with a lower TG level in PL2 shrimp. This result is consistent with a study on dietary LC-PUFA supplements for juvenile grass carp, in which reduced TG in the supplemented animals was explained by down-regulated expression of diacylglycerol O-acyltransferase, encoding the key enzyme that synthesizes TG from DG (Tian et al., 2017). Therefore, the downregulation of conversion from DG to TG might be the reason for the decrease of TG and the accumulation of DG in PL2 shrimp supplemented with the high DHA-containing AL in this study. It is known that DG acts as a second messenger for cell signaling by increasing the affinity of protein kinase C to bind with substrates; DG is thus involved in the signal transduction of several pathways related to immunity, cell activation, regulation, and function (Berg et al., 2002b; Singh and Kambayashi, 2016).

Analysis of the fatty acid distribution in lipid classes and subclasses showed differential accumulation of two distinct types of fatty acids present in AL i.e., SFA (16:0) and n-3 LC-PUFAs (DPA and DHA), among lipid classes of the shrimp. Increased 16:0 levels in the diet were associated with a greater percent of sites esterified with the fatty acid on acylglycerols but not phospholipids and lysophospholipids, consistent with the role of 16:0 fatty acid as a reserve for energy and substrate for the synthesis of other fatty acids (Caers et al., 2000b; Rey et al., 2018). In contrast, both of the LC-PUFAs accumulated on phospholipids and acylglycerols. The percent of DHA- and DPA-esterified sites on phospholipids in PL2 shrimp of the AL supplemented groups were higher than the percent of those of the control group. However, they did not increase further with more AL in the diet (Figures 6B, 7B) whilst the percent of those on acylglycerols increased along with the amount of AL meal replacement (Figures 6A, 7A). This suggests that DHA and DPA were prioritized to be incorporated in phospholipids, which are the main component of cell membrane (Tian et al., 2017; Rey et al., 2018). Nonetheless, if phospholipids with excess LC-PUFA esterified sites are present, they cannot be packed easily into the membrane bilayer structure and may disrupt the membrane’s integrity owing to too much fluidity (Chapelle et al., 1982; Rajamoorthi et al., 2005; Bradbury, 2011; Parisi et al., 2019). The integration of simpler fatty acids into the membrane structure is also required to maintain the membrane’s stability (Huang et al., 2019). From our data, it can be inferred that dietary DHA and DPA were first preferentially deposited in the membrane and the excess was then stored with acylglycerols since the percent of sites esterified with these fatty acids continuously increased with the higher amounts of supplied AL. This result was in agreement with those in Crassostea gigas spat and juvenile Mytilus edulis fed with high LC-PUFA diets (Caers et al., 2000b; Laudicella et al., 2020). In contrast to that of the LC-PUFAs, deposition of 16:0 fatty acid in acylglycerols did not increase further with the higher amounts of supplied AL (Figure 5A). This result additionally suggests that LC-PUFAs were prioritized to be reserved over SFA when both types of fatty acids were present in high amounts in the diet. The preferential accumulation of LC-PUFAs over other types of fatty acids was also observed in other marine larvae (Caers et al., 1999, 2000b). The importance of LC-PUFAs is also corroborated by their selective retention in animals that were starved or fed with DHA-deficient diet (Coutteau et al., 1996; Knauer and Southgate, 1997).

The 16:0 fatty acid was markedly more abundant on PC than DHA or DPA (Figures 5B, 6B, 7B). In contrast, DHA was the most common modification of PE and PS. Moreover, unlike the 16:0 fatty acid, both n-3 LC-PUFAs were not detected to associate with PI, which is a minor component on the cytosolic side of cell membrane. Instead, the percent of 16:0-esterified sites on PI were significantly greater in PL2 shrimp fed with AL (Figure 5B). These results are in line with those in other species. Namely, DHA is more abundant on PE and PS than on PC and PI, and DHA supplementation resulted in the highest DHA accumulation on PE (Gillis and Ballantyne, 1999; Axelsen and Murphy, 2010; Rey et al., 2015; Bascoul-Colombo et al., 2016; Huang et al., 2019). In fact, PE is regarded as the most highly unsaturated phospholipid in marine animals (Ronnestad et al., 1995; Huang et al., 2019). Furthermore, PI is more specific to LC-PUFAs with a Δ5 double bond, like ARA and EPA, than those with a Δ3 double bond, which are DHA and DPA (Bascoul-Colombo et al., 2016). This also agrees with our result, in which the only LC-PUFAs detected to associate with PI were ARA and EPA (see Supplementary Table 2). Additionally, the high content of 16:0 fatty acid in PC coincides with a role of PC as a secondary energy source when TG is nearly depleted (Sasaki et al., 1986).

Cholesteryl ester was detected only in PL2 shrimp supplemented with AL and associated only with DHA (Figure 6D). Esterification of cholesterol with long-chain fatty acids makes cholesterol become more hydrophobic, which can be packed more easily inside the core of lipoproteins for the transportation in plasma (Maranhão and Freitas, 2014). AL supplementation in PL2 shrimp resulted in higher levels of cholesterol and the presence of cholesteryl ester (Figure 4). The presence of the latter indicates cholesterol transport between organs in shrimp supplemented with AL since cholesterol is transported in plasma as cholesteryl esters. Cholesterol is the precursor of steroid hormones that activate molting and growth such as ecdysteroids, which are synthesized specifically in the Y-organ of shrimp (Huberman, 2000; Kumar et al., 2018b). This suggests an additional role of DHA as a chaperone for cholesterol transportation in plasma to the target organ for the steroid hormone synthesis.

In conclusion, supplementation of AL as a source of dietary n-3 LC-PUFAs was demonstrated for the first time in shrimp larvae during the mysis developmental stage. The growth results from this and other previous studies suggests that dietary n-3 LC-PUFAs are more important and beneficial to PL shrimp than to the larvae at earlier stages. Analysis of differential accumulation of different fatty acid types provides an additional evidence on the importance and biological roles of LC-PUFAs in the larval diet.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

VV: experimental design and investigation, data analysis, and manuscript preparation. JK, PC, JS, and SL: shrimp rearing and feeding experiments. SP, AK, TR, KS, LK, and NR: laboratorial experiments. PU: experimental design and investigation. UU: experimental design and investigation, manuscript preparation. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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