Unilateral Eyestalk Ablation Research Articles

Effects of eyestalk ablation and seawater temperature on d-glutamate levels in the reproductive tissues of male kuruma prawn Marsupenaeus japonicus.

D-Glutamate, a novel d-amino acid found in animal tissues, exclusively exists in the male reproductive tissues of the kuruma prawn, Marsupenaeus japonicus. Herein, changes in the d-glutamate content were determined in the male reproductive tissues of M. japonicus during acclimation to breeding seawater temperatures of 18-22°C and unilateral eyestalk ablation. The d-glutamate content in the testis increased with increasing seawater temperature and with unilateral eyestalk ablation. This suggests that both stimulations induced d-glutamate synthesis in the testis. Although the d-alanine content in the testis increased after unilateral eyestalk ablation, it did not change with elevated seawater temperature. Furthermore, we determined the d-glutamate distribution in the M. japonicus spermatophore. This indicates that d-glutamate is crucial in prawn fertilization.

Erratum: Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs.

Erratum: Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs.

Sesquiterpenoid pathway in the mandibular organ of Penaeus monodon: Cloning, expression, characterization of PmJHAMT and its alteration response to eyestalk ablation

Methyl farnesoate (MF), a crustacean equivalent of juvenile hormone (JH) of insects, is known to be produced from the mandibular organ (MO). This study reports transcriptome analysis of Penaeus monodon MO and identifies putative genes encoding enzymes in the sesquiterpenoid pathway. A total of 44,490,420 clean reads were obtained and utilized for subsequent analysis. De novo assembly created 31,201 transcripts and 31,167 unigenes. To archive the functional annotation, all unigenes were annotated with KOG, KEGG, and GO. Putative genes encoding enzymes and regulatory proteins involved in the sesquiterpenoid pathway were obtained from the MO transcriptome data based on the conserved domains and sequence homology. They included S-adenosylmethionine synthetase, farnesyl pyrophosphate synthase, short chain dependent dehydrogenase/reductase (SDR), NAD(P) + -dependent aldehyde dehydrogenase, S-adenosylmethionine-dependent methyltransferases or juvenile hormone acid-O-methyl transferase (JHAMT), farnesoic acid O-methyl transferase (FAMeT), juvenile hormone binding protein, cytochrome C/P-450 family 15 (CRYP15A1)/methylfarnesoate epoxidase (MFE), juvenile hormone epoxide hydrolase (JHEH), and juvenile hormone esterase (JHE). We first identified and characterized JHAMT orthologs inP. monodon(PmJHAMT). The complete cDNA sequence ofPmJHAMTconsisted of 1,221 nt encoded 271 amino acids with a conserved S-adenosyl methionine (SAM) binding domain. Phylogenetic analysis clusteredPmJHAMTinto the group JHAMT with the same clade of the crabPortunus trituberculausJHAMT. Moreover, the predicted three-dimensional structure of PmJHAMT showed remarkable similarity with the recent crystal structure ofthe Bombyx moriJHAMT homodimer. RT–PCR analysis revealed that PmJHAMT was exclusively expressed in MO and initially expressed at stage 3 postlarvae. In situ hybridization with a specific probe to PmJHAMT validated the specific expression of this gene in MO cells. Finally, we evaluated the regulation of MO by eyestalk inhibitory peptides. Diminishing MO inhibitory hormone through unilateral eyestalk ablation resulted in a significantly higher expression ofPmJHAMTin MO by quantitative PCR. This result indicated that the eyestalk inhibitory hormone inhibited MF synthesis byPmJHAMTgene suppression in the MO. This finding provides insight into the crustacean sesquiterpenoid pathway and improves our understanding of crustacean endocrinology.

Isolation and Expression Analysis of cAMP-Dependent Protein Kinase and Adenylyl Cyclase-Associated Protein 1-like cDNAs in the Giant Tiger Shrimp Penaeus monodon

Characterization of genes exhibiting differential expression profiles during ovarian development is important for understanding reproductive maturation of the giant tiger shrimp (Penaeus monodon). Here, the partial cDNAs of P. monodon cAMP-dependent protein kinase, catalytic subunit 1 (PmPkaC1) and adenylyl cyclase-associated protein 1-like (PmCap1-l) were studied. They were more preferentially expressed in ovaries than testes of cultured juveniles and wild broodstock. PmPkaC1 mRNA in ovaries of non-ablated broodstock was significantly increased during vitellogenesis (P<0.05). However, unilateral eyestalk ablation (the removal of one eyestalk) resulted in a significant reduction of its expression (P<0.05). PmCap1-l was not differentially expressed during ovarian development in wild non-ablated broodstock. It was up-regulated in mature ovaries following eyestalk ablation (P<0.05). The PmCap1-l transcript in each ovarian stage of the former was significantly lower than that of the latter (P<0.05). The levels of ovarian PmPkaC1 and vitellogenin 1 (PmVtg1) treated in vitro with 17-20β-DHP (0.1, 1.0 and 10.0 μg/ml for 24 h) was not significantly different from the control (P>0.05). Nevertheless, the expression of PmCap1-l was increased in ovaries treated with 0.1 and 10 μg/ml 17-20β-DHP at 24 hours post treatment (hpt, P<0.05).

Histological and Histochemical Dynamism of Oogenesis in the Cinnamon River Prawn Macrobrachium acanthurus (Caridea: Palaemonidae) Induced by Eyestalk Ablation

Macrobrachium acanthurus is a caridean prawn native to Brazil, and studying techniques to assist in its cultivation is important due to economic interest in it. Eyestalk ablation is commonly used to promote ovarian development and maturation of captive crustaceans, but it can have possible consequences on fertility and brood quality. Histological and histochemical dynamism of oogenesis was analyzed under control (non-ablated females) and unilateral eyestalk ablated females. Females with ovaries in the spent stage of gonadal development were divided into two treatments: unilaterally ablated and non-ablated. A second experiment with the same treatments was conducted using females with mature gonads. Histological and histochemical analyses of the ovaries indicated that the ablation did not affect oogenesis based on ovarian structure, oocyte size, histochemical properties, and atretic oocytes. Ovarian maturation was not accelerated by ablation, either. Survival, nuptial molting, and spawning were also unaffected. From an applied point of view, unilateral eyestalk ablation is not needed in M. acanthurus farming because it does not improve reproductive performance.

The Regulatory Relationships Between the Gonad-Inhibiting Hormone and Insulin-Like Androgenic Gland Hormone-Binding Protein Genes in the Eyestalk-Androgenic Gland-Testis Axis of Macrobrachium rosenbergii

Gonad-inhibiting hormone (GIH) belongs to a family of neuropeptides that are released from the eyestalks of male crustaceans and plays key roles in gonadal maturity, reproduction, and molting. However, the detailed mechanisms underlying the effects of GIH on sexual regulation have yet to be elucidated. In the present study, we aimed to demonstrate how GIH mediate the activity of the androgenic gland (AG) to affect sexual regulation. To do this, we cloned and characterized a GIH sequence from Macrobrachium rosenbergii (MrGIH). The open reading frame (ORF) of MrGIH was 360 bp and codes for a polypeptide of 119 amino acids and a putative protein of 13.56 KDa. Tissue analysis showed that MrGIH is widely expressed in a range of tissues but particularly, the eyestalk, intestine, and nerve cord. Following the dsRNA silencing of MrGIH for 24 h, the expression levels of MrGIH were down-regulated in both the eyestalk and AG when compared with the negative control, but significantly increased the expression of Macrobrachium rosenbergii insulin-like androgenic gland hormone-binding protein (MrIAGBP) in AG, thus suggesting that MrGIH is an inhibitory factor for MrIAGBP. In addition, we found that eyestalk removal on certain days led to increased expression levels of MrIAGBP expression. The expression levels of MrIAGBP peaked at 2 d in the AG after unilateral and bilateral eyestalk ablation, exhibiting a 7.27- and 6.03-fold increase, respectively. Afterward, the expression of GIH protein levels were down-regulated and IAGBP protein levels were up-regulated after GIH silencing using immunohistochemistry method, combined with the increase of IAGBP protein levels after eyestalk ablation, confirming that MrGIH is an inhibitory factor that can moderately regulate AG development and IAGBP expression. Collectively, our findings enriched the mechanisms that control the sexual regulation pathway of male M. rosenbergii, and provided significant information for further explorations of the mechanism underlying sex regulation in other decapod crustaceans.

Impact of unilateral eyestalk ablation on Callinectes arcuatus (Ordway, 1863) under laboratory conditions: behavioral evaluation

Eyestalk ablation allows the removal of neurohormones that inhibit early reproduction and decreases the time to complete the molting cycle. The present study evaluated the impact of unilateral eyestalk ablation on the behavior of blue crab breeders Callinectes arcuatus in the short term under laboratory conditions. Immature male and female crabs and individuals in the intermolt stage were collected in Ohuira Bay, Ahome, Sinaloa, Mexico. Three bioassays were performed: males, females, and male-females each with three treatments; control + xylocaine (50 μL, 5%), squash ablation + xylocaine (50 μL, 5%), ablation by string ligation + xylocaine (50 μL, 5%), each with three replicas and three periods of evaluation after the ablation procedure (10 min, 2 h, and 24 h). Kruskal-Wallis and Chi-square (χ2) tests were used to evaluate significances (P < 0.05) in the behavior of C. arcuatus. The sizes of blue crab C. arcuatus were 9.72 ± 1.0 cm of carapace width. The feeding behavior of the male-females bioassay was significant at 2 h (P < 0.05). The results obtained were satisfactory using two techniques, unilateral eyestalk ablation by squash and ligation in which pain and aggressiveness indicators were mitigated. Disorientation and aggressiveness in males were significant at 2 h (P < 0.05), and in females after 10 min, and 2 h after ablation (P < 0.05). In the male-females bioassay, aggressiveness was significant at 2 h (P < 0.05). Courtship and copulation were recorded in those crabs subjected to unilateral eyestalk squash ablation (13 and 8, respectively).

A Novel Imprinted Gene (Sp-Pol) With Sex-Specific SNP Locus and Sex-Biased Expression Pattern Provides Insights Into the Gonad Development of Mud Crab (Scylla paramamosain)

Identification and exploring the role of novel sex-related genes is a crucial step for understanding the regulation mechanism of sexual development. In the present study, we identified a novel sex-related gene (designated as Sp-Pol) upstream of a sex-specific single nucleotide polymorphism (SNP). SNP1888 together with Sp-Pol were mapped on LG32 (which is a sex-related linkage group) of a high-density genetic map. The full-length cDNA of Sp-Pol consists of 1703 bp with an open reading frame (ORF) encoding 204 amino acids, a 344 bp 5′-UTR and a 744 bp 3′-UTR. Phylogenetic analysis showed that Sp-Pol may need to be classified as a new gene family due to the very low sequence identity with other known genes (less than 25% identity). The sex-biased expression pattern of Sp-Pol starts from crablet stage V (C5) with approximately three- to four-fold higher in males than in females. Sp-Pol was expressed at a higher level in gonads compared to other tissues, with the highest expression level in the testis. In testis, a downward trend was observed in the expression level of Sp-Pol from the testis stage I (T1) to testis stage III (T3). After unilateral eyestalk ablation, the expression level of Sp-Pol significantly increased in testis and hepatopancreas in males, while it was downregulated in the hepatopancreas of females. Fluorescence in situ hybridization (FISH) assay revealed that Sp-Pol transcripts were strongly localized in the epithelia of seminiferous tubules of the testis, and in the ovary, it was detected in the oogonium cells. These findings showed that Sp-Pol may play crucial roles in the gonad development of S. paramamosain.

Transcriptome Analysis Reveals the Endocrine Regulation on the Expression of IAG in Litopenaeus vannamei

The insulin-like androgenic gland hormone (IAG) plays a key role in male sexual differentiation and spermatogenesis in crustaceans. The expression of IAG is usually negatively regulated by neuropeptide hormones through the “eyestalk-AG” endocrine axis. However, the underlying mechanism is still largely unknown. In the present study, we performed a comparative transcriptome analysis on the androgenic gland (AG), the main secretory organ of IAG, of L. vannamei before and after unilateral eyestalk ablation. A total of 67 differentially expressed genes (DEGs) were identified, including some putative genes involved in sexual development. Interestingly, several genes related to molting and endocrine processes were found differentially expressed between the two treatments. Further investigation on the expression profiles of these genes and Lv-IAG showed that their expression patterns were closely correlated throughout the molting cycle. In addition, injection of 20-hydroxyecdysone (20E) could inhibit the expression of Lv-IAG at different molting stages in a dosage-dependent manner. The data indicated that ecdysteroids played important roles in regulating the Lv-IAG expression and might bridge the endocrine axis between eyestalk and AG. The present study provided new insights into understanding the regulation of sexual development in male crustacean.

The significant sex-biased expression pattern of Sp-Wnt4 provides novel insights into the ovarian development of mud crab (Scylla Paramamosain)

The wingless-type MMTV integration site family member-4 (Wnt4), a member of the wingless-related integration site (Wnt) family, is widely accepted as a key regulator of ovarian development in mammals. In this study, a full-length cDNA of Wnt4 (designated as Sp-Wnt4) was cloned, characterized, and functionally studied in mud crab (Scylla paramamosain). The full-length cDNA of Sp-Wnt4 consists of 2659 bp with an open reading frame (ORF) encoding 359 amino acids, a 907 bp 5′-UTR and a 672 bp 3′-UTR. Sp-Wnt4 contains 25 cysteine (Cys) residues and three potential N-glycosylation sites. Sp-Wnt4 protein shared the highest identity (98.9%) to the Wnt4 protein of Portunus trituberculatus. The phylogenetic tree showed that Sp-Wnt4 and Wnt4 protein of Malacostracan crustaceans clustered together, indicating that they had a close genetic distance. Sp-Wnt4 was expressed at a higher level in the ovary compared to other tissues, with the highest expression level at the third stage (O-III) of the ovarian development (P < 0.05). A downward trend was observed in the expression level of Sp-Wnt4 from the embryo stage to crablet stages (P < 0.05). After unilateral eyestalk ablation, the expression level of Sp-Wnt4 significantly increased in testis (14-fold) and downregulated (3.1-fold) in the gill (P < 0.05) of females. In situ hybridization (ISH) assay revealed that Sp-Wnt4 transcripts were mainly localized in the cytoplasm of oocyte cells. These findings showed that Sp-Wnt4 play crucial roles in the ovarian development of S. paramamosain. In conclusion, our study provides novel insights into the evolution and roles of the Wnt4 gene.

Effects of using the biofloc system and eyestalk ablation on reproductive performance and egg quality of Litopenaeus vannamei (Boone, 1931) (Decapoda: Dendrobranchiata: Penaeidae)

The aim of this study was to evaluate egg production and quality of females of the Pacific white shrimp, Litopenaeus vannamei, in which there was or was not unilateral eyestalk ablation after there was pre-maturation culturing in biofloc or clear-water systems. Acylglycerides, cholesterol, glucose and total soluble protein were determined for the hepatopancreas, ovaries, hemolymph and eggs. Females cultured using the biofloc system had a larger number of eggs released per spawning and per gram of spawning specimen body weight. The number of total spawning’s per week was comparable among treatments. Females cultured in the biofloc system in which there was no eyestalk ablation had that greatest concentrations of nutrient reserves in the hepatopancreas (P < 0.05) with the females cultured in the biofloc and clear-water system that had eye stalk ablation having the next most abundant nutrient reserves. There were the least concentrations of nutrient reserves in females with eyestalk ablation cultured in the clear-water system (P < 0.05). There, however, were no difference in nutrient reserve concentrations in the hemolymph and ovaries. In the eggs, there was the same trend among treatments as the hepatopancreas nutrient reserves, indicating that both eyestalk ablation and pre-maturation culture conditions (i.e., either biofloc or clear-water) affected the quality of eggs in L. vannamei.

Reproductive performance of Macrobrachium acanthurus (Crustacea, Palaemonidae) females subjected to unilateral eyestalk ablation

AbstractThe present study investigated the effects of eyestalk ablation on the reproductive performance of females from the native caridean shrimp Macrobrachium acanthurus. The reproduction parameters fertility, fecundity, egg volume, larvae size, loss of embryos and number of females with viable eggs/larvae were evaluated. Females presenting carapace length of 12–15 mm and spent stage of gonadal development were divided into two groups, one undergoing unilateral eyestalk ablation and the other remaining intact. The specimens were kept in aquarium until ovarian maturation, then brought together with males for mating (proportion of two females for each male). Fecundity and fertility were greater in intact females (1,741 eggs and 1,045 larvae) than in ablated ones (1,373 eggs and 741 larvae). Ablation also reduced the percentage of females with viable eggs (68% intact versus 49% ablated) and larvae (82% intact versus 64% ablated), besides increasing the loss of embryos. Egg volume and shape (ellipsoid) and larvae size were similar, as was the duration of embryonic development. The results of this study suggest that, although ablated females exhibit a high rate of survival, their reproductive performance is reduced; therefore, the ablation technique is not recommended in M. acanthurus farming, so as to protect the animals from physiological stress.

Insulin-like androgenic gland hormone from the shrimp Fenneropenaeus merguiensis: Expression, gene organization and transcript variants.

Male sex differentiation in the crustacean is best known to be controlled by the insulin-like androgenic gland hormone (IAG). In this report, the cDNA and gene of the shrimp Fenneropenaeus merguiensis FmIAG were studied and characterized. FmIAG gene shares a high sequence identity in the coding region as well as the promoter region with that of F. chinensis. FmIAG gene is most likely consists of 5 exons and 4 introns. The cDNA reported here is the most abundant transcript that retained cryptic intron 4. The use of different splicing acceptor sites in exon 2 can produce a long-form FmIAG transcript variant with 6 additional amino acids inserted. Splicing of cryptic intron 4 would produce a transcript variant with a different C-terminal end. Therefore 4 different FmIAG transcripts can be produced from the FmIAG gene. During the molt cycle, the expression level of FmIAG was low in the early intermolt, increase steadily towards the late premolt and decreased rapidly in the early postmolt. In addition to the androgenic gland, FmIAG is also expressed in the hepatopancreas and ovary of adult females. Unilateral eyestalk ablation caused a significant increase in FmIAG transcript suggesting that the eyestalk consists of inhibiting factor(s) that suppressesFmIAGexpression. To explore the function of FmIAG in males, injection of FmIAG dsRNA knock-down the expression of FmIAG and up-regulated the expression of the vitellogenin gene in the testis and hepatopancreas. Interestingly a CHH-like gene identified in the androgenic gland was down-regulated. CHH-like gene knock-down resulted in altered expression of FmIAG in males suggesting that the CHH-like may be involved in FmIAG's regulation. RT-PCR with specific primers to the different transcript variant were used to determine if there is an association of different sizes of male and the type of IAG transcript. Results indicated that a high percentage of the large male shrimp expressed the long-form of FmIAG. The results suggested that FmIAG may be useful as a size marker for male shrimp aquaculture. In summary, the results of this study have expanded our knowledge of shrimp insulin-like androgenic gland hormone in male sex development and its potential role as a marker gene for growth regulation in shrimp.

Evaluation of different induced molting methods in Callinectes ornatus (Crustacea, Decapoda, Portunidae) as a tool for the commercial production of soft-shell crabs.

We investigated the effects of eyestalk ablation and cheliped autotomy in inducing molting in Callinectes ornatus. The specimens in intermolt stage were divided into two size classes: 1 (30-50 mm) and 2 (51-70 mm) and were further divided into four experimental groups. In the CA group (Cheliped Autotomy, n=76), crabs were submitted to cheliped autotomy; in the UA group (Unilateral Ablation, n=66) and BA group (Bilateral Ablation, n=66) to unilateral and bilateral eyestalk ablation, respectively. The C group (Control, n=70) was used as control. The animals were individually kept in tanks interconnected to a recirculation system for 30 days. The highest frequency of premolt was recorded in the BA group (32%), followed by the CA group (16%). The premolt frequency of class 1 (28%) was significantly higher (p<0.05) than of class 2 (8%). The mean time until molt in the BA group was significantly lower (p <0.05) than other groups tested. The highest mortality rates were 55% and 25% in crabs from the BA and CA groups, respectively. The results indicate bilateral eyestalk ablation and cheliped autotomy are potentially capable of inducing molt in C. ornatus, but these techniques have limited efficiency for the commercial application of large-scale soft-shell crab.

Increased robustness of postlarvae and juveniles from non-ablated Pacific whiteleg shrimp, Penaeus vannamei, broodstock post-challenged with pathogenic isolates of Vibrio parahaemolyticus (VpAHPND) and white spot disease (WSD)

The maturation and reproduction of Pacific whiteleg shrimp, Penaeus vannamei, through the practice of unilateral eyestalk ablation though common is an animal welfare concern. This study assessed the resilience of offspring from non-ablated P. vannamei when challenged with an isolate of Vibrio parahaemolyticus (Vp) causing acute hepatopancreatic necrosis disease (VpAHPND), and with white spot syndrome virus (WSSV). VpAHPND and WSSV challenges were conducted using PL and juveniles under controlled conditions, with both trials using four groups (i.e. shrimp from either ablated or non-ablated females and then either challenged with the pathogen or not challenged). For the VpAHPND challenge, ten replicate 20 L tanks (five replicates for each population) each containing 100 PL 17 (average weight 14 mg) in 15 ppt, 29.05 ± 0.13 °C water were challenged with 2 mL of 2.0 × 108 CFU mL−1 culture of V. parahaemolyticus. A further ten replicate tanks (five per population) served as the corresponding non-challenged controls. The shrimp mortalities were assessed every 3 h over the following 96 h. For the WSSV challenge, individual 1.4 g (average weight) shrimp (50 individuals per population) were housed in 1 L tanks and fed 0.1 g WSSV infected tissue (av. 2.02 × 109 WSSV). A further 50 shrimp per population served as non-challenged controls. The shrimp were maintained at 15 ppt, 26.3 ± 0.71 °C water and assessed every 3 h post-infection over the subsequent 168 h and mortalities at each time point noted. Postlarvae from non-ablated females had significantly (p = 2.4E-23) better survival (70.4%) than those from ablated females (38.8%) at 96 h post-challenge with VpAHPND. Both challenged populations had significantly (p ≤1.3E-36) lower survival than the control groups. The survival of the juveniles from non-ablated females (62%) at 168 h post-infection with WSSV was not significantly higher than that of the juveniles from ablated female (48%) although the difference was significantly different at 65 to 75 h. Both challenged populations also had significantly (p ≤1.0E-5) lower survival rates than the control groups. The study demonstrates that postlarvae and juveniles from non-ablated females are more resilient to typical pathogens (VpAHPND and WSSV) and may show higher survival rates during a disease outbreak.

Isolation of Differentially Expressed Transcripts by cDNA-AFLPaba and Expression Analysis of DEAD Box ATP-Dependent RNA Helicase 48 in Ovaries of the Giant Tiger Shrimp Penaeus monodon

Genes involved in ovarian development of the giant tiger shrimp (Penaeus monodon) were isolated by cDNA-AFLP analysis. In total, 75 primer combinations were screened against cDNA of different stages of ovaries (premature ovaries of juveniles and previtellogenic, vitellogenic and mature ovaries of wild intact and eyestalk-ablated broodstock; N = 6 for each group). Eighteen cDNA-AFLP fragments were cloned and sequenced. Nucleotide sequences of ten fragments significantly matched EF-1α (7e63), DEAD box ATP-dependent RNA helicase 48 (PmDdx48; 8e-61), tyrosine phosphatase n9 (1e-42), autophagy related protein Atg4-like protein (3e-36), nuclear pore complex protein nup154 (6e-20), GK14382 gene product (1e-20), L-3-hydroxyacryl coenzyme A dehydrogenase short chain (8e-13) and three different hypothetical proteins. The remaining fragments did not match any previously deposited sequence (E-value &gt; 10-4). The full-length cDNA of PmDdx48 was successfully characterized. It was 1646 bp containing an open reading frame (ORF) of 1209 bp corresponding to 402 amino acids. PmDdx48 was up-regulated in late vitellogenic and mature ovaries of wild intact broodstock and in mature ovaries of wild eyestalk-ablated broodstock (P&lt;0.05). Unilateral eyestalk ablation resulted in down-regulation of PmDdx48 in late vitellogenic and mature ovaries compared to that of wild intact broodstock (P&lt;0.05).

Transcriptome analyses reveal the synergistic effects of feeding and eyestalk ablation on ovarian maturation in black tiger shrimp

Unilateral eyestalk ablation in the female black tiger shrimp Penaeus monodon is commonly employed to induce ovarian maturation. However, the importance of complementing this practice with the provision of live feed supplement (such as polychaetes) has not been emphasized in previous studies. Indeed, it has been less emphasized that female broodstock must be fed with live feeds such as polychaetes for this practice to be effective. While the effects of eyestalk ablation have been thoroughly studied in various aspects, the synergistic effects of feeding with live feeds and the ablation have never been elucidated at a transcriptome-wide level. With recent advances in the next-generation sequencing platforms, it is now possible to investigate the effects of eyestalk ablation and live feeds at the transcriptomic levels. This study employed both short-read Illumina RNA sequencing and long-read Pacific Biosciences (PacBio) isoform sequencing (Iso-seq) to generate the first high-quality ovarian reference transcriptome in P. monodon. This novel assembly allowed us to dissect the effects of feeds and eyestalk ablation and reveal their synergistic effects at the transcriptomic level through the regulation of important genes involved in fatty acid regulation, energy production, and hormone-mediated oocyte maturation pathways. The synergistic effects between the polychaete feeding and the eyestalk ablation in the process of ovarian maturation in black tiger shrimp suggest that without having proper nutrients from the polychaetes, female broodstock might not be ready to develop its ovary. However, even with proper nutrients, the eyestalk ablation is still necessary to perhaps manipulate the female endocrine of the black tiger shrimp. These findings shed the light on molecular mechanisms and key molecular pathways that lead to successful ovarian maturation.

Evaluation of Reproductive Capacity in Pond-Reared Penaeus monodon after Unilateral Eyestalk Ablation

Evaluation of Reproductive Capacity in Pond-Reared Penaeus monodon after Unilateral Eyestalk Ablation

Expression patterns of MIH, EcR2, and RXR3 in the moult cycle of the red swamp crayfish, Procambarus clarkii (Girard, 1852) (Decapoda, Cambaridae)

Abstract In arthropods, the moult-inhibiting hormone (MIH), the ecdysone receptor (EcR), and the retinoid X receptor (RXR) are key regulators in moulting. In the present study, the full-length cDNAs of the MIH, EcR2, and RXR3 genes from the red swamp crayfish, Procambarus clarkii (Girard, 1852) (denoted as PcMIH, PcEcR2, and PcRXR3) were cloned. Tissue-specific and moult stage-specific mRNA expression patterns of these genes were detected by real-time quantitative polymerase chain reaction. PcMIH was detected only in the eyestalk, whereas PcEcR2 and PcRXR3 mRNA were expressed in all tissues tested. The highest levels of PcEcR2 and PcRXR3 were detected in the gill and hepatopancreas. Expression of PcMIH mRNA in the eyestalk increased from postmoult to peak in intermoult and then decreased in premoult. Expression of PcEcR2 mRNA in the eyestalk, hepatopancreas, and muscle increased from postmoult to peak in early premoult and then decreased. However, expression of PcEcR2 mRNA in the gill increased from postmoult to reach a maximum in intermoult and then decreased in premoult. Expression of PcRXR3 mRNA also fluctuated in the eyestalk, hepatopancreas, muscle, and gill, with a decrease from postmoult to late premoult. Expression of PcEcR2 and PcRXR3 mRNA increased relative to the control in the hepatopancreas and gill after unilateral and bilateral eyestalk ablation, which suggested that PcMIH can inhibit their mRNA expression. Double-stranded RNA-mediated RNA interference of PcRXR3 caused different changes in mRNA expression of these genes in different tissues and resulted in decreased expression of PcEcR2 mRNA, which suggested a collaborative relationship between PcEcR2 and PcRXR3.

Cloning and expression analysis of a cytoplasmic juvenile hormone-binding protein from the mud crab, Scylla paramamosain (Decapoda, Brachyura, Portunidae)

Abstract Juvenile hormone (JH) has an important role in regulating the development of arthropods, and methyl farnesoate (MF) is the only juvenile hormone found in Crustacea to date. Juvenile hormone-binding proteins (JHBPs) bind to JHs and transport or deliver JH signals. In this study, the complete cDNA sequence of cytoplasmic JHBP (cJHBP) was obtained from Scylla paramamosain, which we named Sp-cJHBP. The putative protein sequence of Sp-cJHBP contains two conserved glyoxalase domains. Expression analysis revealed that Sp-cJHBP expression varied greatly among larval developmental stages, with a significant reduction at the Zoea-5 stage. Sp-cJHBP had the highest expression in the ovary, with low expression in other tissues of the female adult crabs. During ovarian development, the expression of Sp-cJHBP changed significantly in the ovary, though its trends correlated poorly with the ovarian developmental process. While the expression in the mandibular organ (MO) was similar to 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), which is a key gene involved in MF biosynthesis. After unilateral eyestalk ablation (ESA), the expression of Sp-cJHBP altered significantly in the ovary and MO, but not in cerebral ganglion and thoracic ganglion. Altogether, these results suggest that Sp-cJHBP is likely to function primarily in the ovary, probably through binding to MF.