Eyestalk Of Shrimp Research Articles

An eclosion hormone-like gene participates in the molting process of Palaemonid shrimp Exopalaemon carinicauda

Molting behavior is an important physiological process related to metamorphosis, growth, and reproduction in crustaceans. Previous studies indicated that the molting process was controlled by 20-hydroxyecdysone (20E) and upstream hormones, peptides, and environmental factors, which regulate 20E function. Eclosion hormone (EH) in insect is a kind of neuropeptide that is regulated by 20E and triggers ecdysis behavior at the end of molting process. However, the function of eclosion hormone gene during the molting process in crustaceans is still largely unknown. In the present study, an eclosion hormone-like gene EcEHL was identified from Exopalaemon carinicauda. The deduced amino acid sequence of EcEHL contained a signal peptide, a typical eclosion domain, and six conserved cysteine residues forming three putative disulfide bonds. EcEHL was predominantly expressed in the epidermis, gill, and eyestalk of shrimp. In situ hybridization analysis showed that EcEHL transcripts were localized in gill cells and in medulla externa X-organ, medulla terminalis X-organ, sinus gland, and lamina ganglionaris of eyestalks. During the molting process of shrimp, EcEHL showed the highest expression level in shrimp at the premolt stage. The expression level of EcEHL in shrimp at mid premolt stage was up-regulated by injection of exogenous 20E. Silencing of EcEHL using double-stranded RNA delayed both the molting process and ecdysis rate of E. carinicauda. Furthermore, injection of exogenous 20E to shrimp at mid premolt stage (D2) could remarkably speed up the molting process and also raise the ecdysis rate of E. carinicauda. The results revealed that EcEHL might participate in the molting process of shrimp and its expression was regulated by 20E. These data will help us to understand the molecular mechanism of molting in crustacean.

In vivo and in vitro inhibitory action of 17β-estradiol and environmental estrogen 4-nonylphenol on gonad-inhibiting hormone (GIH) expression in the eyestalks of Litopenaeus vannamei.

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.

Comparative immunohistochemistry and cellular distribution of farnesoic acid O-methyltransferase in the shrimp and the crayfish

Farnesoic acid O-methyltransferase (FAMeT) catalyzes the conversion of farnesoic acid (FA) to methylfarnesoate (MF) by the mandibular organ (MO) of crustaceans. Here we report the cellular localization of FAMeT and radiochemical assay of endogenous FAMeT activity in shrimp ( Metapenaeus ensis) and crayfish ( Procambarus clarkii) tissues. As in the eyestalk (ES), FAMeT is concentrated in specific neurosecretory cells of the ventral nerve cord (VNC) whereas only weak FAMeT immunoreactivity was observed in the MO. FAMeT was also detected in the ventral nerve cord, heart (HET), eyestalk, and muscle of the juvenile shrimp. Although the VNC shows the greatest FAMeT immunoreactivity, the heart extract exhibited the highest FAMeT enzymatic activity. These results suggest that FAMeT in the VNC may be inactive or inactivated at the stages of development tested. Contrary to the previous reports in other crustaceans, MO extract in shrimp shows only low FAMeT activity. The eyestalk, epidermis, ovary and testis show appreciable FAMeT activity. The presence of FAMeT in neurosecretory cells of VNC and eyestalk of shrimp and crayfish implies a possible interaction of FAMeT with the eyestalk CHH-family of neuropeptides. The widespread activity of FAMeT suggests that it has a wide spectrum of action in many tissues that contribute to the function and regulation of MF synthesis in shrimp and crayfish.

Characterization of the shrimp eyestalk cDNA encoding a novel fushi tarazu-factor 1 (FTZ-F1)

To study the role of ecdysone and the ecdysone inducible gene in the regulation of molting and development in crustaceans, we have cloned a cDNA encoding an orphan nuclear receptor family member from the eyestalk of the shrimp Metapenaeus ensis. The size of the cDNA is 4.3 kb with the longest open reading frame (ORF) encoding a protein of 545 amino acid residues. The deduced amino acid sequence of the shrimp cDNA consists of regions that are characteristic of those of the nuclear hormone receptors. It shows a high degree of amino acid sequence identity in the DNA binding domain, ligand binding domain and the FTZ box as compared to those of invertebrates and vertebrates. Unlike the insects Drosophila melanogaster and Bombyx mori, an AF2 transactivation domain was present in the shrimp FTZ-F1. Northern blot analysis using total RNA indicated that the FTZ-F1 mRNA could also be detected in the mature ovary. Northern blot analysis and RT-PCR analysis showed that the shrimp FTZ-F1 transcripts could be detected in the ovary, newly hatched nauplius, testis, eyestalk and epidermis of the adult shrimp. Although the cDNA clone was isolated from the eyestalk library, the shrimp FTZ-F1 appeared to express most abundantly in the mature oocytes. The presence of abundant FTZ-F1 specific maternal message in the late vitellogenic ovary and early nauplius indicates that it may be important for the early embryonic and larval development of the shrimp. Interestingly, shrimp FTZ-F1 can also be found in testis of the male shrimp. The presence of FTZ-F1 in other tissues such as epidermis suggests that it may also be involved in other physiological processes such as molting.

PCR cloning and expression of the molt-inhibiting hormone gene for the crab (Charybdis feriatus).

A PCR-based genomic DNA walking technique was used to clone the gene for the molt-inhibiting hormone of the crab, Charybdis feriatus. Several overlapping genomic clones were isolated, and the MIH gene for the crab was reconstructed. DNA sequence determination of the overlapping clone reveals that the MIH gene spans 4.3kb and consists of three exons and two introns. Exons 1 and 2 carry a coding sequence for the signal peptide, and exons 2 and 3 consist of coding sequence for the mature peptide. The exon-intron boundary of the crab MIH gene also follows the 'GT-AG rule' for the splice donor and acceptor. The deduced amino acid sequence of MIH shows the highest overall similarity to those of the crabs, Callinectes sapidus and Carcinus maenas, and the gonad-inhibiting hormone (GIH) of the lobster. The putative polyadenylation signal is approximately 1.0kb 3' downstream of the termination codon (TGA). Genomic Southern blot analysis indicates that few genomic fragments were hybridized to the cDNA probe. The 5' flanking region contains a putative promoter with several putative cis elements similar to some vertebrate neuropeptide genes. The 530-bp flanking region was subcloned separately to two promoterless reporter plasmids carrying either the Green Fluorescent Protein gene (GFP) or the Choramphenicol Acetyltransferase gene (CAT). The DNA constructs were transfected into insect cells (Sf21) and mouse pituitary cells (GH4ZR7), respectively. Green fluorescent protein was detected in some of the transfected insect cells, and expression of the CAT was detected in cells transfected with DNA constructs containing the crab promoter. By RT-PCR, MIH transcripts can be detected in the eyestalk of shrimp in intermolt, early premolt, late premolt stages and females that brood their eggs. It can also be found in the brain, but not in the ovary, hepatopancreas, muscle and epidermis. During early larval development, MIH mRNA can be detected in the pre-hatched and the newly hatched larvae. Unlike the adult, the expression of the MIH in the larvae is exclusively in the brain.

Yolk synthesis in the marine shrimp, Penaeus vannamei

1. 1. In vitro yolk synthesis was measured in fragments of the ovary of developing shrimp, Penaeus vannamei. 2. 2. Progesterone and estradiol stimulated yolk synthesis in vitro, while ecdysterone, testosterone and estrogen had no effect. 3. 3. A peptide factor from the eyestalks of crayfish stimulated yolk synthesis in vitro. A peptide factor from shrimp eyestalks inhibited yolk synthesis in vitro.

Vitellogenesis in the shrimp, Penaeus vannamei: in vitro studies of the isolated hepatopancreas and ovary

Abstract 1. 1. Yolk proteins were isolated from ovaries of the shrimp Penaeus vannamei and used as an antigen for antibody production in rabbits. 2. 2. Protein synthesis was measured for both the hepatopancreas and the ovary in vitro, and proteins present in both tissues were immunoreactive with the antibodies. 3. 3. Extracts of shrimp eyestalks inhibited in vitro protein synthesis by both tissues. The inhibitory factor from the eyestalks was heat stable and had a molecular weight of 3300 daltons.

Endocrine Control of Apolysis in Rhithropanopeus Harrisii Larvae

ABSTRACT 20-Hydroxyecdysone (20-HE) stimulated the initiation of premolt (apolysis) in third and fourth instar Rhithropanopeus harrisii larvae in vivo and in spines explanted from third and fourth instar larvae in vitro. The premolt-initiating activity of 20-HE, in vitro, was inhibited by extracts of third or fourth instar zocae, megalopae, and shrimp eyestalks. The results suggest that apolysis in crab larvae may be controlled by both 20-HE and MIH.

Hormonal control of apolysis in barnacle mantle tissue epidermis, in vitro

AbstractEcdysterone (5–20 μg/ml) stimulated apolysis in inner mantle tissue explants of the barnacle, Balanus amphitrite, maintained under in vitro conditions. Apolysis was noticeable after 12 to 15 hours while complete apolysis was reached by 24 hours. Enlargement of the epidermal cells was also observed in ecdysterone‐stimulated tissues. Ecdysone (10–20 μg/ml) was equally as effective as ecdysterone in stimulating apolysis. In some tissues, which had been cultured with 20 μg/ml ecdysterone for 24 hours, new cuticle formation had occurred following apolysis. The initiation of apolysis by ecdysterone was dependent on RNA and protein synthesis, as indicated by the inhibition of ecdysterone‐promoted apolysis by actinomycin D and cycloheximide. Aqueous extracts of shrimp eyestalks (MIH) (10 es eq/ml), either as a crude extract, or as a low molecular weight fraction from a Sephadex G‐25 column, were active in repressing the apolysis‐stimulating action of ecdysterone. The MIH activity could be overridden by increased levels of ecdysterone. Protease treatment of the MIH from crude and partially purified fractions abolished the inhibitory activity. Molt‐inhibiting hormone did not bind 3H‐ecdysterone. The results demonstrate that the early premolt integumental activities are under the control of ecdysterone and molt‐inhibiting hormone.

Structure of the red-pigment-concentrating hormone of the shrimp, Pandalus borealis

The structure of a crustacean hormone secreted by nerve cells, the red-pigment-concentrating hormone (blanching hormone) of the shrimp, Pandalus borealis, has been determined. About 100 μg of hormone, purified from shrimp eyestalks, was used. The structure, which is < Glu-Leu-Asn-Phe-Ser-Pro-Gly-Trp-NH 2, was revealed by thermolysin digestion and separation by Sephadex G-25 chromatography of the resulting two fragments. One fragment was the NH 2-terminal peptide, < Glu-Leu-Asn, whose structure was determined by carboxypeptidase digestion and identification of the products in the automatic amino acid analyser and by thin-layer chromatography; that of the other, Phe-Ser-Pro-Gly-Trp-NH 2, which was a COOH-terminal fragment, was determined by the dansyl-Edman technique. The COOH-terminal residue was identified by comparison of its 1-dimethylaminonaphthalene-5-sulfonyl (Dns) derivative with synthetic Dns-Trp-NH 2 by polyamide thin-layer chromatography.