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Juvenile hormone (JH) and methyl farnesoate (MF) play essential roles in the development and reproduction of insects and crustaceans respectively. Juvenile hormone acid methyltransferase (JHAMT) catalyzes the methyl esterification in insect JH biosynthesis, while the corresponding step in crustacean MF biosynthesis was long thought to be catalyzed by farnesoic acid O -methyltransferase (FAMeT). However, the new discovery of JHAMT orthologs in crustaceans indicates that JHAMT may also play essential role in the MF biosynthesis in crustaceans. Here we cloned and characterized the full-length cDNA encoding JHAMT in the swimming crab Portunus trituberculatus ( PtJHAMT ). Sequence and structure analysis of PtJHAMT revealed that it was composed of a 6-stranded β sheet with 9 α helices, and contained a signature Sadenosyl-l-methionine (SAM) binding motif, which is the hallmark in all SAM dependent methyltransferases (SAM-MTs). Several active sites that are critical for the interaction of SAM and JH/FA substrate were also conserved in PtJHAMT. The gene expression of PtJHAMT was highly specific to the mandibular organ, which is the sole site of MF synthesis. PtJHAMT expression significantly increased in the late-vitellogenic stage and mature stage, which suggests a possible role of PtJHAMT in modulating ovarian development. The role of PtJHAMT and PtFAMeT in MF biosynthesis was further investigated by RNA interfering (RNAi). Injection of PtJHAMT and PtFAMeT dsRNA both led to a decrease in hemolymph MF titers. Injection of PtHMGR dsRNA caused the decrease in PtJHAMT expression, but had no effect on mRNA level of PtFAMeT . Together these results suggested that JHAMT and FAMeT are both involved in the MF biosynthesis in crustaceans, while the JHAMT is highly specific to FA substrate, and FAMeT may have more catalytic functions.

The phylum Arthropoda contains the largest number of described living animal species, with insects and crustaceans dominating the terrestrial and aquatic environments, respectively. Their successful radiations have long been linked to their rigid exoskeleton in conjunction with their specialized endocrine systems. In order to understand how hormones can contribute to the evolution of these animals, here, we have categorized the sesquiterpenoid and ecdysteroid pathway genes in the noninsect arthropod genomes, which are known to play important roles in the regulation of molting and metamorphosis in insects. In our analyses, the majority of gene homologs involved in the biosynthetic, degradative, and signaling pathways of sesquiterpenoids and ecdysteroids can be identified, implying these two hormonal systems were present in the last common ancestor of arthropods. Moreover, we found that the “Broad-Complex” was specifically gained in the Pancrustacea, and the innovation of juvenile hormone (JH) in the insect linage correlates with the gain of the JH epoxidase (CYP15A1/C1) and the key residue changes in the binding domain of JH receptor (“Methoprene-tolerant”). Furthermore, the gain of “Phantom” differentiates chelicerates from the other arthropods in using ponasterone A rather than 20-hydroxyecdysone as molting hormone. This study establishes a comprehensive framework for interpreting the evolution of these vital hormonal pathways in these most successful animals, the arthropods, for the first time.

Crustacean hyperglycemic hormone (CHH) plays an important role in crustacean. In the present study, a full-length cDNA of CHH was cloned from the eyestalk of Litopenaeus vannamei by RACE approach for the first time. The full-length cDNA of LvCHH was 846 bp, containing a 5' untranslated region (UTR) of 65 bp, a 3' UTR of 436 bp with a canonical polyadenylation signal-sequence AATAA and a poly (A) tail, and an open reading frame (ORF) of 345 bp. The ORF encoded a polypeptide of 114 amino acids including a 24 amino acid signal peptide. The calculated molecular mass of the mature protein (74 amino acids) was 8.76 kDa with an estimated pI of 6.78. The sequence of LvCHH was submitted in NCBI GenBank under the accession number HM748790.2. Phylogenetic analysis revealed that LvCHH was clustered with CHH of other crustaceans. Tissue distribution analysis revealed that the expression of LvCHH mRNA was observed in all tissues but gill, and was highest in heart. Specific primers containing Xho I and BamH I restriction sites respectively, were designed based on the obtained ORF sequence of LvCHH gene and the cloning sites of expression vector pET-32a (+). The recombinant plasmid LvCHH-pET32a, was used to transform Escherichia coli BL21 (DE3). LvCHH was successfully expressed by means of SDS-PAGE and western blot analysis. We detected gill Na(+)/K(+)-ATPase activity after rLvCHH protein injection and found that All the experimental group Na(+)/K(+)-ATPase activity presented peak change among 0-6h, and the peaks of all treated groups occurred in 1 h. 20 and 30 μg/shrimp(-1) groups showed significant increase (P

Bursicon is a heterodimeric neurohormone that acts through a G protein-coupled receptor named rickets (rk), thus inducing an increase in cAMP and the activation of tyrosine hydroxylase, the rate-limiting enzyme in the cuticular tanning pathway. In insects, the role of bursicon in the post-ecdysial tanning of the adult cuticle and wing expansion is well characterized. Here we investigated the roles of the genes encoding the bursicon subunits during the adult cuticle development in the honeybee, Apis mellifera. RNAi-mediated knockdown of AmBurs α and AmBurs β bursicon genes prevented the complete formation and tanning (melanization/sclerotization) of the adult cuticle. A thinner, much less tanned cuticle was produced, and ecdysis toward adult stage was impaired. Consistent with these results, the knockdown of bursicon transcripts also interfered in the expression of genes encoding its receptor, AmRk, structural cuticular proteins, and enzymes in the melanization/sclerotization pathway, thus evidencing roles for bursicon in adult cuticle formation and tanning. Moreover, the expression of AmBurs α, AmBurs β and AmRk is contingent on the declining ecdysteroid titer that triggers the onset of adult cuticle synthesis and deposition. The search for transcripts of AmBurs α, AmBurs β and candidate targets in RNA-seq libraries prepared with brains and integuments strengthened our data on transcript quantification through RT-qPCR. Together, our results support our premise that bursicon has roles in adult cuticle formation and tanning, and are in agreement with other recent studies pointing for roles during the pharate-adult stage, in addition to the classical post-ecdysial ones.

A complete understanding of the biological functions of large signaling peptides (>4 kDa) requires comprehensive characterization of their amino acid sequences and post-translational modifications, which presents significant analytical challenges. In the past decade, there has been great success with mass spectrometry-based de novo sequencing of small neuropeptides. However, these approaches are less applicable to larger neuropeptides because of the inefficient fragmentation of peptides larger than 4 kDa and their lower endogenous abundance. The conventional proteomics approach focuses on large-scale determination of protein identities via database searching, lacking the ability for in-depth elucidation of individual amino acid residues. Here, we present a multifaceted MS approach for identification and characterization of large crustacean hyperglycemic hormone (CHH)-family neuropeptides, a class of peptide hormones that play central roles in the regulation of many important physiological processes of crustaceans. Six crustacean CHH-family neuropeptides (8–9.5 kDa), including two novel peptides with extensive disulfide linkages and PTMs, were fully sequenced without reference to genomic databases. High-definition de novo sequencing was achieved by a combination of bottom-up, off-line top-down, and on-line top-down tandem MS methods. Statistical evaluation indicated that these methods provided complementary information for sequence interpretation and increased the local identification confidence of each amino acid. Further investigations by MALDI imaging MS mapped the spatial distribution and colocalization patterns of various CHH-family neuropeptides in the neuroendocrine organs, revealing that two CHH-subfamilies are involved in distinct signaling pathways.

Drosophila melanogaster flies concentrate behavioral activity around dawn and dusk. This organization of daily activity is controlled by central circadian clock neurons, including the lateral-ventral pacemaker neurons (LN(v)s) that secrete the neuropeptide PDF (pigment dispersing factor). Previous studies have demonstrated the requirement for PDF signaling to PDF receptor (PDFR)-expressing dorsal clock neurons in organizing circadian activity. Although LN(v)s also express functional PDFR, the role of these autoreceptors has remained enigmatic. Here, we show that (1) PDFR activation in LN(v)s shifts the balance of circadian activity from evening to morning, similar to behavioral responses to summer-like environmental conditions, and (2) this shift is mediated by stimulation of the Gα,s-cAMP pathway and a consequent change in PDF/neurotransmitter corelease from the LN(v)s. These results suggest another mechanism for environmental control of the allocation of circadian activity and provide new general insight into the role of neuropeptide autoreceptors in behavioral control circuits.

The pattern of amino acid residue replacement in the components of the bursicon signaling system (involving the BURSα/BURSβ heterodimer and its receptor BURSrec) was reconstructed across a phylogeny of 17 insect species, in order to test for the co-occurrence of replacements at sets of individual sites. Sets of three or more branches with perfectly concordant changes occurred to a greater extent than expected by chance, given the observed level of amino acid change. The latter sites (SPC sites) were found to have distinctive characteristics: (1) the mean number of changes was significantly lower at SPC sites than that at other sites with multiple changes; (2) SPC sites had a significantly greater tendency toward parallel amino acid changes than other sites with multiple changes, but no greater tendency toward convergent changes; and (3) parallel changes tended to involve relatively similar amino acids, as indicated by relatively low mean chemical distances. The results implicated functional constraint, permitting only a limited subset of amino acids in a given site, as a major factor in causing both parallel amino acid replacement and coordinated amino acid changes in different sites of the same protein and of interacting proteins in this system.

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.

We have cloned an earthworm-derived Factor Xa (FXa) inhibitor, with an excellent inhibitory specificity from the midgut of the Eisenia andrei. We designate this inhibitor eisenstasin. An eisenstasin-derived small peptide (ESP) was synthesized and we examined whether ESP played an essential role in FXa inhibition. Compared to antistasin-derived small peptides (ASP) originating from leech, ESP primarily exhibited a high level of FXa inhibition in chromogenic peptide substrate assays and revealed an approximately 2-fold greater inhibition of FXa cleavage of a target protein than ASP. This suggests that ESP could be an effective anti-coagulant that targets FXa during the propagation step of coagulation. ESP also inhibited proteinase-activated receptor 2-mediated FXa activation, which may trigger endothelial inflammation. Endothelial nitric oxide (NO) was significantly reduced by ESP (p

The neuropeptides of the crustacean hyperglycaemic hormone (CHH) family are encoded by a multigene family and are involved in a wide spectrum of essential functions. In order to characterize CHH family peptides in one of the last groups of decapods not yet investigated, CHH was studied in two anomurans: the hermit crab Pagurus bernhardus and the squat lobster Galathea strigosa. Using RT-PCR and 3' and 5' RACE methods, a preproCHH cDNA was cloned from the major neuroendocrine organs (X-organs) of these two species. Hormone precursors deduced from these cDNAs in P. bernhardus and G. strigosa are composed of signal peptides of 29 and 31 amino acids, respectively, and CHH precursor-related peptides (CPRPs) of 50 and 40 amino acids, respectively, followed by a mature hormone of 72 amino acids. The presence of these predicted CHHs and their related CPRPs was confirmed by performing MALDI-TOF mass spectrometry on sinus glands, the main neurohaemal organs of decapods. These analyses also suggest the presence, in sinus glands of both species, of a peptide related to the moult-inhibiting hormone (MIH), another member of the CHH family. Accordingly, immunostaining of the X-organ/sinus gland complex of P. bernhardus with heterologous anti-CHH and anti-MIH sera showed the presence of distinct cells producing CHH and MIH-like proteins. A phylogenetic analysis of CHHs, including anomuran sequences, based on maximum-likelihood methods, was performed. The phylogenetic position of this taxon, as a sister group to Brachyura, is in agreement with previously reported results, and confirms the utility of CHH as a molecular model for understanding inter-taxa relationships. Finally, the paraphyly of penaeid CHHs and the structural diversity of CPRPs are discussed.

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.

Serotonin, a highly conserved neurotransmitter, controls many biological functions in vertebrates, but also in invertebrates. Selective serotonin reuptake inhibitors (SSRIs), such as fluoxetine, are commonly used in human medication to ease depression by affecting serotonin levels. Their residues and metabolites can be detected in the aquatic environment and its biota. They may also alter serotonin levels in aquatic invertebrates, thereby perturbing physiological functions. To investigate whether such perturbations can indeed be expected, shore crabs (Carcinus maenas) were injected either with serotonin, fluoxetine or a combination of both. Dose-dependent effects of fluoxetine ranging from 250 to 750nM were investigated. Gene expression of crustacean hyperglycemic hormone (chh) as well as moult inhibiting hormone (mih) was assessed by RT-qPCR at 2h and 12h after injection. Glucose and ecdysteroid levels in the haemolymph were monitored in regular intervals until 12h. Serotonin led to a rapid increase of chh and mih expression. On the contrary, fluoxetine only affected chh and mih expression after several hours, but kept expression levels significantly elevated. Correspondingly, serotonin rapidly increased glycaemia, which returned to normal or below normal levels after 12h. Fluoxetine, however, resulted in a persistent low-level increase of glycaemia, notably during the period when negative feedback regulation reduced glycaemia in the serotonin treated animals. Ecdysteroid levels were significantly decreased by serotonin and fluoxetine, with the latter showing less pronounced and less rapid, but longer lasting effects. Impacts of fluoxetine on glycaemia and ecdysteroids were mostly observed at higher doses (500 and 750nM) and affected principally the response dynamics, but not the amplitude of glycaemia and ecdysteroid-levels. These results suggest that psychoactive drugs are able to disrupt neuroendocrine control in decapod crustaceans, as they interfere with the normal regulation of the serotonergic system.

International audience; To assess functional importance of the residues in the amino-and carboxyl-termini of crustacean hyperglycemic hormone in the mud crab Scylla olivacea (Sco-CHH), both wild-type and point-mutated CHH peptides were produced with an amidated C-terminal end. Spectral analyses of circular dichroism, chromatographic retention time, and mass spectrometric analysis of the recombinant peptides indicate that they were close in conformation to native CHH and were produced with the intended substitutions. The recombinant peptides were subsequently used for an in vivo hyperglycemic assay. Two mutants (R13A and I69A rSco-CHH) completely lacked hyperglycemic activity, with temporal profiles similar to that of vehicle control. Temporal profiles of hyperglycemic responses elicited by 4 mutants (I2A, F3A, D12A, and D60A Sco-CHH) were different from that elicited by wild-type Sco-CHH; I2A was unique in that it exhibited significantly higher hyperglycemic activity, whereas the remaining 3 mutants showed lower activity. Four mutants (D4A, Q51A, E54A, and V72A rSco-CHH) elicited hyperglycemic responses with temporal profiles similar to those evoked by wild-type Sco-CHH. In contrast, the glycine-extended version of V72A rSco-CHH (V72A rSco-CHH-Gly) completely lost hyperglycemic activity. By comparing our study with previous ones of ion-transport peptide (ITP) and molt-inhibiting hormone (MIH) using deleted or point-mutated mutants, detail discussion is made regarding functionally important residues that are shared by both CHH and ITP (members of Group I of the CHH family), and those that discriminate CHH from ITP, and Group-I from Group-II peptides. Conclusions summarized in the present study provide insights into understanding of how functional diversification occurred within a peptide family of multifunctional members.

This study was aimed at determining the role of the crustacean hyperglycemic hormone (CHH) in the physiological compensation to both saline and thermal stress, in the freshwater crayfish Cherax quadricarinatus. By determining the expression of the CHH gene in the eyestalk of juvenile crayfish, we found that maximal induction of CHH was induced at high salinity (10 g/L) and low temperature (20 °C). In order to investigate the role of CHH in the physiological compensation to such stressful conditions, recombinant CHH was supplied to stressed animals. CHH-injected crayfish showed increased hemolymphatic levels of glucose, in accordance with a significant utilization of glycogen reserves from the hepatopancreas. Furthermore, CHH administration allowed stressed animals to regulate hemolymphatic sodium and potassium at more constant levels than controls. Taken together, these results suggest a relevant role of CHH in increasing the energy available intended for processes involved in the physiological compensation of C. quadricarinatus to both saline and thermal stress. Fil: Prymaczok, Natalia Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina Fil: Pasqualino, Valeria M.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina Fil: Viau, Veronica Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; Argentina Fil: Rodriguez, Enrique Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; Argentina Fil: Medesani, Daniel Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; Argentina

The essential trace elements Cr and Mn are toxic at high concentrations and information about low concentration is insufficient in the literature. In polluted mangroves, the crab Ucides cordatus can represent a useful tool to assess information on the potential impact of trace elements like Cr and Mn on the environment, since this species is comestible and thus, commercially negotiated. Therefore, U. cordatus crabs were exposed in vivo to different concentrations of Cr and Mn solved in seawater and had their tissue distribution and subcellular deposits evaluated. The gill, hepatopancreas and muscle concentrations were determined by atomic absorption spectroscopy and the results showed that Cr and Mn presented the highest values in the gills rather than in the hepatopancreas and muscular tissue. Electron microscopy and analytical X-ray microanalysis revealed Cr precipitates on the gill surface, co-localized with epiphyte bacteria. In addition, since Cr and Mn did not equally accumulate in most of the tissues studied, glycemic rate of animals, which received injections of extracts of eyestalks of the contaminated crabs, were measured in order to evaluate whether the studied concentrations of Cr and Mn could produce any metabolic alteration. The results indicated that extracts of the eyestalks of crabs submitted to Cr and Mn salts and injected into normal crabs markedly influenced crustacean hyperglycemic hormone synthesis and/or release. The results are discussed with respect to sensitivity of the employed methods and the possible significance of the concentrations of Cr and Mn in the organisms.

Peptidic lipopolysaccharide (LPS) antagonists are the subject of intensive research. We report an NMR and modeling study of LALF-14 (GCKPTFRRLKWKYKCG), a synthetic cyclized fragment of the limulus anti-LPS factor (LALF) comprising residues 36-47. In a mixture with LPS we observed the transferred NOE effect and derived the LPS-bound structure of LALF-14. Neither the free nor the LPS-bound peptide displays NOEs indicative of a beta-sheet-like structure that is adopted by the fragment in the full-size protein. However, docking calculations show that the former structure is not a prerequisite for binding of LALF-14 to LPS.

SUMMARYThis study investigates (by means of bioassays and ELISA using an antibody against recombinant cHH) the variation of cHH levels in the eyestalks and haemolymph of Palaemon elegans (Decapoda, Caridea) following exposure to various stresses (heavy metals and lipopolysaccharide), and correlates them with the variation in amount and time course of blood glucose. The dose-relationship between exposure to copper and quick release of cHH from the eyestalk into haemolymph was confirmed by variation of blood glucose with a dose-related hyperglycaemia, that peaked 2 h after immersion in contaminated seawater. Animals exposed to a sublethal concentration of mercury showed the same dose relation between toxicant, release of cHH from the eyestalk,increment of circulating hormone level and subsequent hyperglycaemia as observed for copper contamination. It is of note that although the highest lethal mercury concentration induced the release of cHH from the eyestalk into the haemolymph, it was not followed by a significant variation of blood glucose. Step doses of a bacterial contaminant [such as lipopolysaccharide(LPS) from E. coli injected into shrimps] confirmed the dose-relationship and convergent chain of events that bring about hyperglycaemia. These are the first data that relate the release of cHH from the eyestalk, the circulating hormone level and the consequent glycaemic response to stress. Moreover, they confirm the dose-related pathway that leads to variation of blood glucose as a quantitative biomarker of environmental quality, even at sublethal toxicant concentrations.

Crustacean hyperglycemic hormone (CHH) and molt-inhibiting hormone (MIH) have similar amino acid sequences and therefore comprise a peptide family referred to as the CHH family. All MIHs unexceptionally have an additional glycine residue at position 12, which is lacking in all CHHs. In order to understand the relevance of the absence of the glycine residue for hyperglycemic activity, a mutant CHH having a glycine residue insertion was prepared, and its hyperglycemic activity was assessed. This mutant CHH had the same disulfide bond arrangement as the recombinant CHH produced in Escherichia coli cells, and exhibited a similar circular dichroism spectrum to the recombinant CHH, indicating that the two CHHs possessed similar conformations. The mutant CHH showed a hyperglycemic effect weaker than the recombinant CHH by about one order of magnitude. These results suggest that the insertion of a glycine residue is one of the indices for structural and functional divergence of the CHH family peptides.

An efficient pipeline for peptide discovery accelerates peptidomic analysis and facilitates a better understanding of the functional roles of neuropeptides. However, qualitative and quantitative analysis of large neuropeptides are challenging due to the bigger molecular sizes, multiple post-translational modifications, and interference by homologous isoforms. Herein, we refined two methodologies in the pipeline for highly confident and efficient mass spectrometry (MS)-based peptide discovery. For the qualitative analysis, the so-called “high resolution/accurate mass” measurement on Orbitrap mass spectrometers was integrated with computer-assisted homology search, which was successfully applied to decipher the substituted amino acid residues in large neuropeptides by referring to homologous sequences. For the quantitative analysis, a new isotopic labeling-assisted top-down MS strategy was developed, which enabled direct monitoring of the abundance changes of endogenous large neuropeptides. By using the refined peptide discovery pipeline, one novel crustacean hyperglycemic hormone (CHH) from the Dungeness crab sinus glands was confidently identified and de novo sequenced, and its relative abundance was quantified. Comparative analysis of CHHs in unfed and fed crabs revealed that the peptide abundance in the sinus glands was significantly increased after food intake, suggesting that the release of CHHs might be altered by feeding behavior.

Ghilanten, isolated from the leech Haementeria ghilianii, is a potent two-domain anticoagulant protein homologous to the factor Xa inhibitor antistasin. A synthetic gene encoding the amino-terminal domain of ghilanten (ghilanten-D1) was constructed, expressed in the methylotrophic yeast Pichia pastoris and purified by heparin-Sepharose chromatography. Recombinant ghilanten-D1 inhibits bovine trypsin and human factor Xa with equilibrium inhibition constants (K(i)) of 126 and 1.2 nM, respectively. Ghilanten-D1 has been crystallized in complex with porcine beta-trypsin; three different-looking but isomorphous crystal forms were obtained, each belonging to the orthorhombic space group P2(1)2(1)2(1). These crystals diffracted to beyond 3.6 A resolution using a rotating-anode X-ray source. A data set complete to 3.7 A resolution was collected.

The effects of a dinoflagellate parasite (Hematodinium sp.) on carbohydrate metabolism were examined in the Norway lobster, Nephrops norvegicus. Five stages of infection were observed. These included uninfected (Stage 0), subpatently infected (SP), and patently infected (Stage 1-4) lobsters. During patent infection, the concentration of glucose in the hemolymph was reduced significantly from its value of 180 microg ml(-1) in uninfected (Stage 0) lobsters to 25.3 microg ml(-1) in Stage 3-4. These changes were accompanied by significantly lower levels of hepatopancreatic glycogen in lobsters at Stage 2 (2.01 mg g(-1)) and Stage 3-4 (0.84 mg g(-1)) of infection than in those at Stage 0 (16.19 mg g(-1)) and Stage 1 (14.71 mg g(-1)). Due to disruption of the normal feedback loops which control the release of crustacean hyperglycemic hormone (CHH), plasma concentrations increased with the severity of infection from 32.2 fmol ml(-1) in Stage 0 to 106.6 fmol ml(-1) in Stage 3-4. The increased CHH concentrations occurred concomitantly with reduced concentrations of plasma glucose and tissue glycogen. A significantly increased hemolymph CHH titer (107.7 fmol ml(-1)) was also observed during SP infection. It is concluded that the parasite places a heavy metabolic load on the host lobster.

The crustacean hyperglycemic hormone (CHH) peptide family includes four types of neuropeptide in decapod and isopod crustaceans, and the ion-transport peptide in orthopteran insects. To identify a new member of this family in Insecta, a PCR-based search for cDNAs encoding CHH-family peptides was carried out in the silkworm Bombyx mori. A cDNA, named BmCHHL (Bombyx mori CHH-like protein), with an open reading frame of 110 amino acids was isolated. Sequence analyses suggested that the conceptual protein was a precursor of a peptide of 72 amino acids which was amidated at the carboxy terminus. The BmCHHL sequence exhibited significant similarities to members of the CHH family including the orthopteran ion-transport peptide. BmCHHL expression was detected in five or six cells (per hemisphere) in the frontal area of the brain in day 4 fifth instar larvae.

In an effort to characterize the insect molting hormone bursicon from the cockroach, Periplaneta americana, amino acid sequences with high identity of Cu,Zn-superoxide dismutase (SOD) of Drosophila virilis were identified. Antisera against a conserved region of SOD, and a sequence unique to Periplaneta SOD were produced and used to test whether bursicon might be a form of SOD. Western blots of one- and two-dimensional gels revealed that the dimeric form of SOD and bursicon have a similar molecular mass (30 kDa). The two proteins can be separated, however, according to their different isoelectric points. Bursicon is identified in two-dimensional gels by elution from four unique spots not labeled by the anti-SOD antisera. In sections of Periplaneta nerve cords the antisera labeled glial material surrounding neuronal somata close to the neural sheath. Bursicon, however, is contained in unique cell pairs in the ganglia of the ventral nerve cord. These neurons were labeled with new antisera produced against novel sequences of one of the four above-mentioned bursicon active spots. The results show unequivocally that SOD and bursicon are distinctly different proteins. Furthermore, the anti-SOD antisera provided a tool to isolate and sequence bursicon.

Three chromatophorotropic neuropeptide hormones were purified from an aqueous extract of the sinus glands of the kuruma prawn Penaeus japonicus by two steps of reverse-phase HPLC and their amino acid sequences determined. One of them was found to show pigment concentrating activity and to have an amino acid sequence identical with that of the known red pigment concentrating hormone (RPCH), and therefore it was named Pej-RPCH. The other two peptides showed pigment dispersing hormone (PDH) activity and were named Pej-PDH-I and -II. They both consisted of 18 amino acid residues with a free amino-terminus and an amidated carboxyl-terminus, the sequences of Pej-PDH-I and -II being NSELINSLLGIPKVMTDAamide and NSELINSLLGLPKFMIDAamide, respectively. Three amino acid residues at positions 11, 14, and 16 differed between the two PDHs. Pej-PDH-II was about 5-, 7-, and 10-fold more potent than Pej-PDH-I for erythrophores, xanthophores, and melanophores, respectively. The major reason for the difference in potency between the two PDHs was attributed to differences in residues at position 16. In addition, they were found to be produced by a single individual. The order of sensitivity of the four types of chromatophores to Pej-RPCH and both PDHs was found to be erythrophores = xanthophores > melanophores > leukophores.

The molecular links between membrane excitability and neurohormone synthesis were investigated using a simple model system: the bag cell neurons of Aplysia. We tested the hypothesis that the electrical afterdischarge, which leads to depletion of egg-laying hormone (ELH) by triggering secretion, rapidly stimulates ELH synthesis to replenish bag cell stores of hormone. Newly synthesized peptides were radiolabeled, and ELH peptide was immunoprecipitated. Within 4 h of afterdischarge, there was a 100% increase in ELH synthesis compared with that in unstimulated controls. Northern blot analysis showed that ELH messenger RNA (mRNA) levels were not altered for at least 8 h after the onset of afterdischarge. ELH mRNA levels were also not affected by 4-h treatments that inhibited either transcription (with actinomycin D) or translation (with anisomycin). Further work revealed that ELH mRNA is stable, with a half-life exceeding 32 h. Notably, the stimulatory effect of afterdischarge on ELH synthesis was blocked in response to treatment with the transcription inhibitor actinomycin D. These results suggest that the afterdischarge-induced increase in ELH synthesis is mediated by an increase in the rate of translation of already existing ELH mRNA, and that transcription of a non-ELH gene(s) is required for this effect to occur.

To elucidate the mechanisms of inactivation of the ecdysiostatic peptide trypsin-modulating oostatic factor (Neb-TMOF) in the blue blowfly Calliphora vicina, we investigated its proteolytic degradation. In homogenates and membrane and soluble fractions, this hexapeptide (sequence: NPTNLH) was hydrolyzed into two fragments, NP and TNLH, suggesting the involvement of a proline-specific dipeptidyl peptidase. The dipeptidyl peptidase activity was highest in the late larval stage. It was purified 240-fold from soluble fractions of pupae of mixed age and classified on the basis of several catalytic properties as an invertebrate homologue of mammalian dipeptidyl peptidase IV (EC 3.4.14.5). Fly dipeptidyl peptidase IV has a molecular mass of 200 kDa, showed a pH optimum of 7.5-8.0 with the chromogenic substrate Gly-Pro-4-nitroanilide, and cleaved other chromogenic substrates with penultimate Pro or, with lower activity, Ala. It liberated Xaa-Pro dipeptides from the N-terminus of several bioactive peptides including substance P, neuropeptide Y, and peptide YY but not from bradykinin, indicating that the peptide bond between the two proline residues was resistant to cleavage. Fly dipeptidyl peptidase belongs to the serine class of proteases as the mammalian enzyme does; the fly enzyme, however, is not inhibited by several selective or nonselective inhibitors of its mammalian counterpart. It is suggested that dipeptidyl peptidases exert a regulatory role for the clearance not only of TMOF in files but for other bioactive peptides in various invertebrates.

Blood ingestion by the female mosquito initiates a series of endocrine events that is dominated by juvenile hormone, ecdysteroids, and several peptide hormones, resulting in the maturation of a batch of eggs. The mechanisms of hormone release and their roles during the previtellogenic, vitellogenic, and postvitellogenic phases are discussed. Arch. Insect Biochem. Physiol. 35:491–512, 1997. © 1997 Wiley-Liss, Inc.

Reich, G., K. E. Doble, D. A. Price and M. J. Greenberg. Effects of cardioactive peptides on myocardial cAMP levels in the snail Helix aspersa. Peptides 18 (3) 355–360, 1997.—Several cardioactive peptides from the pulmonate snail Helix aspersa were tested for their effects on myocardial cAMP levels, but only the family of small cardioactive peptides (SCPs) were clearly effective. SCP increased cAMP in a dose dependent manner; the time course was phasic. The structure-activity relations of this effect were examined with a set of 3 synthetic analogs having characteristics, at the carboxyterminal, of both the SCPs and FMRFamide-related peptides. The adenylate cyclase activator forskolin mimicked the mechanical effect of SCPs on the heartbeat. We conclude that the effect of SCPs on the Helix heart may be mediated by cAMP.

Hyperglycemia that is caused by the release of crustacean hyperglycemic hormone (CHH) from the sinus gland to hemolymph is one of the hallmark physiological phenomena, occurring in decapod crustaceans experiencing stressful conditions. However, the mechanism(s) by which such elevated glucose levels return to resting levels is still unknown. Interestingly, noted is a difference in the clearance rate of hemolymph glucose between adult females and adult males of the blue crab, Callinectes sapidus: the former with more rapid clearance than the latter. The presence of an endogenous-insulin-like molecule is suggested in C. sapidus because an injection of bovine insulin, significantly reduces the levels of hemolymph glucose that were previously elevated by emersion stress or the glucose injection. Using 5' and 3' RACE, the full-length cDNA of an insulin-like molecule is isolated from the hepatopancreas of an adult female C. sapidus and shows the same putative sequence of an insulin-like androgenic gland factor (IAG) but differs in 5' and 3' UTR sequences. A knock-down study using five injections of double-stranded RNA of CasIAG-hep (dsRNA-CasIAG-hep, 10μg/injection) over a 10-day period reduces CasIAG-hep expression by ∼50%. The levels of hemolymph glucose are also kept higher in dsRNA-CasIAG-hep injected group than those treated with dsRNA-green fluorescent protein (dsRNA-IAG-hep) or saline. Most importantly, the hepatopancreas of dsRNA-CasIAG-hep injected animals contains amounts of carbohydrate (glucose, trehalose, and glycogen) significantly lower than those of control groups, indicating that the function of CasIAG-hep in carbohydrate metabolism in crustaceans is similar to carbohydrate metabolism in vertebrates.

Temperature is one of the most important environmental factors affecting the biological processes of aquatic species. To investigate the potential effects of temperature on the developmental processes of aquatic invertebrates, we analyzed biological and molecular transcriptional responses during Chironomus riparius development, including five stages spanning from embryo to adult stages. We assessed the temperature change-induced reduction of survival rate, changes in biological development including the male:female ratio in emerged adults, the success rates of pupation and emergence, and the developmental timing of pupation and emergence. The increased temperature induced expression of endocrine signaling genes, such as the ecdysone receptor, ultraspiracle (ortholog of the RXR), and the estrogen-related receptor in the fourth-instar larval and pupal stages of C. riparius development. Altered temperature also affected the activity of antioxidant genes, including catalase, peroxidase, glutathione peroxidase, and superoxide dismutase during the fourth-instar larval to adult stages of C. riparius development, as a result of altered development. Increased temperature during the fourth-instar larval stage increased oxidative stress in pupae and adults. Responses of antioxidant genes to increased temperature occurred in a developmental stage-dependent manner. However, reduced temperature did not induce the expression of antioxidant genes in a developmental stage-dependent manner, although it did induce oxidative stress during C. riparius development. Increased temperature also caused greater toxicity of di-ethylhexyl phthalate (DEHP) in fourth-instar larvae. Our findings suggest that altered temperatures may disturb the invertebrate hormone system and developmental processes by inducing oxidative stress in aquatic environments.

1. Modulatory effects of the four molluscan neuroactive peptides. FMRFamide (Phe-Met-Arg-Phe-NH2), APGW-amide (Ala-Pro-Gly-Trp-NH2), oxytocin and [SER2]-Mytilus inhibitory peptide ([SER2]-MIP) (Gly-Ser-Pro-Met-Phe-Val-NH2) were examined on the inward current (Iin) caused by achatin-I (Gly-D-Phe-Ala-Asp), which has been isolated from the Achatina ganglia. 2. Two Achatina giant neurone types, v-RCDN (ventral-right cerebral distinct neurone) and PON (periodically oscillating neurone), were used. Achatin-I was applied locally to the neurone tested by brief pneumatic pressure ejection, and the other molluscan neuroactive peptides were perfused around the ganglia. 3. FMRFamide, perfused at 3 microM, suppressed markedly the Iin elicited by the achatin-I of both v-RCDN and PON. APGW-amide at 3 microM also suppressed the Iin of v-RCDN, but did not affect that of PON. Oxytocin at 1 microM suppressed the Iin of PON, but did not affect that of v-RCDN. [Ser2]-MIP at 3 microM did not affect the Iin of v-RCDN. 4. The dose-response curves of FMRFamide, APGW-amide and oxytocin, indicated that their respective suppressive effects on the Iin of achatin-I were dose-dependent, and that APGW-amide was slightly more potent than the other peptides. The dose (pressure duration)-response curves of achatin-I (1 kg/cm2, 10(-3) M, 5 min interval), obtained by varying the duration of the achatin-I pressure ejection, were measured in the presence and absence of each of the three peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

A recombinant endotoxin-neutralizing protein, rBPI23, was shown to partially prevent endotoxin-induced activation of the fibrinolytic and coagulation systems in experimental endotoxemia in humans. In a placebo-controlled, blinded crossover study, eight volunteers were challenged twice with an intravenous bolus injection of endotoxin (40 EU/kg of body weight) and concurrently received either rBPI23 (1 mg/kg) or placebo (human serum albumin, 0.2 mg/kg). rBPI23 treatment significantly lowered the endotoxin-induced fibrinolytic response, ie, reduced the release of tissue-type plasminogen activator, urokinase-type plasminogen activator, plasminogen activator inhibitor antigen, and complex formation of plasmin alpha 2-antiplasmin (P = .0078 for each). Plasminogen activator inhibitor activity was also reduced, but not significantly according to the Hochberg method (P = .0304). The endotoxin-induced activation of the procoagulant state as reflected by increase in F1 + 2 fragments and TAT complexes was blunted by rBPI23 infusion (P = .0391 [not significant according to the Hochberg method] and .0078, respectively). These results indicate that rBPI23 is capable of reducing both the activation of the fibrinolytic and the coagulation systems after low-dose endotoxin infusion in humans.

When a larva of the red flour beetle Tribolium freemani (Hinton) was introduced into a vial, the larva pupated within 6 days, whereas when two larvae were introduced, the larvae pupated in 12 days. Crowded larvae delayed production of juvenile-hormone esterase (JHE) and release of ecdysteroids, as measured by radioimmunoassay, compared with the isolated larvae. The highest JHE activity was observed at pupal ecdysis in both groups and there were increases in whole-body ecdysteroid levels during the last part of intermoult before pupation and adult emergence, which coincided with decreases in ecdysteroid titres in both isolated and crowded groups. Cold shock, heat stress, the JHE inhibitor methamidophos, and juvenile hormone (JH) I delayed pupation in isolated larvae. Cold shock, heat stress and methamidophos reduced the JHE activity, and cold shock and JH I reduced ecdysteroid levels in isolated larvae. Light, vibration and a nonsteroidal ecdysteroid agonist RH 5849 stimulated pupation in crowded T. freemani larvae, which was antagonized by JH I. The JHE activity and ecdysteroid levels were higher in crowded larvae reared under 24 h light or treated with vibration, octopamine agonists and precocene II than those control larvae kept in constant darkness.

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.

The crustacean Hyperglycemic Hormone (cHH) is present in many decapods in different isoforms, whose specific biological functions are still poorly understood. Here we report on the first chemical synthesis of three distinct isoforms of the cHH of Astacus leptodactylus carried out by solid phase peptide synthesis coupled to native chemical ligation. The synthetic 72 amino acid long peptide amides, containing L- or D-Phe3 and (Glp1, D-Phe3) were tested for their biological activity by means of homologous in vivo bioassays. The hyperglycemic activity of the D-isoforms was significantly higher than that of the L-isoform, while the presence of the N-terminal Glp residue had no influence on the peptide activity. The results show that the presence of D-Phe3 modifies the cHH functionality, contributing to the diversification of the hormone pool.

LMWP is the low molecular weight protein which copurifies with alpha-latrotoxin, the main neurotoxin from the black widow venom. It contains 70 residues and three disulfides. We found that its primary structure, including its 6 half-cystines, can be aligned with the amino acid sequences of crustacean hyperglycemic hormones (CHHs) which contain 72-73 residues and three disulfides. To further investigate this structural relationship, we produced a recombinant analog of LMWP in which the unique Met was changed in Leu (LMWPM35L). LMWPM35L was produced as a folded fusion protein in the periplasm of Escherichia coli and was generated in vitro by treating the fusion protein with cyanogen bromide. We showed that LMWPM35L and CHHs have an identical disulfide pairing pattern and possess some alpha-helical structure, as deduced from a comparison of their circular dichroism spectra. In addition, LMWPM35L and CHHs are consensually predicted to possess a helical structure within the region 13-17. Together, the data indicate that CHHs are structurally related to LMWPM35L and presumably also to LMWP. Finally, preliminary studies showed that LMWPM35L is not toxic to mice and does not form channels in lipid bilayers, two well-known properties of alpha-latrotoxin preparations.

A purification scheme is described for the isolation of secretory granules containing egg-laying prohormone-related peptides from the atrial gland of Aplysia californica, an exocrine organ in the reproductive tract. Granules were purified by differential centrifugation of atrial gland homogenates followed by centrifugation on continuous Percoll-sucrose gradients. Quantitative enzyme assays in conjunction with electron microscopic analyses demonstrated that secretory granules thus isolated were significantly purified with respect to other subcellular organelles such as mitochondria and lysosomes. Immunoelectron microscopy demonstrated that the majority (approximately 85%) of the purified secretory granules were immunoreactive for A-NTP (N-terminal peptide), a cleavage product of the egg-laying prohormone-related A and A' precursors (residues 22-34). The purified granules represented an enriched source of peptides that were readily resolved by reversed-phase high performance liquid chromatography.

Microcosm studies were undertaken to relate biomarker responses to the toxicities in soil ecosystems contaminated by phenanthrene (Phe) and pyrene (Pyr), both singly and combined. Growth inhibition, enzyme activity, MDA content, sperm count, neutral-red retention time (NRRT) and annetocin and TCTP gene transcriptions were determined in earthworm Eisenia fetida exposed to Phe and Pyr, both singly and combined pollution in microcosm. Exposure to 0.5 and 2.5 mg kg(-1) Phe or 50 and 100 mg kg(-1) Pyr alone significantly decreased E. fetida growth, NRRT and sperm count. Two-way ANOVA analysis shows that the combination of these two compounds decreased growth, SOD activities, NRRT and sperm count synergistically, but increased the CAT activities and MDA content. The highest suppression rate of growth was 48.12%, the lowest levels of SOD activities and NRRT were 51.66% and 45.57% of the control, respectively. The highest increase in CAT activities and MDA content were 120.05% and 121.03% greater than that of the control when exposed to 0.5 (Phe)+100 (Pyr) mg kg(-1) soils. A clear dose-related response with exposure concentration was established for the NRRT. Real-time PCR shows that Phe and Pyr increased the expression levels of annetocin and TCTP gene synergistically. These results demonstrate that earthworms were under physiological stress at field dose of 0.5 (Phe)+100 (Pyr) mg kg(-1) soils. Phe and Pyr synergistically decreased sperm count and NRRT, but antagonistically caused changes in antioxidant enzyme activities to disrupt the detoxification functions and inhibit earthworm growth.

Double labelling experiments were performed on the same tissue section at the electron microscopic level, in order to show the involvement of the opioid leucine-enkephalin (Leu-enk) in the modulation of crustacean hyperglycaemic hormone (CHH) mobilization. Both neuropeptides were stored in distinct axon terminals of the sinus gland of Carcinus maenas. A post-embedding immunogold cytochemical technique for Leu-enk, CHH and the CHH neurohormone related moult inhibiting hormone (MIH) was combined with a scintillator intensified autoradiographic method to demonstrate binding of the opioid antagonist [3H] naloxone. Ultrathin sections were successively incubated with antisera against Leu-enk, CHH or MIH, and the corresponding colloidal gold labelled antisera, followed by autoradiographic processing. At the ultrastructural level [3H] naloxone binding sites were easily recognized by their silver tracks after development. Opioid binding sites for [3H] naloxone were visualized only at membranes of CHH-containing axon terminals. These results provide morphological evidence for direct enkephalinergic control of CHH containing neurons in the sinus gland of C. maenas and are furthermore the first autoradiographic demonstration of opioid binding sites in the nervous system of invertebrates.

The first step in the biosynthesis of ecdysteroids by Manduca sexta prothoracic glands, the conversion of cholesterol to 7-dehydrocholesterol, is mediated by an enzyme with characteristics of a microsomal cytochrome P-450, i.e. sensitivity to CO and fenarimol, and a requirement for NADPH. The enzyme responsible for hydroxylation at C-25 of the putative 3-dehydroecdysone precursor, 14-hydroxy-5 beta-cholest-7-en-3,6-dione, is also microsomal, while those mediating hydroxylations at C-22 and C-2 of 3,14,25-trihydroxy-5 beta-cholest-7-en-6-one are mitochondrial. Indirect evidence revealed that the steps between 7-dehydrocholesterol and the trideoxyecdysteroids occur in the mitochondria, suggesting that extensive shuttling of intermediates between the endoplasmic reticulum and mitochondria takes place in the prothoracic gland cell during ecdysteroid biosynthesis. During the fifth larval instar, cholesterol 7,8-dehydrogenase activity is evident from days 2 to 9, while the conversion to [3H]ecdysteroids is not significant prior to the ecdysteroid commitment peak on day 4. Terminal hydroxylase activity shows little change throughout the instar. These data support the hypothesis that regulation of the biosynthetic pathway by PTTH occurs at the step immediately following the formation of 7-dehydrocholesterol. The steroid biosynthesis inhibitor, fenarimol, has been shown to inhibit each of these P-450 enzymes, as well as fat body ecdysone 20-monooxygenase, with an I50 of 10(-4) M in disrupted glands, suggesting that it is a general P-450 inhibitor. The secretion of ecdysteroids by the glands in vitro is very sensitive to fenarimol, i.e. I50 of 10(-6) M. RH5849, 1,2-dibenzoyl-1-tert-butylhydrazine, fails to inhibit any of these prothoracic gland reactions, yet strongly inhibits fat body ecdysone 20-monooxygenase activity. This suggests that RH5849 is a specific ecdysteroid substrate/product mimic in this reaction.

Antimicrobial peptides (AMPs) serve a major role in host defense systems against microbial invasion. In this study, a novel isoform (ALFSp2) of antilipopolysaccharide factors (ALFs) was cloned from the mud crab, Scylla paramamosain. The open reading frame of the ALFSp2 cDNA is 348 bp and encodes for a predicted 115 amino acid residues (12.92 kDa), and a mature protein of 94 amino acids and a molecular mass of 10.79 kDa. The amino acid sequence of ALFSp2 has an overall similarity of 74%, 66% and 52% to those of Eriocheir sinensis ALF, Penaeus monodon ALFPm3 and S. paramamosain ALFSp1, respectively. The genomic organization of the ALFSp2 gene consists of three exons and two introns, whilst the upstream region contains multiple putative transcription factor binding sites. In healthy crabs, ALFSp2 transcript levels were high in the hemocytes and gill tissues, intermediate levels in the intestine and muscles and at a low level in the hepatopancreas, as determined by RT-PCR. To characterize the in vitro antimicrobial activities of ALFSp2, the 24 amino acid LPS-binding domain encoding peptide was synthesized and revealed an antimicrobial activity against Gram-positive (Aerococcus viridans and Micrococcus luteus) and Gram-negative (Vibrio harveyi and Vibrio anguillarum) bacteria. Altogether these results suggest a potential involvement for ALFSp2 in the defense mechanism of the mud crab, S. paramamosain.

Y-organs of the xanthid crab, Menippe mercenaria, secrete ecdysteroid hormones in vitro, apparently both 3-dehydroecdysone and 25-deoxyecdysone. Studies were initiated on the biosynthetic path(s), in which cholesterol is converted to these ecdysteroids. Crabs were injected with [3H]cholesterol. Y-organs and hemolymph were removed 12 hr later and extracted directly and the extracts were analyzed by HPLC. Both polar and nonpolar sterols were surveyed. The only metabolite of cholesterol detectable in Y-organs was 7-dehydrocholesterol (identified by mass spectrometry). The total amount of 7-dehydrocholesterol and the amount that was labeled were generally greater than for cholesterol and were higher in Y-organs from de-eyestalked crabs than in those from intact crabs. Subcellular fractionation of the Y-organs showed that over 70% of total radioactivity was in cholesterol and 7-dehydrocholesterol of mitochondria and microsomes, distributed about equally between the two organellar fractions. In hemolymph, the only nonpolar sterols present were cholesterol and 7-dehydrocholesterol; the concentration ratio was 20:1. However, 7-dehydrocholesterol was not significantly labeled. Analyses of polar compounds revealed two prominent, uv-absorbing ecdysteroids which coeluted with the authentic standards, 3-dehydro-20-hydroxyecdysone and 25-deoxy-20-hydroxyecdysone (ponasterone A). The radioactivity profile showed, in addition, a third prominent peak that corresponded in retention time with 3-dehydroecdysone. These results indicate that the Y-organs in vivo form 7-dehydrocholesterol from cholesterol and convert the latter to secretion products without accumulation of other intermediates. At least two ecdysteroids are secreted and appear to be converted peripherally in this crab to their respective 20-hydroxy derivatives.

HPLC separation of corpus cardiacum homogenates from Tabanus lineola, T. proximus and T. calens all produced two peaks whose retention times corresponded to the retention times of the synthetic Tabanus atratus adipokinetic and T. atratus hypotrehalosaemic hormones. Corpus cardiacum extracts from T. proximus and from T. lineola produced hyperlipaemic and hyperglycaemic responses when injection into sucrose fed T. lineola. Synthetic T. atratus hypotrehalosaemic hormone induced strong hyperlipaemic and hyperglycaemic responses in T. lineola, but synthetic T. atratus adipokinetic hormone produced only a weak hyperglycaemic response. The current names of the tabanid metabolic peptides do not correspond to their action in T. lineola. Octopamine also elevated both lipids and sugars in the blood of sucrose fed T. lineola, indicating the sensitivity of storage tissues to both biogenic amines and to metabolic peptides. Lack of response to synthetic T. atratus hypotrehalosaemic by starved T. lineola suggests an inhibition of the hyperlipaemic/glycaemic response when the available tissue reserves falls below a given level.

Loss-of-function mutations of the dre4 gene of Drosophila melanogaster caused stage-specific developmental arrest, the stages of arrest coinciding with periods of ecdysteroid (molting hormone) regulated development. Nonconditional mutations resulted in the arrest of larval development in the first instar; embryogenesis was not impaired, and mutant larvae were behaviorally normal and long-lived. At 31 degrees the temperature-sensitive dre4e55 allele caused the arrest of larval development in the first or second instars. When upshifted to 31 degrees at various times during development, dre4e55 mutants exhibited nonpupariation of third-instar larvae, failure of pupal head eversion, failure of adult differentiation, or noneclosion of pharate adults. Under some temperature regimens second-instar larvae pupariated precociously without entering the normally intervening third-instar. Nonpupariation and defects in metamorphosis were associated with the reduction or elimination of ecdysteroid peaks normally associated with late-larval, prepupal, pupal and pharate adult development. Ecdysteroid production by larval ring glands from dre4e55 hemizygous larvae was suppressed after 2 hr of incubation in vitro at 31 degrees, indicating autonomous expression of the dre4 gene in the ring gland. We postulate that the dre4 gene is required for ecdysteroid production at multiple stages of Drosophila development and that the pathologies observed in dre4 mutants reflect developmental consequences of ecdysteroid deficiency.

Prothoracic glands of the locus, Locusta migratoria, incubated in vitro converted tritiated 3-dehydrocetodiol (22,23,24,25 3H4-14 alpha-hydroxy-5 beta-cholest-7-en-3,6-dione) into ecdysteroids and 3-dehydroecdysteroids as far as the final products of the two series, ecdysone and 3-dehydroecdysone. In the two series, the different compounds are formed in the same quantities, except for 2,22-desoxy-products, the nature of which could not have been determined. Converted 3-dehydroecdysone issued from 3-dehydrocetodiol is transformed into ecdysone after several hours incubation with Locusta last instar larvae hemolymph. Till now is has been impossible to determine if the reduction of 3-dehydroecdysteroids took place into the prothoracic glands or in the incubation medium. In no case is 3-dehydrocetodiol converted into cetodiol. Conversion rates of the different compounds, either issued from cetodiol or from 3-dehydrocetodiol as precursors, are of same importance, so that a weak specificity of the hydroxylation enzymes must be considered.

The crustacean hyperglycemic hormone (CHH) is a 72-amino acid residue polypeptide with multiple physiological effects. The X-organ/sinus gland is the primary source for CHH and its family members. However, the amino acid sequence of CHH in Cancer borealis , a premier model system for neuromodulation, has not been characterized. In this study, a novel hybrid strategy combining "bottom-up" and "top-down" methodologies enabled direct sequencing of CHH peptide in the sinus gland of C. borealis . Multiple mass spectrometry (MS)-based techniques were employed to characterize the CHH peptide, including direct tissue analysis by MALDI-FT-ICR-MS, de novo sequencing of tryptic digested CHH by nano-LC/ESI-Q-TOF MS and intact CHH analysis by LC/FT-ICR-MS. In-trap cleaning removed the extensive matrix adducts of CHH in the direct tissue analysis by MALDI-FT-ICR-MS. Fragmentation efficiency of the intact CHH was drastically improved after the reduction-alkylation of the disulfide bonds. The sequence coverage was further enhanced by employing multiple complementary fragmentation techniques. Overall, this example is the largest neuropeptide de novo sequenced in C. borealis by mass spectrometric methods.

The purification and characterization of six isoforms of ornatin, potent glycoprotein IIb-IIIa (GP IIb-IIIa) antagonists and platelet aggregation inhibitors are described. These isoforms were purified from whole leech homogenates of the leech Placobdella ornata, a North American leech commonly known as the turtle leech, by trichloroacetic acid precipitation, Sephadex G-50 size exclusion chromatography, GP IIb-IIIa affinity chromatography, and C18 reverse-phase HPLC. Each of the five completely sequenced isoforms, which range from 41 to 52 residues in length, contains the Arg-Gly-Asp (RGD) sequence, a common recognition sequence in adhesion proteins, as well as 6 cysteine residues; the positions of both of these features are conserved in the primary sequences. The amino acid sequences of ornatin isoforms B, C, D, and E are highly conserved, whereas ornatin A2 and A3 are less similar and lack 9 residues at the N-terminus. The ornatins are approximately 40% identical with decorsin, a GP IIb-IIIa antagonist isolated from the leech Macrobdella decora [Seymour, J. L., Henzel, W. J., Nevins, B., Stults, J. T. & Lazarus, R. A. (1990) J. Biol. Chem. 265, 10143-10147]; furthermore, the RGD sequence and 5 out of 6 cysteine residues are maintained in the same relative positions in both decorsin and ornatin. The ornatin isoforms do not exhibit significant similarity to any members of the snake-venom-derived family of GP IIb-IIIa antagonists [Dennis, M. S., Henzel, W. J., Pitti, R. M., Lipari, M. T., Napier, M. A., Deisher, T. A., Bunting, S. & Lazarus, R. A. (1990) Proc. Natl Acad. Sci. USA 87, 2471-2475] except in the RGD region of these proteins. The ornatin isoforms inhibit the binding of GP IIb-IIIa to immobilized fibrinogen with IC50 values ranging over 2.9-5.3 nM; ornatin isoforms A2, C, and E inhibit ADP-induced human platelet aggregation with IC50 values of about 130, 280, and 440 nM, respectively.

Rhythmic synthesis of moulting hormones (ecdysteroids) by prothoracic glands (PGs) of the insect Rhodnius prolixus during the last larval instar was studied in vitro following explantations every 4-5 hr for up to 96 hr. Ecdysteroid synthesis was measured by radioimmunoassay as the quantity of ecdysteroid produced during 4 hr in vitro. A massive daily rhythm is seen, with synthesis at night being three- to fivefold higher than during the day. This rhythm of ecdysteroid synthesis by PGs free-runs in continuous darkness with a temperature-compensated period length close to 24 hr and is therefore controlled by a circadian system. This is the first report of circadian regulation of synthesis of an invertebrate hormone. The synthesis rhythm also free-runs in continuous light, but with an inverted phase and shorter period length. It is argued that the circadian system controlling synthesis comprises two oscillators which free-run in antiphase, occupy different anatomical locations, and are coupled by a humoral factor, possibly prothoracicotropic hormone. The ecdysteroid synthesis rhythm in PGs appears to drive the previously reported circadian rhythm in the haemolymph ecdysteroid titre. It is concluded that the circadian system controlling synthesis of ecdysteroids constitutes a pacemaker which drives various rhythms in the target cells of ecdysteroids via the rhythm in the haemolymph titre. Ecdysteroids are viewed as "hormonal Zeitgebers," imposing temporal order on development.