Molt-inhibiting Hormone Research Articles

Molt regulation in green and red color morphs of the crab Carcinus maenas: gene expression of molt-inhibiting hormone signaling components

Molt regulation in green and red color morphs of the crab <i>Carcinus maenas</i>: gene expression of molt-inhibiting hormone signaling components

Expansion of the Litopenaeus vannamei and Penaeus monodon peptidomes using transcriptome shotgun assembly sequence data

The shrimp Litopenaeus vannamei and Penaeus monodon are arguably the most important commercially farmed crustaceans. While expansion of their aquaculture has classically relied on improvements to rearing facilities, these options have largely been exhausted, and today a shift in focus is occurring, with increased investment in manipulating the shrimp themselves. Hormonal control is one strategy for increasing aquaculture output. However, to use it, one must first understand an animal’s native hormonal systems. Here, transcriptome shotgun assembly (TSA) data were used to expand the peptidomes for L. vannamei and P. monodon. Via an established bioinformatics workflow, 41 L. vannamei and 25 P. monodon pre/preprohormone-encoding transcripts were identified, allowing for the prediction of 158 and 106 distinct peptide structures for these species, respectively. The identified peptides included isoforms of allatostatin A, B and C, as well as members the bursicon, CAPA, CCHamide, crustacean cardioactive peptide, crustacean hyperglycemic hormone, diuretic hormone 31, eclosion hormone, FLRFamide, GSEFLamide, intocin, leucokinin, molt-inhibiting hormone, myosuppressin, neuroparsin, neuropeptide F, orcokinin, orcomyotropin, pigment dispersing hormone, proctolin, red pigment concentrating hormone, RYamide, SIFamide, short neuropeptide F and tachykinin-related peptide families. While some of the predicted peptides are known L. vannamei and/or P. monodon isoforms (which vet the structures of many peptides identified previously via mass spectrometry and other means), most are described here for the first time. These data more than double the extant catalogs of L. vannamei and P. monodon peptides and provide platforms from which to launch future physiological studies of peptidergic signaling in these two commercially important species.

Molt-inhibiting hormone (MIH) gene from the green mud crabScylla paramamosainand its expression during the molting and ovarian cycle

Mud crab is a group of commercially important species that are found throughout the Indo-Pacific region. In this study, the full-length cDNA of molt-inhibiting hormone (Scp-MIH) was cloned from the green mud crab, Scylla paramamosain. The conceptually translated peptide precursor consists of a 78-residue mature peptide, preceded by a 35-residue signal peptide. The mature peptide shares a high identity with the other known MIHs, and contains six conserved cysteine residues that have been proposed to be functionally critical for MIH activity. Analysis of genomic sequence revealed that Scp-MIH gene was organized in a 3 exons/2 introns manner. Quantitative real-time PCR showed that Scp-MIH transcripts were detected mainly in the eyestalk ganglion and brain. During the molting cycle, Scp-MIH mRNA increased significantly from postmolt stage to intermolt stage, then decreased sharply at premolt stage. It implies that MIH is closely related to ecdysteroid production in S. paramamosain. In the ovarian cycle, Scp-MIH transcripts in some neural tissues (eyestalk ganglion and brain) increased from stage I to stage II, and reached the peak value at stage III, then declined at later stage. It suggests that MIH might be involved in the ovarian maturation in S. paramamosain.

CRUSTACEAN HYPERGLYCEMIC HORMONE: STRUCTURAL VARIANTS, PHYSIOLOGICAL FUNCTION, AND CELLULAR MECHANISM OF ACTION

Crustacean hyperglycemic hormone (CHH) is a peptide hormone originally identified in the X-organ/sinus gland (XO/ SG) complex of the eyestalks. It belongs to the CHH family which also includes molt-inhibiting hormone (MIH), vitellogenesis-inhibiting hormone (VIH), and mandibular organ-inhibiting hormone (MOIH), and ion transport peptide (ITP). Multiple molecular variants of CHH are generated by both post-transcriptional and post-translational mechanisms. In addition to the XO/SG complex, CHH gene is widely expressed in many extra-eyestalk tissues. Functionally, available data indicate that CHH is involved with the regulation of carbohydrate metabolism and stress-induced hyperglycemia. Several other physiological processes, including molting, ion and water balance, reproduction, and immunity, are likely also regulated by CHH. The functional role(s) of the alternatively splice form CHH-like peptide (CHH-L) are different from those of CHH and remain unknown. Combined results showed that cyclic guanosine 3',5'-monophosphate (cGMP) plays important roles in mediating the effects of CHH on carbohydrate metabolism. Recent studies of receptor guanylyl cyclase (rGC) may lead to characterization and identification of CHH receptor(s). Future efforts are needed to fully understand the functional roles of the different CHH variants; identification of CHH receptor(s) hold the promise of revealing in greater depth the structure/functional relationships of the various structural variants.

Nitric oxide production and sequestration in the sinus gland of the green shore crab Carcinus maenas.

Molting in decapod crustaceans is regulated by molt-inhibiting hormone (MIH), a neuropeptide produced in the X-organ (XO)/sinus gland (SG) complex of the eyestalk ganglia (ESG). Pulsatile release of MIH from the SG suppresses ecdysteroidogenesis by the molting gland or Y-organ (YO). The hypothesis is that nitric oxide (NO), a neuromodulator that controls neurotransmitter release at presynaptic membranes, depresses the frequency and/or amount of MIH pulses to induce molting. NO synthase (NOS) mRNA was present in Carcinus maenas ESG and other tissues and NOS protein was present in the SG. A copper based ligand (CuFL), which reacts with NO to form a highly fluorescent product (NO-FL), was used to image NO in the ESG and SG and quantify the effects of NO scavenger (cPTIO), NOS inhibitor (l-NAME), and sodium azide (NaN3) on NO production in the SG. Pre-incubation with cPTIO prior to CuFL loading decreased NO-FL fluorescence ~30%; including l-NAME had no additional effect. Incubating SG with l-NAME during pre-incubation and loading decreased NO-FL fluorescence ~40%, indicating that over half of the NO release was not directly dependent on NOS activity. Azide, which reacts with NO-binding metal groups in proteins, reduced NO-FL fluorescence to near background levels without extensive cell death. Spectral shift analysis showed that azide displaced NO from a soluble protein in SG extract. These data suggest that the SG contains NO-binding protein(s) that sequester NO and releases it over a prolonged period. This NO release may modulate neuropeptide secretion from the axon termini in the SG.

Computational analysis of molt-inhibiting hormone from selected crustaceans

Molt-inhibiting hormone (MIH) is a principal endocrine hormone regulating the growth in crustaceans. In total, nine MIH peptide sequences representing members of the family Penaeidae (Penaeus monodon, Litopenaeus vannamei, Marsupenaeus japonicus), Portunidae (Portunus trituberculatus, Charybdis japonica, Charybdis feriata), Cambaridae (Procambarus bouvieri), Parastacidae (Cherax quadricarinatus) and Varunidae (Eriocheir sinensis) were selected for our study. In order to develop a structure based phylogeny, predict functionally important regions and to define stability changes upon single site mutations, the 3D structure of MIH for the crustaceans were built by using homology modeling based on the known structure of MIH from M. japonicus (1J0T). Structure based phylogeny showed a close relationship between P. bouvieri and C. japonica. ConSurf server analysis showed that the residues Cys8, Arg15, Cys25, Asp27, Cys28, Asn30, Arg33, Cys41, Cys45, Phe51, and Cys54 may be functionally significant among the MIH of crustaceans. Single amino acid substitutions ‘Y’ and ‘G’ at the positions 71 and 72 of the MIH C-terminal region showed an alteration in the stability indicating that a change in this region may alter the function of MIH. In conclusion, we proposed a computational approach to analyze the structure, phylogeny and stability of MIH from crustaceans.

Ecdysone and retinoid-X receptors of the blue crab, Callinectes sapidus: Cloning and their expression patterns in eyestalks and Y-organs during the molt cycle

Crustacean molting is known to be regulated largely by ecdysteroids and crustacean hyperglycemic hormone (CHH) neuropeptide family including molt-inhibiting hormone (MIH) and CHH. The surge of 20-OH ecdysone and/or ponasterone A initiates the molting process through binding to its conserved heterodimeric nuclear receptor: Ecdysone Receptor (EcR) and Ultraspiracle (USP)/Retinoid-X Receptor (RXR). To better understand the role of ecdysteroids in the molt regulation, the full-length cDNAs of the blue crab, Callinectes sapidus EcR1 and RXR1 were isolated from the Y-organs and their expression levels were determined in both Y-organs and eyestalks at various molt stages. Y-organs show the expression of four putative isoforms of CasEcRs and CasRXRs which differ in the length of the open reading frame but share the same domain structures as in typical nuclear receptors: AF1, DBD, HR, LBD, and AF2. The putative CasEcR isoforms are derived from a 27-aa insert in the HR and a 49-aa residue substitution in the LBD. In contrast, an insertion of a 5-aa and/or a 45-aa in the DBD and LBD gives rise to CasRXR isoforms. The eyestalks and Y-organs show the co-expression of CasEcRs and CasRXRs but at the different levels. In the eyestalks, the expression levels of CasRXRs are 3–5 times higher than those of CasEcRs, while in Y-organs, CasRXRs are 2.5–4 times higher than CasEcRs. A tissue-specific response to the changes in the levels of hemolymphatic ecdysteroids indicates that these tissues may have differences in the sensitivity or responsiveness to ecdysteroids. The presence of upstream open reading frame and internal ribosome entry site in 5′ UTR sequences of C. sapidus and other arthropod EcR/RXR/USP analyzed by in silico indicates a plausible, strong control(s) of the translation of these receptors.

Moult inhibiting hormone: a new approach to the discovery and design of growth promoters in crustaceans?

AbstractSeveral chemicals have been tested as growth promoters in aquaculture but they cannot be endorsed for commercial processes due to their residual effects in the body of prawns, lobsters and crabs. The concern over environmental hazard, human health and food safety have led to a search for alternative growth promoters significantly to improve the growth of crustaceans with no such effects. Neurohormones, regulatory signalling molecules of crustaceans that coordinate multiple developmental and physiological processes, are major determinants underlying phenotypic integration. Competitive inhibitors for the moult inhibiting hormone receptor are expected to have a direct growth promoting effect on crustaceans by direct involvement in interference with the receptor in the Y‐organ, which can cause a surge the production of ecdysteroids. Competitive inhibitors therefore can be regarded as growth promoters in crustacean fishery in addition to various other benefits. This review emphasizes the manifold effects of moult inhibiting hormone, a most versatile animal hormone, with an emphasis on the target sites and competitive inhibition.

Cloning and molecular structure analysis of crustacean hyperglycemic hormone(Ers-CHH)in Eriocheir sinensis

Crustacean hyperglycemic hormone(CHH)is a peptide hormone originally identified in a crustacean neurosecretory complex,the X-organ/sinus gland complex,and located within the eyestalks.CHH is placed in the crustacean hyperglycemic hormone family,which also includes molt-inhibiting hormone(MIH),vitellogenesis-inhibiting hormone(VIH)or gonad-inhibiting hormone(GIH),mandibular organ-inhibiting hormone(MOIH)and insect ion transport peptide(ITP).CHH plays a significant role in the growth,molting and reproduction of crustaceans.By using degenerate primers which were designed according to the N-terminal amino acid sequences of CHH isolated by our lab and the sequences of homologous species CHH,a novel cDNA of crustacean hyperglycemic hormone gene(Ers-CHH,GenBank Accession No:JX485664)was cloned from Chinese mitten crab(Eriocheir sinensis).The full length cDNA consists of 585 bp with a 453 bp open reading frame,encoding 150 amino acids,containing a 30 amino acids signal peptide,and a 42 amino acids CHH precursor-related peptide,a 78 amino acids mature peptide.The mature peptide contains 6 conserved cysteines which formed three disulfide bonds C7-C43、C23-C39、C26-C52.There was an arthropod CHH/MIH/GIH neurohormones family signature in this domain.The three-dimensional structure prediction showed that Ers-CHH mature peptide consisted of 4 α-helixes,and not with the β-sheet structure.5 Prosite patterns,which were analyzed by a Swiss-PdbViewer(v4.0.4),are P5:Protein kinase C phosphorylation site(Ser6,Lys8);P6:Casein kinase Ⅱ phosphorylation site(Ser17,Glu20);P8:N-myristoylation site(Gly38,Cys39,Asn42,Cys43);PS00342:Microbodies C-terminal targeting signal(Ser17,Lys18,Leu19);PS01250:Arthropod CHH/MIH/GIH neurohormones family(Val35,Cys39,Arg40,Asn42,Cys43,Tyr44,Asn46,Phe49,Cys52).The results of the structural model comparison show that the structure of Ers-CHH has high similarity with other CHHs and Ers-CHH.They have no β-sheet structure,except that CHH has 4α-helixes,MIH has 5α-helixes.Although α1 helix of MIH could not be found in CHHs,the position and the amino acid number of α2-α5 helixes in MIH molecular were same as the α1-α4 helixes in CHH.It might be able to interpret the function similarity between CHH and MIH.Multiple alignment results showed that Ers-CHH has the highest identity with Ptychognathus pusillus CHH(92%),and it also shared high identities with Neohelice granulata(86%)and Pachygrapsus marmoratus(72%).However,it showed lower identity with CHH from crayfishes,such as Nephrops norvegicus(49%)and Homarus gammarus(46%).Multiple alignments of Ers-CHH and MIH from other crustaceans showed that only six amino acid residues(Arg13,Asp25,Asn28,Arg31,Arg40,Phe49)in Ers-CHH sequence were same with MIH sequences.It may be helpful for further research on regulation mechanism of CHH in the process of crab growth and carbohydrate metabolism.

Molt regulation in green and red color morphs of the crab,Carcinus maenas: gene expression of molt-inhibiting hormone signaling components

In decapod crustaceans, regulation of molting is controlled by the X-organ/sinus gland complex in the eyestalks. The complex secretes molt-inhibiting hormone (MIH), which suppresses production of ecdysteroids by the Y-organ (YO). MIH signaling involves nitric oxide and cGMP in the YO, which expresses nitric oxide synthase (NOS) and NO-sensitive guanylyl cyclase (GC-I). Molting can generally be induced by eyestalk ablation (ESA), which removes the primary source of MIH, or by multiple leg autotomy (MLA). In our work on Carcinus maenas, however, ESA has limited effects on hemolymph ecdysteroid titers and animals remain in intermolt at 7 days post-ESA, suggesting that adults are refractory to molt induction techniques. Consequently, the effects of ESA and MLA on molting and YO gene expression in C. maenas green and red color morphotypes were determined at intermediate (16 and 24 days) and long-term (~90 days) intervals. In intermediate-interval experiments, ESA of intermolt animals caused transient twofold to fourfold increases in hemolymph ecdysteroid titers during the first 2 weeks. In intermolt animals, long-term ESA increased hemolymph ecdysteroid titers fourfold to fivefold by 28 days post treatment, but there was no late premolt peak (>400 pg μl(-1)) characteristic of late premolt animals and animals did not molt by 90 days post-ESA. There was no effect of ESA or MLA on the expression of Cm-elongation factor 2 (EF2), Cm-NOS, the beta subunit of GC-I (Cm-GC-Iβ), a membrane receptor GC (Cm-GC-II) and a soluble NO-insensitive GC (Cm-GC-III) in green morphs. Red morphs were affected by prolonged ESA and MLA treatments, as indicated by large decreases in Cm-EF2, Cm-GC-II and Cm-GC-III mRNA levels. ESA accelerated the transition of green morphs to the red phenotype in intermolt animals. ESA delayed molting in premolt green morphs, whereas intact and MLA animals molted by 30 days post treatment. There were significant effects on YO gene expression in intact animals: Cm-GC-Iβ mRNA increased during premolt and Cm-GC-III mRNA decreased during premolt and increased during postmolt. Cm-MIH transcripts were detected in eyestalk ganglia, the brain and the thoracic ganglion from green intermolt animals, suggesing that MIH in the brain and thoracic ganglion prevents molt induction in green ESA animals.

The ex vivo effects of eyestalk peptides on ovarian vitellogenin gene expression in the kuruma prawn Marsupenaeus japonicus

Seven major peptides belonging to the crustacean hyperglycemic hormone family were purified from the sinus gland located in the eyestalk of the kuruma prawn Marsupenaeus japonicus, and their effects on vitellogenin gene expression were examined using the ex vivo ovary incubation system. Six molecular species of crustacean hyperglycemic hormone, Pej-SGP-I, -II, -III, -V, VI, and VII, displayed significant inhibitory effects on vg expression with almost the same efficacies, whereas Pej-SGP-IV (known as molt-inhibiting hormone) did not. Two chromatophorotropic peptides, red pigment-concentrating hormone and pigment-dispersing hormone, which were also present in the sinus glands, did not have a clear effect on the gene expression levels in this incubation system. These results suggest that the six crustacean hyperglycemic hormones are potentially capable of acting as vitellogenesis-inhibiting hormones in M. japonicus.

Cloning of the crustacean hyperglycemic hormone and evidence for molt-inhibiting hormone within the central nervous system of the blue crab Portunus pelagicus

The crustacean X-organ–sinus gland (XO–SG) complex controls molt-inhibiting hormone (MIH) production, although extra expression sites for MIH have been postulated. Therefore, to explore the expression of MIH and distinguish between the crustacean hyperglycemic hormone (CHH) superfamily, and MIH immunoreactive sites (ir) in the central nervous system (CNS), we cloned a CHH gene sequence for the crab Portunus pelagicus (Ppel-CHH), and compared it with crab CHH-type I and II peptides. Employing multiple sequence alignments and phylogenic analysis, the mature Ppel-CHH peptide exhibited residues common to both CHH-type I and II peptides, and a high degree of identity to the type-I group, but little homology between Ppel-CHH and Ppel-MIH (a type II peptide). This sequence identification then allowed for the use of MIH antisera to further confirm the identity and existence of a MIH-ir 9kDa protein in all neural organs tested by Western blotting, and through immunohistochemistry, MIH-ir in the XO, optic nerve, neuronal cluster 17 of the supraesophageal ganglion, the ventral nerve cord, and cell cluster 22 of the thoracic ganglion. The presence of MIH protein within such a diversity of sites in the CNS, and external to the XO–SG, raises new questions concerning the established mode of MIH action.

Stage-specific changes in calcium concentration in crustacean (Callinectes sapidus) Y-organs during a natural molting cycle, and their relation to the hemolymphatic ecdysteroid titer

Secretion of ecdysteroid molting hormones by crustacean Y-organs is suppressed by molt-inhibiting hormone (MIH). The suppressive effect of MIH on ecdysteroidogenesis is mediated by one or more cyclic nucleotide second messengers. In addition, existing data indicate that ecdysteroidogenesis is positively regulated (stimulated) by intracellular Ca++. Despite the apparent critical role of calcium in regulating ecdysteroidogenesis, the level of Ca++ in Y-organ cells has not been previously measured during a natural molting cycle for any crustacean species. In studies reported here, a fluorescent calcium indicator (Fluo-4) was used to measure Ca++ levels in Y-organs during a molting cycle of the blue crab, Callinectes sapidus. Mean calcium fluorescence increased 5.8-fold between intermolt (C4) and stage D3 of premolt, and then dropped abruptly, reaching a level in postmolt (A) that was not significantly different from that in intermolt (P>0.05). The level of ecdysteroids in hemolymph of Y-organ donor crabs (measured by radioimmunoassay) showed an overall pattern similar to that observed for calcium fluorescence, rising from 2.9ng/mL in intermolt to 357.1ng/mL in D3 (P<0.05), and then dropping to 55.3ng/mL in D4 (P<0.05). The combined results are consistent with the hypothesis that ecdysteroidogenesis is stimulated by an increase in intracellular Ca++.

Stimulation of molt by RNA interference of the molt-inhibiting hormone in the crayfish Cherax quadricarinatus

In crustaceans, molting is known to be under the control of neuropeptide hormones synthesized and secreted from the eyestalk ganglia. While the role of molt-inhibiting hormone (MIH) in regulating molting has been described in several species using classical methods, an in vivo specific MIH targeted manipulation has not been described yet. In the present study, an MIH cDNA was isolated and sequenced from the eyestalk ganglia of the Australian freshwater red claw crayfish Cherax quadricarinatus (Cq) by 5′ and 3′ RACE. We analyzed the putative Cq-MIH based on sequence homology, a three dimensional structure model and transcript’s tissue specificity. We further examined the involvement of Cq-MIH in the control of molt in the crayfish through RNAi by in vivo injections of Cq-MIH double-stranded RNA, which resulted in, similarly to eyestalk ablation, acceleration of molt cycles. This acceleration was reflected by a significant reduction (up to 32%) in molt interval and an increased rate in molt mineralization index (MMI), which correlated with the induction of ecdysteroid hormones compared to control. Altogether, this study provides a proof of function for the involvement of the Cq-MIH gene in molt regulation in the crayfish.

Decreased level of crustacean hyperglycemic hormone (CHH) in black tiger shrimp Penaeus monodon suffering from Monodon Slow-Growth Syndrome (MSGS)

Monodon Slow-Growth Syndrome (MSGS), a pathological condition in the black tiger shrimp Penaeus monodon, is associated with Laem–Singh virus (LSNV) infection. Infected shrimp grow slowly when the virus invades the part of the retina called the zona fasciculata. Since molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH) are synthesized in the optic lobe and are related to molting activity and growth of the shrimp, the purpose of this study is therefore to determine the levels of these hormones and related parameters in MSGS shrimp. P. monodon juveniles were sampled from normal and MSGS ponds and were divided into small-negative, large-negative, small-positive and large-positive groups, depending on the size of the shrimp and whether they were LSNV-negative or LSNV-positive. Individual shrimp were measured for duration of each molt stage and molt interval. Levels of MIH1 and CHH1 transcripts were determined from the optic lobe, using real-time reverse transcriptase–polymerase chain reaction. The results revealed that the small-positive and the small-negative shrimp did not differ in durations of molt stages or molt intervals, as well as on the levels of MIH1 transcript in the optic lobe, MIH1 peptide in the optic lobe and hemolymph, or ecdysteroids in the hemolymph. Differences in molting activities and related transcript and hormones were observed between the small- and large-sized shrimp, but not in the status of LSNV infection. While levels of CHH1 transcript in the optic lobe of both small-negative and small-positive shrimp did not differ, levels of CHH1 peptide, as well glucose, in the hemolymph of the small-positive shrimp were significantly lower than those of the small-negative group. Levels of glycogen in the hepatopancreas of the small-positive shrimp were also significantly higher than that of the small-negative ones. The results suggest that growth retardation in MSGS shrimp is related to the suppression of the release of CHH1 peptide by LSNV invasion in the zona fasciculata, consequently causing decreased hepatopancreatic glycogenolysis and persistent hypoglycemia, resulting in growth stunting.

Neuropeptide signaling mechanisms in crustacean and insect molting glands

Growth in arthropods requires the periodic synthesis of a new exoskeleton and shedding, or molting, of the old exoskeleton. Both processes are initiated and coordinated by ecdysteroid molting hormones synthesized and secreted by a pair of molting glands: Y-organ (YO) in decapod crustaceans and the prothoracic gland (PG) in insects. The primary regulation of arthropod molting glands is mediated by brain neuropeptides that either stimulate (prothoracicotropic hormone – PTTH – in insects) or inhibit (molt-inhibiting hormone – MIH – in crustaceans) ecdysteroidogenesis. Both neuropeptide signaling pathways involve Ca2+/calmodulin (CaM) and cyclic nucleotides. In the crustacean YO, the MIH signaling pathway is proposed to have two phases: a cAMP/CaM-dependent “triggering” phase that activates a NO/cGMP-dependent “summation” phase to repress ecdysteroidogenesis during most of the intermolt period. A model is presented that incorporates the following data: (1) there is a pulsatile release of MIH from the eyestalk neurosecretory center; (2) MIH has a short half-life (5–10 min) in the hemolymph; and (3) there is a decrease in sensitivity of the YO to MIH that occurs during the premolt period. Molting is initiated by a temporary reduction in MIH titers due to a decrease in the amount and/or frequency of MIH released. During premolt, an increase in MIH release that is linked to a cAMP-dependent and cGMP-independent stimulation of protein synthesis drives an increase in molting hormone synthesis. By contrast, PTTH stimulates PG ecdysteroidogenesis by a Ca2+-dependent activation of cAMP and mitogen-activated protein (MAP) kinase pathways. A hypothetical scheme of how these two diametrical regulatory mechanisms may have originated and evolved is presented. It assumes that both systems evolved from a common ecdysozoan ancestor that had a cAMP-dependent stimulatory pathway antagonized by a NO/cGMP-dependent inhibitory pathway. This “parallel” arrangement of the two pathways regulates ecdysteroidogenesis in the gonad of adult insects. In insect PG, cAMP-dependent signaling became the dominant pathway and NO/cGMP-dependent signaling was lost. MAP kinase signaling was later co-opted as an ancillary pathway. This dual pathway occurs in lepidopteran species (Manduca sexta and Bombyx mori), but MAP kinase signaling is the dominant pathway in Drosophila melanogaster. In crustacean YO, the cAMP- and NO/cGMP-dependent pathways became arranged in series such that a transient increase in cAMP triggers a large and sustained increase in cGMP, which leads to prolonged repression of ecdysteroidogenesis during the intermolt period. It is now recognized that the control of molting involves a constant communication between the nervous system and the molting gland mediated by a variety of endocrine factors. Both the YO and PG undergo changes in sensitivity to MIH and PTTH, respectively, that are associated with the commitment to molt. It is not surprising, however, that the changes in sensitivities of the two glands to their respective neuropeptides are opposite: the PG is the most sensitive to PTTH, whereas the YO is least sensitive to MIH, prior to and during the large peak in hemolymph ecdysteroid titers that triggers molting. As more research is conducted, we anticipate that more similarities in the neuropeptide signaling pathways of crustacean and insect molting glands will be revealed.

Gene Expression Profiling of the Cephalothorax and Eyestalk in Penaeus Monodon during Ovarian Maturation

In crustaceans, a range of physiological processes involved in ovarian maturation occurs in organs of the cephalothorax including the hepatopancrease, mandibular and Y-organ. Additionally, reproduction is regulated by neuropeptide hormones and other proteins released from secretory sites within the eyestalk. Reproductive dysfunction in captive-reared prawns, Penaeus monodon, is believed to be due to deficiencies in these factors. In this study, we investigated the expression of gene transcripts in the cephalothorax and eyestalk from wild-caught and captive-reared animals throughout ovarian maturation using custom oligonucleotide microarray screening. We have isolated numerous transcripts that appear to be differentially expressed throughout ovarian maturation and between wild-caught and captive-reared animals. In the cephalothorax, differentially expressed genes included the 1,3-β-D-glucan-binding high-density lipoprotein, 2/3-oxoacyl-CoA thiolase and vitellogenin. In the eyestalk, these include gene transcripts that encode a protein that modulates G-protein coupled receptor activity and another that encodes an architectural transcription factor. Each may regulate the expression of reproductive neuropeptides, such as the crustacean hyperglycaemic hormone and molt-inhibiting hormone. We could not identify differentially expressed transcripts encoding known reproductive neuropeptides in the eyestalk of either wild-caught or captive-reared prawns at any ovarian maturation stage, however, this result may be attributed to low relative expression levels of these transcripts. In summary, this study provides a foundation for the study of target genes involved in regulating penaeid reproduction.

Molt-inhibiting hormone from Chinese mitten crab (Eriocheir sinensis): Cloning, tissue expression and effects of recombinant peptide on ecdysteroid secretion of YOs

Molt-inhibiting hormone (MIH), a member of the crustacean hyperglycemic hormone (CHH) family, inhibits the synthesis of ecdysteroid in Y-organ (YO) and plays a significant role in the regulation of molting and growth of crustaceans. A complete cDNA sequence encoding MIH (Ers-MIH, GenBank Accession No.: DQ341280) was cloned from eyestalk of Chinese mitten crab (Eriocheir sinensis) by 5′ and 3′ RACEs and PCR cloning. The full-length cDNA consists of 1457bp with a 330bp open reading frame, encoding 110 amino acids, containing a 75 amino acid mature peptide. The deduced amino acid sequence contains a typical CHH domain. Transcripts of Ers-MIH mRNA were detected in eyestalk by Northern blotting. The production of purified recombinant Ers-MIH (rErs-MIH) expressed in Escherichia coli was 0.3g/L. The LC–ESI-MS analysis showed that two peptide fragments of the recombinant protein were identical to the deduced amino acid sequence of Ers-MIH. By in vitro assay on E. sinensis YOs, a cGMP mediated suppression of rErs-MIH on ecdysteroidogenesis could be observed. Accumulation of cGMP in YOs showed a concentration-dependent manner within 0.01–1nmol/mL of rErs-MIH; ecdysteroid secretion was inhibited significantly at the range of 0.01–100nmol/mL rErs-MIH; furthermore, a significant inhibition effect on ecdysteroid releasing was shown when cGMP analog (8-Br-cGMP) concentration rose up to 100nmol/mL. This study would facilitate to investigate the roles of MIH in molt cycle regulation.

Genomic analyses of the Daphnia pulex peptidome

Genome mining has provided a valuable tool for peptide discovery in many species, yet no crustacean has undergone this analysis. Currently, the only crustacean with a sequenced genome is the cladoceran Daphnia pulex, a model organism in many fields of biology. Here, we have mined the D. pulex genome for peptide-encoding genes. For each gene identified, the encoded precursor protein was deduced, and its mature peptides predicted. Twenty-four peptide-encoding genes were identified, including ones predicted to produce members of the A-type allatostatin, B-type allatostatin, C-type allatostatin, allatotropin (ATR), bursicon α, bursicon β, calcitonin-like diuretic hormone, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone, ecdysis-triggering hormone, eclosion hormone (EH), insulin-like peptide (ILP), molt-inhibiting hormone, neuropeptide F, orcokinin (two genes), pigment-dispersing hormone, proctolin, red pigment concentrating hormone/adipokinetic hormone (RPCH/AKH), short neuropeptide F, SIFamide, sulfakinin, and tachykinin-related peptide (TRP) families/subfamilies. In total, 96 peptides were predicted from these genes. Our identification of isoforms of corazonin, EH, ILP, proctolin, RPCH/AKH, sulfakinin and TRP are the first for D. pulex, while our prediction of ATR from this species is the first from any crustacean. The number of peptides predicted in our study shows the power of genome mining for peptide discovery, and provides a model for future genomic analyses of the peptidomes of other crustaceans. In addition, the data presented in our study provide foundations for future molecular, biochemical, anatomical, and physiological investigation of peptidergic signaling in D. pulex and other cladoceran species.

Computational analysis and structure predictions of CHH-related peptides from Litopenaeus vannamei

The crustaceans produce several related peptides that belong to the crustacean hyperglycemic hormone (CHH) family. While these peptides have similar amino acid sequences, they have diverse biological functions that must arise, in part, from differences in the 3D shape of these peptides. However, it is generally accepted that peptides with a high degree of sequence similarity also have a similar 3-D structure. We used the solution structure of one peptide in the crustacean hyperglycemic hormone family, the molt-inhibiting hormone of the kuruma prawn (Marsupenaeus japonicus), to predict the shape of the five known peptides related to CHH in the Pacific white shrimp, Litopenaeus vannamei. The high similarity of the 3-D structures of these peptides suggests a common fold for the entire family. Nevertheless, minor differences in the shape of these peptides were observed, which may be the basis for their different biological properties.