FABP gene family members are evolutionarily conserved, with extensive and interdependent functions in the regulation of fatty acid oxidation/metabolism . Although much research has focused on the classification, structure and function of vertebrate FABPs, these proteins remain unidentified in many invertebrate species. The current study reports the cloning and gene structure of a FABP family member in E. sinensis, Es-FABP, and presents evidence of functionality in fatty acid metabolism.
The full-length Es-FABP cDNA reported in the present paper encoded a putative FABP protein of 131 amino acids, which falls within the size range of other reported FABPs (127-136 amino acids) . The ClustalX alignment of Es-FABP and fourteen other reported vertebrate and invertebrate FABP sequences revealed high identity among invertebrate sequences (56-62%) although percent identity remained lower when comparing within invertebrate and mammalian taxa (79-90%) . Three-dimensional homology modeling revealed that several key tertiary structures of Es-FABP were similar to those of vertebrate FABP, such as the 10 anti-parallel β-strands, their resultant barrel with a clamshell-like structure, and the barrel cap comprised of a pair of α-helices, which enclose the FABP lipid-binding site cavity .
The cloned Es-FABP gene was 9108 bp in length, and was comprised of four exons interrupted by three introns, a conserved genomic organization among both species [31, 32] and FABP superfamily members. Comparison of the exon/intron organization of the FABP genes revealed that, the genes are organized in four exons and three introns located in conserved positions, although varying markedly in size. And the second intron of Es-FABP was substantially longer than reported for other species. In general, while large introns are not unusual in organisms with very large genomes, they are less common among heavily transcribed genes, like the locust FABP. Long introns might be favored because they enhance recombination, thus introducing mutations in adjacent exons  or intra- and inter- gene recombination , all of which would be subjected to selection pressures.
Phylogenetic analysis revealed two distinct sister groups within our constructed NJ tree as previously reported [8, 35]: (1) group 1 contained FABP1, 6, 10, and (2) group 2 contained FABP2, 3, 4, 5, 7, 8, 9, 11, 12. Schaap et. al. estimated that FABP1, FABP6 and FABP10 diverged from a common ancestral gene ~679 million years ago (mya) . FABP6 is an ileal-type FABP , while FABP1 and FABP10 are paralogs, and are thus evolutionarily related liver-type FABPs, which possibly arose by a gene duplication event . Es-FABP segregated within group 1, clustering in close proximity to Fc-FABP10 and LvFABP10. ClustalX alignment results suggest that Es-FABP shares a high percent identity with FABP10. Based on collective phylogenetic evidence, we hypothesize that the Es-FABP we cloned from hepatopancreas is a FABP10.
We hypothesized that Es-FABP tissue expression profiles may provide useful cues when speculating gene function. In the present study, Es-FABP transcripts were primarily detected in hepatopancreas, hemocytes, and ovary. The hepatopancreas is generally regarded as a major storage organ of lipids, which are transported to reproductive organs and tissues during a period of reproductive activity . Interestingly, the crustacean ovary contains a high lipid content compared to other organs [37, 38], which is a key nutrient required for ovarian development and appropriate egg hatching rate and larval survival [38–41]. Further, the crustacean ovary is rich in long-chain poly unsaturated fatty acids (PUFA) . As intracellular transporters of PUFA, FABP may serve as mediators of their physiological function, availability and access to intracellular targeted systems . The high Es-FABP expression levels observed in these organs suggest that lipid nutrients, especially fatty acids, are transported from the hepatopancreas to the ovary in great supply via Es-FABP expressing hemocytes during the stage of rapid ovarian development. Es-FABP transported lipids are then stored in the ovaries in preparation for the significant and impendent energy expenditure required during reproduction .
Real-time qRT-PCR analysis revealed Es-FABP expression levels were dependent on the stage of ovarian development in all three organs. Fatty acids (FA) in general, including the ubiquitous palmitic and oleic acids, can regulate gene expression at the transcriptional level [43, 44]. Furthermore, long-chain FA and long-chain dicarboxylic acids can act as inducers of FABP1 expression [45, 46], as tissue FABP content is related to the rate of FA uptake and/or utilization [47–49]. Similarly, manipulations altering the rate of tissue FA metabolism appear to be associated with concomitant changes in FABP content . Therefore, a direct or indirect feedback loop between FA and FABP may exist. In the present study, ovarian Es-FABP expression increased with the developmental progression of the ovary, mirroring lipid nutritional requirements. The ovary requires substantial accumulation of lipids, especially fatty acids, during developmental stages [51–53]. Interestingly, the rapid increase in ovarian lipid deposition was associated with a gradual decline in Es-FABP expression in hepatopancreas, suggesting hepatopancreas-mediated lipid transport may be occurring during ovarian development. Determining the existence or absence of a hepatopancreas-ovary feedback loop requires further study.