In the present study, there was a rotifer DHA dose-dependent effect on DHA accumulation in 10 dph BFT larval tissue, which was positively associated with rotifer ingestion rate. The 8–9 dph BFT larvae ingesting the high DHA rotifers showed very good rotifer feeding rates reaching over 50 mastaxes 90 min−1. This is much higher compared to other species such as gilthead sea bream (Sparus aurata), where 13 dph larvae ingested about 20 mastaxes 90 min−12 and agrees well with our previous publication on BFT9.In support of this, there was a marked (P< 0.05) increase in larval length in 2–9 dph fish, that was associated with larval DHA content and indicative of normal growth at this developmental stage22. In addition, 15 dph BFT larvae fed the highest DHA rotifers demonstrated significantly (P< 0.05) improved growth, as previously reported by our group9. Moreover, the results obtained by larvae RNAseq analysis demonstrated the influence of DHA on a broad range of biological processes, that were age-dependent, and emphasized the upregulation of genes associated with neurogenesis, synaptogenesis, and synapse function in BFT larvae. Interestingly, we observed a major expression shift in 67 DHA down-regulated genes in 5 dph larvae that were up-regulated in 10 dph fish, highlighting the importance of DHA at specific stages of larval development.The critical importance of DHA in the prey of marine fish larvae has been well documented1,23 and is widely believed to be a more effective larval growth promoter during development than the other essential fatty acids; eicosapentaenoic acid (EPA, 20:5n-3) and arachidonic acid (ARA; 20:4n- 6)1,24,25. However, how DHA contributes to growth in marine fish larvae appears to be broadly linked to its physiological function in the cellular membranes of the eyes, brain, and other neural tissues. DHA impacts the structural development and function of the nervous system and promotes neurogenesis, synaptogenesis, and synapse function in fish larvae. This suggests that DHA greatly enhances the biological processes of visual acuity, learning and neuromuscular function, which are all necessary for successful prey recognition, behavior, and capture. This was demonstrated by several authors that reported increased dietary DHA or n-3 long chain polyunsaturated fatty acids (LCPUFAs) improved feeding behavior in herring larvae3 and larval prey consumption in gilthead sea bream (Sparus aurata)26 as well as bluefin tuna (Thunnus thynnus)9.A common thread tightly woven into the benefits of DHA is the contribution of its unique 22 carbon, 6 double bond structure, which resists orderly packing, to increased membrane fluidity. In phototransduction, membrane fluidity facilitates the rate of photopigment conformational change27,28 and a more rapid processing of light. In addition, the abundant levels of DHA in neuronal membranes affect the activity of neurotransmitter receptors and ion channels, which modulate neurotransmission and synaptic plasticity. DHA can also modify the physical organization of lipid rafts, within cell membranes, that are comprised of cholesterol, glycophospholipids and receptors that serve as organizing centers for the assembly of signaling molecules29. In addition, DHA has anti-inflammatory properties and can modulate immune responses such as the production of cytokines. Moreover, DHA can prevent oxidative stress-induced apoptosis in photoreceptors through its conversion to neuroprotectin D1 30 and/or its upregulation of Bcl-2 proteins, which inactivate pro-apoptotic proteins31.However, the effect of DHA on biological processes and pathways is not always linked to membranal or signaling function but can be more complex and often overlaps with its role in modulating gene expression. In the present study, the transcriptome in BFT larvae fed various levels of DHA at different larval ages demonstrated (PCA plot) significant (P< 0.05) variability in both of these covariants and points to a central role for DHA in BFT larval development. Comparing the DEGs in 5 and 10 dph BFT larvae, the highest variability was between the low and medium DHA treatments, while the largest variability in 15 dph fish was between the medium and high DHA treatments. This suggests that the high DHA treatment provided an excessive amount of DHA during early larval development, in terms of regulating genes, and didn’t demonstrate significant variability in DEGs between these treatments in 5 and 10 dph fish. Conceivably, the excess DHA was retro-converted to EPA, a 20 carbon LCPUFA that may be less effective at regulating the expression of specific genes or may exert its action through different mechanisms other than DHA. Allam-Ndoul et al.32 reported that EPA and DHA regulate genes involved in cell cycle regulation, apoptosis, immune response, inflammation, and oxidative stress in a differential and dose-dependent manner. This is not to say that excess DHA wouldn’t be contributing, simultaneously with its gene modulation, to membranal function and improved physiological processes. Having said that, in older larvae the rate of development may be more rapid and DHA demand higher as a modulator of gene expression. This might explain why there was a significant change in larval DEGs only between medium and high DHA treatments in 15 dph fish. On the other hand, the lack of a significant change in larval DEGs between the low and high DHA treatments was likely due to one of the low DHA replicates being expressed close to the cluster of high DHA replicates in the PCA plot, which reduced the DEGS variability between these treatments.The 5 dph MA plot showed a greater number of significantly down-regulated (78) than up- regulated genes (12), while the MA plot of 10 dph larvae exhibited an impressive abundance of DHA up-regulated genes (491) and only 43 DHA down-regulated genes. In 15 dph larvae, the MA plot presented relatively similar numbers of up- and down-regulated genes. The emerging picture is that DHA modulates different genes, albeit with some overlap, at different developmental stages. This further strengthens the notion that the sequence and timing of different stages of development dictates which genes are modulated and are highly conserved, while the degree to which they are modulated is influenced by DHA. It is noteworthy that gene ontology analysis showed that the down-regulated genes in 5 dph larvae were mainly linked with neuron development and synaptic synthesis that included pre- and post-synaptic membrane potential, axonogenesis as well as neuron plasticity. Conversely, many of these down-regulated genes associated with neurons and synaptic biological processes, were then significantly up-regulated in 10 dph fish. In fact, there were 67 shared genes between 5 dph down-regulated and 10 dph up-regulated genes. The delay of DHA modulated expression of key genes in neural development in 5 dph BFT reinforces our previous notion that the sequence of developmental events in this species is strictly adhered to.DHA modulated genes predominantly influenced neural development in 10 dph BFT larvae through various essential biological processes, which are supported in a number of mammalian and human studies. DHA is important for brain development and function33, particularly the hippocampus of the mammalian brain34, which is responsible for storing short term memories and transferring them to long term storage. Although fish lack a hippocampus, BFT larvae appear to share some of these mammalian genes and biological processes expressed in different parts of the larval fish brain. Studies in mammals found that DHA increased synaptic vesicle density, hippocampal neurite growth, dendritic spine density16, synaptic proteins35 as well as cell migration36. Cao et al.16 studying mice hippocampal neurons in culture reported that DHA influenced neurite growth, synaptogenesis, synapsin proteins and two types of glutamate receptors: N-methyl-D-aspartic acid (NMDA) and a-amino-3-hydroxy-5-methylisoxazole (AMPA). Glutamate receptors mediate fast excitatory synaptic transmission in the central nervous system and regulate a broad spectrum of processes in the brain, spinal cord, retina, and peripheral nervous system.One of the biological processes up-regulated in 10 dph larvae was the detection of visible light. Gaon et al.2 and Koven et al.9 reported that rotifer DHA contributed to opsin biosynthesis in the gilthead sea bream and BFT larvae, respectively, in a dose dependent manner, which was correlated with prey consumption. In the current study, rotifer DHA stimulated the up-regulation of the opsin (OPN5) for ultraviolet A (UVA) and blue light in 10 dph BFT larvae. However, DHA in BFT larvae also up-regulated genes involved at different stages of the phototransduction process. This included the genes activating voltage dependent calcium channels (Cacna1f), the G protein coupled receptor signaling pathway (Gpr52), the synthesis of cGMP (Guc2f), the glutamate receptor signaling pathway (Gnaq), ion channel modulating, and the G protein-coupled receptor signaling pathway (Gna11). Interestingly, the latter two genes were also involved in the entrainment of the circadian clock.The biological process of atrial cardiac muscle cell action potential includes the genes Cacnb2, Scn5a and Kcna5, which modulate calcium, sodium, and potassium transport in excitable membranes, respectively, as well as the involvement of Ank2 in signal transduction. The DHA up-regulated genes; Mmp24, Grik2, Ephb1, Ano3, which are involved in neuron biological processes such as the glutamate receptor signaling pathway, modulation of neuronal action potential, synaptic transmission, angiogenesis, eye morphogenesis, axonogenesis, dendritic spine development, immunological synapse formation, optic nerve morphogenesis and retinal ganglion cell axon guidance. The biological process: cytoplasmic translation, which was linked to the 67 up-regulated genes found in 10 dph larvae, had the highest probability (-log10 (P-value)) of being associated with cytoplasmic ribosomal mRNA translation and the synthesis of a large array of proteins.DHA modulated genes in 15 dph BFT larvae continued to modify neural development as well as influencing an increasing number of developing organs. In the biological process of otic placode formation, DHA up-regulated Prox1. In mammals, this gene is involved in brain development particularly the hippocampus37 and the cerebellar cortex38. However, in fish the otic placode forms the inner ear, which is responsible for hearing and balance. Studies have shown that fish larvae can detect physically and biologically generated sounds from specific habitats such as thriving coral reefs through the interaction of the otolith and the neuromast sensory bed of the inner ear. This allows the larvae to orient themselves towards such favorable ecosystems that are rich in prey and provide shelter39,40. Tuna larvae, even at the early flexion stage, were 100 times more abundant in near reef waters than in the open sea41,42,43. In addition, Prox1 mediates uniquely a dual function of initially promoting neuronal differentiation of neural progenitor cells, and then suppressing neurite and axon outgrowth in nascent neurons, a critical process for neuronal maturation during central nervous system development44.The DHA up-regulated gene Tfap2a is broadly involved in eye development and elicited the biological processes of optic cup structural organization, optic vesicle morphogenesis and oculomotor nerve formation45. Moreover, DHA-modulated Tfap2a likely up-regulated bmp7a46. This gene promotes differentiation of stato-acustic ganglion (SAG) precursors in the inner ear47, which contribute to balance and hearing.A subset in Table 1of 31 genes regulating neural growth and development is derived from the 67 genes, that were DHA down-regulated in 5 dph larvae but DHA up-regulated in 10 dph. These genes were associated with GO biological processes that were mainly involved in synaptic function in terms of pre-synaptic vesicle formation, exocytosis, and binding to glutamate receptors. Interestingly, these genes were also involved in human cognitive impairment but their modulation by DHA has not yet been investigated in humans suggesting a promising line of research in future studies on neurodegenerative diseases. Although early developing fish are clearly not afflicted with age related cognitive decline or neural synaptic dysfunction, a DHA deficiency in BFT larvae would likely affect their behavior, vision, hearing, learning and memory. All of these attributes would be necessary for mastering the learned process of larval prey recognition and capture48.It is noteworthy that, in contrast to the normal growth experienced by larvae feeding on the high DHA rotifer treatment, fish consuming lower DHA rotifers likely exhibited transcriptomes directly linked to reduced neural, brain, muscle, vision and hearing functions leading to restricted rotifer feeding and a potential state of malnutrition and starvation. Although these are secondary factors, they can also modulate transcriptome expression. However, the evidence strongly suggests that the DHA modulation of genes is a key and major driving force affecting the BFT larval transcriptome.Taken together, the picture emerging is that DHA modulated genes were intimately involved in a wide array of critical biological processes occurring in a range of developing organs in different age BFT larvae. These results shed light on the essential role of DHA modulated gene expression in the promotion of BFT larval growth.
