A novel G protein-coupled receptor for starfish gonadotropic hormone, relaxin-like gonad-stimulating peptide.

Gonadotropic hormones play important regulatory roles in reproduction. Relaxin-like gonad-stimulating peptide (RGP) is a gonadotropin-like hormone in starfish. However, a receptor for RGP remains to be identified. Here, we describe the identification of an authentic receptor for RGP (RGPR) in the starfish, Patiria pectinifera. A binding assay using radioiodinated P. pectinifera RGP (PpeRGP) revealed that RGPR was expressed in ovarian follicle cells. A RGPR candidate was identified by homology-searching of transcriptome data of P. pectinifera follicle cells. Based on the contig sequences, a putative 947-amino acid PpeRGPR was cloned from follicle cells. Like the vertebrate relaxin family peptide receptors (RXFP 1 and 2), PpeRGPR was a G protein-coupled receptor that harbored a low-density lipoprotein-receptor class A motif and leucine-rich repeat sequences in the extracellular domain of the N-terminal region. Sf9 cells transfected with Gαq16-fused PpeRGPR activated calcium ion mobilization in response to PpeRGP, but not to RGP of another starfish Asterias amurensis, in a dose-dependent fashion. These results confirmed the species-specific reactivity of RGP and the cognate receptor. Thus, the present study provides evidence that PpeRGPR is a specific receptor for PpeRGP. To the best of our knowledge, this is the first report on the identification of a receptor for echinoderm RGP.

Defining the Neuropeptidome of the Spiny Lobster Panulirus interruptus Brain Using a Multidimensional Mass Spectrometry-Based Platform.

Decapod crustaceans are important animal models for neurobiologists due to their relatively simple nervous systems with well-defined neural circuits and extensive neuromodulation by a diverse set of signaling peptides. However, biochemical characterization of these endogenous neuropeptides is often challenging due to limited sequence information about these neuropeptide genes and the encoded preprohormones. By taking advantage of sequence homology in neuropeptides observed in related species using a home-built crustacean neuropeptide database, we developed a semi-automated sequencing strategy to characterize the neuropeptidome of Panulirus interruptus, an important aquaculture species, with few known neuropeptide preprohormone sequences. Our streamlined process searched the high mass accuracy and high-resolution data acquired on a LTQ-Orbitrap with a flexible algorithm in ProSight that allows for sequence discrepancy from reported sequences in our database, resulting in the detection of 32 neuropeptides, including 19 novel ones. We further improved the overall coverage to 51 neuropeptides with our multidimensional platform that employed multiple analytical techniques including dimethylation-assisted fragmentation, de novo sequencing using nanoliquid chromatography-electrospray ionization-quadrupole-time-of-flight (nanoLC-ESI-Q-TOF), direct tissue analysis, and mass spectrometry imaging on matrix-assisted laser desorption/ionization (MALDI)-TOF/TOF. The high discovery rate from this unsequenced model organism demonstrated the utility of our neuropeptide discovery pipeline and highlighted the advantage of utilizing multiple sequencing strategies. Collectively, our study expands the catalog of crustacean neuropeptides and more importantly presents an approach that can be adapted to exploring neuropeptidome from species that possess limited sequence information.

Identification, cloning, characterization and recombinant expression of an anti-lipopolysaccharide factor from the hemocytes of Indian mud crab, Scylla serrata.

Anti-lipopolysaccharide factors (ALF) are a group of small basic proteins which are released into the hemolymph as a result of rapid degranulation of hemocytes in response to bacterial lipopolysaccharide (LPS). In the present study, using a combined approach of degenerate and RACE PCR, the gene coding for Scylla serrata anti-lipopolysaccharide factor (SsALF) was cloned and characterized. The full-length SsALF cDNA sequence consists of 607 bp and encodes a polypeptide of 97 amino acids, constituting a molecular mass of 11172 Da with an estimated pI of 10.01. The SsALF protein showed upto 92% similarity with ALF from Scylla paramamosain and about 33-53% amino acid sequence identity with other known ALF sequences. SsALF protein sequence demonstrated the presence of two highly conserved cysteine residues and putative LPS binding domain. An in vivo expression study showed that SsALF mRNA was expressed predominantly in hemocytes, heart and muscle of healthy mud crabs. The recombinant form of SsALF protein (rSsALF) was expressed with a Histag, in Escherichia coli, using the pTriEx-4 Ek/LIC vector. The purified rSsALF protein demonstrated antimicrobial activity against both Gram-positive and Gram-negative bacteria. The recombinant protein was able to significantly neutralize LPS-induced expression on SsALF in vivo as demonstrated by real-time PCR. rSsALF was able to permeabilize artificial phospholipid membranes as demonstrated by calcein enclosed liposome model. These studies strongly suggest that SsALF is one among the important antimicrobial factors produced in the crab during a microbial invasion.

Gonad-stimulating substance-like molecule from the radial nerve of the sea cucumber.

Gonad-stimulating substance-like molecule (GSSL) was isolated from the radial nerve of the sea cucumber, Apostichopus japonicus (Aj-GSSL), and its partial DNA and protein sequences were characterized. The smaller part of the molecule that also retains GSSL activity was estimated. Radial nerve extract (RNE) induced germinal vesicle breakdown (GVBD) at 3 mg/ml in 85% of immature ovarian oocytes. Similar intensity of GSSL activity to RNE was seen in a fraction that contained peptides between 3 kDa and 10 kDa (3-10 kDa-fraction) separated by ultrafiltlation membrane. MALDI-TOF MS analysis and silver-stained 18% SDS-PAGE slab gels identified a major peptide at around 4.6 kDa in a 3-10 kDa-fraction, and that was subjected to internal protein sequencing. The resulting 12-amino acid sequence was not found in the BLAST database to date. Immunohistochemistry using antiserum raised against the 12-amino acid peptide located the peptide to granular cells in the hyponeural part of the radial nerve and in the epineural sinus beneath the radial nerve. Sequence data was obtained using degenerate primers designed from the 12-amino acid sequence and 5 and 3 RACEs. These resulted in a 148 bp cDNA that coded a 43-amino acid sequence of H2N-VLSKQAHHHHHEGWSLPGVPAEIDDLAGNIDYNIFKEQREKIK-COOH. The synthetic 43-amino acid Aj-GSSL generated from this sequence induced GVBD in 50% of immature ovarian oocytes at 6 microM. An N-terminal 21-amino acid peptide of the synthetic partial Aj-GSSL (Aj-GSSL-P1) induced GVBD to 80% of immature ovarian oocytes at 12 microM. This indicated that Aj-GSSL-P1 is of sufficient length for GSSL activity.

Bursicon, the tanning hormone of insects: recent advances following the discovery of its molecular identity.

Bursicon was identified in 1965 as a peptide neurohormone that initiates the tanning of the insect cuticle immediately after the shedding of the old one during the final stages of the molting process. Its molecular identity as an approximately 30 kDa bioactive heterodimer consisting of two cystine knot proteins resisted elucidation for 43 years. The sequence of the two bursicon subunits is highly conserved among arthropods, and this conservation extends even to echinoderms. We review the efforts leading to bursicon's characterization, the identification of its leucine-rich repeat-containing, G protein-coupled receptor (LGR2), and the progress towards revealing its various functions. It is now clear that bursicon regulates different aspects of wing inflation in Drosophila melanogaster besides being involved at various points in the cuticle tanning process in different insects. We also describe the current knowledge of the expression of bursicon in the central nervous system of different insects in large homologous neurosecretory cells, and the changes in its expression during the development of Manduca sexta and D. melanogaster. Although much remains to be learned, the elucidation of its molecular identity and that of its receptor has provided the breakthrough needed for investigating the diverse actions of this critical insect neurohormone.

Functional aspects of cHH C-terminal amidation in crayfish species.

The crustacean hyperglycemic hormone is the most abundant neuropeptide present in the eyestalk of Crustacea and its main role is to control the glucose level in the hemolymph. Our study was aimed at assessing the importance of C-terminal amidation for its biological activity. Two recombinant peptides were produced, Asl-rcHH-Gly with a free carboxyl terminus and Asl-rcHH-amide with an amidated C-terminus. Homologous bioassays performed on the astacid crayfish Astacus leptodactylus showed that the amidated peptide had a stronger hyperglycemic effect compared to the non-amidated peptide. To assess the relevance of amidation also in other decapods and how much the differences in the cHH amino acid sequence can affect the functionality of the peptides, we carried out heterologous bioassays on the cambarid Procambarus clarkii and palaemonid Palaemon elegans. The Asl-rcHH-amide elicited a good response in P. clarkii and in P. elegans. The injection of Asl-rcHH-Gly evoked a weak response in both species. These results prove the importance of C-terminal amidation for the biological activity of cHH in crayfish as well as the role of the peptide primary sequence for the species-specificity hormone-receptor recognition.

Characterization of a molt-inhibiting hormone (MIH) receptor in the Y-organ of the kuruma prawn, Marsupenaeus japonicus.

To characterize a receptor for molt-inhibiting hormone (MIH) in the kuruma prawn, Marsupenaeus japonicus, we performed a binding assay and chemical cross-linking experiments using radiolabeled recombinant MIH ([(125)I]rMIH). The specific binding of [(125)I]rMIH was found in the membrane fraction of the Y-organ (K(d) = 4.76 x 10(-10) M and B(max) = 5.51 x 10(-12) M). Chemical cross-linking between [(125)I]rMIH and the membrane fraction of the Y-organ revealed that the MIH receptor protein has a molecular size of approximately 70 kDa.

Cloning and characterization of a molt-inhibiting hormone-like peptide from the prawn Marsupenaeus japonicus.

Recently, it was demonstrated by PCR amplification that an additional molt-inhibiting hormone (MIH)-like peptide was present in the kuruma prawn Marsupenaeus japonicus. In this study, a cDNA encoding this peptide designated Pej-MIH-B was cloned. The Pej-MIH-B gene was expressed strongly in the nerve cord, and weakly in the eyestalk. It was possible to isolate Pej-MIH-B from the sinus glands in the eyestalks. The recombinant Pej-MIH-B expressed in Escherichia coli showed low molt-inhibiting activity, but did not exhibit hyperglycemic activity. These results suggest that Pej-MIH-B does not function as MIH or CHH intrinsically, but may have some unknown functions.

Expression of biologically active crustacean hyperglycemic hormone (CHH) of Penaeus monodon in Pichia pastoris.

Crustacean hyperglycemic hormone (CHH), molt-inhibiting hormone (MIH), and gonad-inhibiting hormone (GIH) are members of a major peptide family produced from the X-organ sinus gland complex in the eyestalk of crustaceans. This peptide family plays important roles in controlling several physiologic processes such as regulation of growth and reproduction. In this study the complementary DNA encoding a peptide related to the CHH/MIH/GIH family (so-called Pem-CMG) of the black tiger prawn Penaeus monodon was successfully expressed in the yeast Pichia pastoris under the control of the AOX1 promoter. The recombinant Pem-CMG was secreted into the culture medium using the alpha-factor signal sequence; of Saccharomyces cerevisiae without the Glu-Ala-Glu-Ala spacer peptide. The amino terminus of the recombinant Pem-CMG was correctly processed as evidenced by amino-terminal peptide sequencing. The recombinant Pem-CMG was purified by reverse-phase high-performance liquid chromotography and used in a biological assay for CHH activity. The final yield of the recombinant Pem-CMG after purification was 260 micro g/L of the culture medium. Both crude and purified recombinant Pem-CMG produced from P. pastoris showed the ability to elevate the glucose level in the hemolymph of eyestalk-ablated P. monodon, which demonstrates that Pem-CMG peptide functions as hyperglycemic hormone in P. monodon.

Production of recombinant endotoxin neutralizing protein in Pichia pastoris and methods for its purification.

Production of recombinant Limulus endotoxin neutralizing protein (rENP) was attained with the GS115 methylotrophic strain of Pichia pastoris transformed with a plasmid, bearing multiple ENP gene copies. The synthetic gene for Limulus ENP was cloned into the integrative plasmid pAO815 under the control of a methanol-inducible promoter. Clones containing a single enp insert were used to construct cassettes bearing 2 and 3 tandem copies of enp. These were then integrated at the HIS locus of P. pastoris GS115 (his4). Clones were chosen for their ability to produce rENP upon methanol induction in shaker flasks, and then the 1x, 2x, and 3x-enp strains were analyzed by Southern blot for the presence of the ENP gene(s). Isolate 3 x 5q, containing a 3x-enp cassette, was the best producer of rENP. Under optimal conditions this strain grown in a fed-batch mode produced yields of >500 mg rENP/L with an average of 5.46 mg rENP/g DCW. Purification of rENP from the clarified broth resulted in a yield of 35% and a purity of >86%. Glycosylated rENP, the main contaminant, was removed with a concanavalin-A column and characterized. The pure rENP neutralized lipopolysaccharide and had the mass, amino-acid composition and N-terminal sequence expected from the cloned gene.

Cellular localization of bursicon using antisera against partial peptide sequences of this insect cuticle-sclerotizing neurohormone.

Bursicon is the final neurohormone released at the end of the molting cycle. It triggers the sclerotization (tanning) of the insect cuticle. Until now, its existence has been verified only by bioassays. In an attempt to identify this important neurohormone, bursicon was purified from homogenates of 2,850 nerve cords of the cockroach Periplaneta americana by using high performance liquid chromatography technology and two-dimensional gel electrophoresis. Bursicon bioactivity was found in four distinct protein spots at approximately 30 kDa between pH 5.3 and 5.9. The protein of one of these spots at pH 5.7 was subsequently microsequenced, and five partial amino acid sequences were retrieved. Evidence is presented that two of these sequences are derived from bursicon. Antibodies raised against the two sequences labeled bursicon-containing neurons in the central nervous systems of P. americana. One of these antisera labeled bursicon-containing neurons in the crickets Teleogryllus commodus and Gryllus bimaculatus, and the moth Manduca sexta. A cluster of four bilaterally paired neurons in the brain of Drososphila melanogaster was also labeled. In addition, this antiserum detected three spots corresponding to bursicon in Western blots of two-dimensional gels. The 12-amino acid sequence detected by this antiserum, thus, seems to be conserved even among species that are distantly related.

The enterins: a novel family of neuropeptides isolated from the enteric nervous system and CNS of Aplysia.

To identify neuropeptides that have a broad spectrum of actions on the feeding system of Aplysia, we searched for bioactive peptides that are present in both the gut and the CNS. We identified a family of structurally related nonapeptides and decapeptides (enterins) that are present in the gut and CNS of Aplysia, and most of which share the HSFVamide sequence at the C terminus. The structure of the enterin precursor deduced from cDNA cloning predicts 35 copies of 20 different enterins. Northern analysis, in situ hybridization, and immunocytochemistry show that the enterins are abundantly present in the CNS and the gut of Aplysia. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry we characterized the enterin-precursor processing, demonstrated that all of the precursor-predicted enterins are present, and determined post-translational modifications of various enterins. Enterin-positive neuronal somata and processes were found in the gut, and enterins inhibited contractions of the gut. In the CNS, the cerebral and buccal ganglia, which control feeding, contained the enterins. Enterin was also present in the nerve that connects these two ganglia. Enterins reduced the firing of interneurons B4/5 during feeding motor programs. Such enterin-induced reduction of firing also occurred when excitability of B4/5 was tested directly. Because reduction of B4/5 activity corresponds to a switch from egestive to ingestive behaviors, enterin may contribute to such program switching. Furthermore, because enterins are present throughout the nervous system, they may also play a regulatory role in nonfeeding behaviors of Aplysia.

Proteinase inhibitors from the medicinal leech Hirudo medicinalis.

The medicinal leech Hirudo medicinalis produces various types of proteinase inhibitors: bdellins (inhibitors of trypsin, plasmin, and acrosin), hirustasin (inhibitor of tissue kallikrein, trypsin, alpha-chymotrypsin, and granulocyte cathepsin G), tryptase inhibitor, eglins (inhibitors of alpha-chymotrypsin, subtilisin, and chymasin and the granulocyte proteinases elastase and cathepsin G), inhibitor of factor Xa, hirudin (thrombin inhibitor), inhibitor of carboxypeptidase, and inhibitor of complement component C1s. This review summarizes data on their primary and tertiary structures, action mechanisms, and biological activities.

Crustacean hyperglycaemic hormone (CHH)-like peptides and CHH-precursor-related peptides from pericardial organ neurosecretory cells in the shore crab, Carcinus maenas, are putatively spliced and modified products of multiple genes.

About 24 intrinsic neurosecretory neurons within the pericardial organs (POs) of the crab Carcinus maenas produce a novel crustacean hyperglycaemic hormone (CHH)-like peptide (PO-CHH) and two CHH-precursor-related peptides (PO-CPRP I and II) as identified immunochemically and by peptide chemistry. Edman sequencing and MS revealed PO-CHH as a 73 amino acid peptide (8630 Da) with a free C-terminus. PO-CHH and sinus gland CHH (SG-CHH) share an identical N-terminal sequence, positions 1-40, but the remaining sequence, positions 41-73 or 41-72, differs considerably. PO-CHH may have different precursors, as cDNA cloning of PO-derived mRNAs has revealed several similar forms, one exactly encoding the peptide. All PO-CHH cDNAs contain a nucleotide stretch coding for the SG-CHH(41-76) sequence in the 3'-untranslated region (UTR). Cloning of crab testis genomic DNA revealed at least four CHH genes, the structure of which suggest that PO-CHH and SG-CHH arise by alternative splicing of precursors and possibly post-transcriptional modification of PO-CHH. The genes encode four exons, separated by three variable introns, encoding part of a signal peptide (exon I), the remaining signal peptide residues, a CPRP, the PO-CHH(1-40)/SG-CHH(1-40) sequences (exon II), the remaining PO-CHH residues (exon III) and the remaining SG-CHH residues and a 3'-UTR (exon IV). Precursor and gene structures are more closely related to those encoding related insect ion-transport peptides than to penaeid shrimp CHH genes. PO-CHH neither exhibits hyperglycaemic activity in vivo, nor does it inhibit Y-organ ecdysteroid synthesis in vitro. From the morphology of the neurons it seems likely that novel functions remain to be discovered.

Characterization and sequence elucidation of a novel peptide with molt-inhibiting activity from the South African spiny lobster, Jasus lalandii.

We have isolated a peptide from extracts of sinus glands from a South African spiny lobster species, Jasus lalandii, by high-performance liquid chromatography (HPLC) and identified it as a putative molt-inhibiting hormone (MIH) by (i) an in vitro assay with J. lalandii Y-organs to measure the inhibition of ecdysteroid synthesis and (ii) an immunoassay using antiserum raised against MIH of the edible crab. The MIH of J. lalandii has 74 amino acid residues, a molecular mass of 9006 Da, a free N-terminus and an amidated C-terminus. The full primary sequence has been obtained from sequencing various digest fragments (tryptic, endoproteinase Asp-N, cyanogen bromide) of the unreduced (native) peptide: RFTFDCPGMMGQRYLYEQVEQVCDDCYNLYREEKIAVNCRENCFLNSWFTVCLQATMREHETPRFDIWR SIILKA-NH(2). Structural comparisons with other peptides show that the J. lalandii MIH belongs to the peptide family which includes the crustacean hyperglycemic hormone, molt-inhibiting hormone and vitellogenesis-inhibiting hormone (cHH/MIH/VIH). This novel peptide has 36-43% sequence identity to putative MIHs from other decapod crustaceans and 32-34% identity to the two cHH peptides previously identified in this spiny lobster species. This is the first report of a peptide with MIH activity in the Palinuridae infraorder.

Purification and characterization of biliverdin-binding protein from larval hemolymph of the swallowtail butterfly, Papilio xuthus L.

The biliverdin-binding protein from the larval hemolymph of the swallowtail butterfly, Papilio xuthus L., was purified and characterized. The crude biliverdin-binding protein, obtained by ammonium sulfate fractionation, was purified in two steps, the first one by gel filtration chromatography and the second one by ion-exchange chromatography. The molecular mass of the purified protein was analyzed by SDS-polyacrylamide gel electrophoresis and estimated to be 21 kDa. The Namino terminal sequence of P. xuthus biliverdin-binding protein analyzed up to the 19th residue showed that 42% of the amino acid sequence are sequence similarity to the bilin-binding protein from Pieris brassicae. These results suggest that the P. xuthus biliverdin-binding protein belongs to the insecticyanin-type.

A hyperglycemic peptide hormone from the Caribbean shrimp Penaeus (litopenaeus) schmitti.

From a crude extract of the sinus glands of the shrimp Penaeus (litopenaeus) schmitti a peptide with hyperglycemic activity in a homologous bioassay was isolated and characterized by a combination of automatic Edman degradation, enzymatic digestions, TLC of dansyl-amino acids, and mass spectrometry. Its M(r) is 8359.4 Da by MS, which coincides with the deduced sequence. Its N-terminus is free and its C-terminus is amidated. It has 6 Cys residues in conserved positions compared with other known CHHs. This is the first sinus gland hormone from an Atlantic Ocean shrimp characterized to date. It has a remarkable 90% sequence similarity to the Indo-Pacific shrimp P. (marsupenaeus) japonicus Pej-VII hyperglycemic hormone.

Primary structure of CHH/MIH/GIH-like peptides in sinus gland extracts from Penaeus vannamei.

Peptides belonging to the CHH/MIH/GIH-family of crustacean hormones were isolated from acetic acid extracts of sinus glands isolated from eyestalks of the shrimp, Penaeus vannamei. The peptides were isolated by chromatography and molecular weights determined by MALDI mass spectrometry. Peptides in the range of 7-9 kDa and containing three disulfide bridges were selected for amino acid sequence analysis. Three peptides with the requisite properties were present in sufficient amounts for sequence analysis. Two peptides had unique sequences similar to CHH/MIH/GIH peptides from other crustaceans. A third peptide seemed to be a truncated form of one of the previous sequences.

Ecdysteroid hormones and metabolites of the stone crab, Menippe mercenaria.

The Y-organs of the xanthid crab Menippe mercenaria secrete the ecdysteroids, 3-dehydroecdysone (3DE) and lesser amounts of 3-dehydro (or 2-dehydro)-25-deoxyecdysone (3D25dE) in vitro. These ecdysteroids were identified by elution-time comparisons with authentic standards, mass spectrography, and, for 3D25dE, infrared spectrometry. Tissues were incubated 18 hr with [(3)H]3DE. Activities representing 3beta-reductase and 20-hydroxylase generally were present, evidenced by finding in the tissue/medium extract labeled ecdysone (E) and 20-hydroxyecdysone (20E). Labeled 3-dehydro-20-hydroxyecdysone (3D20E) also appeared to be present. Tissue blanks and hemolymph were devoid of activity. Muscle was low, hypodermis was intermediate, and hindgut and gonads were high in activity of the enzymes. Consistent with the presence of these enzymes in peripheral tissues, ecdysteroid products identified in the hemolymph were 20E, 3D20E, and 25-deoxy-20-hydroxyecdysone (25d20E; ponasterone A). Structures of 20E and 3D20E were confirmed by co-elution with authentic standards in high-performance liquid chromatography (HPLC), co-elution of derivatives in gas chromatography, and mass spectroscopy. Ponasterone A (identified by HPLC co-elution with the standard), like 20E is present in the hemolymph in prominent amounts. These data indicate that Menippe, among crustaceans thus far studied, secretes a unique combination of ecdysteroid hormones, namely, a 3- (or 2-) oxo compound and a 25-deoxy compound. This represents a different kind of branch point from 5beta-diketol in ecdysteroid biosynthesis, in which the intermediate, 5beta-ketodiol is bypassed. A result is the joint appearance in the circulation of the hormones, 20E and ponasterone A, which in other species are singly prominent.

Molecular characterization of an additional shrimp hyperglycemic hormone: cDNA cloning, gene organization, expression and biological assay of recombinant proteins.

The crustacean eyestalk CHH/MIH/GIH neurohormone gene family represents a unique group of neuropeptides identified mainly in crustaceans. In this study, we report the cloning and characterization of the cDNA and the gene encoding the hyperglycemic hormone (MeCHH-B) of the shrimp Metapenaeus ensis. The amino acid sequence of MeCHH-B shows 85% identity to that of MeCHH-A (formerly MeCHH-like neuropeptide). Two separate but identical MeCHH-B genes were identified in the genome of shrimp by library screening and they are located on different CHH gene clusters. The organization of the MeCHH-B gene is identical to other members of the CHH/MIH/GIH neurohormone family. MeCHH-B is expressed at a constant level in the eyestalks of juveniles and mature females. Unlike the MeCHH-A gene, a low level of MeCHH-B transcripts can also be detected in the central nervous system. Interestingly, the expression pattern of MeCHH-B in the eyestalk of vitellogenic females is reversed to that of the MeCHH-A gene. At the middle stage of gonad maturation, a minimum level of MeCHH-B transcript was recorded and a maximum level of MeCHH-A transcript was detected. Recombinant proteins for MeCHH-A and MeCHH-B were produced by a bacterial expression system. The hemolymph glucose level of bilaterally eyestalk-ablated shrimp increased two-fold 1 h after the rCHH injection and then returned to normal after 2 h. The hyperglycemic effect of these fusion proteins is comparable to that of de-stalked shrimp injected with crude extract from a single sinus gland.