Suppression of a Novel Vitellogenesis-Inhibiting Hormone Significantly Increases Ovarian Vitellogenesis in the Black Tiger Shrimp, Penaeus monodon

P. monodon female broodstock in previtellogenic stage (80-85 grams body weight, n = 5) and unilateral eyestalk-ablated female broodstock (at day 5 after eyestalk ablation, n = 3) were obtained from Shrimp Genetic Improvement Center, Suratthani, Thailand. Subadult female shrimp (20-30 grams body weight, in premolt stage, n = 30) were obtained from commercial farms at Pathumthani, Thailand. All shrimp were checked for pathogens, including White spot syndrome virus (WSSV), Yellow head virus (YHV), Taura syndrome virus (TSV), Monodon baculovirus (MBV), P. monodon densovirus (PemDNV), Infectious hypodermal and hematopoietic necrosis virus (IHHNV), Decapod iridescent virus 1 (DIV1) and Vibrio parahaemolyticus. They undergo cold narcosis in ice-cold water for 5 min prior to dissection. The central nervous system (CNS), hepatopancreas (HP), muscle (Mus), ovary and gill were collected. The CNS was further subdivided into the eyestalk (ES), brain (Br), thoracic ventral nerve cord (VNC-TG), and abdominal ventral nerve cord (VNC-AbG). Note that the VNC-TG included commissural ganglion (CoG), subesophageal ganglion (SEG), and thoracic ganglion (TG). Tissue samples were individually immersed in TriPure isolation reagent (Roche, Santa Clara, CA) and stored at -20°C until RNA isolation. All animal experiments were reviewed and approved by the Animal Ethics Committee, Faculty of Science, Mahidol University (MUSC63-019-527).

Nucleotide sequence of PemVIH (GenBank accession no. MW847946↗) was obtained from our transcriptome data prepared from P. monodon central nervous tissue (unpublished data). It was identified by BLASTs searching tools and later named PemVIH based on its sequence homology and function. A primer pair, PemVIH-01-F and R (Table 1), was designed and used to amplify the PemVIH transcript by reverse transcription (RT)-PCR. The PCR amplicon was subcloned into pGEM-T Easy (pGEM-T Easy Vector Systems, Promega, Madison, WI) for sequencing. Bioinformatic analysis of PemVIH was performed using The European Bioinformatics Institute (EBI) database (https://www.ebi.ac.uk/ena↗). Open reading frame (ORF) for the prepropeptide was predicted using Open Reading Frame Finder online software (https://www.ncbi.nlm.nih.gov/orffinder↗). A putative signal peptide region was predicted by SignalP-5.0 online server (http://www.cbs.dtu.dk/services/SignalP↗). The available amino acid sequences from the CHH superfamily in other crustacean species were searched in GenBank (https://www.ncbi.nlm.nih.gov/genbank↗) for multiple sequence alignment analysis using The Hidden Markov Model (HMM)-HMM alignment (HHalign) method of Clustal Omega via Uniprot website (https://www.uniprot.org/align↗). Accession numbers and abbreviations of crustacean hormones used for the analyses are shown in Table 2. A phylogenetic tree was constructed using the Maximum Likelihood analyses with Molecular Evolutionary Genetics Analysis (MEGA-X) program.

The putative three-dimensional structure of PemVIH was predicted using the SWISS MODEL server (https://www.swissmodel.expasy.org↗). The server assembled PemVIH by referencing the Protein Data Bank (PDB). Hormone structure was visualized via UCSF Chimera 1.13.1 program.

To extract RNA, tissue samples were homogenized in TriPure reagent (Roche) and incubated in chloroform for 10 min and centrifuged at 4°C, 13,200 ×g for 15 min. Supernatant containing total RNA was precipitated using isopropanol, washed twice with ethanol, and dissolved in RNase-free water. RNA samples were quantified using a nanodrop spectrophotometer (ThermoScientific, Waltham, MA), and RNA integrity was assessed by formaldehyde agarose gel electrophoresis. One microgram of RNA was treated with 1U of DNaseI at 37°C for 1 h to eliminate genomic DNA. cDNA was synthesized using a RevertAid First-Strand cDNA synthesis kit (#K1612, ThermoScientific) according to the manufacturer’s instructions and stored at -80°C until use.

RT-PCR was performed using i-Taq Plus DNA Polymerase kit (#25151, INtRON, Gyeonggi-do, South Korea). The PCR cocktail (10X PCR buffer, 0.2 µM/µl, primers mixture, 0.2 mM dNTPs, and 0.05 U/µl of Tag polymerase) was prepared as a master-mix and added batchwise to each cDNA template sample (100 ng). The cDNA template for actin amplification was diluted two times. Specific primers for PemVIH and a reference gene are listed in Table 1. PCR conditions were 94°C for 15 s, followed by 40 cycles at 94°C for 15s, 58°C for 30s, and 72°C for 40s, and lastly with a final extension step at 72°C for 7min. Negative controls were amplified using sterile water instead of cDNA template under the same RT-PCR protocol. PCR products were subjected to agarose gel electrophoresis and stained with ethidium bromide. PCR products were visualized using Geldoc (Syngene, Frederick, MD).

For quantitative PCR (qPCR), PemVIH expression was determined in VNC-TG, whereas vitellogenin (PemVg) expression was determined in HP and ovary. Primers for PemVg were designed according to accession no. EE332453.1↗ of GenBank database (20). Actin was used as the reference gene. The cDNA template was diluted at 1:4 for PemVIH and PemVg, and 1:16 for actin amplification. A master mix for qPCR (0.4 μl of primers mix, and 2X master mix of KAPA SYBR buffer (#KK4600, Roche) was prepared, and the assay was performed according to the recommended protocol (Roche).

Relative gene expression was calculated according to Livak and Schmittgen (2001) (21). Differences in relative gene expression between control and experimental groups were analyzed by Student’s t-test with α = 0.05. Statistical analysis was performed using Prism version 5 (GraphPad, San Diego, CA). All experiments were performed in triplicates.

A rabbit polyclonal antiserum against PemVIH was generated by Genscript Biotech, Piscataway, NJ. Briefly, a synthetic peptide CIMASERHAEVEQFN was designed from a hydrophilic domain of the predicted PemVIH amino acid sequence (87^th – 101^st), conjugated to chicken ovalbumin via the cysteine residue, and used for three rounds of immunizations. The resulting serum from the final bleed was collected and used for immunofluorescence study described below.

To localize PemVIH in shrimp tissues, whole mount immunofluorescence (WM-IF) was performed as described previously by Kruangkum et al. (2013) (22). Dissected shrimp tissues were fixed in 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) for 24 h, then washed in PBS at 4°C. The fixed CNS tissues, including the brain and the entire ventral nerve cord spanning thoracic and abdominal regions, were dissected and immersed in PBS containing 0.2% Triton-X100 (PBST) overnight at 4°C. Tissues were subsequently permeabilized using Dent’s fixative (mixture solution of 80% Ethanol and 20% DMSO) for 8 h at -20°C, washed with cold PBS and PBST at 4°C for 15 min, 3 times each. The CNS tissues were then incubated in the anti-PemVIH polyclonal antiserum (1:500) in a blocking solution (10% normal goat serum in PBST), and gently shaken at 4°C for 5 days. Negative controls included CNS tissues incubated with the anti-PemVIH antiserum preadsorbed with 10 µM immunizing peptide. The tissue samples were then washed separately three times each in PBST and PBS, incubated in an Alexa 488-conjugated goat anti-rabbit antibody (Santa Cruz, Dallas, TX; 1:1,000) and a nuclear staining probe TO-PRO-3 (Invitrogen, Waltham, MA; 1:2,000) in blocking solution at 4°C for 3 days. Afterwards, the specimens were washed 3 times each with PBST and PBS, gradually dehydrated in increasing concentrations of ethanol (50% to 100%), and cleared in methyl salicylate overnight. Tissues were observed and photographed under an inverted Olympus FV1000 confocal microscope.

dsRNA specific to PemVIH (dsPemVIH) was synthesized using a bacterial cell culture system previously described (23). Briefly, DNA templates for generating dsPemVIH and EGFP (green fluorescence protein) were amplified with gene specific primers (Table 1) using our RT-PCR protocol. PCR amplicons were ligated into pGEM-T Easy vector and pDrive cloning vector, and subjected to sequencing analysis. A sense strand was further digested and recombined into an antisense strand-containing plasmid prior to transformation into E. coil (HT115). The dsRNA was isolated from cells using TriPure reagent. Any contaminated DNA and single stranded RNA was eliminated by 1 U of DNaseI and 1 U of RNase A digestion at 37°C for 1 h. The amount and integrity of dsRNA was evaluated by a nanodrop spectrophotometer and agarose gel electrophoresis, respectively.

Twenty-four subadult female shrimp were used in an in vivo knockdown bioassay. Shrimp were divided into 2 groups (12 shrimp/group): 1) an experimental group treated with dsPemVIH (10 μg/g body weight), and 2) a control group treated with dsEGFP (10 μg/g body weight). All shrimp were injected intramuscularly once with 80-100 μl of the dsRNA dissolved in PBS, kept in individual tanks for 4 days under the pre-injection environment, and fed commercial food twice a day. Afterwards, all shrimp were sacrificed for tissue collection. Since vitellogenesis takes place especially in ovary and hepatopancreas (HP), PemVg expression was determined in both ovary and HP. The relative expression of PemVIH and PemVg was evaluated by qPCR as described above.

A full-length 1,022 bp cDNA of PemVIH was identified in transcriptomic data of P. monodon CNS. The nucleotide sequence consisted of 275 bp of 5′untranslated region (UTR), 339 bp of ORF and 408 bp of 3′ UTR. The ORF encoded a peptide of 112 amino acids with a conserved domain exhibiting significant sequence similarity to other CHH superfamily members (Figure 1). A signal peptide prediction showed a significant cleavage position at Ala³⁴, and a proposed dibasic cleavage site at Arg¹¹¹-Lys¹¹² with amidation site at Gly¹¹⁰giving rise to a mature peptide of 75 amino acids with a calculated molecular mass of 8.85 kDa (see Supplement 1).

Multiple alignment classified the putative PemVIH as a member of the CHH superfamily based on the presence of six highly conserved cysteine residues (Cys⁴¹, Cys⁵⁸, Cys⁶¹, Cys⁷⁴, Cys⁷⁸ and Cys⁸⁷) characteristic of this superfamily. Moreover, CHH precursor-related peptide (CPRP), a characteristic of CHH-type I family, was not found in PemVIH. Instead, it contained a glycine residue (Gly⁴⁶) 5 amino acids downstream of the first cysteine residue (Cys⁴¹), indicating that the PemVIH belonged to the CHH-type II family (Figure 1).

Percent similarity of PemVIH with other crustacean hormones is shown in Figure 1. The putative mature peptide of PemVIH showed the highest percent similarity to Macrobrachium rosenbergii sinus gland peptide A (MarSGPA) at 66.67%. Focusing on hormones in Penaeid shrimp, PemVIH showed the highest percent similarity to Peneaus japonicus molt-inhibiting hormone (PejMIH) and P. monodon molt-inhibiting hormone 1 (PemMIH1) at 60%, followed by PemMIH 2 and P. monodon gonad-inhibiting hormone (PemGIH) at 57.33% and 53.33%, respectively. A maximum likelihood phylogenetic analysis also grouped PemVIH into the CHH-type II family (bootstrap value 100%) and suggested a monophyletic clade of PemVIH in relation to other VIHs (bootstrap value 78%) (Figure 2). This suggested that PemVIH is a novel P. monodon CHH-type II.

A putative three-dimensional structure of PemVIH was constructed and compared against PejMIH. As a member of CHH-type II, the conserved six cysteine residues were shown in both hormones at position 8^th, 25^th, 28^th, 41^st, 45^th and 54^th (Figure 3A). However, based on the PDB ID (code 1jot) and our sequence, the putative structure of PemVIH had five predicted α-helices (Figure 3B), whereas PejMIH only had four (Figure 3C). The lengths of α-helices were also different between the two peptides. The α₂-helix of PemVIH was shorter than that of PejMIH, whereas α₃- and α₄-helices of PemVIH were extended compared to PejMIH.

In addition, the three-dimensional structures of P. monodon CHH-type II family, including PemGIH, PemMIH1 and PemMIH2, were constructed and compared with PemVIH. All of them showed five α-helices and the root-mean square distance (RMSD) values between them were ~ 2 Å suggesting structural similarity of the P. monodon CHH-type II (see Supplement 2). Moreover, the putative surface structure of PemVIH and PemGIH was compared. Area comparison of acidic, basic, and hydrophobic residues in the two peptides and electrostatic potential mapped onto the surface area was demonstrated (see Supplement 3). The surface representations showed the similar surface area properties of the two peptides.

Expression of PemVIH transcript was determined in CNS and non-CNS tissues of female subadults and broodstock. The CNS tissues included ES, Br, VNC-TG, and VNC-AbG, whereas the non-CNS tissues included HP, Mus, ovary, and gill. RT-PCR revealed the expression of PemVIH transcript as a 659-bp amplicon only in the Br, VNC-TG, and VNC-AbG, but not in the ES and non-CNS tissues of both female subadult (Figure 4A) and broodstock shrimp (Figure 4B).

Localization of PemVIH was elucidated using WM-IF staining. PemVIH immunoreactivity (-ir) was detected in CNS tissues including the Br, commissural ganglion (CoG), subesophageal ganglion (SEG), thoracic ganglion (TG) and abdominal ganglion (AbG) (Figure 5, left panels).

In the brain, PemVIH-ir was observed in neuronal cluster number 9/11 of deuterocerebrum (Figures 5A, B) and neurons in cluster 17 of tritocerebrum (Figure 5C, magnified in the inset). Surprisingly, the intensive PemVIH-ir was detected in a pair of CoG, a component of circumesophageal commissures (CEC). The fluorescent signal was present not only in nerve fibers linking medial and lateral neurons (Figure 5D, arrowheads), but also localized in the medial and lateral neurons of the CoG (Figures 5E, F, arrowheads).

In additon, PemVIH-ir was shown in neuropils of subesophageal ganglion (SEG) (Figures 5G, H). It was found in the dorsolateral cluster (DLC) (Figures 5G, H, arrowheads) and ventromedial cluster (VMC) of visceral sensory neuropils (VSN) (Figure 5I, arrowheads). More posteriorly, PemVIH-ir was present in the DLC of the 1^st TG (Figure 5J) and scattered in the neuropils of ventral ganglia including TG (Figures 5K, M, O). PemVIH-ir was also found in the VMC of 2^nd and 3^rd TG, as shown in Figures 5L, N, respectively. Lastly, diffused PemVIH-ir was found in the DLC (Figure 5P, arrowheads) and neuropils of AbG (Figure 5Q). The negative control using the preadsorbed antiserum showed no immunoreactivity (Figure 5R).

To determine the effect of PemVIH on vitellogenesis, a single dose of dsPemVIH was injected into female subadults at a concentration of 10 μg/g body weight to suppress the expression of PemVIH. We first evaluated the efficiency and specificity of the synthetic dsRNA administration on PemVIH suppression in VNC-TG. The expression of both PemVIH and PemGIH was determined after dsPemVIH and dsEGFP administration. PemVIH was significantly downregulated in the dsPemVIH-treated group compared to the dsEGFP-treated group (Figure 6A). In contrast, PemGIH expression level was not significantly different between dsPemVIH- and dsEGFP-treated groups (Figure 6B). Since both HP and ovary produced vitellogenin, the expression of PemVg was determined in both tissues after dsPemVIH and dsEGFP administrations. Results showed that the expression of PemVg in HP was not significantly different between both groups (Figure 6C). However, in the ovary, the expression of PemVg was significantly upregulated by three-fold in the dsPemVIH-injected group compared to the dsEGFP-injected group (Figure 6D). In addition, an effect of eyestalk ablation on PemVIH expression was evaluated in broodstocks. A significant decrease of PemVIH expression level was shown in Br and VNC-AbG of the eyestalk-ablated group (Figure 7). However, in VNC-TG, PemVIH expression level was lower in the eyestalk-ablated group, but it was not statistically significantly different.