Feedback between a retinoid-related nuclear receptor and the let-7 microRNAs controls the pace and number of molting cycles in C. elegans.

Animal development requires coordination among cyclic processes, sequential cell fate specifications, and once-a-lifetime morphogenic events, but the underlying timing mechanisms are not well understood. Caenorhabditis elegans undergoes four molts at regular 8 to 10 hour intervals. The pace of the cycle is governed by PERIOD/lin-42 and other as-yet unknown factors. Cessation of the cycle in young adults is controlled by the let-7 family of microRNAs and downstream transcription factors in the heterochronic pathway. Here, we characterize a negative feedback loop between NHR-23, the worm homolog of mammalian retinoid-related orphan receptors (RORs), and the let-7 family of microRNAs that regulates both the frequency and finite number of molts. The molting cycle is decelerated in nhr-23 knockdowns and accelerated in let-7(-) mutants, but timed similarly in let-7(-) nhr-23(-) double mutants and wild-type animals. NHR-23 binds response elements (ROREs) in the let-7 promoter and activates transcription. In turn, let-7 dampens nhr-23 expression across development via a complementary let-7-binding site (LCS) in the nhr-23 3' UTR. The molecular interactions between NHR-23 and let-7 hold true for other let-7 family microRNAs. Either derepression of nhr-23 transcripts by LCS deletion or high gene dosage of nhr-23 leads to protracted behavioral quiescence and extra molts in adults. NHR-23 and let-7 also coregulate scores of genes required for execution of the molts, including lin-42. In addition, ROREs and LCSs isolated from mammalian ROR and let-7 genes function in C. elegans, suggesting conservation of this feedback mechanism. We propose that this feedback loop unites the molting timer and the heterochronic gene regulatory network, possibly by functioning as a cycle counter.

RNAi suppression of the nuclear receptor FTZ-F1 impaired ecdysis, pupation, and reproduction in the 28-spotted potato ladybeetle, Henosepilachna vigintioctopunctata.

Fushi-tarazu factor 1 (FTZF1) is an ecdysone-inducible transcription factor that plays a vital role during the metamorphosis in insects. In this study, we functionally characterized HvFTZ-F1 in H. vigintioctopunctata, a dreadful solanaceous crop pest, by using a dietary RNA interference technique. The HvFTZ-F1 expression levels were elevated in the 1st and 2nd-instars before molting and declined immediately after ecdysis. The HvFTZ-F1 silencing led to high mortality in the 1st instars, while the expression of the osmosis-regulative gene, HvAQPAn.G, was significantly increased in the 1st instars. HvFTZ-F1 silencing downregulated the Halloween and 20E-related genes, decreased the ecdysteroids titer, suppressed the expression of pigmentation-related genes, and reduced the catecholamines titer. In the 4th instars, HvFTZ-F1 silencing caused 100% mortality by arresting the development at the prepupal stage and preventing new abdominal cuticle formation. In the female adults, HvFTZ-F1 silencing caused an evident decrease in fecundity, prolonged the pre-oviposition period, reduced the number of eggs and hatching rate, severely atrophied the ovaries. Moreover, the 20E-related genes and the dopamine synthesis genes were suppressed in the dsHvFTZ-F1-treated females. Overall, our results revealed that HvFTZ-F1 regulates ecdysis, pupation, and reproduction in H. vigintioctopunctata, thereby could be a promising molecular target for the development of RNAi-based biopesticides to control H. vigintioctopunctata.

Knockdown of ecdysteroid synthesis genes results in impaired molting and high mortality in Bactericera cockerelli (Hemiptera: Triozidae).

BACKGROUND: RNA-mediated interference (RNAi) has become a promising biopesticide technology with which to direct sequence-specific gene knockdown of key targets in the potato psyllid (PoP) Bactericera cockerelli, resulting in significant mortality. In this study, three strategically selected target genes, ATF4, C7 and D24, essential for the biosynthesis and regulation of ecdysteroids, were evaluated for knockdown and mortality using oral delivery of individual, paired and all three double-stranded RNAs (dsRNAs), in five replicated experiments. Knockdown was determined as the fold-change in gene expression using a quantitative polymerase chain reaction. RESULTS: Knockdown of the D24 target, at 39%-45%, resulted in 51% PoP mortality by 10 days post-ingestion (dpi) of dsRNA. Knockdown of C7, at 38%-61%, resulted in 53% mortality by 10 dpi, whereas dsD24 ingestion resulted in 65% mortality by 10 dpi when dsD24 and dsC7 were co-delivered. Three phenotypes, INCOMEC, PREMEC and SWOLLEN, were observed at a frequency of 4%-12%, and are consistent with incomplete ecdysis in immature and/or adult PoP. Adult PoP exhibiting INCOMEC survived for several days but were unable to mate or fly, whereas SWOLLEN and PREMEC were lethal to the immature instars. Knockdown of ATF4 did not result in the mortality or malformations in immature and adult PoP. CONCLUSIONS: Compared with knockdown of individual D24 and C7 targets, significantly greater RNAi penetrance was achieved following delivery of combined dsRNAs. The highest knockdown that resulted in incomplete ecdysis and/or mortality was obtained for targets with predicted involvement in the same or interacting pathway(s). Knockdown of ATF4 was apparently "rescued" by uncharacterized compensatory gene(s) or effects. © 2022 Society of Chemical Industry.

Calcium signaling and regulation of ecdysteroidogenesis in crustacean Y-organs.

Crustacean Y-organs secrete ecdysteroid molting hormones. Ecdysteroids are released in increased amount during premolt, circulate in hemolymph, and stimulate the events in target cells that lead to molting. During much of the molting cycle, ecdysteroid production is suppressed by molt-inhibiting hormone (MIH), a peptide neurohormone produced in the eyestalks. The suppressive effect of MIH is mediated by a cyclic nucleotide second messenger. A decrease in circulating MIH is associated with an increase in the hemolymphatic ecdysteroid titer during pre-molt. Nevertheless, it has long been hypothesized that a positive regulatory signal or stimulus is also involved in promoting ecdysteroidogenensis during premolt. Data reviewed here are consistent with the hypothesis that an intracellular Ca2+ signal provides that stimulus. Pharmacological agents that increase intracellular Ca2+ in Y-organs promote ecdysteroidogenesis, while agents that lower intracellular Ca2+ or disrupt Ca2+ signaling suppress ecdysteroidogenesis. Further, an increase in the hemolymphatic ecdysteroid titer after eyestalk ablation or during natural premolt is associated with an increase in intracellular free Ca2+ in Y-organ cells. Several lines of evidence suggest elevated intracellular calcium is linked to enhanced ecdysteroidogenesis through activation of Ca2+/calmodulin dependent cyclic nucleotide phosphodiesterase, thereby lowering intracellular cyclic nucleotide second messenger levels and promoting ecdysteroidogenesis. Results of transcriptomic studies show genes involved in Ca2+ signaling are well represented in Y-organs. Several recent studies have focused on Ca2+ transport proteins in Y-organs. Complementary DNAs encoding a plasma membrane Ca2+ ATPase (PMCA) and a sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) have been cloned from crab Y-organs. The relative abundance of PMCA and SERCA transcripts in Y-organs is elevated during premolt, a time when Ca2+ levels in Y-organs are likewise elevated. The results are consistent with the notion that these transport proteins act to maintain the Ca2+ gradient across the cell membrane and re-set the cell for future Ca2+ signals.

From sporadic single genes to a broader transcriptomic approach: Insights into the formation of the biomineralized exoskeleton in decapod crustaceans.

One fundamental character common to pancrustaceans (Crustacea and Hexapoda) is a mineralized rigid exoskeleton whose principal organic components are chitin and proteins. In contrast to traditional research in the field that has been devoted to the structural and physicochemical aspects of biomineralization, the present study explores transcriptomic aspects of biomineralization as a first step towards adding a complementary molecular layer to this field. The rigidity of the exoskeleton in pancrustaceans dictates essential molt cycles enabling morphological changes and growth. Thus, formation and mineralization of the exoskeleton are concomitant to the timeline of the molt cycle. Skeletal proteinaceous toolkit elements have been discovered in previous studies using innovative molt-related binary gene expression patterns derived from transcriptomic libraries representing the major stages comprising the molt cycle of the decapod crustacean Cherax quadricarinatus. Here, we revisited some prominent exoskeleton-related structural proteins encoding and, using the above molt-related binary pattern methodology, enlarged the transcriptomic database of C. quadricarinatus. The latter was done by establishing a new transcriptomic library of the cuticle forming epithelium and molar tooth at four different molt stages (i.e., inter-molt, early pre-molt, late pre-molt and post-molt) and incorporating it to a previous transcriptome derived from the gastroliths and mandible. The wider multigenic approach facilitated by the newly expanded transcriptomic database not only revisited single genes of the molecular toolkit, but also provided both scattered and specific information that broaden the overview of proteins and gene clusters which are involved in the construction and biomineralization of the exoskeleton in decapod crustaceans.

De novo transcriptome assembly and functional annotation for Y-organs of the blue crab (Callinectes sapidus), and analysis of differentially expressed genes during pre-molt.

Blue crabs (Callinectes sapidus) undergo incremental growth involving the shedding (molting) of the old exoskeleton, and subsequent expansion and re-calcification of the newly synthesized one. The cellular events that lead to molting are triggered by steroid hormones termed ecdysteroids released from Y-organs, paired endocrine glands located in the anterior cephalothorax. The regulatory pathways leading to increased synthesis and release of ecdysteroids are not fully understood, and no transcriptome has yet been published for blue crab Y-organs. Here we report de novo transcriptome assembly and annotation for adult blue crab Y-organs, and differential gene expression (DGE) analysis between Y-organs of intermolt and premolt crabs. After trimming and quality assessment, a total of 91,819,458 reads from four cDNA libraries were assembled using Trinity to form the reference transcriptome. Trinity produced a total of 171,530 contigs coding for 150,388 predicted genes with an average contig length of 613 and an N50 of 940. Of these, TransDecoder predicted 31,661 open reading frames (ORFs), and 10,210 produced non-redundant blastx results through Trinotate annotation. Genes involved in multiple cell signaling pathways, including Ca2+ signaling, cGMP signaling, cAMP signaling, and mTOR signaling were present in the annotated reference transcriptome. DGE analysis showed in premolt Y-organs up-regulated genes involved in energy production, cholesterol metabolism, and exocytosis. The results provide insights into the transcriptome of blue crab Y-organs during a natural (rather than experimentally induced) molting cycle, and constitute a step forward in understanding the cellular mechanisms that underlie stage-specific changes in the synthesis and secretion of ecdysteroids by Y-organs.

Transgenic potato lines expressing hairpin RNAi construct of molting-associated EcR gene exhibit enhanced resistance against Colorado potato beetle (Leptinotarsa decemlineata, Say).

Most of the commercialized insect resistant transgenic crops express cry gene(s) isolated from Bacillus thuringiensis; however, intensive cultivation of Bt crops over almost two decades has been questioned regarding its sustainability and durability in pest management. The present study focused on silencing of highly specific molting-associated Ecdysone receptor (EcR) gene of Colorado potato beetle (CPB) using RNA interference (RNAi) approach. The partial cDNA of EcR gene of CPB was amplified using specific primers in sense and anti-sense orientations, and cloned in pRNAi-GG vector flanked by an intronic sequence (pdk). Leaf and internodal explants of Agria and Lady Olympia potato cultivars were infected with Agrobacterium strain LBA4404 harboring constructs under the control of CaMV 35S promoter. Standard molecular analysis of primary transformants showed proper integration of T-DNA in plant genome. The transgenic plants of both cultivars were evaluated for their efficacy against first, second and third instar CPB larvae. The leaf biotoxicity assays revealed 15-80% of CPB mortality. A significantly lower fold-change (0.87-4.14×) in larval weight was observed in insects fed on transgenic plants compared to the ones fed on control plants (1.87-6.53×). Furthermore, CPB larvae fed on transgenic plants exhibited reduced EcR transcripts, indicating the functionality of dsRNA EcR in silencing EcR gene expression. This study is an excellent example of the integration of an alternative, effective and reliable method to cope with potato insect pests that incur significant losses to potato production in the world.

Crustacean cardioactive peptide (CCAP) of the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae): Molecular characterization, distribution and its potential roles in larva-pupa ecdysis.

Insects must undergo ecdysis for successful development and growth, and the crustacean cardioactive peptide (CCAP) is one of the most important hormone in this process. Here we reported a cDNA encoding for the CCAP precursor cloned from the oriental fruit fly, Bactrocera dorsalis, a most destructive insect pest of agriculture. The CCAP mature peptide (PFCNAFTGC-NH2) of B. dorsalis was generated by post-translational processing and found to be highly comparable with other insects. RT-qPCR showed that mRNA of CCAP in B. dorsalis (BdCCAP) was predominantly expressed in the central nervous system (CNS) and midgut of 3rd-instar larvae. By using immunohistochemical analysis, we also localized the endocrine cells that produce CCAP in the CNS, ring gland and midgut of 3rd-instar larvae of B. dorsalis. The synthetic CCAP mature peptide could induce the expression of mRNA of adipokinetic hormone (AKH), the metabolic neuropeptides in insects. The expression of BdCCAP mRNA in the CNS, but not in the midgut, could be upregulated in the response to the challenge of insect molting hormone, 20-hydroxyecdysone.

[Exploration of Insect Growth Regulators Targeting Noppera-bo, an Enzyme Involved in Ecdysteroid Biosynthesis].

Insect growth regulators (IGRs) are chemicals that adversely affect the physiological processes associated with insect development and cause abnormalities that impair insect survival. Ecdysone, an insect steroid hormone originally identified as a molting hormone, plays an essential role in developmental transition, such as during molting and metamorphosis. Recently, a member of the epsilon class of glutathione S-transferases (GST), GSTe14, also called Noppera-bo (Nobo), has been identified as essential for regulating the biosynthesis of ecdysone. Knockout or knockdown of the nobo gene causes ecdysone deficiency, leading to either death or arrested phenotype development at the larval stage. It is therefore considered that Nobo is potentially well suited as a target for novel IGRs. In this review, we focus on the development of a high-throughput screening strategy for Nobo inhibitors using a GST fluorogenic substrate.

Identification and expression of nuclear receptor genes and ecdysteroid titers during nymphal development in the spider Agelena silvatica.

Ecdysteroids play an essential role in the regulation of the molting processes of arthropods. Nuclear receptors of the spider Agelena silvatica that showed high homology with other arthropods especially in the functional domains were identified, two isoforms of ecdysone receptor (AsEcRA, AsEcRB), retinoid X receptor (AsRXR) and two isoforms of E75 (AsE75A, AsE75D). AsEcR and AsRXR mRNA did not show major changes in expression but occurred throughout the third instar nymphal stage. AsE75DBD was low or non-existent at first then showed a sudden increase from D7 to D10. On the other hand, AsE75D was expressed in the first half and decreased from D6 to D10. Ecdysteroid titers showed a peak on D6 in A. silvatica third instar nymphs. LC-MS/MS analysis of the ecdysteroid peak revealed only 20-hydroxyecdysone (20E) was present. The 20E peak on D6 and increase in AsE75DBD from D7 is likely a result of ecdysteroids binding to the heterodimer formed with constant expression of the AsEcR and AsRXR receptors. These findings indicate the mechanisms regulating molting widely conserved in insects and other arthropods also similarly function in spiders.

WAFs lead molting retardation of naupliar stages with down-regulated expression profiles of chitin metabolic pathway and related genes in the copepod Tigriopus japonicus.

Oil pollution is considered being disastrous to marine organisms and ecosystems. As molting is critical in the developmental process of arthropods in general and copepods, in particular, the impact will be adverse if the target of spilled oil is on molting. Thus, we investigated the harmful effects of water accommodated fractions (WAFs) of crude oil with an emphasis on inhibition of chitin metabolic pathways related genes and developmental retardation in the copepod Tigriopus japonicus. Also, we analysed the ontology and domain of chitin metabolic pathway genes and mRNA expression patterns of developmental stage-specific genes. Further, the developmental retardation followed by transcriptional modulations in nuclear receptor genes (NR) and chitin metabolic pathway-related genes were observed in the WAFs-exposed T. japonicus. As a result, the developmental time was found significantly (P<0.05) delayed in response to 40% WAFs in comparison with that of control. Moreover, the NR gene, HR3 and chitinases (CHT9 and CHT10) were up-regulated in N4-5 stages, while chitin synthase genes (CHS-1, CHS-2-1, and CHS-2-2) down-regulated in response to WAFs. In brief, a high concentration of WAFs repressed nuclear receptor genes but elicited activation of some of the transcription factors at low concentration of WAFs, resulting in suppression of chitin synthesis. Thus, we suggest that WAF can lead molting retardation of naupliar stages in T. japonicus through down-regulations of chitin metabolism. These findings will provide a better understanding of the mode of action of chitin biosynthesis associated with molting mechanism in WAF-exposed T. japonicus.

Rapid Communication: MicroRNA co-expression network reveals apoptosis in the reproductive tract during molting in laying hens.

The aim of this study was to determine the regulatory mechanisms of molting and recrudescence via studying the micro-RNA (miRNA) expression in the oviduct of laying hens. We performed a cDNA microarray analysis in the magnum tissue from the oviduct to identify the whole miRNA profiles through the molting and recrudescence periods. A total of 35 laying hens (47-wk-old) were divided into 7 groups (0 d: a control group; 6 and 12 d: 2 molting-period groups fed on a high-zinc diet; and 20, 25, 30, and 35 d: 4 recrudescence-period groups fed on a normal diet after a 12-d period on a high-zinc diet). An miRNA co-expression network (miRCN) was generated using the differentially expressed miRNA (DEM) according to the entire data integration. The significantly co-expressed miRNA ( = 111) were highly differentially expressed from 12 to 20 d, which was a transition period between molting and recrudescence, while their expression patterns were contrary to the estrogen changes. The targets of highly connected miRNA ( = 12) indicated the significant biological pathways and gene ontology (GO) terms, such as MAPK and Wnt signaling and magnesium-ion binding, which are associated with apoptotic activities. These results suggest that the miRNA of the miRCN might play a role in the apoptotic progression of the reproductive tract during molting.

Transcriptome analysis of the molting gland (Y-organ) from the blackback land crab, Gecarcinus lateralis.

In decapod crustaceans, arthropod steroid hormones or ecdysteroids regulate molting. These hormones are synthesized and released from a pair of molting glands called the Y-organs (YO). Cyclic nucleotide, mTOR, and TGFß/Smad signaling pathways mediate molt cycle-dependent phase transitions in the YO. To further identify the genes involved in the regulation of molting, a YO transcriptome was generated from three biological replicates of intermolt blackback land crab, Gecarcinus lateralis. Illumina sequencing of cDNA libraries generated 227,811,829 100-base pair (bp) paired-end reads; following trimming, 90% of the reads were used for further analyses. The trimmed reads were assembled de novo using Trinity software to generate 288,673 contigs with a mean length of 872 bp and a median length of 1842 bp. Redundancy among contig sequences was reduced by CD-HIT-EST, and the output constituted the baseline transcriptome database. Using Bowtie2, 92% to 93% of the reads were mapped back to the transcriptome. Individual contigs were annotated using BLAST, HMMER, TMHMM, SignalP, and Trinotate, resulting in assignments of 20% of the contigs. Functional and pathway annotations were carried out via gene ontology (GO) and KEGG orthology (KO) analyses; 58% and 44% of the contigs with BLASTx hits were assigned to GO and KO terms, respectively. The gene expression profile was similar to a crayfish YO transcriptome database, and the relative abundance of each contig was highly correlated among the three G. lateralis replicates. Signal transduction pathway orthologs were well represented, including those in the mTOR, TGFß, cyclic nucleotide, MAP kinase, calcium, VEGF, phosphatidylinositol, ErbB, Wnt, Hedgehog, Jak-STAT, and Notch pathways.

Molecular characterization of a cDNA encoding red pigment-concentrating hormone in black tiger shrimp Penaeus monodon: Implication of its function in molt and osmoregulation.

Red pigment-concentrating hormone (RPCH) is a member of the AKH/RPCH peptide family present mainly in crustaceans and insects. Insect AKH is responsible for metabolic functions whereas RPCH plays a major role in the aggregation of red chromatophores in crustaceans. In this study, a full-length cDNA of RPCH of the black tiger shrimp, Penaeus monodon (PmRPCH) was cloned by Rapid Amplification of cDNA Ends strategies from the eyestalk RNA. A 770 bp full-length PmRPCH cDNA harbored 279 bp of an open reading frame encoding a signal peptide of 21 amino acid residues, an 8 amino acid mature RPCH peptide, followed by 61 amino acid residues of a RPCH precursor-related peptide. The highest levels of PmRPCH mRNA expression were detected in eyestalks while lower expression was found in other nervous tissues i.e. brain, thoracic ganglia and abdominal nerve cord. Expression of PmRPCH was transiently stimulated upon hypersalinity change within 12 h suggesting its osmoregulatory function. During the molting cycle, PmRPCH in the eyestalk was expressed at the lowest level in the early pre-molt stage (D0), then gradually increased over the pre-molt period and reached the highest level in the late pre-molt (D4) and post-molt (AB) stages. RPCH peptide at a dose of 100 pmol also increased gill Na(+)/K(+) ATPase activity in 36-48 h after injection. However, PmRPCH did not accelerate the duration of molting cycle. Our results provide the first evidence on the potential function of PmRPCH in molting, probably by mediating hemolymph osmolality and ion transport enzymes during the late pre-molt stage.

Calcium-calmodulin dependent protein kinase I from Macrobrachium nipponense: cDNA cloning and involvement in molting.

Calcium-calmodulin dependent protein kinase I is a component of a calmodulin-dependent protein kinase cascade and involved in many physiological processes. The full-length cDNA of calcium-calmodulin dependent protein kinase I (MnCaMKI) was cloned from the freshwater prawn Macrobrachium nipponense and its expression pattern during the molt cycle and after eyestalk ablation is described. The full-length cDNA of MnCaMKI is 3,262 bp in length and has an open reading frame (ORF) of 1,038 bp, encoding a 345 amino acid protein. The expression of MnCaMKI in three examined tissues was upregulated in the premolt stage of the molt cycle. Its expression was induced after eyestalk ablation (ESA): the highest expression level was reached 1 day after ESA in hepatopancreas, and 3 days after ESA in muscle. By dsRNA-mediated RNA interference assay, expression of MnCaMKI and ecydone receptor gene (MnEcR) was significantly decreased in prawns treated by injection of dsMnCaMKI, while expression of these two genes was also significantly decreased in prawns treated by injection of dsMnEcR, demonstrating a close correlation between the expression of these two genes. These results suggest that CaMKI in M. nipponense is involved in molting.

Cloning of prophenoloxidase from hemocytes of the blue crab, Callinectes sapidus and its expression and enzyme activity during the molt cycle.

The arthropods cuticle undergoes dramatic morphological and biochemical changes from being soft to hardness through each molting process. Prophenoloxidase (PPO) known as a key enzyme in the arthropod innate immune system involved in the melanization reaction, has been related with the initial shell-hardening process, specifically in the sclerotization of the protein matrix in the new cuticle. Since hemocytes have been reported as the main PPO source in arthropods, the transport of hemocyte PPO into the newly laid, soft cuticle has been proposed for shell-hardening occurring during and immediately after ecdysis. In order to define the role of hemocyte PPO in the shell-hardening of crustaceans, the full-length cDNA sequence (2806 nt) of hemocytes PPO of the blue crab Callinectes sapidus (CasPPO-hemo) is isolated using degenerate PCR and 5'-3' RACE. CasPPO-hemo encodes a putative PPO (672 aa) showing three hemocyanin domains: N, M, and C in order and two copper binding sites (CuA & CuB). The sequence analysis identifies the putative CasPPO-hemo as zymogen which requires the cleavage at the N-terminus for its activation. Hemocyte extract (CasHLS) contains the PO, the activity of which depends on the in vitro activation of trypsin. The expression levels of CasPPO-hemo are kept constant during the molt cycle. The increase in the number of hemocytes at early premolt correlates with the elevated PO activity, while at late premolt, the increment in hemocyte numbers does not reflect on the PO activity. The functional importance of the changes in the levels of CasHLS-PO activity during molt cycle is discussed in relation to cuticle hardening process.

Molecular cloning of a plasma membrane Ca²âº ATPase (PMCA) from Y-organs of the blue crab (Callinectes sapidus), and determination of spatial and temporal patterns of PMCA gene expression.

Existing data indicate that a stage-specific increase in intracellular free Ca(2+) stimulates ecdysteroid production by crustacean molting glands (Y-organs). The concentration of Ca(2+) in cytosol is controlled mainly by proteins intrinsic to the plasma membrane and to the membranes of organelles. Several families of proteins are involved, including Ca(2+) channels, Ca(2+) pumps (ATPases), and Ca(2+) exchangers. The family of Ca(2+) pumps includes plasma membrane calcium ATPases (PMCAs). As a step toward understanding the involvement of calcium signaling in regulation of ecdysteroidogenesis, we used a PCR-based cloning strategy (RT-PCR followed by 3'- and 5'-RACE) to clone from Y-organs of the blue crab (Callinectes sapidus) a cDNA encoding a putative PMCA. The 4292 base pair (bp) cDNA includes a 3510 bp open reading frame encoding a 1170-residue protein (Cas-PMCA). The conceptually translated protein has a relative molecular mass of 128.8×10(3) and contains all signature domains of an authentic PMCA, including ten transmembrane domains and a calmodulin binding site. The predicted membrane topography of Cas-PMCA is as expected for an authentic PMCA protein. A phylogenetic analysis of nonredundant amino acid sequences of PMCA proteins from different species showed Cas-PMCA clusters with other arthropod PMCA proteins. An assessment of tissue distribution showed the Cas-PMCA transcript to be broadly distributed in both neural and non-neural tissues. Studies using quantitative real-time PCR revealed stage-specific changes in Cas-PMCA abundance during the molting cycle, with peak expression occurring during premolt stage D2, a pattern consistent with the hypothesis that Cas-PMCA functions to maintain cellular Ca(2+) homeostasis in Y-organs.

Effects of vitamins, probiotics, and protein level on semen traits and some seminal plasma macro- and microminerals of male broiler breeders after zinc-induced molting.

This study was conducted to investigate the effects of vitamin E, vitamin C, probiotics, dietary protein level, and their combination on semen traits and seminal plasma macro- and microminerals in 65-week-old male broiler breeders after zinc-induced molting. One hundred eighty birds were induced to molt by mixing zinc oxide (3,000 mg/kg) in the diet. The birds were divided into six groups (five replicates) by completely randomized design. One group was kept as control (16% CP), while the other five were supplemented with vitamin E (100 IU/kg feed), vitamin C (500 IU/kg feed) probiotics (50 mg/L), protein level (14% CP), and their combination. Semen samples were weekly collected for determination of semen volume, sperm concentration, motility, and dead sperm percentage. Analyses of Na, K, Ca and Mg, Zn, Fe, Mn, and Cu in seminal plasma were also performed. Overall, mean semen volume was significantly high in vitamin E and C supplemented groups compared to control. Overall mean sperm motility was significantly higher in vitamin E supplemented group, whereas dead sperm percentage was significantly lower in the vitamin C group compared to control. Mineral analyses revealed that overall mean seminal plasma Mg increased significantly in vitamin E and C supplemented groups compared to control. Similarly, significantly high overall mean seminal plasma Cu concentration was observed in vitamins E and C and combination groups. It can be concluded that vitamins have a vital role in improving semen quality and bioavailability of Mg and Cu in seminal plasma of the post-molt cockerels.

Identification and cloning of a selenophosphate synthetase (SPS) from tiger shrimp, Penaeus monodon, and its transcription in relation to molt stages and following pathogen infection.

Complementary (c)DNA encoding selenophosphate synthetase (SPS) messenger (m)RNA of the tiger shrimp Penaeus monodon, designated PmSPS, was obtained from the hepatopancreas by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The 1582-bp cDNA contained an open reading frame (ORF) of 1248 bp, a 103-bp 5'-untranslated region (UTR), and a 231-bp 3'-UTR, which contained a conserved selenocysteine insertion sequence (SECIS) element, a conventional polyadenylation signal, and a poly A tail. The molecular mass of the deduced amino acid (aa) sequence (416 aa) was 45.5 kDa with an estimated pI of 4.85. It contained a putative selenocysteine residue which was encoded by the unusual stop codon, (275)TGA(277), which formed at the active site with residues Sec(58) and Lys(61). A comparison of amino acid sequences showed that PmSPS was more closely related to invertebrate SPS1, such as those of Heliothis virescens and Drosophila melanogaster a and b. PmSPS cDNA was synthesized in all tested tissues, especially in the hepatopancreas. PmSPS in the hepatopancreas of shrimp significantly increased after an injection with either Photobacterium damsela or white spot syndrome virus (WSSV) in order to protect cells against damage from oxidation, and enhance the recycling of selenocysteine or selenium metabolism, indicating that PmSPS is involved in the disease-resistance response. The PmSPS expression by hemocytes significantly increased in stage C, and then gradually decreased until stage A, suggesting that the cloned PmSPS in hemocytes might play a role in viability by renewing hemocytes and antioxidative stress response for new exoskeleton synthesis during the molt cycle of shrimp.

Identification and cloning of a transglutaminase from giant freshwater prawn, Macrobrachium rosenbergii, and its transcription during pathogen infection and moulting.

Complementary (c)DNA encoding transglutaminase (TG) messenger (m)RNA of the giant freshwater prawn, Macrobrachium rosenbergii, was cloned from haemocytes by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) using oligonucleotide primers based on the TG sequence of the horseshoe crab, Tachypleus tridentatus; tiger shrimp, Penaeus monodon; kuruma shrimp, Marsupenaeus japonicus; and crayfish, Pacifastacus leniusculus. The 2722-bp cDNA contained an open reading frame (ORF) of 2334 bp, a 72-bp 5'-untranslated region (UTR), and a 316-bp 3'-UTR containing a stop codon and a poly A tail. The molecular mass of the deduced amino acid (aa) sequence (778 aa) was 86.67 kDa with an estimated pI of 5.4. The M. rosenbergii TG (abbreviated MrTG, accession no.: JF309296) contains a typical transglutaminase-like homologue, two putative integrin-binding motifs (RGD), ten glycosylation sites, and four calcium-binding sites; a catalytic triad is present as in arthropod TGs. Sequence comparison and a phylogenetic analysis revealed that shrimp TG can be separated into three subgroups, penaeid TG1, freshwater crustacean TG2 and marine crustacean TG2, and MrTG was more closely related to TG2 than to TG1. MrTG mRNA and TG activities were detected in all tested tissues of M. rosenbergii, with MrTG mainly being synthesised by haemocytes. There was a negative correlation between clotting time of haemolymph, and MrTG expression and TG activity of haemocytes in prawn injected with Lactococcus garvieae. The pattern of MrTG mRNA expression and TG activity in haemocytes exhibited a contrary tendency with clotting time of haemolymph during the moult stages. Those results indicate that cloned MrTG is involved in the defence response, and is probably the major functional TG for haemolymph coagulation in M. rosenbergii.