Diversity and evolution of serotonergic cells in taste buds of elasmobranchs and ancestral actinopterygian fish.

A subset of gustatory cells are serotonin immunoreactive (ir) in the mammalian taste bud. In the taste bud of lamprey, elongated gustatory-like cells are also serotonin-ir. In contrast, flattened serotonin-ir cells are located only in the basal region of the taste buds in the teleosts and amphibians. These serotonin-ir cells are termed as basal cells. To evaluate the evolution and diversity of serotonergic cells in the taste bud of amniote animals, we explored the distribution and morphology of serotonin-ir cells in the taste buds of ancestral actinopterygian fish (spotted gar, sturgeon, Polypterus senegalus) and elasmobranch (stingray). In all examined animals, the taste buds contained serotonin-ir cells in their basal part. The number of serotonin-ir basal cells in each taste bud was different between these fish species. They were highest in the stingray and decreased in the order of the Polypterus, sturgeon, and gar. While serotonin immunoreactivity was observed only in the basal cells in the taste buds of the ancestral actinopterygian fish, some elongated cells were also serotonin-ir in addition to the basal cells in the stingray taste buds. mRNA of tryptophan hydroxylase 1 (tph1), a rate-limiting enzyme of the serotonin synthesis, is expressed in both the elongated and basal cells of stingray taste buds, indicating that these cells synthesize the serotonin by themselves. These results suggest that the serotonin-ir basal cells arose from the ancestor of the cartilaginous fish, and serotonin-ir cells in the elasmobranch taste bud exhibit an intermediate aspect between the lamprey and actinopterygian fish.

Chemical synthesis of insulin-like peptide 1 and its potential role in vitellogenesis of the kuruma prawn Marsupenaeus japonicus.

The insulin superfamily comprises a group of peptides with diverse physiological functions and is conserved across the animal kingdom. Insulin-like peptides (ILPs) of crustaceans are classified into four major types: insulin, relaxin, gonadulin, and androgenic gland hormone (AGH)/insulin-like androgenic gland factor (IAG). Of these, the physiological functions of AGH/IAG have been clarified to be the regulation of male sex differentiation, but those of the other types have not been uncovered. In this study, we chemically synthesized Maj-ILP1, an ILP identified in the ovary of the kuruma prawn Marsupenaeus japonicus, using a combination of solid-phase peptide synthesis and regioselective disulfide bond formation reactions. As the circular dichroism spectral pattern of synthetic Maj-ILP1 is typical of other ILPs reported thus far, the synthetic peptide likely possessed the proper conformation. Functional analysis using ex vivo tissue incubation revealed that Maj-ILP1 significantly increased the expression of the yolk protein genes Maj-Vg1 and Maj-Vg2 in the hepatopancreas and Maj-Vg1 in the ovary of adolescent prawns. This is the first report on the synthesis of a crustacean ILP other than IAGs and also shows the positive relationship between the reproductive process and female-dominant ILP.

Characterization of the expression of gastrin-releasing peptide and its receptor in the trigeminal and spinal somatosensory systems of Japanese macaque monkeys: Insight into humans.

Gastrin-releasing peptide (GRP) and its receptor (GRPR) have been identified as itch mediators in the spinal and trigeminal somatosensory systems in rodents. In primates, there are few reports of GRP/GRPR expression or function in the spinal sensory system and virtually nothing is known in the trigeminal system. The aim of the present study was to characterize GRP and GRPR in the trigeminal and spinal somatosensory system of Japanese macaque monkeys (Macaca fuscata). cDNA encoding GRP was isolated from the macaque dorsal root ganglion (DRG) and exhibited an amino acid sequence that was highly conserved among mammals and especially in primates. Immunohistochemical analysis demonstrated that GRP was expressed mainly in the small-sized trigeminal ganglion and DRG in adult macaque monkeys. Densely stained GRP-immunoreactive (ir) fibers were observed in superficial layers of the spinal trigeminal nucleus caudalis (Sp5C) and the spinal cord. In contrast, GRP-ir fibers were rarely observed in the principal sensory trigeminal nucleus and oral and interpolar divisions of the spinal trigeminal nucleus. cDNA cloning, in situ hybridization, and Western blot revealed substantial expression of GRPR mRNA and GRPR protein in the macaque spinal dorsal horn and Sp5C. Our Western ligand blot and ligand derivative stain for GRPR revealed that GRP directly bound in the macaque Sp5C and spinal dorsal horn as reported in rodents. Finally, GRP-ir fibers were also detected in the human spinal dorsal horn. The spinal and trigeminal itch neural circuits labeled with GRP and GRPR appear to function also in primates.

Vasopressin-oxytocin-type signaling is ancient and has a conserved water homeostasis role in euryhaline marine planarians.

Vasopressin/oxytocin (VP/OT)-related peptides are essential for mammalian antidiuresis, sociosexual behavior, and reproduction. However, the evolutionary origin of this peptide system is still uncertain. Here, we identify orthologous genes to those for VP/OT in Platyhelminthes, intertidal planarians that have a simple bilaterian body structure but lack a coelom and body-fluid circulatory system. We report a comprehensive characterization of the neuropeptide derived from this VP/OT-type gene, identifying its functional receptor, and name it the "platytocin" system. Our experiments with these euryhaline planarians, living where environmental salinities fluctuate due to evaporation and rainfall, suggest that platytocin functions as an "antidiuretic hormone" and also organizes diverse actions including reproduction and chemosensory-associated behavior. We propose that bilaterians acquired physiological adaptations to amphibious lives by such regulation of the body fluids. This neuropeptide-secreting system clearly became indispensable for life even without the development of a vascular circulatory system or relevant synapses.

Transcriptomic Analysis of the Kuruma Prawn Marsupenaeus japonicus Reveals Possible Peripheral Regulation of the Ovary.

Crustacean reproduction has been hypothesized to be under complex endocrinological regulation by peptide hormones. To further improve our understanding of the mechanisms underlying this complex regulation, knowledge is needed regarding the hormones not only of the central nervous system (CNS) such as the X-organ/sinus gland (XOSG), brain, and thoracic ganglia, but also the peripheral gonadal tissues. For example, in vertebrates, some gonadal peptide hormones including activin, inhibin, follistatin, and relaxin are known to be involved in the reproductive physiology. Therefore, it is highly likely that some peptide factors from the ovary are serving as the signals among peripheral tissues and central nervous tissues in crustaceans. In this work, we sought to find gonadal peptide hormones and peptide hormone receptors by analyzing the transcriptome of the ovary of the kuruma prawn Marsupenaeus japonicus. The generated ovarian transcriptome data led to the identification of five possible peptide hormones, including bursicon-α and -ß, the crustacean hyperglycemic hormone (CHH)-like peptide, insulin-like peptide (ILP), and neuroparsin-like peptide (NPLP). Dominant gene expressions for the bursicons were observed in the thoracic ganglia and the ovary, in the CNS for the CHH-like peptide, in the heart for NPLP, and in the ovary for ILP. Since the gene expressions of CHH-like peptide and NPLP were affected by a CHH (Penaeus japonicus sinus gland peptide-I) from XOSG, we produced recombinant peptides for CHH-like peptide and NPLP using Escherichia coli expression system to examine their possible peripheral regulation. As a result, we found that the recombinant NPLP increased vitellogenin gene expression in incubated ovarian tissue fragments. Moreover, contigs encoding putative receptors for insulin-like androgenic gland factor, insulin, neuroparsin, and neuropeptide Y/F, as well as several contigs encoding orphan G-protein coupled receptors and receptor-type guanylyl cyclases were also identified in the ovarian transcriptome. These results suggest that reproductive physiology in crustaceans is regulated by various gonadal peptide hormones, akin to vertebrates.

Characterization of distinct ovarian isoform of crustacean female sex hormone in the kuruma prawn Marsupenaeus japonicus.

The eyestalk hormone, crustacean female sex hormone (CFSH), regulates the development of female secondary sexual characteristics in the blue crab Callinectes sapidus. After its discovery, several CFSH gene orthologs have been identified in some species of the suborder Pleocyemata as well. Similarly, in species of another suborder (Dendrobranchiata), an ortholog (Maj-CFSH) has been characterized as an eyestalk factor expressed in both females and males of the kuruma prawn, Marsupenaeus japonicus. In this study, another novel CFSH isoform was identified in the same species using cDNA cloning, expression analysis, and recombinant protein production. The isoform has "CFSH-family" structural characteristics but is dominantly expressed in the ovary, and was therefore designated as Maj-CFSH-ov. Its mRNA and protein levels in vitellogenic ovaries are higher than those in non-vitellogenic ovaries. In the vitellogenic ovary, both mRNA and protein expression of Maj-CFSH-ov are localized to oogonia and previtellogenic oocytes that occupy a small portion of vitellogenic ovaries, but not to the major developing oocytes. A vitellogenesis-inhibiting peptide of M. japonicus (Pej-SGP-I) reduced the expression of vitellogenin in incubated ovarian fragments, but not that of Maj-CFSH-ov. These results indicate that M. japonicus possesses two CFSH isoforms that are derived from distinct tissues, the central X-organ/sinus gland complex and peripheral ovaries. The expression profile of Maj-CFSH-ov suggests its involvement in some reproductive process other than vitellogenesis.

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

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

Crystal structure of a crustacean hyperglycemic hormone (CHH) precursor suggests structural variety in the C-terminal regions of CHH superfamily members.

The crustacean hyperglycemic hormone (CHH) is one of the major hormones in crustaceans, and peptides belonging to the CHH superfamily have been found in diverse ecdysozoans. Although the basic function of CHH is to control energy metabolism, it also plays various roles in crustacean species, such as in molting and vitellogenesis. Here, we present the crystal structure of Pej-SGP-I-Gly, a partially active precursor of CHH from the kuruma prawn Marsupenaeus japonicus, which has an additional Gly residue in place of the C-terminal amide group of the mature Pej-SGP-I. The 1.6-angstrom crystal structure showed not only the common CHH superfamily scaffold comprising three α-helices, three disulfide bridges, and a hydrophobic core but also revealed that the C-terminal part has a variant backbone fold that is specific to Pej-SGP-I-Gly. The α-helix 4 of Pej-SGP-I-Gly was much longer than that of molt-inhibiting hormone (Pej-MIH) from the same species, and as a result, the following C-terminal helix, corresponding to α-helix 5 in MIH, was not formed. Unlike monomeric Pej-MIH, Pej-SGP-I-Gly forms a homodimer in the crystal structure via its unique α-helix 4. The unexpected dissimilar folds between Pej-SGP-I-Gly and Pej-MIH appear to be the result of their distinct C-terminal amino acid sequences. Variations in amino acid sequences and lengths and the resulting variety of backbone folds allow the C-terminal and sterically adjoining regions to confer different hormonal activities in diverse CHH superfamily members. DATABASE: Structural data are available in the PDB under the accession number 5B5I.

Seasonal variations in reproductive activity of the blue crab, Callinectes sapidus: Vitellogenin expression and levels of vitellogenin in the hemolymph during ovarian development.

In general, season affects the physiology and behavior of most animals. Warmer temperatures accelerate growth and reproduction of ectotherms, whereas these processes are slowed or halted in colder temperatures. Female blue crabs, Callinectes sapidus inhabiting the Chesapeake Bay, exhibit a seasonal migratory behavior that is closely tied with spawning and the release of larvae. To better understand reproductive activities of the migratory adult females, we examined two reproductive parameters of these crabs sampled monthly (April-December, 2006): the levels of vitellogenin (VtG) in the hemolymph and VtG expression in the hepatopancreas and ovary. The full-length cDNA of VtG (CasVtG-ova) has been isolated from the ovary. The putative CasVtG sequence found in the ovary is >99% identical to that of the hepatopancreas and is related most closely to the sequences reported in other crab species. In female C. sapidus, the hepatopancreas produces over 99% of the total VtG toward the ovarian development. Ovarian stages 2 and 3 in the sampled females are characterized by significant high levels of VtG in hemolymph and VtG expression in both the hepatopancreas and ovary. However, during the southbound migration in fall, females at ovarian stages 2 and 3 have decreased VtG levels, compared to those in spring and summer. The decreased vitellogenesis activity during the fall migration suggests seasonal adaptation to ensure successful spawning and the larval release.

Direct evidence for the function of crustacean insulin-like androgenic gland factor (IAG): total chemical synthesis of IAG.

Insulin-like androgenic gland factor (IAG) is presumed to be a sex differentiation factor so-called androgenic gland hormone (AGH) in decapod crustacean, although the function of IAG peptide has not yet been reported. In this study, we synthesized IAG from the prawn, Marsupenaeus japonicus, and its function was assessed by an in vitro bioassay. As a result, IAG with the insulin-type disulfide bond arrangement showed biological activity, whereas its disulfide isomer did not. These results strongly suggest that the native IAG peptide has an insulin-type disulfide, and it is the decapod AGH.

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

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

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

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

A novel putative lipoprotein receptor (CasLpR) in the hemocytes of the blue crab, Callinectes sapidus: cloning and up-regulated expression after the injection of LPS and LTA.

The full-length cDNA encoding a putative lipoprotein receptor (CasLpR) was isolated from the hemocytes of Callinectes sapidus using 5' and 3' RACEs. The open reading frame for CasLpR contains a precursor of putative CasLpR consisting of 1710 amino acid residues including 22 amino acid residues of the signal peptide (22 amino acids). Mature CasLpR (1688 amino acids with 5.6% of phosphorylation sites) has multiple, putative functional domains: five low-density lipoprotein receptor domains in the N-terminus, and a G-protein-coupled receptor proteolysis site domain and a 7 transmembrane receptor (secretin family) domain in the C-terminus. To date, there are no proteins with a similar domain structure in the GenBank. The expression pattern of CasLpR was exclusive in hemocytes among all tested tissues obtained from a juvenile female at intermolt stage: brain, eyestalk ganglia, pericardial organs, and thoracic ganglia complex (nervous system); hepatopancreas (digestive system); heart, artery and hemocytes (circulatory system); gill and antennal gland (excretory system), hypodermis; and Y-organ (endocrine organ). There was no CasLpR expression in the ovary of an adult female. A putative function of CasLpR was examined after challenges of lipopolysaccharides (LPS) and lipoteichoic acid (LTA) in vivo using qRT-PCR assays. Animals at 24 h after injection of LPS or LTA up-regulated the expression of CasLpR in hemocytes by ∼3.5 and 1.4 folds, respectively, compared to the controls that received saline injection. LPS challenge also caused the greatest increment (∼55 folds) of heat shock protein 90 (Hsp90) expression in these samples. These data indicate that putative CasLpR and CasHsp90 may be involved in the defense system or the stress response of C. sapidus.

Crystallization and preliminary X-ray analysis of crustacean hyperglycaemic hormone from the kuruma prawn Marsupenaeus japonicus in its weakly active precursor form.

Crustacean hyperglycaemic hormone (CHH) plays a pivotal role in the regulation of glucose metabolism in crustaceans. Pej-SGP-I, one of the six known CHHs in the kuruma prawn Marsupenaeus japonicus, was heterologously expressed in Escherichia coli as an N-terminally His-tagged and Nus-tagged protein in its weakly active precursor form, Pej-SGP-I-Gly, which has an extra glycine residue at the C-terminus. The recombinant peptide was subjected to affinity purification, tag removal, further purification and crystallization by the sitting-drop vapour-diffusion method using NaCl as the main precipitant. The crystals diffracted to 1.95 Å resolution and the space group was assigned as primitive orthorhombic P2(1)2(1)2(1), with unit-cell parameters a = 40.19, b = 53.65, c = 53.63 Å. The Matthews coefficient (V(M) = 1.73 Å(3) Da(-1)) indicated that the crystal contained two Pej-SGP-I-Gly molecules per asymmetric unit, with a solvent content of 29.0%.

Identification and characterisation of a calcium carbonate-binding protein, blue mussel shell protein (BMSP), from the nacreous layer.

The nacreous layer of molluscan shells consists of a highly organised, layered structure comprising calcium carbonate aragonite crystals, each surrounded by an organic matrix. In the Japanese pearl oyster Pinctada fucata, the Pif protein from the nacreous layer functions in aragonite binding, and plays a key role in nacre formation. Here, we investigated whether the blue mussel Mytilus galloprovincialis also has a protein with similar functions in the nacreous layer. By using a calcium carbonate-binding assay, we identified the novel protein blue mussel shell protein (BMSP) 100 that can bind calcium carbonate crystals of both aragonite and calcite. When the entire sequence of a cDNA encoding BMSP 100 was determined, it was found that BMSP is a preproprotein consisting of a signal peptide and two proteins, BMSP 120 and BMSP 100. BMSP 120 contains four von Willebrand factor A (VWA) domains and one chitin-binding domain, thus suggesting that it has a role in maintaining structure within the matrix. Immunohistochemical analysis revealed that BMSP 100 is present throughout the nacreous layer with dense localisation in the myostracum. Posttranslational modification analysis indicated that BMSP 100 is phosphorylated and glycosylated. These results suggest that there is a common molecular mechanism between P. fucata and M. galloprovincialis that underlies the nacreous layer formation.

Purification of sinus gland peptides having vitellogenesis-inhibiting activity from the whiteleg shrimp Litopenaeus vannamei.

Vitellogenesis-inhibiting hormone (VIH) in Crustacea belongs to the crustacean hyperglycemic hormone (CHH)-family. To characterize multiple VIH molecules in the whiteleg shrimp Litopenaeus vannamei, seven CHH-family peptides designated as Liv-SGP-A, -B, -C, -D, -E, -F, and -G were purified by reversed-phase HPLC and identified by N-terminal amino acid sequencing. The dose-response effects of these peptides on vitellogenin mRNA levels were examined using in vitro incubation of ovarian fragments of the kuruma prawn Marsupenaeus japonicus. Liv-SGP-D showed no significant inhibitory activities, while the other six peptides significantly reduced vitellogenin mRNA levels, however, with differing efficacies, in the order of Liv-SGP-C, -F, -G > -A, -B > -E. Liv-SGP-G was the most abundant CHH-family peptide in the sinus gland and showed strong vitellogenesis-inhibiting activity. As a result of detailed structural analysis, its complete primary structure was determined; it consisted of 72 amino acid residues and possesses an amidated C-terminus.

Isolation and characterization of two pigment-dispersing hormones from the whiteleg shrimp, Litopenaeus vannamei.

In crustaceans, the pigment-dispersing hormone (PDH) is released from the X-organ/sinus gland complex located in the eyestalks, and controls pigment dispersion in the chromatophores. Knowledge concerning the structure and activity of PDH in penaeid shrimps is remains limited, since natural PDH has been purified from only the Kuruma prawn, Marsupenaeus japonicus. In this study, two PDHs (Liv-PDH-A and -B) were purified from the sinus gland extracts of another penaeid species, the whiteleg shrimp, Litopenaeus vannamei, by two steps of reversed-phase HPLC, and their amino acid sequences were determined. They both consist of 18 amino acid residues, with a free N-terminus and an amidated C-terminus, the sequences of Liv-PDH-A and -B being NSELINSLLGIPKVMNDAamide and NSELINSLLGLPKVMNDAamide, respectively. These sequences are identical to those of mature PDHs deduced from cDNAs encoding L. vannamei PDH precursors cloned previously by other workers. Liv-PDH-A and -B showed significant pigment-dispersing activity in melanophores by in vivo bioassay.

Preparation of two recombinant crustacean hyperglycemic hormones from the giant freshwater prawn, Macrobrachium rosenbergii, and their hyperglycemic activities.

Crustacean hyperglycemic hormone (CHH) is released from the X-organ/sinus gland complex located in the eyestalks, and regulates glucose levels in the hemolymph. In the giant freshwater prawn (Macrobrachium rosenbergii), two cDNAs encoding different CHH molecules were previously cloned by other workers. One of these (Mar-CHH-2) was expressed only in the eyestalks, whereas the other (Mar-CHH-L) was expressed in the heart, gills, antennal gland, and thoracic ganglion, but not in the eyestalks. However, their biological activities had not yet been characterized. Therefore, in this study, recombinant Mar-CHH-2 (rMar-CHH-2) and Mar-CHH-L (rMar-CHH-L) were produced using an E. coli expression system, by expression in bacterial cells and recovery in the insoluble fraction. Thereafter, rMar-CHH-2 and rMar-CHH-L were subjected to refolding and were subsequently purified by reversed-phase HPLC. The rMar-CHH-2 and rMar-CHH-L thus obtained exhibited the same disulfide bond arrangements as those of other CHHs reported previously, indicative of natural conformation. In in vivo bioassay, rMar-CHH-2 showed significant hyperglycemic activity, whereas rMar-CHH-L had no effect. These results indicate that Mar-CHH-L does not function as a CHH, but may have some other, unknown function.

Dynamics of vitellogenin synthesis in juvenile giant freshwater prawn Macrobrachium rosenbergii.

The dynamics of vitellogenin (Vg) mRNA expression and patterns of Vg and vitellin distribution in the hepatopancreas and ovary of juvenile Macrobrachium rosenbergii were examined using real-time RT-PCR and immunohistochemical methods. Eyestalk ablation was seen to induce rapid development of the gonads and Vg synthesis in females. In the female hepatopancreas, Vg mRNA expression was observed several days following ablation, after which levels increased gradually with increasing gonadosomatic index (GSI). Vitellin accumulation in the oocytes also increased with increasing Vg mRNA synthesis; expression was however negligible in the ovary. Hemolymph Vg levels in females ranged from 0.04 to 2.2 mg/ml. SDS PAGE/Western blotting analysis of hemolymph samples revealed that juvenile Vg was composed of 199 and 90 kDa subunits; the 102 kDa subunit present in adult female Vg (Okuno et al., 2002. J Exp Zool 292:417-429) could not be detected at any stage of vitellogenesis in juveniles. Vg was not detectable in non-ablated juveniles. The results of this study confirmed that the mode of involvement of eyestalk factors in regulating vitellogenesis is intrinsic to both juveniles and adults, and that a basic pattern of Vg synthesis and processing is conserved. However, the fact that juveniles are not able to produce the same Vg levels observed in adult females, and do not reach high GSI levels culminating in spawning suggests that other factors and physiological conditions specific to adult females are necessary to demonstrate full reproductive ability.