Gender-Specific Metabolic Responses of Crassostrea hongkongensis to Infection with Vibrio harveyi and Lipopolysaccharide

Abstract

Gender differences in the hemocyte immune response of Hong Kong oyster Crassostrea hongkongensis to Vibrio harveyi and lipopolysaccharide (LPS) infection exist. To determine if a gender difference also exists, we use a ¹H NMR-based metabolomics method to investigate responses in C. hongkongensis hepatopancreas tissues to V. harveyi and LPS infection. Both infections induced pronounced gender- and immune-specific metabolic responses in hepatopancreas tissues. Responses are mainly presented in changes in substances involved in energy metabolism (decreased glucose, ATP, and AMP in males and increased ATP and AMP in LPS-infected females), oxidative stress (decreased glutathione in males and decreased tryptophan and phenylalanine and increased choline and proline in LPS-infected females), tricarboxylic acid (TCA) cycle (decreased α-ketoglutarate acid and increased fumarate in LPS-infected males, and decreased fumarate in LPS-infected females), and osmotic regulation (decreased trigonelline and increased taurine in V. harveyi-infected males and decreased betaine in V. harveyi-infected females). Results suggest that post-spawning-phase male oysters have a more significant energy metabolic response and greater ability to cope with oxidative stress than female oysters. We propose that the impact of oyster gender should be taken into consideration in the aftermath of oyster farming or oyster disease in natural seas.

Keywords: Crassostrea hongkongensis; gender-based difference; hepatopancreas; metabolomics.

Figure 1

¹H NMR spectra of Crassostrea hongkongensis hepatopancreas extract under infected (control) condition, with spectral regions 0.0–4.8 ppm amplified by a factor of 4, and 4.8–9.9 ppm by 10. Key: Ace, acetate; Ade, adenine; Ads, adenosine; Ala, alanine; AMP, adenosine monophosphate; Ara, arabinose; Arg, arginine; Asn, asparagine; Asp, aspartate; ATP, adenosine triphosphate; Bet, betaine; Chl, chlorogenate; Cho, choline; Cyt, cytosine; DSS, dextran sulfate sodium; Fum, fumarate; Glc, glucose; Glc-1-p, glucose-1-phosphate; Glu, glutamate; Glucur, glucuronate; Gly, glycine; GSH, glutathione; GTP, guanosine triphosphate; Guo, guanosine; Hom, homarine; Hyd, hydroxyacetone; Ile, isoleucine; Ino, inosine; Leu, leucine; Lys, lysine; Mal, malonate; Met, methionine; N-Ace, N-acetylornithine; Orn, ornithine; Pan, pantothenate; Phe, phenylalanine; Pro, proline; Pyr, pyridoxine; Rib, ribose; Ribo, riboflavin; Sar, sarcosine; Tau, taurine; TMA, trimethylamine; TGL, trigonelline; Tre, trehalose; Try, tryptophan; Tyr, tyrosine; UDP-Glc, UDP-glucose; UDP-N-Ace, UDP-N-acetylglucosamine; UMP, uridine monophosphate; Ura, uracil; Urd, uridine; Val, valine; 3-Ami, 3-aminoisobutyrate; α-Ket, α-ketoglutarate acid; β-Ala, β-alanine.

Figure 2

Multivariate statistical analysis plot based on ¹H NMR spectra of C. hongkongensis hepatopancreas tissues. (A) PLS-DA plots for all treatments; (B) OPLS-DA score plots of male and female oysters (R²X = 68.8%, R²Y = 0.927, Q² = 0.565, p < 0.003); (C) permutation test for the model in (B). Groups: F, female; M, male; C, control; B, Vibrio harveyi infection; L, LPS infection.

Figure 3

OPLS-DA score plots of ¹H NMR spectra of C. hongkongensis hepatopancreas extract from different paired groups (left panel) and the corresponding model permutation test chart (middle panel) and its corresponding volcano map (right panel). (A) F_B vs F_C. (B) M_B vs M_C. (C) F_L vs F_C. (D) M_C vs M_L. Groups: F, female; M, male; C, control; B, V. harveyi infection; L, LPS infection.

Figure 4

Upset diagram of C. hongkongensis hepatopancreas extracts from different paired groups. Increased metabolites (A), decreased metabolites (B). Groups: F, female; M, male; C, control; B, V. harveyi infection; L, LPS infection.

Figure 5

KEGG function analysis based on the differential metabolites from different paired groups. Pathways with p values < 0.05 are shown. Groups: F, female; M, male; C, control; B, V. harveyi infection; L, LPS infection.

Figure 6

Molecular mechanism of the hepatopancreas response in male and female C. hongkongensis to V. harveyi and LPS infection according to KEGG. Blue (down-regulated), red (up-regulated), and white (unchanged) boxes reveal changes in levels of differential metabolites compared to controls; ellipses with different background colors indicate different interconnecting pathways. Groups: F, female; M, male; C, control; B, V. harveyi infection; L, LPS infection.

Figure 7

Differential metabolites and immune factors correlated by Spearman’s correlation analysis. A color gradient denoting Spearman’s correlation coefficient is shown for pairwise comparisons of metabolites. Spearman’s correlation coefficients are depicted using line size and line color denotes statistical significance. Lines: dashed, positive correlation; solid, negative connection. Apo, apoptotic ratio; Ca, calcium content; Est, esterase activity; Lyso, lysosome mass; Mito, mitochondrial mass; Mor, mortality; NO, nitric oxide level; Pha, phagocytic ratio; ROS, reactive oxygen species level. *** p < 0.001, ** p < 0.01, * p < 0.05.