Ernest S. Chang

Methyl farnesoate and its role in crustacean reproduction and development

Studies with Libinia emarginata suggest that methyl farnesoate (MF), a product of the mandibular organs (MOs), may be a crustacean juvenile hormone. In order to better understand the significance of this compound in crustacean physiology, we first investigated the presence of MF in other decapods. MF was synthesized and secreted by MOs from all species tested. However, large differences in the level of MF secretion were observed between species and also between individuals of a species. For example, the level of secretion by MOs from L. emarginata was 100-fold greater than that observed in MOs from Homarus americanus. Analysis of hemolymph from these two species by GC-MS indicated comparable differences in the amount of MF present. Differences in the level of MF secretion by MOs from individuals of a species appear to reflect the physiological roles of this compound. For example, a close relationship was seen between MF secretion and gametogenesis in females of L. emarginata. Finally, treatment of lobster larvae with seawater containing MF caused a small but significant delay in their metamorphosis when compared with untreated larvae. These data suggest that MF affects reproduction in a manner similar to the effects of JH on insects, and may also have effects on the development of crustacean larvae. Taken together, these data support the classification of MF as a crustacean JH.

Amino acid sequence of a peptide with both molt-inhibiting and hyperglycemic activities in the lobster, Homarus americanus

A hydrophobic peptide of 71 residues was isolated from lobster sinus gland extracts that prolonged intermolt periods and lowered ecdysteroid titers in juvenile lobsters. Removal of the N-terminal pyroglutamyl residue allowed sequencing of 30 of the first 36 residues. Additional data were obtained from HPLC-purified fragments from endoproteinase cleavages (Lys-C, Glu-C, Arg-C, Asp-N), and carboxypeptidase Y digestion. This is the first reported amino acid sequence of a crustacean molt-inhibiting hormone. This peptide also has significant hyperglycemic activity.

Regulation of crustacean molting: a review and our perspectives.

Molting is a highly complex process that requires precise coordination to be successful. We describe the early classical endocrinological experiments that elucidated the hormones and glands responsible for this process. We then describe the more recent experiments that have provided information on the cellular and molecular aspects of molting. In addition to providing a review of the scientific literature, we have also included our perspectives.

Stressed-Out Lobsters: Crustacean Hyperglycemic Hormone and Stress Proteins

Abstract Organisms in natural habitats must frequently respond to changes in their environments through various physiological mechanisms. My laboratory has developed several methods for the quantification of stress in crustaceans. An ELISA was developed for the crustacean hyperglycemic hormone (CHH) from the American lobster (Homarus americanus). It is sensitive to as little as 0.2 fmol of peptide. Increases in hemolymph CHH were observed under conditions of acute hypoxia, elevated temperature, and altered salinity. In addition, elevated CHH concentrations were observed in Norway lobsters (Nephrops norvegicus) that were parasitized with the dinoflagellate Hematodinium sp. Stress proteins, also known as heat-shock proteins (HSPs), comprise a highly conserved class of proteins that display elevated transcription during periods of stress. Using homologous molecular probes, my collaborators and I have examined the influence of heat-shock, osmotic stress, and the molt cycle upon HSP expression at the protein and mRNA levels. We observed a significant elevation in HSP mRNA expression after 1 hr of heat-shock or after 0.5 hr of osmotic stress. When comparing claw and abdominal muscles during molting, we observed a tissue-specific HSP response. Quantification of these different stress responses may serve as early indicators of the degradation of environmental health.

CRUSTACEAN HYPERGLYCEMIC HORMONE IN THE LOBSTER NERVOUS SYSTEM : LOCALIZATION AND RELEASE FROM CELLS IN THE SUBESOPHAGEAL GANGLION AND THORACIC SECOND ROOTS

Crustacean hyperglycemic hormones (CHHs) are neuropeptides involved in the regulation of hemolymph glucose. The primary source of CHHs has been identified as the neurosecretory neurons of the eyestalk X‐organ and its associated neurohemal organ, the sinus gland. We have identified another source of CHH‐like peptides in the nervous system. With the use of immunocytochemistry, cells in the second roots of the thoracic ganglia have been observed to stain positively for CHH‐reactive material. We also identified a pair of cells in the subesophageal ganglion that contain large amounts of CHH‐reactive material. Depolarization of these cells with elevated potassium mediates a calcium‐dependent release of CHH‐like material from the ganglion as quantified with an enzyme‐linked immunosorbent assay (ELISA). J. Comp. Neurol. 414:50–56, 1999.

Osmotic Induction of Stress-Responsive Gene Expression in the Lobster Homarus americanus

The American lobster, Homarus americanus, encounters osmotic stress throughout its life cycle. To understand the molecular basis of osmotic stress responses in vivo, we used homologous cDNA probes to characterize the mRNA patterns of lobster HSP70 (=70-kDa heat-shock protein), HSP90 (=90-kDa heat-shock protein), and polyubiquitin during hypo- and hyper-osmotic stress in abdominal muscle and hepatopancreas (a digestive tissue) at 30, 60, and 120 min of osmotic stress. Hypo- and hyper-osmotic stress significantly increased the levels of the mRNAs encoding HSP70 and HSP90 in abdominal muscle. Hyper-osmotic stress increased HSP90 mRNA levels in hepatopancreas, but hypo-osmotic stress did not. Both abdominal muscle and hepatopancreas exhibited significant changes in polyubiquitin gene expression during osmotic stress. In abdominal muscle, polyubiquitin mRNA levels increased during both hypo- and hyper-osmotic stress. Hepatopancreas, however, showed a significant elevation in polyubiquitin mRNA only during hypo-osmotic stress.

Methyl Farnesoate: Crustacean Juvenile Hormone in Search of Functions

Abstract The sesquiterpenoid methyl farnesoate (MF) is synthesized by crustacean mandibular organs and is present in the hemolymph. The chemical structure of MF is nearly identical to that of insect juvenile hormone III, differing only by the absence of an epoxide group. The functions of MF in crustaceans have been elusive, in part because the mandibular organs of most species are difficult to ablate. However, three roles of MF have emerged from a growing body of literature. They are (a) stimulation of general protein synthesis; (b) promotion of the molt cycle; and (c) reproduction in both males and females. These functions are not universal; rather, they reflect the diversity of the crustaceans that have been studied to date. This review will synthesize aspects of what is known about MF in crustaceans and outline some current avenues of research.

Ecdysteroids in relation to the molt cycle of the American lobster, Homarus americanus: I. Hemolymph titers and metabolites

Hemolymph ecdysteroid (Ecd) titers were measured using radioimmunoassay (RIA) during the molt cycle of the American lobster, Homarus americanus. Individual animals showed small, transitory rises of Ecds which increased in magnitude with the onset of premolt and culminated in a large premolt peak at morphological stages D2(2)-D3(1). Male lobsters had significant postmolt peaks and late premolt titers that remained high until ecdysis. In females, postmolt peaks were absent and late premolt titers reached basal levels before ecdysis. At least seven different Ecd metabolites were identified by high-performance liquid chromatography-RIA analyses. High polarity products (HP) were the most abundant metabolites in virtually every molt stage. Titers of HP were significantly higher in males during late postmolt-early intermolt and in late premolt. Levels of 20-hydroxyecdysone (20E) were equivalent in both sexes and correlated with the morphological changes associated with premolt. Evidence was also obtained for the presence of ecdysone, ponasterone A, and other as yet unidentified metabolites. The pattern of Ecd metabolites in the hemolymph supports other data indicative of 20E as the major molting hormone. Metabolism of 20E is primarily toward more polar compounds, including conjugates.

Purification and amino acid composition of a peptide with molt-inhibiting activity from the lobster, Homarus americanus.

A peptide was isolated and purified from sinus glands of the lobster, Homarus americanus, that was able to decrease circulating titers of ecdysteroids and increase the molt interval of eyestalk-ablated juvenile lobsters. This molt-inhibiting activity was demonstrated to consist of two very closely related peptides by means of high-performance liquid chromatography and gel electrophoresis. By means of amino acid analyses, a molecular weight of approximately 8700 was obtained.

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

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