Bilateral Eyestalk Ablation Research Articles

Mechanisms of neurocentral-eyestalk-intestinal immunotoxicity in whiteleg shrimp Litopenaeus vannamei under ammonia nitrogen exposure

Ammonia-N, as the most toxic nitrogenous waste, has high toxicity to marine animals. However, the interplay between ammonia-induced neuroendocrine toxicity and intestinal immune homeostasis has been largely overlooked. Here, a significant concordance of metabolome and transcriptome–based “cholinergic synapse” supports that plasma metabolites acetylcholine (ACh) plays an important role during NH4Cl exposure. After blocking the ACh signal transduction, the release of dopamine (DA) and 5-hydroxytryptamine (5-HT) in the cerebral ganglia increased, while the release of NPF in the thoracic ganglia and NE in the abdominal ganglia, and crustacean hyperglycemic hormone (CHH) and neuropeptide F (NPF) in the eyestalk decreased, finally the intestinal immunity was enhanced. After bilateral eyestalk ablation, the neuroendocrine system of shrimp was disturbed, more neuroendocrine factors, such as corticotropin releasing hormone (CRH), adrenocorticotropic-hormone (ACTH), ACh, DA, 5-HT, and norepinephrine (NE) were released into the plasma, and further decreased intestinal immunity. Subsequently, these neuroendocrine factors reach the intestine through endocrine or neural pathways and bind to their receptors to affect downstream signaling pathway factors to regulate intestinal immune homeostasis. Combined with different doses of ammonia-N exposure experiment, these findings suggest that NH4Cl may exert intestinal toxicity on shrimp by disrupting the cerebral ganglion-eyestalk axis and the cerebral ganglion-thoracic ganglion-abdominal ganglion axis, thereby damaging intestinal barrier function and inducing inflammatory response.

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

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

Molecular identification and putative role of insulin growth factor binding protein-related protein (IGFBP-rp) in the swimming crab Portunus trituberculatus

The insulin-like growth factor/insulin-like polypeptide (IGF/ILP) signaling is vital for growth, physiological metabolism, development, and reproduction. Insulin-like growth factor-binding protein (IGFBP) is involved in the insulin signaling pathway in both vertebrates and invertebrates and is critical for various physiology functions. Herein, we cloned and characterized the full-length cDNA of IGFBP-rp in the swimming crab, Portunus trituberculatus (PtIGFBP-rp). The deduced amino acid sequence of PtIGFBP-rp was found to contain three key domains (insulin-like binding (IB) domain, the kazale-type serine protease inhibitor (KAZAL) domain, and the immunoglobulin-like C2 (IGc2) domain). Results showed that PtIGFBP-rp shared the same expression pattern as P. trituberculatus insulin androgenic gland hormone (PtIAG) transcripts during the embryonic larval, juvenile crab stage and the androgenic gland (AG) developmental cycle. Moreover, PtIGFBP-rp transcripts were also present in high abundance in hepatopancreas, muscle, and androgenic glands. The regulatory relationship between PtIGFBP-rp and PtIAG was investigated by RNA interference and co-localization assays, which showed a co-localization relationship and feedback regulation between them. Bilateral eye stalk ablation (ESA) increased the expression of PtIGFBP-rp in the AG at 7 d after surgery. These results demonstrate the involvement of PtIGFBP-rp in the signaling regulatory network of IAG in P. trituberculatus.

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.

Examination of the potential role of CHH in regulating the expression of IAGBP gene through the eyestalk-testis pathway

Crustacean hyperglycemic hormone (CHH) is a peptide hormone of the CHH family produced by the X-organ/sinus gland complex and plays role in metabolism, osmoregulation, molting, and reproduction. However, the mechanism by which CHH regulates crustacean sexual behavior remains unclear. To elucidate the mechanism of CHH-mediated sexual regulation in this study, the giant freshwater prawn (Macrobrachium rosenbergii) CHH gene (MrCHH) was cloned and characterized. The MrCHH open reading frame was 408 bp in length and was found to encode a 135–amino acid putative protein of 15.42 kDa. In normal prawns, MrCHH mRNA transcripts were abundant in the examined tissues, as follows: eyestalk > intestine > androgenic gland > testis > ventral nerve cord > stomach > gills > heart > brain. In prawns injected with dsCHH RNA, MrCHH mRNA and protein expression were down-regulated at 24 h in the eyestalk and testis compared with prawns injected with negative control dsGFP. Moreover, MrCHH dsRNA increased mRNA and protein expression of M. rosenbergii insulin-like androgenic gland hormone–binding protein (MrIAGBP), indicating that MrCHH inhibits MrIAGBP expression. Additionally, after unilateral or bilateral eyestalk ablation, expression of both insulin-like androgenic gland hormone (IAG) and IAGBP in the prawn testis was significantly up-regulated. Immunohistochemical analyses showed that after unilateral or bilateral eyestalk ablation, MrIAGBP protein levels also increased in the testis, further suggesting that MrCHH is an inhibitor that negatively regulates the expression of genes that play roles in male gonadal development. Our findings thus suggest that MrCHH serves to enrich the “eyestalk-androgenic gland-testis” endocrine axis involved in IAG signaling, providing significant information for further explorations of the mechanism underlying sex regulation in other decapod crustaceans.

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.

Molecular Characterization of the Insulin-Like Androgenic Gland Hormone in the Swimming Crab, Portunus trituberculatus, and Its Involvement in the Insulin Signaling System.

The insulin-like androgenic gland hormone (IAG) is mainly produced in the androgenic gland (AG) of the male crustaceans and is a crucial regulator in male sexual differentiation. In the current study, the full-length cDNA of IAG in the swimming crab, Portunus trituberculatus (Pt-IAG), was cloned and characterized. Similar to other reported IAGs, the deduced amino acid sequence of Pt-IAG consists of signal peptide, B chain, C peptide, and A chain, containing six conserved cysteines that form two interchain disulfide bonds and one intra-B chain disulfide bond. Tissue distribution analysis suggested that the Pt-IAG cDNA was highly expressed in the AG and was slightly expressed in several other tissues. A short-term silencing of PtIAG with double-stranded RNA was found to reduce the transcript levels of insulin receptor (Pt-IR) and insulin-like growth factor-binding protein (Pt-IGFBP), suggesting the Pt-IAG might perform its biological function through the insulin family-based signaling system. Bilateral eyestalk ablation (ESA) induced the expression of Pt-IAG in the AG at 4 and 7 days after surgery, while the transcript levels of Pt-IR in the AG and testis and Pt-IGFBP in the muscle, testis, and thoracalia ganglia were significantly decreased from 1 day after surgery. The results suggested that the Pt-IR and Pt-IGFBP might also be the targets of eyestalk neuropeptides and responded to the ESA independent of IAG regulation.

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.

The serotonin or dopamine by cyclic adenosine monophosphate-protein kinase A pathway involved in the agonistic behaviour of Chinese mitten crab, Eriocheir sinensis

Agonistic behaviour is common in an encounter between two crustaceans. It often causes limb disability and consumes a lot of energy, which is harmful for the growth and survival of commercially important crustaceans. In the present study, we mainly focused on the agonistic behaviour of the Chinese mitten crab, Eriocheir sinensis, which is an important species of the aquaculture industry in China. We recorded agnostic behaviour with a high-definition camera and preliminarily evaluated the role of serotonin (5-HT) or dopamine (DA)-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway and eyestalk in the behaviour. The results showed that agonistic behaviour in E. sinensis consisted of three stages: approach, contact and fight. We found that the number of fights and cumulative time of fight were significantly higher in the male vs. male group than in the female vs. female and female vs. male groups (P < 0.05). After 1 h of agonistic behaviour, 5-HT concentration showed a significant increase and DA concentration showed a significant decrease when compared with the control group (no encounter; P < 0.05). 5-HT1B and 5-HT2B mRNA levels showed a significant increase in the eyestalk (P < 0.05). 5-HT7 mRNA levels showed significant downregulation in the thoracic ganglia and DA1A mRNA levels showed upregulation in the intestine (P < 0.05). DA2 mRNA levels showed a significant decrease in the eyestalk (P < 0.05). These changes were accompanied by a significant increase in cAMP level and significant decrease in PKA level in the haemolymph (P < 0.05). In addition, a significant decrease in glucose levels was detected after the agonistic behaviour. Crustacean hyperglycemic hormone (CHH) mRNA levels showed significant upregulation in the eyestalk and significant downregulation in the intestine (P < 0.05). The number of fights and cumulative time of fight in the left eyestalk ablation (L-X vs. L-X) group were more and longer than those in the intact eyestalk (C vs. C), right eyestalk ablation (R-X vs. R-X) and bilateral eyestalk ablation (D-X vs. D-X) groups. In short, E. sinensis shows special agonistic behaviour modulated by 5-HT or DA-cAMP-PKA pathway and eyestalk, especially the left eyestalk.

Hemolymph transcriptome analysis of Chinese mitten crab (Eriocheir sinensis) with intact, left cheliped autotomy and bilateral eyestalk ablation

In the pond culture of Eriocheir sinensis, high limb-autotomy seriously affects the quality and culture's economic efficiency. Based on our previous studies, limb autotomy can induce the changes of hematological immune response in E. sinensis hemolymph. Eyestalk ablation can accelerate the regeneration of limbs after autotomy. To detect the important functional genes related to the hematological molecular immunity of E. sinensis, we compared and analyzed the hemolymph transcriptome data of the intact crab, left cheliped autotomized crabs and bilateral eyestalk ablation crabs with high-throughput sequencing techniques. The results showed that the three groups obtained 62 172 414, 68 143 682, and 67 811 618 clean reads, respectively. A total of 9567 differentially expressed genes were obtained by multiple comparison of the three groups' libraries. Gene ontology (GO) functional classification analysis shows that the differential genes belong to 42 categories of biological process, cellular components and molecular function. The differentially expressed genes in the three libraries were enriched to 344 specific KEGG metabolic pathways by KEGG enrichment analysis, such as the up-regulated gene (dual oxidase (Duox), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (YWHAQ)) in MAPK signaling pathway, the up-regulated gene (aldehyde dehydrogenase 1 (ALDH 1)) and down-regulated gene (UDP-glucuronosyltransferase 2 (UGT 2)) in metabolism of the xenobiotics by cytochrome P450 pathway, the down-regulated gene (actin gene (AG), heat shock protein 90 (HSP 90)) in fluid shear stress and atherosclerosis pathway. To verify the expression levels of DEGs identified by RNA-Seq, the above six hematological immune-related genes were selected for qRT-PCR validation, the qRT-PCR results were consistent with the DEGs results. Our research obtained abundant E. sinensis hemolymph transcriptome information by RNA-Seq, which provides multi-level information for the cloning of novel genes and the study of hemolymph molecular immunology mechanisms of E. sinensis.

The Hyperglycemic Effect of Melatonin in the Chinese Mitten Crab, Eriocheir sinensis.

Melatonin has been identified in a variety of invertebrate species, but its function is not as well understood as in crustaceans. The effects of melatonin on hemolymph glucose levels and tissue carbohydrate metabolism in the Chinese mitten crab, Eriocheir sinensis, were fully investigated in this study. Moreover, whether the eyestalk (an important endocrine center in invertebrate species) involves in this process or not, also were clarified. Analysis revealed that eyestalk ablation, especially bilateral, caused a significant decrease in the hemolymph glucose level. Moreover, injection of melatonin induced hyperglycemia in a dose-dependent manner both in intact and ablated crabs. Based on the expression of CHH mRNA in the 10 different tissues, eyestalk, thoracic ganglion, intestinal tract and hemolymph were selected to estimate the effect of melatonin on the expression of CHH mRNA. Bilateral eyestalk ablation caused a significant increase in the expression of CHH mRNA in the thoracic ganglion, intestinal tract and hemolymph compared with the controls. In addition, injection of melatonin into intact or ablated crabs elevated the CHH mRNA level in the eyestalk, thoracic ganglion and intestinal tract tissues compared with controls. The hemolymph CHH mRNA after melatonin injection was elevated only in ablated crabs. Administration of melatonin resulted in a significant decrease in total carbohydrates and glycogen levels with an increase in phosphorylase activity levels in the hepatopancreas and muscle in intact and ablated crabs. Our findings demonstrated that melatonin can induce hyperglycemic effects in both intact and ablated crabs, suggesting that this effect is probably not mediated solely via eyestalk.

The Effect of Bilateral Eyestalk Ablation on Signal Transduction Pathways of Ion Regulation of Litopenaeus vannamei

AbstractA study was performed on the effects of bilateral eyestalk ablation on signal transduction pathways of ion regulation of Litopenaeus vannamei. The study included three treatments (starvation group, bilateral eyestalk ablation, and starvation and bilateral eyestalk ablation) in addition to a control group. The shrimp were sampled at 0, 12, 24, and 48 h. Results showed that the ablation of bilateral eyestalk had significant effects on the contents of three kinds of biogenic amines (BAs), cyclic guanosine monophosphate (cGMP), and the activities of three kinds of ion‐transport enzymes (P &lt; 0.05). According to these results, bilateral eyestalk ablation had significant effects on the ion signal pathway of L. vannamei. The same changes were observed in 5‐hydroxytryptamine (5‐HT) contents, Na+‐K+‐ATPase, and HCO3−‐ATPase activities, suggesting that crustacean hyperglycemic hormone (CHH) regulated the changes in ion‐transport enzymes, mediated by BAs and cGMP. The specific pathways may be 5‐HT → cGMP → Na+‐K+‐ATPase, HCO3−‐ATPase, and BAs → cGMP → V‐ATPase. 5‐HT contents, Na+‐K+‐ATPase, and HCO3−‐ATPase activities in the starvation group were ultimately higher than those in the bilateral eyestalk ablation group, while the cyclic adenosine monophosphate (cAMP) contents increased slightly. Study results suggested that under the situation of bilateral eyestalk ablation, the shrimp could also use feed or its metabolites to increase 5‐HT or cAMP contents to regulate the Na+‐K+‐ATPase and HCO3−‐ATPase activities in gills.

Molt and reproduction enhancement together with hemolymph ecdysteroid elevation under eyestalk ablation in the female fiddler crab, Uca triangularis (Brachyura: Decapoda)

We present the results of eyestalk extirpation experiments performed on the fiddler crab, Uca triangularis at seasons of molting and reproduction, with a view to have a better understanding of the regulatory mechanisms of these two highly energy-demanding processes. Bilateral eyestalk ablation resulted in precocious acceleration of both molting and reproduction, irrespective of the season at which each experiment was conducted. The rate of accelerated ovarian growth, however, was maximum if the eyestalk ablation was conducted during August–January, the breeding season in the wild, or in February–May (molting-reproductive season), wherein a section of the wild population would be engaged in molting and another section in breeding. The highest degree of precocious molt acceleration, on the other hand, was obtained during June–July when the population was primarily engaged in molting, but with no reproductive activity. The precocious oocyte maturation (due to de-eyestalking) was minimal in June–July. Significantly, the eyestalk ablation also resulted in a dramatic increase in the hemolymph ecdysteroid titer, revealing that a high ecdysteroid titer would have no restraining influence on vitellogenesis. No spawning was, however, observed among de-eyestalked females, even though their final oocyte size surpassed the size of the normal mature oocytes, implying that spawning is not exclusively under the control of eyestalk hormones. A comparative study performed on the vitelline components of the experimentals and the controls revealed that the precociously incorporated yolk under eyestalk ablation was biochemically impoverished. These results indicate that throughout the annual cycle, both the somatic and the reproductive growth of U. triangularis are under the influence of inhibitory principles from the eyestalks. It is also revealing that mere deprival of the inhibitory principles does not culminate into successful vitellogenesis. Arguably, the inhibitory influence from the eyestalks could be a prerequisite for normal healthy maturation of the oocytes and spawning.

Effect of bilateral eyestalk ablation on ovarian development and moulting in early and late intermoult stages of female spiny lobster Panulirus homarus (Linnaeus, 1758)

The present study describes the effect of bilateral eyestalk ablation (BESA) on reproduction and moulting of spiny lobster Panulirus homarus females in their early and late intermoult stages. The lobsters obtained from the wild were conditioned for the experiment. The experiments were conducted at the Calicut research centre of Central Marine Fisheries Research Institute, India. Eyestalk ablation of females was done by ligation. The responses of the bilaterally eyestalk-ablated lobsters were statistically analysed. BESA conducted on the early and late intermoult stages resulted in the simultaneous acceleration of the somatic growth and reproductive processes with higher emphasis for oogenesis in lobsters ablated in the early intermoult phase and comparatively lower activity in those ablated in the late intermoult phase. Ablation in late intermoult phase resulted in faster entry into the premoult stage compared to the control.

Impact of eyestalk ablation on androgenic gland activity in the freshwater crab Travancoriana schirnerae Bott, 1969 (Decapoda: Gecarcinucidae)

The current study investigated the impact of eyestalk ablation on androgenic gland activity in the freshwater crab Travancoriana schirnerae Bott, 1969 (Decapoda: Gecarcinucidae). Bilateral eyestalk ablation during active phase (March-June) induced hypertrophy and hyperplasia of the gland as evidenced by increased width of the gland, thickness of the connective tissue layer, increase in cell and nuclear diameter with predominance of type I cells. This hypertrophy and hyperplasia was not followed by secretory activity during active phase which possibly suggests the non-functional nature of hypertrophy and hyperplasia. Unilateral destalkation during active phase neither caused hypertrophy nor hyperplasia of the gland cells probably because these crabs may still receive the inhibitory principle from the remaining intact eyestalk causing the gland to remain analogous to that of the unablated controls. The androgenic gland of control crabs during revival phase (January-February) was discerned with a few patches of proliferated type I cells scattered among vacuoles and degenerating cells. The most prominent feature of the gland of bilaterally ablated crabs during revival phase was the extensive proliferation of type I cells. Not much significant change was observed in the morphology and histology of the gland of unilaterally ablated crabs of revival phase.

13-Cis-Retinoic Acid-Induced Hyperglycemia in the Fresh Water Edible Crab, Oziothelphusa Senex Senex is mediated by Triggering Release of Hyperglycemic Hormone from Eyestalks

The present study was aimed to investigate the effect of 13-cis-retinoic acid (13-CRA) on hemolymph glucose levels in the fresh water edible crab, Oziothelphusa senex senex. Injection of 13-CRA significantly increased hemolymph glucose levels in a dose-dependent manner in intact crabs. Bilateral eyestalk ablation (ESX) resulted in significant decrease in hemolymph glucose levels. Injection of 13-CRA in to ESX crabs did not cause any significant changes in hemolymph glucose level as compared to ESX crabs suggesting that the effect of 13-CRA could be on the neuroendocrine system in the eyestalks increasing secretion of hyperglycemic hormone. To test this hypothesis, eyestalks were collected from control and 13-CRA injected crabs, and tested for hyperglycemic effect and also for the hyperglycemic hormone levels. The levels of hyperglycemic hormone and the hyperglycemic effect were significantly low in the eyestalks collected from 13-CRA injected crabs when compared with eyestalks from control crabs. From results, it is hypothesized that 13-CRA-induced hyperglycemia in the crab, O. senex, is mediated by triggering the release of hyperglycemic hormone from the eyestalk.

Growth and Survival of Bilateral Eyestalk Ablated Small Male Freshwater Prawn Macrobrachium rosenbergii (DeMan)

The freshwater prawn Macrobrachium rosenbergii exhibits three male morphotypes: Blue Claw (BC) male, Orange Claw (OC) male and Small Male (SM). Effects of bilateral eyestalk ablation on growth and survival of SM freshwater prawn were determined in monosex culture. The SM juveniles (9 g) were used in treatment bilateral eyestalk ablation and control (intact/unablated) at a hatchery. Ablated prawns were stocked at 5 prawn m-2 for 80 days. At the end of the experimental period, the average weight increased from 8.96±1.06 to 40.95±4.01 g, with bilaterally ablated prawns attaining the greatest growth. The final body weight of prawn at harvest was 40.95± 4.01 g in bilateral ablated SM and 22.69±2.41 g in unablated SM. The specific growth rate of bilaterally ablated prawns (1.90%) was significantly higher than unablated prawns (1.15%). The growth trend of SM prawn was increased significantly from the controls. The wet weight of ablated prawns doubled compared to the control but yield was not significantly different due to mortality. The survival of bilaterally ablated prawn was significantly low 50%. The potency of bilateral eyestalk ablation in enhancing growth rates may be limited due to high mortalities of ablated prawn.

Mode of action of dopamine in inducing hyperglycemia in the fresh water edible crab, Oziothelphusa senex senex.

The objective of this study was to investigate the mode of action of dopamine in regulating hemolymph sugar level in the fresh water edible crab, Oziothelphusa senex senex. Injection of dopamine produced hyperglycemia in a dose-dependent manner in intact crabs but not in eyestalkless crabs. Administration of dopamine resulted in a significant decrease in total carbohydrates and glycogen levels with a significant increase in glycogen phosphorylase activity levels in hepatopancreas and muscle of intact crabs, indicating dopamine-induced glycogenolysis resulting in hyperglycemia. Bilateral eyestalk ablation resulted in significant increase in the total carbohydrates and glycogen levels with a significant decrease in the activity levels of phosphorylase in the hepatopancreas and muscle of the crabs. Eyestalk ablation resulted in significant decrease in hemolymph hyperglycemic hormone levels. The levels of hyperglycemic hormone in the hemolymph of dopamine injected crabs were significantly higher than in control crabs. However, no significant changes in the levels of hemolymph hyperglycemic hormone and sugar and tissue carbohydrate and phosphorylase activity were observed in dopamine injected eyestalk ablated crabs when compared with eyestalk ablated crabs. These results support an earlier hypothesis in crustaceans that dopamine acts as a neurotransmitter and induces hyperglycemia by triggering the release of hyperglycemic hormone in the crab, O. senex senex.

Dynamics of Vitellogenin and Vitellogenesis-Inhibiting Hormone Levels in Adult and Subadult Whiteleg Shrimp, Litopenaeus vannamei: Relation to Molting and Eyestalk Ablation1

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.

Alterations of pattern in immune response and vitellogenesis during induced ovarian development by unilateral and bilateral ablation in Litopenaeus vannamei

Eyestalk ablation is often used to induce ovarian development in female shrimp, but it possibly alters the animal’s hormonal balance, and affects its physiology and immune system. Therefore, this study was conducted to investigate alterations to the immune response during induced ovarian development by unilateral and bilateral ablation in Litopenaeus vannamei. Sub-adult female shrimp could be induced to undergo ovarian development over a 20-day period by both unilateral and bilateral eyestalk ablation; however, ovaries did not reach the final mature stage in which cortical rods are observed. In terms of immune response, total hemocyte count decreased significantly at 4 h after unilateral ablation, and at 20 days after bilateral ablation. After 20 days, expression in the hemocytes of the immune-related genes prophenoloxidase and peroxinectin in bilaterally ablated shrimp increased significantly, whereas that for penaeidin, for lipopolysaccharide- and β-glucan-binding protein, and for transglutaminase mRNA expression did not change significantly. The results of this study suggest that, while eyestalk ablation induces ovarian development, it may also bring about a hormonal imbalance that leads to immune-related genes being activated.