X-organ Sinus Gland Complex Research Articles

Immunocytochemical Localization of Enzymes Involved in Dopamine, Serotonin, and Acetylcholine Synthesis in the Optic Neuropils and Neuroendocrine System of Eyestalks of Paralithodes camtschaticus.

Identifying the neurotransmitters secreted by specific neurons in crustacean eyestalks is crucial to understanding their physiological roles. Here, we combined immunocytochemistry with confocal microscopy and identified the neurotransmitters dopamine (DA), serotonin (5-HT), and acetylcholine (ACh) in the optic neuropils and X-organ sinus gland (XO-SG) complex of the eyestalks of Paralithodes camtschaticus (red king crab). The distribution of Ach neurons was studied by choline acetyltransferase (ChAT) immunohistochemistry and compared with that of DA neurons examined in the same or adjacent sections by tyrosine hydroxylase (TH) immunohistochemistry. We detected 5-HT, TH, and ChAT in columnar, amacrine, and tangential neurons in the optic neuropils and established the presence of immunoreactive fibers and neurons in the terminal medulla in the XO region of the lateral protocerebrum. Additionally, we detected ChAT and 5-HT in the endogenous cells of the SG of P. camtschaticus for the first time. Furthermore, localization of 5-HT- and ChAT-positive cells in the SG indicated that these neurotransmitters locally modulate the secretion of neurohormones that are synthesized in the XO. These findings establish the presence of several neurotransmitters in the XO-SG complex of P. camtschaticus.

Relative expression of three key genes involved in the hormonal cycle of the freshwater amphipod, Gammarus pulex.

Ecdysteroids and sesquiterpenoids are the two groups of hormones controlling molt and growth processes in amphipods, and are mostly represented by ecdysone (i.e., 20-hydroxyecdysone) and methyl farnesoate (MF), respectively. The endocrine system responsible for their syntheses is located in the cephalothorax and is composed of three main organs: the X-organ/sinus gland complex (XO), the Y-organ (YO) and the mandibular organ. Ecdysone synthesis is negatively controlled by the molt-inhibiting hormone (MIH) produced by XO, and its signal is mediated through the ecdysteroid receptor (EcR); whereas the MF production is limited by the farnesoic acid O-methyltransferase enzyme (FAMeT). As little is known on MIH, EcR, and FAMeT in amphipods, this study focused on the evaluation of the expression variations of the gene of these three proteins in Gammarus pulex, during the embryonic development and the molt cycle of females. Results highlighted the presence of ecr and famet genes from the first stages of the embryogenesis, suggesting key roles in the embryo development. The mih gene was only measured from Stage 3 of embryogenesis, probably related to the gastrulation and the cephalothorax development. Regardless of the gene, a strong overexpression was also measured at the hatch time. Besides, differential expression levels of mih, ecr, and famet genes through the molt cycle were observed. The highest expressions of the three genes were measured at the premolt stage, confirming key roles of MIH and EcR in the ecdysteroid pathways, and also suggesting the involvement of the FAMeT enzyme during the ecdysis in G. pulex.

Molecular aspects of eye development and regeneration in the Australian redclaw crayfish, Cherax quadricarinatus

The compound eye evolved over 500 million years ago and enables mosaic vision in most arthropod species. The molecular regulation of the development of the compound eye has been primarily studied in the fruit fly Drosophila melanogaster. However, due to the nature of holometabolous insects halting growth after their terminal metamorphosis into the adult form, they lack the capacity to regenerate. Crustaceans, unlike holometabolous insects, continue to grow during adulthood, achieved through regular shedding of their exoskeleton, in a cyclic process known as molting. This therefore offers crustaceans as a highly suitable model to study ocular regeneration in the adult arthropod eye. We have assessed the regenerative capacity of the retinal section of the Cherax quadricarinatus (red-claw crayfish) eye, following ablation and successive post-metamorphic molts. This work then provides a transcriptomic description of the outer, pigmented retinal tissue (the ommatidia and lamina ganglionaris) and the basal, non-pigmented neuroendocrine ocular tissue (the X-organ Sinus Gland complex, hemiellipsoid body and optic nerve). Using comparative analysis, we identified all the transcripts in the C. quadricarinatus ocular transcriptome that are known to function in compound eye development in D. melanogaster. Differentially and uniquely transcribed genes of the retina are described, suggesting proposed mechanisms that may regulate ocular regeneration in decapod Crustacea. This research exemplifies the application C. quadricarinatus holds as an optimal model to study the regulation of ocular regeneration. Further in-depth transcriptomic analyses are now required, sampled throughout the regeneration process to better define the regulatory mechanism.

Quantitative analysis of hypertrophy and hyperactivity in the androgenic gland of eyestalk-ablated male Pacific white shrimp Litopenaeus vannamei during molt stages

Quantitative analysis of hypertrophy and hyperactivity in tissue of the androgenic gland of eyestalk-ablated male Pacific white shrimp Litopenaeus vannamei was made during the molt cycle by measuring the area of androgenic gland cells located in the subterminal region of the terminal ampoule and measuring the expression of the insulin-like androgenic gland hormone precursor gene (Lv-IAG) in the distal vas deferens and terminal ampoule, respectively. Data were compared to intact shrimp at the same molt stages. In eyestalk-ablated shrimp, the lack of negative control from the X-organ–sinus gland complex produced hypertrophy in androgenic gland cells, because the area of the cells folded approximately 1.5 times up to the intermolt stage, when compared to the intact group. It also produced hyperactivity because the relative expression of Lv-IAG was significantly higher than the intact group at all molt stages. After gathering data from the vas deferens and terminal ampoule, the relative expression of Lv-IAG was maintained at higher levels, without a significant difference during the molt stages. However, after each tissue was analyzed independently, a bell-shape curve pattern occurred, according to the molt cycle, as also occurred in intact shrimp. This suggests that in the absence of eyestalks, the brain and nervous system are operating secondarily to synchronize Lv-IAG expression into the molt cycle. The relative expression of Lv-IAG was significantly greater in the terminal ampoule than in the distal vas deferens. In summary, these findings support a reproductive endocrine axis in L. vannamei, as an eyestalk–androgenic gland linkage concomitant with the molt cycle.

A novel localization of molt-inhibiting hormone in the tegumental glands of shrimp Penaeus monodon and its possible role in shrimp molting

Abstract Molt-inhibiting hormone (MIH) is commonly localized in the X-organ sinus gland complex (XOSG) of the crustacean. The present study aimed to elucidate the expression of MIH at the subcellular and cellular levels in the eyestalks and integument of juvenile Penaeus monodon during the molting cycle. Gene expression of Pem-MIH1 in the optic lobes showed a single PCR product at 172 bp, and was restricted only to the eyestalk XOSG but not detected in pleopods, cephalothorax integument, muscle, hepatopancreas or retina. Quantitative analysis of gene expression in the eyestalks demonstrated no significant alteration of Pem-MIH1 mRNA in XOSG during the molt cycle. Immunohistochemistry using antibody against recombinant molt-inhibiting hormone-like peptide (anti-MIH-like) revealed variable staining intensities of individual MTXOs but were most persistently intense in SGs. During the molt cycle, the mean numbers of XO immunoreactive cells were slightly oscillated but not statistically different. The quantitative immunohistochemistry measured from XOSG illustrated minimal fluctuations of the values obtained suggesting the periodical synthesis and release of MIH from XOSG system during the molt cycle. The positive reaction was also detected in the tegumental glands located at the retina and the integument of walking leg, base of eyestalk and cephalothorax of the shrimp. The large variations of immunostaining and amount of reactive tegumental glands in the eyestalk were noted throughout the molt cycle. The numbers of the MIH-like immunoreactive glands obtained from the retina as well as the integument base did not significantly change during the molt cycle but tend to increase during postmolt to intermolt and decrease during premolt. These results suggest that the MIH-like peptides in the tegumental glands are probably released mostly during the late premolt. Our findings thus propose the novel storage site of MIH besides SG of the eyestalk including a new target tissue, an epidermal cell, the role of which may link to the complicated molting regulation and/or the dark-adaptation of shrimp.

In vivo and in vitro inhibitory action of 17β-estradiol and environmental estrogen 4-nonylphenol on gonad-inhibiting hormone (GIH) expression in the eyestalks of Litopenaeus vannamei.

The gonad-inhibiting hormone (GIH) belongs to a neuropeptide family synthesized and released in an X-organ sinus gland complex of crustacean eyestalks. GIH inhibits crustacean ovarian maturation by suppressing vitellogenin (Vtg) synthesis, whereas estrogen is responsible for the stimulation of vitellogenesis (not established). In this study, the effects of 17β-estradiol (E2, 10(-6) M), estrogen receptor antagonist tamoxifen (TAM, 10(-6), 10(-7), and 10(-8) M), and the environmental estrogen nonylphenol (NP, 1 μg/L and 100 μg/L) on LvGIH expression in the eyestalks of shrimp were determined by quantitative real-time PCR. Results showed that LvGIH expression decreased significantly during the L. vannamei ovarian maturation cycle. E2 and NP significantly reduced LvGIH transcripts in vivo, but TAM neutralized the inhibitory action of E2 in a dose-dependent manner (P < 0.05). In addition, the LvGIH expression levels decreased significantly in a time-dependent manner (P < 0.05) when ovary fragments were cultured in vitro with E2. The results of this study suggested that estrogen regulates GIH expression in L. vannamei eyestalks. E2 promoted ovarian development not only by directly upregulating vitellogenesis in the hepatopancreas, but it was also capable of downregulating LvGIH expression, which indirectly resulted in the stimulation of L. vannamei vitellogenesis.

Cadmium inhibits molting of the freshwater crab Sinopotamon henanense by reducing the hemolymph ecdysteroid content and the activities of chitinase and N-acetyl-β-glucosaminidase in the epidermis

Molting is an essential process during the growth of crustaceans, which is coordinated by ecdysteroids secreted by the Y-organ, molting inhibiting hormone secreted by the X-organ sinus-gland complex, as well as chitinase and N-acetyl-β-glucosaminidase synthesized by the epidermis. Cadmium is one of the toxic metals in the aquatic environment. However, the endocrine effects of cadmium on the molting of freshwater crabs and the underlying mechanisms are unknown. To investigate these, freshwater crabs (Sinopotamon henanense) were acutely exposed to 0, 7.25, 14.5 and 29mg/l Cd for 3, 4, 5days or in some experiments for 4days after eyestalk-ablation. The concentration of hemolymph ecdysone and the activities of the molting enzymes chitinase and NAG were measured. Histological changes in the epidermal tissues were documented. Our results showed that eyestalk ablation increased the ecdysteroid content as well as the activities of chitinase and NAG, which were inhibited by cadmium in a concentration-dependent manner; histological examinations demonstrated that eyestalk ablation produced storage particles in the epidermal tissues, which was also reduced by cadmium in a concentration-dependent manner. Our data suggest that cadmium disrupts endocrine function through inhibiting the secretion of ecdysteroids by the Y-organ and altering with the regulation of chitinase and NAG activity in the epidermis. This work provides new insights into the mechanisms underlying the molting inhibition effect of cadmium on the crabs.

Cloning of the crustacean hyperglycemic hormone gene promoter of Astacus leptodactylus

The crustacean Hyperglycemic Hormone (cHH) belongs to a family of neuropeptides synthesized and released by the X-organ sinus gland complex, which is located in the eyestalks. The cHH is a pleiotropic hormone regulating many physiological processes like glucose metabolism, molting, and reproduction. We cloned a 176 bp fragment from the transcription starting site of the cHH gene of Astacus leptodactylus Eschscholtz, 1823. The identified sequence contains several putative core transcription factor binding sites including a TATA box, a CCAAT box, a GC box, as well as binding sites for a hypoxia responsive element, estrogen receptor, and ecdysone receptor. To assess promoter functionality the promoter sequence was ligated to an expression vector bearing a luciferase reporter gene, and promoter activity was recorded using a luciferase expression assay, which showed that the cloned promoter was functional, being able to drive the expression of the reporter gene.

Changes in crustacean hyperglycemic hormones in Pacific whiteleg shrimp Litopenaeus vannamei subjected to air-exposure and low-salinity stresses

Changes in crustacean hyperglycemic hormone (CHH)-family peptides in response to stress were investigated in Litopenaeus vannamei. Stress treatments consisted of air exposure and low salinity. High-performance liquid chromatography was used to quantify CHH-family peptides in the X-organ–sinus gland complex (XO–SG) in the eyestalks. Among the CHH-family peptides analyzed, only the level of sinus gland peptide-G (SGP-G) in the XO–SG was decreased. SGP-G was also detectable by Western blotting analysis in the hemolymph of animals subjected to stress. These results suggest that SGP-G was secreted from the XO–SG into the hemolymph during stress. Glucose levels in the hemolymph increased under conditions during which SGP-G was detected in the hemolymph. Hyperglycemia was also observed when SGP-G was injected. SGP-G may function to shift energy use to deal with stress.

The Effects of Eyestalk Ablation on the Reproductive and Immune Function of Female Macrobrachium americanum

Eyestalk ablation is the most common procedure used to induce crustacean reproduction in commercial hatcheries. However, other physiological and metabolic processes are affected by the removal of the X-organ sinus gland complex located in the eyestalk. In this study, the effect of unilateral eyestalk ablation on the concentration of several hemolymph metabolites, reproduction and immune function in female Macrobrachium americanum was investigated. Molting cycle time was not significantly (P=0.17) shortened in eyestalk-ablated females. However, an increased number of molts was observed in the eyestalk-ablated group because survival was significantly higher due to increased passivity and lowered aggression. As M. americanum has closed thelyca, reproduction was not accelerated after eyestalk ablation compared to control prawns. Food intake (P=0.007) and oxygen consumption (P=0.047) were both higher in eyestalk-ablated females. Concentrations of protein and glucose in hemolymph were not affected (P=0.54 and P=0.19, respectively), indicating that the demand for these metabolites was met. Glucose was metabolized by aerobic metabolism as shown by the lower lactate concentration (P=0.02) and higher respiration rate after ablation. Protein was metabolized for growth as revealed by the 100% higher growth rate in eyestalkablated females (P=0.017). Triglycerides were more highly concentrated (P=0.02) in eyestalk-ablated females. This difference was most likely due to being transported from the digestive gland to the gonad.By analyzing the immune system it was observed that only the total hemocyte count was higher (P=0.002) in eyestalk-ablated females. The protein content in hemocytes, the prophenol oxidase system and the coagulation time were not affected. In conclusion, the eyestalk ablation procedure did not cause female M. americanum to have more reproductive events over time. Despite this failure to accelerate reproduction, eyestalk ablation reduced aggression, and thus increased survival, and promoted superior growth. This result suggests that eyestalk ablation is promising for the cultivation of M. americanum.

Induction of Ovarian Maturation and Spawning in Penaeus monodon Broodstock by Double-Stranded RNA

Ovarian maturation in crustacean is under the control of gonad-inhibiting hormone (GIH); a neuropeptide secreted from X-organ sinus gland complex in eyestalks. Unilateral eyestalk ablation that partially destroys GIH source is therefore a general practice in Penaeus monodon hatchery to induce ovarian maturation and spawning. Our previous report showed that silencing of GIH expression by GIH-specific double-stranded RNA (GIH-dsRNA) resulted in an increased expression level of vitellogenin in P. monodon, thus suggesting that GIH-dsRNA could be an alternative method to induce ovarian maturation in female P. monodon broodstock. In this study, we further demonstrated that a single injection of GIH-dsRNA into previtellogenic female P. monodon at the concentration of 3µg GIH-dsRNA per gram body weight of shrimp was able to inhibit GIH expression for a minimum of 30days. This dsRNA-mediated GIH silencing led to ovarian maturation and eventual spawning in both domesticated and wild female broodstock, particularly with a comparable effect to eyestalk ablation in wild shrimp. This is the first report that demonstrates a potential strategy to induce ovarian maturation in female P. monodon broodstock by GIH-dsRNA and thus provides a possible substitute for the cruel and detrimental eyestalk ablation practice.

CIRCADIAN MODULATION OF CRUSTACEAN HYPERGLYCEMIC HORMONE IN CRAYFISH EYESTALK AND RETINA

Previous studies suggested the retina could be a putative locus of daily crustacean hyperglycemic hormone (CHH) secretion, as it possesses its own metabolic machinery and is independent of the well-known CHH eyestalk locus responsible for the circadian secretion of this peptide. However, it has been proposed that hemolymph glucose and lactate concentrations play a dual role in the regulation of CHH in crayfish. To elucidate the temporal relationship between these two different CHH production loci and to examine their relationship with glucose regulation, we investigated the expression of CHH daily and circadian rhythms in the eyestalk and retina of crayfish using biochemical methods and time series analysis. We wanted to determine whether (1) putative retina and eyestalk CHH rhythmic expressions are correlated and if the oscillations of the two metabolic products of lactate and glucose in the blood due to CHH action on the target tissue correlate, and (2) retina CHH (RCHH) and the possible retinal substrate glycogen and its product glucose are temporally correlated. We found a negative correlation between daily and circadian changes of relative CHH abundance in the retina and eyestalk. This correlation and the cross-correlation values found between eyestalk CHH and hemolymph and glucose confirm that CHH produced by the X-organ sinus gland complex is under the previously proposed dual feedback control system over the 24 h time period. However, the presence of both glycogen and glucose in the retina, the cross-correlation values found between these parameters and hemolymph lactate and glucose, as well as RCHH and hemolymph and retina metabolic markers suggest RCHH is not under the same temporal metabolic control as eyestalk CHH. Nonetheless, their expression may be linked to common rhythms-generating processes. (Author correspondence: mlfm@hp.fciencias.unam.mx; mfajul@gmail.com)

Neuroendocrine and metabolic responses of Pacific whiteleg shrimp Litopenaeus vannamei exposed to acute handling stress

The neuroendocrine mechanisms mediating stress in crustaceans are different and less understood than those of vertebrates, although indirect evidence suggests the participation of catecholamines (CA). However, few studies have analyzed their levels during stress response of crustaceans. We examined hemolymph and tissue levels of catecholamines and other biochemical indicators, in response to handling stress in whiteleg shrimp Litopenaeus vannamei. Shrimp were stressed by transfer, chasing, and confinement and the responses were analyzed at several intervals (10–240 min). Lactate levels in hemolymph increased from 10 to 30 min, glucose increased at 60 min, and both returned to baseline levels by 240 min. An increase in lactate levels before hyperglycemia suggests a possible activation of gluconeogenesis as a main clearance pathway for lactate. Epinephrine increased in hepatopancreas from 10 to 20 min, which indicates it could mediate lactate uptake, glucogenogenesis, and lipid catabolism in this organ. Norepinephrine levels in the eyestalk decreased by 92% between 20 and 30 min and a concomitant 10-fold increase in levels of dopamine was observed. This response suggested that the putative norepinephrine release in the X-organ sinus gland complex could mediate lactate effects in this organ (e.g. lactate positive feedback of crustacean hyperglycemic hormone release).

Effect of unilateral and bilateral eyestalk ablation in Litopenaeus vannamei male and female on several metabolic and immunologic variables

Eyestalk ablation is the most common procedure to induce gonadic maturation in commercial hatcheries of penaeid shrimp. In addition to reproduction, other physiological and metabolic processes are affected by removal of the X-organ sinus gland complex located in the eyestalk. In this study, the effect of unilateral and bilateral eyestalk ablation on the concentration of several hemolymph metabolites and phenoloxidase system in female and male shrimp was investigated. As a consequence of reducing or suppressing molt-inhibiting hormone (MIH) production, the duration of the molting cycle was significantly shorter in eyestalk-ablated shrimp: bilaterally (10 days), unilaterally (17 days), and shrimp that were not ablated (24 days). Mortality was significantly higher in unilaterally (35%) and bilaterally (68%) ablated shrimp than in untreated shrimp (2%), probably caused by impairment of several physiological functions mediated by hormones from the eyestalk and direct injury of the nervous system. Males and females were affected differently by eyestalk ablation in terms of concentrations of glucose, triglycerides, and protein in hemolymph. Glucose and lactate levels were lower in bilaterally ablated shrimp, as expected by the role of crustacean hyperglycemic hormone in glucose metabolism. Cholesterol and hemocyte count were not significantly different among the three treatments. Prophenoloxidase and phenoloxidase activities were significantly lower in bilaterally, but not in unilaterally ablated shrimp. This could suggest an endocrine control of this mechanism of the effector immune response or reflect the level of physiological trauma caused by bilateral eyestalk ablation in this species.

Serotonin modulation of hemolymph glucose and crustacean hyperglycemic hormone titers in Fenneropenaeus indicus

Crustacean hyperglycemic hormone (CHH), a neurohormone synthesized and released from the X-organ sinus gland complex, is primarily involved in carbohydrate metabolism. Biogenic amines and peptidergic neuroregulators are known to modulate the release of CHH. Although mechanisms involved in hormone release in the shrimp, Fenneropenaeus indicus is poorly understood. Daily variations in the levels of crustacean hyperglycemic hormone and serotonin in the shrimp F. indicus were studied. Both CHH and glucose levels increase during the first hours after the beginning of darkness. The animals were injected with serotonin and glycemic and CHH levels were determined after 2 h of injection. Serotonin caused significant hyperglycemia ( P < 0.0.5) in both intact and eyestalk ablated shrimp. These results support the hypothesis that serotonin produces hyperglycemia in crustaceans by triggering release of hyperglycemic hormone both from the eyestalk and from the other sites of its release.

Search for hepatopancreatic ecdysteroid-responsive genes during the crayfish molt cycle: from a single gene to multigenicity

The expression of the vitellogenin gene of the red-claw crayfish Cherax quadricarinatus (CqVg) was previously demonstrated in male crayfish during an endocrinologically induced molt cycle. The hypothesis that this expression is under the direct control of ecdysteroids was tested in this study both in vivo and in vitro. Unlike vitellogenin of insects, CqVg was not found to be ecdysteroid-responsive. Thus, a multigenic approach was employed for the identification of other hepatopancreatic ecdysteroid-responsive genes by a cDNA microarray. For the purposes of this study, a multi-parametric molt-staging technique, based on X-ray detection of gastrolith growth, was developed. To identify ecdysteroid-responsive genes during premolt, the molt cycle was induced by two manipulations, 20-hydroxyecdysone administration and X-organ-sinus gland complex removal; both resulted in significant elevation of ecdysteroids. Two clusters of affected genes (129 and 122 genes, respectively) were revealed by the microarray. It is suggested that only genes belonging to similarly responsive (up- or downregulated) gene clusters in both manipulations (102 genes) could be considered putative ecdysteroid-responsive genes. Some of these ecdysteroid-responsive genes showed homology to genes controlling chitin metabolism, proteases and other cellular activities, while 56.8% were unknown. The majority of the genes were downregulated, presumably by an energetic shift of the hepatopancreas prior to ecdysis. The effect of 20-hydroxyecdysone on representative genes from this group was confirmed in vitro using a hepatopancreas tissue culture. This approach for ecdysteroid-responsive gene identification could also be implemented in other tissues for the elucidation of ecdysteroid-specific signaling pathways during the crustacean molt cycle.

Molecular mechanisms regulating molting in a crustacean

Crustacean growth and development is characterized by periodic shedding (ecdysis) and replacement of the rigid exoskeleton. Secretions of the X-organ sinus gland complex control the cellular events that lead to growth and molting. Western blot and ELISA results showed a progressive increase in growth arrest-specific protein (Gas7) from early postmolt stage to a maximum at late postmolt stage. Phosphorylation of ERK2, a downstream signaling protein, was also identified in the subsequent stages. ERK2 phosphorylation resulted in the expression of molt-inhibiting hormone (MIH). Specific ERK inhibitors (PD98059 and UO126) exhibited the ability to reduce the molting duration of Fenneropenaeus indicus from 12-14 days to 7-8 days, suggesting that the ERK1/2 signaling pathway is responsible for the expression of MIH, which controls the molt cycle. We have identified the stage-specific expression of Gas7 (approximately 48 kDa) in the X-organ sinus gland complex of eyestalk which is involved in the downstream signaling of the ERK1/2 pathway regulating the expression of MIH during the molt cycle of the white shrimp, F. indicus. These are the first data showing an association between the Gas7 signal-transduction process and regulation of the molt cycle and provides an alternative molecular intervention mechanism to the traditional eyestalk ablation in crustaceans.

Involvement of norepinephrine in the hyperglycemic responses of the freshwater giant prawn, Macrobrachium rosenbergii, under cold shock

Hyperglycemic response of freshwater giant prawn, Macrobrachium rosenbergii, under acute cold shock was investigated, and the involvement and stimulatory pathways of norepinephrine (NE) on induced-glycemia were further examined. Remarkable elevations in hemolymph glucose at comparable magnitude were observed in both intact and eyestalkless prawn under cold treatments, suggesting that hyperglycemic response of this species is not solely mediated through the actions of crustacean hyperglycemic hormone released from X-organ sinus gland complex on the target tissues, but NE is involved. Positive and significant correlations were noted between the hemolymph glucose titers and NE contents in both thoracic ganglia and the hemolymph, suggesting that NE plays a significant role in the hyperglycemic responses of this species under cold. Depressive effects of various adrenoceptor antagonists monitored in vivo and in vitro further suggest that the action of NE is primarily mediated through both α 1- and β 1-adrenoceptors.

Expression of vitellogenin and cortical rod proteins during induced ovarian development by eyestalk ablation in the kuruma prawn, Marsupenaeus japonicus

In penaeid shrimp species, ovarian development is characterized by the accumulation of a major yolk protein (vitellin) and the formation of cortical rods in the oocytes. The process is considered to be under the control of a neuroendocrine organ in the eyestalk (the X-organ sinus gland complex). In the present study, the synthesis of vitellogenin (VTG, precursor of vitellin) and two kinds of cortical rod proteins (cortical rod protein, CRP; thrombospondin, MjTSP) was induced by bilateral eyestalk ablation (removal of the X-organ sinus gland complex) in immature female kuruma prawn, Marsupenaeus japonicus, and the synthesis process was monitored over a 7-day period after the ablation. The ovarian weight and hemolymph VTG levels increased in the ablated females. The VTG mRNA levels in the ovary increased concomitantly with vitellin accumulation in the ovary after eyestalk ablation. On the other hand, the CRP and MjTSP protein levels in the ovary increased after eyestalk ablation, whereas the CRP and MjTSP mRNA levels in the ovary did not change concomitantly. The results suggest that the regulatory mechanism of gene expression by eyestalk hormones is different between VTG (transcriptional control) and CRP-MjTSP (translational control).

Complete sequence of Litopenaeus vannamei (Crustacea: Decapoda) vitellogenin cDNA and its expression in endocrinologically induced sub-adult females

The gene that encodes vitellogenin (Vg), the precursor of the major yolk protein, vitellin, is expressed during vitellogenesis in decapod crustaceans. In this study, we sequenced the full-length cDNA from the Pacific white shrimp Litopenaeus vannamei Vg gene ( LvVg). This is the first open thelycum penaeid shrimp Vg cDNA to be sequenced. The transcript encodes a 2587 amino acid polypeptide with up to 85% identity to Vg of different penaeid species. Peptide mass fingerprints (PMFs) of the vitelline polypeptides suggest that the predicted endoprotease cleavage site at amino acids 725–728 does indeed undergo cleavage. Five prominent high-density lipoprotein polypeptides of masses 179, 113, 78, 61, and 42 kDa were isolated from vitellogenic ovary, and their PMFs were determined by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) spectrometry. It is likely that these polypeptides are all products of the LvVg gene. Removal of the X-organ sinus gland complex (XO-SG), which secretes the neurohormones that control the endocrine system regulating molt and reproduction, can induce both these processes. During the course of a number of molt cycles in induced sub-adult females, periodic ovarian growth and resorption were observed. Ovary growth correlated with LvVg expression in both the hepatopancreas and the ovary. Expression in ovaries of induced intermolt–early premolt females was significantly higher compared to all other sub-groups. Expression in ovaries of induced females was significantly higher compared to hepatopancreas at all molt cycle stages. Periodicity of molt and vitellogenesis in endocrinologically induced sub-adult shrimps may serve as a model to study alternate regulation of gene expression during these two processes.