Characterization of heat shock protein 90 in the shrimp Metapenaeus ensis: Evidence for its role in the regulation of vitellogenin synthesis

Estrogen hormones play a vital role in the regulation of female reproductive maturation. In oviparous vertebrates, the synthesis of vitellogenin (VTG) is tightly controlled by estrogen hormone signal transduction pathway, which is mediated by estrogen receptor and heat shock protein 90 (Hsp90). In order to investigate whether a similar mechanism exists in crustaceans, the Hsp90 gene was cloned and isolated from the shrimp Metapenaeus ensis by homology cloning strategy. The Hsp90 is 2,524 bp in length, containing an open reading frame of 2,163 bp that encodes a 720 amino acid polypeptide (83 kD). The Hsp90-coding region is interrupted by four introns. MeHsp90 is differentially expressed in eyestalk, ovary, and hepatopancreas at different ovarian maturation stages, and consistently expressed in other tissues including heart, gill, gut, muscle, and central nervous system. In vitro ovary explant assay reveals that MeHsp90 expression in immature ovary can be induced by the addition of exogenous estradiol-17beta, but expression in fully mature ovary exhibits no response to estradiol-17beta treatment. In situ hybridization shows that MeHsp90 is highly expressed in previtellogenic oocytes and its expression decreases with the progress of maturation, and finally stops in late-vitellogenic oocytes. Our results indicate a strong correlation between estrogen hormones and Hsp90 expression in shrimp, suggesting that the expression of VTG may be under the regulation of estrogen hormones through a mechanism similar to that in vertebrates. The result provides insights on the control of vitellogenesis in invertebrates.     

A change of heart: cardiovascular development in the shrimp Metapenaeus ensis

The larval development of penaeid shrimp is among the most complicated in crustaceans. In Metapenaeus ensis, there are six naupliar, three protozoeal and three mysid larval instars, followed by postlarval development. Irregular heartbeat begins late in naupliar instar 6. Co-ordinated beating at 400-600 beats min(-1) commences in the first protozoeal instar and continues throughout larval life. Initially, the contractile region is located more posteriorly in the cephalothorax and has a single pair of ostia, and the arterial distribution is limited to a single anterior vessel. In later mysid instars, a second cardiac pumping site develops posterior to, but connected with, the original site. This extension is more muscular, contains additional ostia and develops additional distribution vessels supplying the cephalothorax and abdominal areas. The original site is gradually merged into the new extension and only small refinements in the circulation occur in postlarval and juvenile life. Changes in physiological responses of the heart also occur throughout development. Responses to intra-pericardial microinjection of 5-hydroxytryptamine change drastically during development, as do cardiac responses to ambient hypoxia. Similarly, heartbeat of later juvenile instars is inhibited by injection of tetrodotoxin, while heartbeat of larval and early juvenile instars is not, suggesting that neurogenic regulation via the cardiac ganglion arises later in development. Our present studies attempt to integrate the anatomical and physiological changes in the development of the crustacean heart.     

Modelling population dynamics of the pelagic larval shrimp Metapenaeus ensis in the Osaka Bay estuary

Aiming at providing numerical tools useful to assess the impacts of coastal development pressures on marine life, a Lagrangian model for population dynamics of pelagic eggs and larvae of the greasyback shrimp, Metapenaeus ensis, in Osaka Bay was developed and improved based on laboratory experiments. The model was combined with a 3-D Eulerian numerical model that predicts physical and biological conditions of the habitat. A series of numerical experiments was done to investigate their transport processes from the spawning grounds, and the concentration process of full-grown larvae towards the mouth of Yodo River that flows into the innermost basin. Model results revealed, as a general tendency, that the early larval developmental stages through nauplius to zoea continue to drift like a passive tracer, and begin to concentrate at the river mouth area after the mysis stage, mainly avoiding low saline environment in the surface layer. This suggests that the full-grown larvae may be favored with chance to take advantage of the river-induced gravitational circulation that dominates the innermost estuarine basin. In addition, it was found that tidal condition around the spawning period also exerts a considerable influence upon the fate of pelagic larvae.     

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.     

Function and cellular localization of farnesoic acid O-methyltransferase (FAMeT) in the shrimp, Metapenaeus ensis.

The isoprenoid methyl farnesoate (MF) has been implicated in the regulation of crustacean development and reproduction in conjunction with eyestalk molt inhibiting hormones and ecdysteroids. Farnesoic acid O-methyltransferase (FAMeT) catalyzes the methylation of farnesoic acid (FA) to produce MF in the terminal step of MF synthesis. We have previously cloned and characterized the shrimp FAMeT. In the present study, recombinant FAMeT (rFAMeT) was produced for bioassay and antiserum generation. FAMeT is widely distributed in shrimp tissues with the highest concentration observed in the ventral nerve cord. Interestingly, an additional larger protein in the eyestalk also showed immunoreactivity to anti-FAMeT serum. FAMeT was localized in the neurosecretory cells of the X-organ-sinus gland complex of the eyestalk. As shown by RT-PCR, FAMeT mRNA is constitutively expressed throughout the molt cycle in the eyestalk and the ventral nerve cord. To show that our cloned gene product had FAMeT activity, we demonstrated that expressed rFAMeT gene product catalyzed the conversion of FA to MF in a radiochemical assay. The ubiquitous distribution of FAMeT suggests that this enzyme is involved in physiological processes in addition to gametogenesis, oocyte maturation and development and metamorphosis of the shrimp. We hypothesize that FAMeT directly or indirectly (through MF) modulates the reproduction and growth of crustaceans by interacting with the eyestalk neuropeptides as a consequence of its presence in the neurosecretory cells of the X-organ-sinus gland.     

gamma-Butyrobetaine hydroxylase and the protective role of glutathione peroxidase

The selenoenzyme glutathione peroxidase in the presence of GSH effectively replaced catalase in the in vitro assay for gamma-butyrobetaine hydroxylase. Quantitatively, glutathione peroxidase was an order of magnitude more efficient than catalase, with maximal activity at less than 0.1 microM glutathione peroxidase in a standard reaction. Glutathione peroxidase prevented the loss of gamma-butyrobetaine hydroxylase during preliminary incubation with ferrous ions but without other substrates as well as in the course of the reaction. Regardless of whether glutathione peroxidase or catalase was present in the assay, the ascorbate concentrations needed to achieve half-maximal rates were similar (about 1 mM). Phosphate stimulated the rate of L-carnitine synthesis. Ferrous ion saturation indicated a pronounced effect of phosphate on the maximal velocity of the enzyme-catalyzed reaction, but its mechanism of action remains to be elucidated. Based on the subcellular distribution of gamma-butyrobetaine hydroxylase, catalase, and glutathione peroxidase, the role of glutathione peroxidase assumes importance. However, initial studies indicated that the assayable activity of liver gamma-butyrobetaine hydroxylase and L-carnitine concentrations in liver, blood plasma, and muscle were not significantly altered in selenium-deficient rats.     

Physiological role of phospholipid hydroperoxide glutathione peroxidase in mammals

The redox enzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx) has emerged as one of the most significant selenoenzymes in mammals, corroborated by early embryonic lethality of PHGPx null mice. PHGPx is one of five selenium-dependent glutathione peroxidases and the second glutathione peroxidase to be discovered in 1982. PHGPx has a particular position within this family owing to its peculiar structural and catalytic properties, its multifaceted roles during male gametogenesis, and its necessity for early mouse development. Interestingly, mice devoid of endogenous glutathione die at the same embryonic stage as PHGPx-deficient mice compatible with the hypothesis that a similar phenotype of embryonic lethality may be provoked by PHGPx deficiency and lack of its reducing substrate glutathione. Various gain- and loss-of-function approaches in mice have provided some insights into the physiological functions of PHGPx. These include a protective role for PHGPx in response to irradiation, increased resistance of transgenic PHGPx mice to toxin-induced liver damage, a putative role in various steps of embryogenesis, and a contribution to sperm chromatin condensation. The expression of three forms of PHGPx and early embryonic lethality call for more specific studies, such as tissue-specific disruption of PHGPx, to precisely understand the contribution of PHGPx to mammalian physiology and under pathological conditions.     

Molecular and Functional Characterization of an Anti-lipopolysaccharide Factor Mm-ALF from Speckled Shrimp Metapenaeus monoceros

Probiotics and Antimicrobial Proteins - Anti-lipopolysaccharide factors (ALFs) are antimicrobial peptides of approximately 100 amino acid residues with a broad spectrum of antimicrobial activity....     

Size and diel differences in activity patterns of Metapenaeus ensis,penaeus latisulcatus and P. merguiensis

The nursery function of mangroves as shelter has been postulated to explain the positive correlation between shrimp catches and mangrove area. This study was undertaken to document shelter use and other activities in mangrove‐associated penaeids and to determine diel and size differences relating to these activities. Juvenile Metapenaeus ensis, Penaeus latisulcatus and P. merguiensis collected from mangrove areas in Guimaras, central Philippines and stocked individually in glass tanks (= replicates) provided with artificial shelters and sand substrate were observed every hour for 25 h. Size classes tested were very small (1–5.9 mm carapace length); small (6–10.9mm CL); medium (11–15.9mm CL); large (16–20.9mm CL); and very large (21–25.9 mm CL). Due to limited juvenile supply, only 3 sizes were tested for each species: P. merguiensis (very small to medium), M. ensis (small to large) and P. latisulcatus (medium to very large) with 5 replicate animals (one per tank) per size. Juvenile M. ensis and P. latisulcatus showed a strong diel periodicity of daytime burial and nocturnal activity. In contrast, P. merguiensis showed active swimming and feeding throughout the day and night. Very small to small P. merguiensis were observed on the shelters, but burying was exhibited only by a few medium‐sized juveniles. This shelter use is consistent with observations of small P. merguiensis entering the mangrove forest (where roots, twigs, etc. contribute to structural complexity) on the flood tide and concentrating in the shallow, turbid waters of adjoining creeks during ebb tide and slack water.     

Characterization and partial amino acid sequence of human plasma glutathione peroxidase

Human plasma glutathione peroxidase was purified to homogeneity and partially sequenced. Overlapping peptide fragments from three endopeptidase digests permitted the determination of one sequence of 32 contiguous amino acids and one sequence of 23 contiguous amino acids. Five additional unique peptide sequences without obvious overlaps were obtained. The sequence of 32 amino acid residues aligns with positions 82-113 of human cytosolic glutathione peroxidase with nine mismatches without gaps or insertions. The sequence of 23 amino acid residues aligns with positions 157-178 with six mismatches and an insertion of one residue. Three additional peptide sequences with no obvious sequence homology to glutathione peroxidase can be aligned based on the sequence of a cDNA clone encoding plasma glutathione peroxidase that was isolated from a human placental library. The plasma enzyme is a homotetramer composed of 21-kDa subunits which cannot reduce phospholipid hydroperoxides. These results indicate that the plasma glutathione peroxidase is distinct from both the classical cytosolic enzyme and the monomeric phospholipid hydroperoxide glutathione peroxidase. Only a negligible amount of glutathione peroxidase activity was detected in bile, indicating that the liver exports plasma glutathione peroxidase exclusively to the circulation.