Fig 1.
Vg mRNA expression in the hepatopancreas of L. vannamei during ovarian development.
A: Hepatopancreatic Vg transcript levels of sexually immature male shrimp and female shrimp. The data presented are expressed as the mean±S.E. (n = 6), and significant differences between male and female shrimp were assessed using Student’s t-test (* P<0.05, ** P<0.01 and *** P<0.001). B: Hepatopancreatic Vg transcript levels of female shrimp in different ovarian developmental stages. The data presented are expressed as the mean±S.E. (n = 6), and experimental groups denoted by the same letter represent a similar level (P>0.05, ANOVA followed by Fisher’s LSD test). C: Histological observation of ovarian developmental stages based on H/E staining. The selected ovarian maturation stages of female shrimp include: stage I (previtellogenesis), stage II (primary vitellogenesis), stage III (secondary vitellogenesis), and stage IV (maturation).
Fig 2.
Effects of VIH-2 administration on NO, cGMP and cAMP production in the hepatopancreas of L. vannamei.
A-C: The NO (A), cGMP (B) and cAMP (C) production in the shrimp hepatopancreas after VIH-2 (300 ng/g bwt) injection. D-F: The time-dependent NO (D), cGMP (E) and cAMP (F) productions by VIH-2 treatment in shrimp hepatopancreatic cell cultures. G-I: Dose-dependent NO (G), cGMP (H) and cAMP (I) productions by VIH-2 treatment in shrimp hepatopancreatic cell cultures. For in vitro experiments, hepatopancreatic cells were pretreated with IBMX (0.1 mM) at ~20 min before the substrates were added. In this study, the data presented are expressed as the mean±S.E. (n = 6 and 4 for in vivo and in vitro experiments, respectively). Experimental groups denoted by the same letter represent a similar level (P>0.05, ANOVA followed by Fisher’s LSD test).
Fig 3.
Involvement of cGMP-dependent signaling in VIH-2 suppressed Vg mRNA expression in the hepatopancreas of L. vannamei.
A-C: The effects of the NO donor (A), the GC activator (B) and the cGMP analog (C) on Vg mRNA expression in shrimp hepatopancreatic cell cultures. The hepatopancreatic cells were incubated for 12 h with increasing doses of NOC-18 (0.01–100 μM), A-350619 (0.01–100 μM) or 8-Br-cGMP (0.1–1000 nM). D-F: Effects of NOS inhibitor (D), GC inhibitor (E) or PKG inhibitor (F) on VIH-2-inhibited Vg mRNA expression in shrimp hepatopancreatic cell cultures. The hepatopancreatic cells were incubated for 12 h with VIH-2 (1 μM) in the presence or absence of L-NAME (300 μM), Zn(II)PPIX (5 μM) or Rp-8-Br-PET-cGMPS (5 μM). In this study, the data presented are expressed as the mean±S.E. (n = 4). Experimental groups denoted by the same letter represent a similar level (P>0.05, ANOVA followed by Fisher’s LSD test).
Fig 4.
Involvement of cAMP-dependent signaling in VIH-2 suppressed Vg mRNA expression in the hepatopancreas of L. vannamei.
A & B: The effects of the AC activator (A) and the cAMP analog (B) on Vg mRNA expressions in shrimp hepatopancreatic cell cultures. The hepatopancreatic cells were incubated for 12 h with increasing doses of forskolin (0.01–1000 nM) or 8-Br-cAMP (0.1–1000 nM). C: Interaction of cAMP-dependent signal pathways and VIH-2 on Vg mRNA expression in shrimp hepatopancreatic cell cultures. The hepatopancreatic cells were incubated for 12 h with VIH-2 (1 μM) in the presence or absence of forskolin (1 μM) or 8-Br-cAMP (1 μM). In this study, the data presented are expressed as the mean±S.E. (n = 4). Experimental groups denoted by the same letter represent a similar level (P>0.05, ANOVA followed by Fisher’s LSD test).
Fig 5.
Effects of VIH-2 administration on the phosphorylation and mRNA expression of three MAPKs in the hepatopancreas of L. vannamei.
A: Effects of VIH-2 injection on the phosphorylation of P38MAPK, ERK and JNK in shrimp hepatopancreas. B: Effects of VIH-2 treatment on the phosphorylation of P38MAPK, ERK and JNK in shrimp hepatopancreatic cell cultures. C-E: Effects of VIH-2 injection on the P38MAPK (C), ERK (D) and JNK (E) mRNA expressions in the shrimp hepatopancreas. F-H: Effects of VIH-2 treatment on the P38MAPK (F), ERK (G) and JNK (H) mRNA expression in shrimp hepatopancreatic cell cultures. For in vivo experiments, the hepatopancreas were sampled at 0, 1.5, 3, 6, 12 and 24 h after injection of VIH-2 (300 ng/g bwt). For in vitro experiments, the hepatopancreatic cells were treated with VIH-2 (1000 nM) for 0, 0.5, 1, 1.5, 3, 6, 12 and 24 h. In this study, the data presented are expressed as the mean±S.E. (n = 6 and 4 for in vivo and in vitro experiments, respectively), and significant differences between treated and untreated groups were assessed using Student’s t-test (* P<0.05, ** P<0.01 and *** P<0.001).
Fig 6.
Involvement of MAPK-dependent signaling in VIH-2 suppressed Vg mRNA expression in the hepatopancreas of L. vannamei.
A-C: Effects of the P38MAPK inhibitor (A), the ERK inhibitor (B) or the JNK inhibitor (C) on VIH-2-inhibited Vg mRNA expression in shrimp hepatopancreatic cell cultures. The hepatopancreatic cells were incubated for 12 h or 36 h with VIH-2 (1 μM) in the presence or absence of PD169316 (100 nM), or SB02190 (100 nM), PD98059 (10 μM) or U0126 (1 μM), and SP600125 (10 μM) or BI-87G3 (10 μM). D: Interaction of the P38MAPK inhibitor and the JNK inhibitor on VIH-2 inhibited Vg mRNA expression in shrimp hepatopancreatic cell cultures. The hepatopancreatic cells were incubated for 12 h or 36 h with VIH-2 (1 μM) in the presence or absence of PD169316 (100 nM) or SP600125 (10 μM) alone, or PD169316 (100 nM) and SP600125 (10 μM) together. In this study, the data presented are expressed as the mean±S.E. (n = 4). Experimental groups denoted by the same letter represent a similar level (P>0.05, ANOVA followed by Fisher’s LSD test).
Fig 7.
Crosstalk of the GC/cGMP pathway with MAPK-dependent cascades in VIH-2 suppressed Vg mRNA expression in the hepatopancreas of L. vannamei.
A: Effects of the GC activator and the cGMP analog on the JNK phosphorylation in shrimp hepatopancreatic cell cultures. The hepatopancreatic cells were incubated for 1 h with increasing doses of A-350619 (0.01–100 μM) or 8-Br-cGMP (0.1–1000 nM). B: Effects of the GC inhibitor and the PKG inhibitor on the VIH-2-induced JNK phosphorylation in shrimp hepatopancreatic cell cultures. The hepatopancreatic cells were incubated for 1 h with VIH-2 (1 μM) in the presence or absence of Zn(II)PPIX (5 μM) or Rp-8-Br-PET-cGMPS (5 μM). C & D: Effects of GC activator (C) and cGMP analog (D) on the P38MAPK mRNA expression in shrimp hepatopancreatic cell cultures. The hepatopancreatic cells were incubated for 3 h with increasing doses of A-350619 (0.01–100 μM) or 8-Br-cGMP (0.1–1000 nM). E & F: Effects of the GC inhibitor and the PKG inhibitor on the VIH-2 induced P38MAPK mRNA expression in shrimp hepatopancreatic cell cultures. The hepatopancreatic cells were incubated for 3 h with VIH-2 (1 μM) in the presence or absence of Zn(II)PPIX (5 μM) or Rp-8-Br-PET-cGMPS (5 μM). G-I: Effects of the P38MAPK inhibitor (G), the ERK inhibitor (H) and the JNK inhibitor (I) on the VIH-2-induced cGMP productions in shrimp hepatopancreatic cell cultures. The hepatopancreatic cells were incubated for 3 h with VIH-2 (1 μM) in the presence or absence of PD169316 (100 nM), or SB02190 (100 nM), PD98059 (10 μM) or U0126 (1 μM), and SP600125 (10 μM) or BI-87G3 (10 μM). In this study, the data presented are expressed as the mean±S.E. (n = 4). Experimental groups denoted by the same letter represent a similar level (P>0.05, ANOVA followed by Fisher’s LSD test).
Fig 8.
Working model for the signal transduction mechanism of VIH-2 suppressed Vg mRNA expression in the shrimp hepatopancreas.
In this case, a membrane receptor GC activation by VIH-2 can generate cGMP and subsequently activate PKG. The downstream MAPK-dependent cascades are then activated by cooperation of JNK phosphorylation and P38MAPK content increase, to inhibit the gene expression of Vg in the hepatopancreas of L. vannamei.