Assessment of the Effects of Double-Stranded RNAs Corresponding to Multiple Vitellogenesis-Inhibiting Hormone Subtype I Peptides in Subadult Female Whiteleg Shrimp, Litopenaeus vannamei.

Vitellogenesis-inhibiting hormone (VIH) negatively regulates reproduction in shrimp and other decapod crustaceans. In order to assess the effects of transcriptional silencing by multiple VIH subtype I sinus gland peptides (SGPs) on ovarian maturation in female whiteleg shrimp, Litopenaeus vannamei, we synthesized five dsRNAs targeting Liv-SGP-A, -B, -C, -F, and -G and injected them into subadults. The following treatments were employed: sgpG-dsRNA (targeting Liv-SGP-G), sgpC-dsRNA (targeting Liv-SGP-C), and mixed-dsRNA (targeting Liv-SGP-A, -B, and -F). The expression of Liv-SGP-G in eyestalks was significantly decreased at 10, 20, and 30 days after the injection of sgpG-dsRNA In addition, it was significantly decreased at 10 and 30 days after the injection of mixed-dsRNA. The expression of vitellogenin (Vg) gene expression in the ovaries, and concentrations of Vg protein in the hemolymph, were not changed by the administration of any dsRNA treatment (the ovaries remained immature in all treated individuals and contained mostly oogonia and previtellogenic oocytes). Although the administration of dsRNAs corresponding to multiple VIHs did not promote ovarian maturation, this is the first report of the co-transcriptional repression of Liv-SGP-G by the injection of dsRNA for homologous genes (Liv-SGP-A, -B, and -F). These results indicate that subadults can respond to the techniques of transcriptional silencing.

Ontogeny of Cheliped Laterality and Mechanisms of Reversal of Handedness in the Durophagous Gazami Crab, Portunus trituberculatus.

The paired claws in Gazami crabs, Portunus trituberculatus, are bilaterally asymmetrical, and asymmetry is remarkable on the distal two segments of the first pereiopod, that is, the dactylus and propodus. Shells are exclusively cracked by use of the right chela, representing handedness. In Gazami crabs, handedness is reversed after autotomy of the right chela. Our study focused on the ontogeny of handedness and the mechanism of handedness reversal. Morphologically, asymmetry was first detected in megalopa larvae where the right propodus was significantly larger than the left, as was the canine at the base of the right dactylus. Presumably, the rate of chelagenesis differed between the left and right chelae. With these morphological features, the right chela functioned as a crusher. The crusher exerted a closing force two to three times that of the cutter. With loss of the right crusher, the left chela was bigger than the regenerated right chela and was converted to the crusher. In contrast, the performance of the regenerated right chela deteriorated compared to that of the original right crusher, and exertion of full closing force was inhibited by the more active left chela. Furthermore, crabs with two crusher chelae did not clearly show handedness. A decrease in size and performance of the regenerated right chela can be explained by a default program hypothesis. In conclusion, a difference in the chelagenesis rate results in bilateral asymmetry of the two chelipeds, and then handedness is generated by neural regulation in the thoracic ganglion innervating these claws. Since handedness is reversed after autotomy, the thoracic ganglion would not be lateralized in Gazami crabs. A default program hypothesis is proposed to explain the ontogeny of bilateral chela asymmetry and handedness reversal.

Variation of protein kinase C-α expression in eyestalk removal-activated ovaries in whiteleg shrimp, Litopenaeus vannamei.

The regulation of female reproduction in crustaceans is controlled by a variety of hormones. Previous studies of hormone-initiated cellular mechanisms controlling ovarian maturation focused mainly on those initiated by inhibitory hormones. In order to facilitate research on ovarian development, it is necessary to gain a better understanding of factors that promote ovarian growth on the cellular level. Here, we used eyestalk ablation to firstly induce a state of ovarian maturation in Litopenaeus vannamei. Gonadosomatic index, hemolymph vitellogenin (Vg) concentrations, and Vg gene transcript levels in the ovaries were significantly elevated at 1 and 2 weeks after eyestalk ablation (P < 0.05). Correspondingly, immunoblot analysis revealed a remarkable decrease in anti-PKC-α immunoreactivity in both cytosol and membrane fractions of ovarian tissue homogenates: it was strongly apparent in intact animals, but decreased with time after eyestalk ablation, showing a stronger tendency to do so in the membrane fraction than in the cytosol fraction. Considered overall, the data presented strongly suggest that PKC-α isoform plays a role in the regulation of ovarian growth in L. vannamei through a negative-based regulating mechanism.

Involvement of second messengers in the signaling pathway of vitellogenesis-inhibiting hormone and their effects on vitellogenin mRNA expression in the whiteleg shrimp, Litopenaeus vannamei.

We incubated fragments of Litopenaeus vannamei ovary to investigate second messengers involved in the regulation of vitellogenin (vg) mRNA levels. The use of 100nM recombinant vitellogenesis-inhibiting hormone (VIH) (corresponding to recombinant L. vannamei sinus gland peptide-G: rLiv-SGP-G) significantly reduced vg mRNA expression in sub-adults after 8h incubation to less than 20% of the control. The concentration of intracellular cyclic guanosine monophosphate (cGMP) increased 3.2-fold relative to the control after 2h incubation with rLiv-SGP-G. However, it reached levels 18-fold relative to the control after 0.5h incubation with rLiv-SGP-G where 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor) was also added. Moreover, vg mRNA expression was significantly reduced to less than 50% of the control after 24h incubation with 1µM A23187 (a calcium ionophore). Thus, rLiv-SGP-G and calcium ionophore reduced vg mRNA expression in in vitro-cultured ovary, and cGMP may be involved in the signaling pathway of VIH. Overall, the above results suggest that vg mRNA expression might be inhibited in vitro by increasing intracellular cGMP and Ca2+ in L. vannamei ovary.

Dynamics of vitellogenin and vitellogenesis-inhibiting hormone levels in adult and subadult whiteleg shrimp, Litopenaeus vannamei: relation to molting and eyestalk ablation.

Levels of vitellogenin (VG) and vitellogenesis-inhibiting hormone (VIH) in the whiteleg shrimp, Litopenaeus vannamei, were measured by time-resolved fluoroimmunoassay in relation to the molting cycle and ovarian maturation induced by eyestalk ablation. During the molt cycle, VG mRNA expression levels and VG concentrations showed similar patterns of fluctuation. VG levels increased significantly at early intermolt (stage C0) in adults, but not in subadults. Unilateral and bilateral eyestalk ablation increased VG levels in adults, whereas only bilateral eyestalk ablation affected subadults. VIH levels showed contrasting patterns between adults and subadults. In adults, levels were high in late postmolt adults (stage B) and then low thereafter, whereas they increased from postmolt (stage A) to intermolt (stage C0) in subadults and remained high. Unilateral eyestalk ablation increased VIH levels 10 days following ablation in adults, after which levels decreased at 20 days. VIH levels decreased from 10 to 20 days after bilateral ablation. Both unilateral and bilateral ablation led to increased VIH levels in subadults. Eyestalk ablation induced ovarian maturation, but did not reduce VIH concentrations in the hemolymph. This phenomenon was perhaps due to other crustacean hyperglycemic hormone peptides having cross-reactivity with VIH antibodies. This is the first report to quantify concentrations of VG and VIH together in L. vannamei hemolymph, and to examine their relative dynamics.

Molecular cloning of prophenoloxidase and the effects of dietary ß-glucan and rutin on immune response in hemocytes of the fleshy shrimp, Fenneropenaeus chinensis.

To investigate the effects of dietary ß-glucan (0.5 or 1 g kg⁻¹ diet: 0.5-BG, 1-BG) and rutin (0.5 or 1 g kg⁻¹ diet: 0.5-RT, 1-RT) after 10 days in the absence of pathogen challenge on the immune response of Fenneropenaeus chinensis, we determined total hemocyte count (THC) and the expression of four immune-related genes in hemocytes: those for prophenoloxidase (proPO), peroxinectin (PX), lipopolysaccharide and/or ß-glucan binding protein (LGBP), and c-type lectin (CL). As a prerequisite for subsequent experiments, cDNA encoding proPO of the fleshy shrimp, Fenneropenaeus chinensis (f-proPO) was obtained from hemocytes; it had a full length of 3023 bp, with an open reading frame (ORF) of 2061bp, a 105-bp 5'-untranslated region, and a 906-bp 3'-untranslated region containing the poly A signal. The THCs of shrimp fed ß-glucan of 1 g kg⁻¹ diet, and rutin of 1 g kg⁻¹ diet were significantly higher than that of the control (P < 0.05). The expression of proPO mRNA was slightly downregulated and that of LGBP mRNA was upregulated (except in 1-RT). PX and CL mRNA remained constitutively expressed in all groups. Our results reveal that ß-glucan and rutin dietary supplements have minimal effect on immune response in the absence of pathogen challenge.