Bursicon homodimers induce the innate immunity via Relish in Procambarus clarkii.

Bursicon (burs) is a neuropeptide hormone consisting of two cystine-knot proteins (burs α and burs ß), and burs α-ß is responsible for cuticle tanning in insects. Further studies show that burs homodimers induce prophylactic immunity. Here, we investigated the hypothesis that burs homodimers act in regulating immunity in the red swamp crayfish Procambarus clarkii. We found that burs α and burs ß are expressed in neural system of crayfish. Treating crayfish with recombinant burs-homodimer proteins led to up-regulation of several anti-microbial peptide (AMP) genes, and RNAi-mediated knockdown of burs led to decreased expression of AMP genes. The burs proteins also facilitated bacterial clearance and decreased crayfish mortality upon bacterial infection. Furthermore, burs proteins activated the transcriptional factor Relish, and knockdown of Relish abolished the influence of recombinant burs homodimers on AMP induction. We infer the burs homodimers induce expression of AMP genes via Relish in crayfish and this study extends this immune signaling pathway from insects to crustaceans.

A novel crustacean hyperglycemic hormone (CHH) from the mud crab Scylla paramamosain regulating carbohydrate metabolism.

Crustacean hyperglycemic hormone (CHH) plays a crucial role in regulating carbohydrate metabolism in crustaceans. In this study, a new cDNA encoding type I CHH peptide, termed Sp-CHH3, was isolated from the mud crab Scylla paramamosain and its potential functions were investigated. The full length cDNA of Sp-CHH3 was identified as encoding a 127-aa precursor composed of a 27-aa signal peptide, a 23-aa CHH precursor-related peptide and a 75-aa mature peptide with a typical motif of CHH. Phylogenic analysis suggested that, Sp-CHH3 is a previously unreported CHH from S. paramamosain. Tissue distribution analysis showed that Sp-CHH3 was mainly expressed in the eyestalk ganglia, thoracic ganglia, stomach and the ovary. A RNA interference experiments showed that after injection of Sp-CHH3-targeted dsRNA, both the level of Sp-CHH3 expression in the eyestalk ganglia and hemolymph glucose level decreased significantly. A further short-term starvation experiments demonstrated that, the level of Sp-CHH3 detected in the eyestalk ganglia was significantly up-regulated at the 12th h of starvation, it then fell back at the 24th h of starvation and subsequently remained relative stability between the 24th to 96th h of starvation. The hemolymph glucose level decreased significantly (P < .05) at each sampling time during the 96 h starvation duration when compared to that of 0 h (prior to starvation) and the overall trend was largely correlated with the level of Sp-CHH3 expression in the eyestalk ganglia. In summary, the results suggest that Sp-CHH3 plays a functional role in regulating carbohydrate metabolism in S. paramamosain.

Neurotransmitters in hermatypic coral, Acropora spp., and its contribution to synchronous spawning during reproductive event.

Synchronous spawning as mass reproduction is well known to occur in many hermatypic corals, which is one of the mysterious life birth events. However, its contributing mechanism has not yet been clarified. This study placed focus on elucidating a neurotransmitter as endocrine signals that contribute to the synchronous spawning. First, the determination method of the neurotransmitters in coral was established by LC/MS in the selective ion mode together with a solid phase extraction method. As a result, the similar contents of the neurotransmitters for dopamine (DA), adrenaline (AD) and noradrenaline (NR) were detected in both the hermatypic corals of Acropora intermedia and Acropora digitifera. More interestingly, these neurotransmitters increased through the reproductive event during the synchronous spawning of A. intermedia, particularly, remarkable changes in the NR and DA were observed. In addition, hydrogen peroxide is known as the spawning stimulant and the metabolic by-product of the neurotransmitters, which was exposed to A. digitifera, then the neurotransmitters increased as well as those of the synchronization of spawning. All of the results suggested that the neurotransmitters contribute to the synchronous spawning in the hermatypic corals.

Signaling through the G-protein-coupled receptor Rickets is important for polarity, detachment, and migration of the border cells in Drosophila.

Cell migration plays crucial roles during development. An excellent model to study coordinated cell movements is provided by the migration of border cell clusters within a developing Drosophila egg chamber. In a mutagenesis screen, we isolated two alleles of the gene rickets (rk) encoding a G-protein-coupled receptor. The rk alleles result in border cell migration defects in a significant fraction of egg chambers. In rk mutants, border cells are properly specified and express the marker Slbo. Yet, analysis of both fixed as well as live samples revealed that some single border cells lag behind the main border cell cluster during migration, or, in other cases, the entire border cell cluster can remain tethered to the anterior epithelium as it migrates. These defects are observed significantly more often in mosaic border cell clusters, than in full mutant clusters. Reduction of the Rk ligand, Bursicon, in the border cell cluster also resulted in migration defects, strongly suggesting that Rk signaling is utilized for communication within the border cell cluster itself. The mutant border cell clusters show defects in localization of the adhesion protein E-cadherin, and apical polarity proteins during migration. E-cadherin mislocalization occurs in mosaic clusters, but not in full mutant clusters, correlating well with the rk border cell migration phenotype. Our work has identified a receptor with a previously unknown role in border cell migration that appears to regulate detachment and polarity of the border cell cluster coordinating processes within the cells of the cluster themselves.

Isolation and Tissue Distribution of an Insulin-Like Androgenic Gland Hormone (IAG) of the Male Red Deep-Sea Crab, Chaceon quinquedens.

The insulin-like androgenic gland hormone (IAG) found in decapod crustaceans is known to regulate sexual development in males. IAG is produced in the male-specific endocrine tissue, the androgenic gland (AG); however, IAG expression has been also observed in other tissues of decapod crustacean species including Callinectes sapidus and Scylla paramamosain. This study aimed to isolate the full-length cDNA sequence of IAG from the AG of male red deep-sea crabs, Chaceon quinquedens (ChqIAG), and to examine its tissue distribution. To this end, we employed polymerase chain reaction cloning with degenerate primers and 5' and 3' rapid amplification of cDNA ends (RACE). The full-length ChqIAG cDNA sequence (1555 nt) includes a 366 nt 5' untranslated region a 453 nt open reading frame encoding 151 amino acids, and a relatively long 3' UTR of 733 nt. The ORF consists of a 19 aa signal peptide, 32 aa B chain, 56 aa C chain, and 44 aa A chain. The putative ChqIAG amino acid sequence is most similar to those found in other crab species, including C. sapidus and S. paramamosain, which are clustered together phylogenetically.

Evidence for participation of GCS1 in fertilization of the starlet sea anemone Nematostella vectensis: implication of a common mechanism of sperm-egg fusion in plants and animals.

It has been reported that GCS1 (Generative Cell Specific 1) is a transmembrane protein that is exclusively expressed in sperm cells and is essential for gamete fusion in flowering plants. The GCS1 gene is present not only in angiosperms but also in unicellular organisms and animals, implying the occurrence of a common or ancestral mechanism of GCS1-mediated gamete fusion. In order to elucidate the common mechanism, we investigated the role of GCS1 in animal fertilization using a sea anemone (Cnidaria), Nematostella vectensis. Although the existence of the GCS1 gene in N. vectensis has been reported, the expression of GCS1 in sperm and the role of GCS1 in fertilization are not known. In this study, we showed that the GCS1 gene is expressed in the testis and that GCS1 protein exists in sperm by in situ hybridization and proteomic analysis, respectively. Then we made four peptide antibodies against the N-terminal extracellular region of NvGCS1. These antibodies specifically reacted to NvGCS1 among sperm proteins on the basis of Western analysis and potently inhibited fertilization in a concentration-dependent manner. These results indicate that sperm GCS1 plays a pivotal role in fertilization, most probably in sperm-egg fusion, in a starlet sea anemone, suggesting a common gamete-fusion mechanism shared by eukaryotic organisms.

Pacific white shrimp (Litopenaeus vannamei) vitellogenesis-inhibiting hormone (VIH) is predominantly expressed in the brain and negatively regulates hepatopancreatic vitellogenin (VTG) gene expression.

Ovarian maturation in crustaceans is temporally orchestrated by two processes: oogenesis and vitellogenesis. The peptide hormone vitellogenesis-inhibiting hormone (VIH), by far the most potent negative regulator of crustacean reproduction known, critically modulates crustacean ovarian maturation by suppressing vitellogenin (VTG) synthesis. In this study, cDNA encoding VIH was cloned from the eyestalk of Pacific white shrimp, Litopenaeus vannamei, a highly significant commercial culture species. Phylogenetic analysis suggests that L. vannamei VIH (lvVIH) can be classified as a member of the type II crustacean hyperglycemic hormone family. Northern blot and RT-PCR results reveal that both the brain and eyestalk were the major sources for lvVIH mRNA expression. In in vitro experiments on primary culture of shrimp hepatopancreatic cells, it was confirmed that some endogenous inhibitory factors existed in L. vannamei hemolymph, brain, and eyestalk that suppressed hepatopancreatic VTG gene expression. Purified recombinant lvVIH protein was effective in inhibiting VTG mRNA expression in both in vitro primary hepatopancreatic cell culture and in vivo injection experiments. Injection of recombinant VIH could also reverse ovarian growth induced by eyestalk ablation. Furthermore, unilateral eyestalk ablation reduced the mRNA level of lvVIH in the brain but not in the remaining contralateral eyestalk. Our study, as a whole, provides new insights on VIH regulation of shrimp reproduction: 1) the brain and eyestalk are both important sites of VIH expression and therefore possible coregulators of hepatopancreatic VTG mRNA expression and 2) eyestalk ablation could increase hepatopancreatic VTG expression by transcriptionally abolishing eyestalk-derived VIH and diminishing brain-derived VIH.

Targeted inactivation of the rickets receptor in muscle compromises Drosophila viability.

Bursicon is a hormone that modulates wing expansion, cuticle hardening and melanization in Drosophila melanogaster. Bursicon activity is mediated through its cognate G protein-coupled receptor (GPCR), rickets. We have developed a membrane-tethered bursicon construct that enables spatial modulation of rickets-mediated physiology in transgenic flies. Ubiquitous expression of tethered bursicon throughout development results in arrest at the pupal stage. The few organisms that eclose fail to undergo wing expansion. These phenotypes suggest that expression of tethered bursicon inhibits rickets-mediated function. Consistent with this hypothesis, we show in vitro that sustained stimulation of rickets by tethered bursicon leads to receptor desensitization. Furthermore, tissue-specific expression of the tethered bursicon inhibitor unraveled a critical role for rickets in a subset of adult muscles. Taken together, our findings highlight the utility of membrane-tethered inhibitors as important genetic/pharmacological tools to dissect the tissue-specific roles of GPCRs in vivo.

Genetic analysis of ecdysis behavior in Drosophila reveals partially overlapping functions of two unrelated neuropeptides.

Ecdysis behavior allows insects to shed their old exoskeleton at the end of every molt. It is controlled by a suite of interacting hormones and neuropeptides, and has served as a useful behavior for understanding how bioactive peptides regulate CNS function. Previous findings suggest that crustacean cardioactive peptide (CCAP) activates the ecdysis motor program; the hormone bursicon is believed to then act downstream of CCAP to inflate, pigment, and harden the exoskeleton of the next stage. However, the exact roles of these signaling molecules in regulating ecdysis remain unclear. Here we use a genetic approach to investigate the functions of CCAP and bursicon in Drosophila ecdysis. We show that null mutants in CCAP express no apparent defects in ecdysis and postecdysis, producing normal adults. By contrast, a substantial fraction of flies genetically null for one of the two subunits of bursicon [encoded by the partner of bursicon gene (pburs)] show severe defects in ecdysis, with escaper adults exhibiting the expected failures in wing expansion and exoskeleton pigmentation and hardening. Furthermore, flies lacking both CCAP and bursicon show much more severe defects at ecdysis than do animals null for either neuropeptide alone. Our results show that the functions thought to be subserved by CCAP are partially effected by bursicon, and that bursicon plays an important and heretofore undescribed role in ecdysis behavior itself. These findings have important implications for understanding the regulation of this vital insect behavior and the mechanisms by which hormones and neuropeptides control the physiology and behavior of animals.

Membrane tethered bursicon constructs as heterodimeric modulators of the Drosophila G protein-coupled receptor rickets.

The study of complex heterodimeric peptide ligands has been hampered by a paucity of pharmacological tools. To facilitate such investigations, we have explored the utility of membrane tethered ligands (MTLs). Feasibility of this recombinant approach was explored with a focus on Drosophila bursicon, a heterodimeric cystine-knot protein that activates the G protein-coupled receptor rickets (rk). Rk/bursicon signaling is an evolutionarily conserved pathway in insects required for wing expansion, cuticle hardening, and melanization during development. We initially engineered two distinct MTL constructs, each composed of a type II transmembrane domain, a peptide linker, and a C terminal extracellular ligand that corresponded to either the α or ß bursicon subunit. Coexpression of the two complementary bursicon MTLs triggered rk-mediated signaling in vitro. We were then able to generate functionally active bursicon MTLs in which the two subunits were fused into a single heterodimeric peptide, oriented as either α-ß or ß-α. Carboxy-terminal deletion of 32 amino acids in the ß-α MTL construct resulted in loss of agonist activity. Coexpression of this construct with rk inhibited receptor-mediated signaling by soluble bursicon. We have thus generated membrane-anchored bursicon constructs that can activate or inhibit rk signaling. These probes can be used in future studies to explore the tissue and/or developmental stage-dependent effects of bursicon in the genetically tractable Drosophila model organism. In addition, our success in generating functionally diverse bursicon MTLs offers promise that such technology can be broadly applied to other complex ligands, including the family of mammalian cystine-knot proteins.

A brief summary of neuroendocrine regulation of reproduction in sea stars.

Over than fifty years starfishes have been widely used as model for studying the mechanisms of cell cycle regulation, oocyte maturation and fertilization. Besides, significant work has been done to investigate the role of nervous system in the control of reproduction and spawning in these animals. Nowadays, sea stars represent one of the most thoroughly studied model for hormonal regulation of reproduction among invertebrates. However, while the general picture of neuroendocrine control of asteroid reproduction can be drawn easily, our knowledge concerning the details of this process still has some gaps. Filling these gaps is essential for studying the diversity of hormonal mechanisms involved in regulation of animal reproduction. The present paper aims to briefly summarize current data on hormonal regulation of reproduction in sea stars and to highlight existing gaps in our knowledge on the details of this process.

Conservation of progesterone hormone function in invertebrate reproduction.

Steroids play fundamental roles regulating mammalian reproduction and development. Although sex steroids and their receptors are well characterized in vertebrates and several arthropod invertebrates, little is known about the hormones and receptors regulating reproduction in other invertebrate species. Evolutionary insights into ancient endocrine pathways can be gained by elucidating the hormones and receptors functioning in invertebrate reproduction. Using a combination of genomic analyses, receptor imaging, ligand identification, target elucidation, and exploration of function through receptor knockdown, we now show that comparable progesterone chemoreception exists in the invertebrate monogonont rotifer Brachionus manjavacas, suggesting an ancient origin of the signal transduction systems commonly associated with the development and integration of sexual behavior in mammals.

Role of the gene Miniature in Drosophila wing maturation.

Miniature is an extracellular zona pellucida domain-containing protein, required for flattening of pupal wing epithelia in Drosophila. Here, we show that Miniature also plays an important role in the post-eclosion wing maturation processes triggered by the neurohormone bursicon. Wing expansion and epithelial apoptosis are drastically delayed in miniature loss-of-function mutants, and sped up upon overexpression of the protein in wings. Miniature acts upstream from the heterotrimeric Gs protein transducing the bursicon signal in wing epithelia. We propose that Miniature interacts with bursicon and regulates its diffusion through or stability within the wing tissue.

The CHH-superfamily of multifunctional peptide hormones controlling crustacean metabolism, osmoregulation, moulting, and reproduction.

Apart from providing an up-to-date review of the literature, considerable emphasis was placed in this article on the historical development of the field of "crustacean eyestalk hormones". A role of the neurosecretory eyestalk structures of crustaceans in endocrine regulation was recognized about 80 years ago, but it took another half a century until the first peptide hormones were identified. Following the identification of crustacean hyperglycaemic hormone (CHH) and moult-inhibiting hormone (MIH), a large number of homologous peptides have been identified to this date. They comprise a family of multifunctional peptides which can be divided, according to sequences and precursor structure, into two subfamilies, type-I and -II. Recent results on peptide sequences, structure of genes and precursors are described here. The best studied biological activities include metabolic control, moulting, gonad maturation, ionic and osmotic regulation and methyl farnesoate synthesis in mandibular glands. Accordingly, the names CHH, MIH, and GIH/VIH (gonad/vitellogenesis-inhibiting hormone), MOIH (mandibular organ-inhibiting hormone) were coined. The identification of ITP (ion transport peptide) in insects showed, for the first time, that CHH-family peptides are not restricted to crustaceans, and data mining has recently inferred their occurrence in other ecdysozoan clades as well. The long-held tenet of exclusive association with the eyestalk X-organ-sinus gland tract has been challenged by the finding of several extra nervous system sites of expression of CHH-family peptides. Concerning mode of action and the question of target tissues, second messenger mechanisms are discussed, as well as binding sites and receptors. Future challenges are highlighted.

The eyes have it: A brief history of crustacean neuroendocrinology.

To help celebrate the 50th anniversary of General and Comparative Endocrinology, the history of only a small portion of crustacean endocrinology is presented here. The field of crustacean endocrinology dates back to the decades prior to the establishment of General and Comparative Endocrinology and the first article about crustacean endocrinology published in this journal was concerned with the anatomy of neurosecretory and neurohemal structures in brachyuran crabs. This review looks at the history of neuroendocrinology in crustaceans during that time and tries to put perspective on the future of this field.

Molt-inhibiting hormone from Chinese mitten crab (Eriocheir sinensis): Cloning, tissue expression and effects of recombinant peptide on ecdysteroid secretion of YOs.

Molt-inhibiting hormone (MIH), a member of the crustacean hyperglycemic hormone (CHH) family, inhibits the synthesis of ecdysteroid in Y-organ (YO) and plays a significant role in the regulation of molting and growth of crustaceans. A complete cDNA sequence encoding MIH (Ers-MIH, GenBank Accession No.: DQ341280) was cloned from eyestalk of Chinese mitten crab (Eriocheir sinensis) by 5' and 3' RACEs and PCR cloning. The full-length cDNA consists of 1457 bp with a 330 bp open reading frame, encoding 110 amino acids, containing a 75 amino acid mature peptide. The deduced amino acid sequence contains a typical CHH domain. Transcripts of Ers-MIH mRNA were detected in eyestalk by Northern blotting. The production of purified recombinant Ers-MIH (rErs-MIH) expressed in Escherichia coli was 0.3g/L. The LC-ESI-MS analysis showed that two peptide fragments of the recombinant protein were identical to the deduced amino acid sequence of Ers-MIH. By in vitro assay on E. sinensis YOs, a cGMP mediated suppression of rErs-MIH on ecdysteroidogenesis could be observed. Accumulation of cGMP in YOs showed a concentration-dependent manner within 0.01-1 nmol/mL of rErs-MIH; ecdysteroid secretion was inhibited significantly at the range of 0.01-100 nmol/mL rErs-MIH; furthermore, a significant inhibition effect on ecdysteroid releasing was shown when cGMP analog (8-Br-cGMP) concentration rose up to 100 nmol/mL. This study would facilitate to investigate the roles of MIH in molt cycle regulation.

Pupal behavior emerges from unstructured muscle activity in response to neuromodulation in Drosophila.

Identifying neural substrates of behavior requires defining actions in terms that map onto brain activity. Brain and muscle activity naturally correlate via the output of motor neurons, but apart from simple movements it has been difficult to define behavior in terms of muscle contractions. By mapping the musculature of the pupal fruit fly and comprehensively imaging muscle activation at single-cell resolution, we here describe a multiphasic behavioral sequence in Drosophila. Our characterization identifies a previously undescribed behavioral phase and permits extraction of major movements by a convolutional neural network. We deconstruct movements into a syllabary of co-active muscles and identify specific syllables that are sensitive to neuromodulatory manipulations. We find that muscle activity shows considerable variability, with sequential increases in stereotypy dependent upon neuromodulation. Our work provides a platform for studying whole-animal behavior, quantifying its variability across multiple spatiotemporal scales, and analyzing its neuromodulatory regulation at cellular resolution.

How do we find out how the brain works? One way is to use imaging techniques to visualise an animal's brain in action as it performs simple behaviours: as the animal moves, parts of its brain light up under the microscope. For laboratory animals like fruit flies, which have relatively small brains, this lets us observe their brain activity right down to the level of individual brain cells. The brain directs movements via collective activity of the body's muscles. Our ability to track the activity of individual muscles is, however, more limited than our ability to observe single brain cells: even modern imaging technology still cannot monitor the activity of all the muscle cells in an animal's body as it moves about. Yet this is precisely the information that scientists need to fully understand how the brain generates behaviour. Fruit flies perform specific behaviours at certain stages of their life cycle. When the fly pupa begins to metamorphose into an adult insect, it performs a fixed sequence of movements involving a set number of muscles, which is called the pupal ecdysis sequence. This initial movement sequence and the rest of metamorphosis both occur within the confines of the pupal case, which is a small, hardened shell surrounding the whole animal. Elliott et al. set out to determine if the fruit fly pupa's ecdysis sequence could be used as a kind of model, to describe a simple behaviour at the level of individual muscles. Imaging experiments used fly pupae that were genetically engineered to produce an activity-dependent fluorescent protein in their muscle cells. Pupal cases were treated with a chemical to make them transparent, allowing easy observation of their visually 'labelled' muscles. This yielded a near-complete record of muscle activity during metamorphosis. Initially, individual muscles became active in small groups. The groups then synchronised with each other over the different regions of the pupa's body to form distinct movements, much as syllables join to form words. This synchronisation was key to progression through metamorphosis and was co-ordinated at each step by specialised nerve cells that produce or respond to specific hormones. These results reveal how the brain might direct muscle activity to produce movement patterns. In the future, Elliott et al. hope to compare data on muscle activity with comprehensive records of brain cell activity, to shed new light on how the brain, muscles, and other factors work together to control behaviour.

Photoperiodic control of electrophysiological properties of the caudo-dorsal cells in the pond snail, Lymnaea stagnalis.

Egg laying in the pond snail, Lymnaea stagnalis is regulated by the photoperiod; long-day conditions (16L8D) promote egg laying whereas medium-day conditions (12L12D) suppress it. In this snail, a caudo-dorsal cell hormone (CDCH) is produced by neurosecretory cells, CDCs in the cerebral ganglion (CG), and its release triggers ovulation and subsequent egg laying. However, the physiological basis for photoperiod-dependent egg laying remains unraveled. Here, we compared electrophysiological properties of CDCs between 16L8D and 12L12D using intracellular recording, and found that CDC excitability is higher in 16L8D than in 12L12D. Striking differences are as follows: (1) a shallower resting membrane potential in 16L8D than in 12L12D, and (2) a smaller threshold voltage (minimum depolarization from rest to elicit action potentials) in 16L8D than in 12L12D. Switching of the excitability can be a physiological basis of a photoperiod-dependent CDCH release. Simultaneous intracellular dye injection identified two morphological subtypes of CDCs, validating a previous report. Both types bear short lateral extensions in CG, some of which probably function as integration sites of photoperiodic inputs. In addition, we found two novel CDCH-immunoreactive cell groups (CDCCOM and SCm ) in the CG besides conventional CDCs and small cells expressing CDCH. The CDCCOM with cell bodies and fibers in the neurohemal commissure may be involved in triggering ovulation. Notably, the total number of CDCs is larger than that previously reported, the right CDC cluster with more cells than the left. Our findings are instructive in following the neurophysiology of photoperiodism in L. stagnalis.

Insulin-like androgenic gland hormone from the shrimp Fenneropenaeus merguiensis: Expression, gene organization and transcript variants.

Male sex differentiation in the crustacean is best known to be controlled by the insulin-like androgenic gland hormone (IAG). In this report, the cDNA and gene of the shrimp Fenneropenaeus merguiensis FmIAG were studied and characterized. FmIAG gene shares a high sequence identity in the coding region as well as the promoter region with that of F. chinensis. FmIAG gene is most likely consists of 5 exons and 4 introns. The cDNA reported here is the most abundant transcript that retained cryptic intron 4. The use of different splicing acceptor sites in exon 2 can produce a long-form FmIAG transcript variant with 6 additional amino acids inserted. Splicing of cryptic intron 4 would produce a transcript variant with a different C-terminal end. Therefore 4 different FmIAG transcripts can be produced from the FmIAG gene. During the molt cycle, the expression level of FmIAG was low in the early intermolt, increase steadily towards the late premolt and decreased rapidly in the early postmolt. In addition to the androgenic gland, FmIAG is also expressed in the hepatopancreas and ovary of adult females. Unilateral eyestalk ablation caused a significant increase in FmIAG transcript suggesting that the eyestalk consists of inhibiting factor(s) that suppressesFmIAGexpression. To explore the function of FmIAG in males, injection of FmIAG dsRNA knock-down the expression of FmIAG and up-regulated the expression of the vitellogenin gene in the testis and hepatopancreas. Interestingly a CHH-like gene identified in the androgenic gland was down-regulated. CHH-like gene knock-down resulted in altered expression of FmIAG in males suggesting that the CHH-like may be involved in FmIAG's regulation. RT-PCR with specific primers to the different transcript variant were used to determine if there is an association of different sizes of male and the type of IAG transcript. Results indicated that a high percentage of the large male shrimp expressed the long-form of FmIAG. The results suggested that FmIAG may be useful as a size marker for male shrimp aquaculture. In summary, the results of this study have expanded our knowledge of shrimp insulin-like androgenic gland hormone in male sex development and its potential role as a marker gene for growth regulation in shrimp.

Formation of diapause cyst shell in brine shrimp, Artemia parthenogenetica, and its resistance role in environmental stresses.

Artemia has attracted much attention for its ability to produce encysted embryos wrapped in a protective shell when subject to extremely harsh environmental conditions. However, what the cyst shell is synthesized from and how the formative process is performed remains, as yet, largely unknown. Over 20 oviparous specifically expressed genes were identified through screening the subtracted cDNA library enriched between oviparous and ovoviviparous Artemia ovisacs. Among them, a shell gland-specifically expressed gene (SGEG) has been found to be involved in the cyst shell formation. Lacking SGEG protein (by RNA interference) caused the cyst shell to become translucent and the chorion layer of the shell to become less compact and pultaceous and to show a marked decrease of iron composition within the shell. The RNA interference induced defective diapause cysts with a totally compromised resistibility to UV irradiation, extremely large temperature differences, osmotic pressure, dryness, and organic solvent stresses. In contrast, the natural cyst would provide adequate protection from all such factors. SGEG contains a 345-bp open reading frame, and its consequentially translated peptide consists of a 33-amino acid residue putative signal peptide and an 81-amino acid residue mature peptide. The results of Northern blotting and in situ hybridization indicate that the gene is specifically expressed in the cells of shell glands during the period of diapause cyst formation of oviparous Artemia. This investigation adds strong insight into the mechanism of cyst shell formation of Artemia and may be applicable to other areas of research in extremophile biology.