Late Premoult Research Articles

Identifying the genes involved in the egg-carrying ovigerous hair development of the female blue crab Callinectes sapidus: transcriptomic and genomic expression analyses

BackgroundCrustacean female sex hormone (CFSH) controls gradually developing adult female-specific morphological features essential for mating and brood care. Specifically, ovigerous hairs are developed during the prepuberty molt cycle of the blue crab Callinectes sapidus that are essential for carrying the eggs until they finish development. Reduced CFSH transcripts by CFSH-dsRNA injections result in fewer and shorter ovigerous hairs than the control. This study aimed to identify the specific genes responsible for ovigerous hair formation using transcriptomic, genomic and expression analyses of the ovigerous setae at three stages: prepuberty at early (OE) and late premolt (OL), and adult (AO) stages.ResultsThe de novo Trinity assembly on filtered sequence reads produced 96,684 Trinity genes and 124,128 transcripts with an N50 of 1,615 bp. About 27.3% of the assembled Trinity genes are annotated to the public protein sequence databases (i.e., NR, Swiss-Prot, COG, KEGG, and GO databases). The OE vs. OL, OL vs. AO, and OE vs. AO comparisons resulted in 6,547, 7,793, and 7,481 differentially expressed genes, respectively, at a log2-fold difference. Specifically, the genes involved in the Wnt signaling and cell cycle pathways are positively associated with ovigerous hair development. Moreover, the transcripts of ten cuticle protein genes containing chitin-binding domains are most significantly changed by transcriptomic analysis and RT-qPCR assays, which shows a molt-stage specific, down-up-down mode across the OE-OL-AO stages. Furthermore, the expression of the cuticle genes with the chitin-binding domain, Rebers and Riddiford domain (RR)-1 appears at early premolt, followed by RR-2 at late premolt stage. Mapping these 10 cuticle protein sequences to the C. sapidus genome reveals that two scaffolds with a 549.5Kb region and 35 with a 1.19 Mb region harbor 21 RR1 and 20 RR2 cuticle protein genes, respectively. With these findings, a putative mode of CFSH action in decapod crustaceans is proposed.ConclusionsThe present study describes a first step in understanding the mechanism underlying ovigerous hair formation in C. sapidus at the molecular level. Overall, demonstrating the first transcriptome analysis of crustacean ovigerous setae, our results may facilitate future studies into the decapod female reproduction belonging to the suborder Pleocyemata.

Transcriptome responses of RNAi-mediated ETH knockdown in Scylla paramamosain at different premolt substages.

Ecdysis triggering hormone (ETH) plays an important role in molting, reproduction, and courtship behavior in insects. To investigate the potential downstream pathways and genes of ETH in Scylla paramamosain, RNA interference (RNAi) was conducted on crabs at early (D0) and late (D2) premolt substages, and the transcriptome profiles of each group were compared by RNA sequencing. Real-time quantitative polymerase chain reaction (RT-qPCR) and semiquantitative polymerase chain reaction (RT-PCR) results showed a significant knockdown of ETH at D0 stage, whereas a significant increase was shown conversely in crabs at D2 substage after the injection of dsETH. A total of 242,979 transcripts were assembled, and 44,012 unigenes were identified. Transcriptomic comparison between crabs at D2 and D0 substages showed 2,683 differentially expressed genes (DEGs); these genes were enriched in ribosome and pathways related to transcription factor complex and cell part. Twenty DEGs were identified between dsETH-injected and dsGFP-injected crabs at D0 substage; these DEGs were involved in carbohydrate metabolism, one carbon pool by folate, and chitin binding. Twenty-six DEGs were identified between dsETH-injected and dsGFP-injected crabs at D2 substage; these DEGs were involved in calcium channel inhibitor activity, fat digestion and absorption, and cardiac muscle contraction. RT-qPCR verified the differential expression of the selected genes. In conclusion, crabs at D0 substage are more active in preparing the macromolecular complex that is needed for molting. Moreover, ETH has potential roles in carbohydrate metabolism, one carbon pool by folate, and chitin binding for crabs at D0 substage, while the role of ETH turns to be involved in calcium channel inhibitor activity, fat digestion and absorption, and cardiac muscle contraction at D2 substage to facilitate the occurrence of molting. The selected DEGs provide valuable insight into the role of ETH in the regulation of crustacean molting.

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.

Biphasic moult cycle of the parasitic isopod Norileca indica (H. Milne Edwards, 1840) (Isopoda: Cymothoidea): stage-wise characterisation and haemolymph ecdysteroids titre

AbstractNorileca indica (H. Milne Edwards, 1840) is a protandrous hermaphroditic and obligatory parasitic isopod of scombrid fishes. The posterior and anterior biphasic moult cycles in the adult phases (male, transitional, and female) of this species are characterised based on the microscopic changes reflected in the uropod exopodite and maxillule, respectively. Biphasic parturial moult as well as the moult removing the old brood pouch characteristic of ovigerous females was recorded. Assay of haemolymph ecdysteroids titre with respect to different biphasic moult cycle stages appears to be first report of its kind among aquatic and/or parasitic isopods. The ecdysteroids titre showed significant fluctuation in relation to the moult cycle stages, while the parasite was at a biphasically intermoult stage, titre was estimated to be 13.64 ± 2.09 ng ml–1. By the onset of the premoult at the posterior body region, titre showed a gradual increase and reached a maximum level (328.54 ± 70.17 ng ml–1; P = 0.0060) when the parasite was at late premoult at the posterior body part and mid premoult stage at the anterior body part. There was a subsequent sharp decline (92.8 ± 12.95 ng ml–1; P = 0.002) soon after the ecdysis of the posterior part by the time premoult changes in the anterior part advanced as late premoult and about to moult. The level of ecdysteroids titre was 55.56 ± 14.47 ng ml–1 when the parasite completed anterior ecdysis and reached postmoult. Data on the monthly occurrence of biphasic moult cycles revealed that a mean average of 23.26% of the adult population undergoes premoult/ecdysis/postmoult activities throughout the year. The rate of moulting showed a decline (16.12 ± 0.60%) during the monsoon months (June-August) when compared to pre-summer/summer (February-May) (24.83 ± 2.42%) and post-monsoon/winter (September-January) (26.28 ± 1.75; P = 0.005).

Expression patterns of MIH, EcR2, and RXR3 in the moult cycle of the red swamp crayfish, Procambarus clarkii (Girard, 1852) (Decapoda, Cambaridae)

Abstract In arthropods, the moult-inhibiting hormone (MIH), the ecdysone receptor (EcR), and the retinoid X receptor (RXR) are key regulators in moulting. In the present study, the full-length cDNAs of the MIH, EcR2, and RXR3 genes from the red swamp crayfish, Procambarus clarkii (Girard, 1852) (denoted as PcMIH, PcEcR2, and PcRXR3) were cloned. Tissue-specific and moult stage-specific mRNA expression patterns of these genes were detected by real-time quantitative polymerase chain reaction. PcMIH was detected only in the eyestalk, whereas PcEcR2 and PcRXR3 mRNA were expressed in all tissues tested. The highest levels of PcEcR2 and PcRXR3 were detected in the gill and hepatopancreas. Expression of PcMIH mRNA in the eyestalk increased from postmoult to peak in intermoult and then decreased in premoult. Expression of PcEcR2 mRNA in the eyestalk, hepatopancreas, and muscle increased from postmoult to peak in early premoult and then decreased. However, expression of PcEcR2 mRNA in the gill increased from postmoult to reach a maximum in intermoult and then decreased in premoult. Expression of PcRXR3 mRNA also fluctuated in the eyestalk, hepatopancreas, muscle, and gill, with a decrease from postmoult to late premoult. Expression of PcEcR2 and PcRXR3 mRNA increased relative to the control in the hepatopancreas and gill after unilateral and bilateral eyestalk ablation, which suggested that PcMIH can inhibit their mRNA expression. Double-stranded RNA-mediated RNA interference of PcRXR3 caused different changes in mRNA expression of these genes in different tissues and resulted in decreased expression of PcEcR2 mRNA, which suggested a collaborative relationship between PcEcR2 and PcRXR3.

Identification of a S6 kinase transcript in the Chinese mitten crab Eriocheir sinensis and its molting-related expression in muscle tissues

Ribosomal protein S6 kinases (S6Ks) are key targets downstream of the mechanistic target of rapamycin (mTOR) signaling pathway that are activated by mTOR complex 1 phosphorylation to promote protein synthesis, and are involved in muscle growth and pathology. In this study, we cloned a 1560-base pair (bp) complementary DNA sequence encoding a unique S6K1, designated EsS6K1, in the Chinese mitten crab Eriocheir sinensis. EsS6K1 contains a 54-bp 5′ untranslated region, a 246-bp 3′ untranslated region and a 1260-bp coding sequence that encodes 419 amino acids. EsS6K1 contains a conserved kinase domain, a TOR signaling motif, and a turn motif; it lacks a C-terminal autoinhibitory domain, and the conventional threonine in the hydrophobic motif is replaced with serine. Real-time quantitative reverse transcription–polymerase chain reaction experiments showed that EsS6K1 expression was highest in the ovaries, followed by the testes and the eyestalks, in mature crabs. In juvenile crabs, the relative messenger RNA levels of EsS6K1 in the claw, walking leg and abdominal muscles were significantly higher in the late premolt (D3-4) stage than in the intermolt (C) stage. After ecdysis, the expression of EsS6K1 in the walking leg and abdominal muscles declined to the level in the C stage, but the expression in claw muscles remained as high as that in the D3-4 stage. The findings showed that EsS6K1 likely plays roles in muscle changes during crustacean molting. These results provide valuable insights into the mechanism underlying crustacean muscle growth induced by molting.

Transcriptomic analysis of differentially expressed genes in the molting gland (Y-organ) of the blackback land crab, Gecarcinus lateralis, during molt-cycle stage transitions

A transcriptome of the Gecarcinus lateralis molting gland (Y-organ or YO) contained 48,590 contiguous sequences (contigs) from intermolt (IM), early premolt (EP), mid premolt (MP), late premolt (LP), and postmolt (PM) stages. The YO is kept in the basal state in IM by molt-inhibiting hormone (MIH)/cyclic nucleotide-dependent signaling. YO activation in EP requires down-regulation of MIH signaling and activation of mechanistic target of rapamycin (mTOR)-dependent protein synthesis. Transition of the YO to the committed state in MP requires activin/transforming growth factor-beta (TGFβ) signaling. YO repression occurs at the end of LP. A total of 28,179 contigs (58%) showed molt stage-specific changes in gene expression. The largest number of differentially-expressed genes (DEGs) were at the IM/EP (16,142 contigs), LP/PM (18,161 contigs), and PM/IM (8290 contigs) transitions. By contrast, the numbers of DEGs were 372 and 1502 contigs for the EP/MP and MP/LP transitions, respectively. DEG analysis of 23 signal transduction pathways showed significant changes in MIH, mTOR, activin/TGFβ, Notch, MAP kinase, and Wnt signaling. Down-regulation of MIH signaling genes in premolt is consistent with reduced MIH sensitivity in MP and LP. Up-regulation of mTOR signaling genes in IM and premolt stages is consistent with its role in YO activation and sustained ecdysteroidogenesis. Up-regulation of activin/TGFβ signaling genes in EP and MP is consistent with the role of a myostatin/activin-like factor in YO commitment. Notch, MAP kinase, and Wnt DEG analysis may indicate possible crosstalk with the MIH, mTOR, and activin/TGFβ pathways to integrate other inputs to control YO ecdysteroidogenesis.

A novel localization of molt-inhibiting hormone in the tegumental glands of shrimp Penaeus monodon and its possible role in shrimp molting

Abstract Molt-inhibiting hormone (MIH) is commonly localized in the X-organ sinus gland complex (XOSG) of the crustacean. The present study aimed to elucidate the expression of MIH at the subcellular and cellular levels in the eyestalks and integument of juvenile Penaeus monodon during the molting cycle. Gene expression of Pem-MIH1 in the optic lobes showed a single PCR product at 172 bp, and was restricted only to the eyestalk XOSG but not detected in pleopods, cephalothorax integument, muscle, hepatopancreas or retina. Quantitative analysis of gene expression in the eyestalks demonstrated no significant alteration of Pem-MIH1 mRNA in XOSG during the molt cycle. Immunohistochemistry using antibody against recombinant molt-inhibiting hormone-like peptide (anti-MIH-like) revealed variable staining intensities of individual MTXOs but were most persistently intense in SGs. During the molt cycle, the mean numbers of XO immunoreactive cells were slightly oscillated but not statistically different. The quantitative immunohistochemistry measured from XOSG illustrated minimal fluctuations of the values obtained suggesting the periodical synthesis and release of MIH from XOSG system during the molt cycle. The positive reaction was also detected in the tegumental glands located at the retina and the integument of walking leg, base of eyestalk and cephalothorax of the shrimp. The large variations of immunostaining and amount of reactive tegumental glands in the eyestalk were noted throughout the molt cycle. The numbers of the MIH-like immunoreactive glands obtained from the retina as well as the integument base did not significantly change during the molt cycle but tend to increase during postmolt to intermolt and decrease during premolt. These results suggest that the MIH-like peptides in the tegumental glands are probably released mostly during the late premolt. Our findings thus propose the novel storage site of MIH besides SG of the eyestalk including a new target tissue, an epidermal cell, the role of which may link to the complicated molting regulation and/or the dark-adaptation of shrimp.

Food consumption pattern in the snow crab Chionoecetes opilio (Fabricius, 1788) (Decapoda, Majoidea) megalopae under laboratory conditions

The food consumption pattern in megalopae of the snow crab Chionoecetes opilio (Fabricius, 1788) was investigated in the laboratory. Ten megalopae were individually cultured and given an excess of Artemia nauplii each day. All megalopae moulted into first-instar crabs 28-34 days after metamorphosis. The mean total number and total weight of Artemia consumed during the megalopal stage were 1920 individuals and 5.2 mg, respectively. Hence, the food requirement of snow crab megalopae was estimated as ∼190% of the dry body weight of the first-instar crab. Initially, the number of Artemia consumed was nearly constant or decreased only slightly but, later, Artemia consumption decreased with development days. Two-segmented regressions provided good fits to the relationship between the number of days after metamorphosis and the cumulative number of Artemia consumed by individual megalopae. The mean value of the time after metamorphosis of the breakpoint in the rate of food consumption was estimated as 69% of the stage duration, which corresponds to the intermediate of late premoult. Crab sizes (carapace width, wet and dry body weight) were not significantly dependent on the number of Artemia consumed during the entire megalopal period although a positive correlation between these variables was observed. These results provide useful information on the appropriate feeding schedule and management practice for culturing snow crab megalopae and contribute to the understanding of megalopal growth efficiency to the first-instar crab in their natural habitat.

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.

Molt cycle related changes and effect of short term starvation on the biochemical constituents of the blue swimmer crab Portunus pelagicus

Synthesis and hardening of a new exoskeleton are essential to the arthropod molting process. The present study emphasizes the variations in the levels of hemolymph total free sugars, hepatopancreas glycogen and cuticular proteins during the molting stages of Portunus pelagicus. It also reports the effect of short-term starvation conditions on the biochemical constituents of the hemolymph. Intermolt crabs were subjected to 6days of starvation and hemolymph samples were taken. Standard biochemical procedures were followed toward the quantification of total proteins, total free sugars and total lipids. The total free sugar level in the hemolymph of P. pelagicus was observed to increase during early premolt D0 (3.108±0.032g/ml) and a gradual decrease till late postmolt B stage (0.552±0.124g/ml), suggesting the need for total free sugars to provide energy for the apolysis process. Increase in the levels of hepatopancreas glycogen was observed from 1225±0.04μg/mg in early premolt D0 to 1700±0.3μg/mg in late premolt D2–3. This is in correlation with the decreased levels of free sugars during premolt stages, suggesting an increase in the storage of glycogen reserves in the hepatopancreas. Cuticular proteins increased during stage B (2.702±0.093g/ml) and stage C (3.065±0.012g/ml), indicating exoskeleton hardening and mineralization. Results of the starvation studies clearly showed a steady decline in the level of total free sugars till day 6 (0.099±0.00g/ml) when compared to the control (8.646±0.08g/ml). Gradual decrease of total lipids was also observed from the first day of the experiment (6.088±2.44g/ml) to the last day of the study (0.401±0.20g/ml) which was 85% lesser than the control (8.450±0.49g/ml)suggesting the efficient usage of total sugars to consolidate the loss of energy reserves during starvation. The knowledge of Molt-cycle events can be used as a tool for the evaluation of the developmental state providing a morphological reference system for physiological and biochemical studies related to crab aquaculture. Starvation studies enlightens that increasing carbohydrate levels in crab feed together with good protein content could alleviate the natural effects of starvation, improve farm productivity and reduce the deleterious impact of nitrogen pollution generated by rich-protein feeds used in crab farming.

Molting and cuticle deposition in the subterranean trichoniscid Titanethes albus (Crustacea, Isopoda)

Terrestrial isopods are a suitable group for the study of cuticle synthesis and calcium dynamics because they molt frequently and have evolved means to store calcium during molt. Little data is currently available on molting in Synocheta and subterranean isopods. We studied the molting dynamics in the subterranean trichoniscid Titanethes albus under laboratory conditions and performed a microscopic investigation of sternal CaCO3 deposits and the tergal epithelium during molt in this species. In accordance with its lower metabolic rate, molting in the laboratory is roughly 2–3 times less frequent in Titanethes albus than would be expected for an epigean isopod under similar conditions. Animals assumed characteristic postures following the molt of each body half and did not consume the posterior exuviae after posterior molt. The structure of sternal calcium deposits and the ultrastructural characteristics of the epidermis during cuticle formation in Titanethes albus are similar to those described in representatives of Ligiidae. During the deposition of the exocuticle, the apical plasma membrane of epidermal cells forms finger-like extensions and numerous invaginations. In the ecdysial space of individuals in late premolt we observed cellular extensions surrounded by bundles of tubules.

Molt cycle regulation of protein synthesis in skeletal muscle of the blackback land crab, Gecarcinus lateralis, and the differential expression of a myostatin-like factor during atrophy induced by molting or unweighting

In decapod crustaceans, claw muscle undergoes atrophy in response to elevated ecdysteroids while thoracic muscle undergoes atrophy in response to unweighting. The signaling pathways that regulate muscle atrophy in crustaceans are largely unknown. Myostatin is a negative regulator of muscle growth in mammals, and a myostatin-like cDNA is preferentially expressed in muscle of the land crab, Gecarcinus lateralis (Gl-Mstn). Contrary to prediction, levels of Gl-Mstn mRNA decreased dramatically in both the claw closer and weighted thoracic muscles when molting was induced by either eyestalk ablation (ESA) or multiple limb autotomy (MLA). However, the effect of molt induction was greater in the claw muscle. By late premolt, Gl-Mstn mRNA in the claw muscle decreased 81% and 94% in ESA and MLA animals, respectively, and was negatively correlated with ecdysteroids. Gl-Mstn mRNA in thoracic muscle decreased 68% and 82% in ESA and MLA animals, respectively, but was only weakly correlated with ecdysteroid. Claw and thoracic muscles also differed to varying extents in the expression of ecdysteroid receptor (Gl-EcR and Gl-RXR), elongation factor-2 (Gl-EF-2), and calpain T (Gl-CalpT) in response to molt induction, but levels of the four transcripts were not correlated with ecdysteroid. The downregulation of Gl-Mstn expression in premolt claw muscle coincided with 11- and 13-fold increases in protein synthesis in the myofibrillar and soluble protein fractions, respectively. Furthermore, the rate of the increase in the synthesis of soluble proteins was greater than that of myofibrillar proteins during early premolt (1.4:1, soluble:myofibrillar), but the two were equivalent during late premolt. By contrast, Gl-Mstn mRNA increased 3-fold and Gl-CalpT mRNA decreased 40% in unweighted thoracic muscle; there was little or no effect on Gl-EF-2, Gl-EcR, and Gl-RXR mRNA levels. These data indicate that Gl-Mstn expression is negatively regulated by both ecdysteroids and load-bearing contractile activity. The downregulation of Gl-Mstn in claw muscle may induce the elevated protein turnover associated with remodeling of the contractile apparatus during molt-induced atrophy. The upregulation of Gl-Mstn in unweighted thoracic muscle suggests that this factor is also involved in disuse atrophy when hemolymph ecdysteroid levels are low.

Post-Ecdysial Change in the Permeability of the Exoskeleton of the Blue Crab, Callinectes Sapidus

Crustaceans must replace their old exoskeleton with a new, larger one in order to grow and differentiate. During that process a new (preexuvial) cuticle is deposited beneath the old cuticle. Since breakdown products from the old cuticle have been reported to be removed from the old cuticle and transported across the new cuticle and hypodermis into the hemolymph, the preexuvial cuticle is presumed to be permeable. However, it has also been reported that intermolt cuticle is a highly impermeable structure that prevents loss of water and ions from the cuticle to the external environment. This study was designed to determine the timing of any change in the permeability of the cuticle of the blue crab, Callinectes sapidus, from the period just preceding ecdysis through the early postmolt period. To test for any changes in permeability pilot studies were performed on pieces of dorsobranchial cuticle from late premolt (D4) and early postmolt crabs (15 min, 2 h, 12 h and 18 h post-ecdysis) using 3 H2O and an Ussing chamber. Additional permeability studies were done monitoring the movement of p-nitrophenol (pNP) across the cuticle using a more restricted post-exuvial time course (0, 5, 10, 15 and 30 min. postecdysis) as compared to premolt cuticle. Results from these studies as well as wash-out studies using tissues pre-loaded with pNP and staining of 1 h post-molt cuticle with OsO4 suggest that the epicuticle undergoes a transition at or soon after ecdysis that renders it impermeable to water and small molecules. While the precise mechanism for this transition is unknown, it is clear that the resultant alteration in epicuticle permeability during early post-ecdysis allows for the formation of a barrier that prevents osmotic and ionic exchange, allows for the generation of significant hydrostatic pressure, and provides a suitable microenvironment for calcification.

Ecdysteroids in female shore crabs Carcinus maenas during the moulting cycle and oocyte development

The ecdysteroids ecdysone (E), 20-hydroxyecdysone (20E) and ponasterone A (PoA) were measured in the haemolymph, hepatopancreas and oocytes of female shore crabs Carcinus maenas during the moulting cycle and during oocyte maturation using HPLC-MS. In the haemolymph, ecdysteroid titres varied over the moulting cycle with high levels during premoult and low levels during postmoult and intermoult, however, no significant change in haemolymph ecdysteroid titres was observed in relation to oocyte development. In the hepatopancreas, PoA levels were high during premoult but low during postmoult and intermoult. This is in contrast to E and 20E where levels remained high from early intermoult (C1) until late premoult (D3) and only decreased during postmoult. In the oocytes, ecdysteroid levels were low during postmoult and for 20E and PoA also during late premoult D2 and D3. In contrast, all three ecdysteroids were observed to increase in the oocytes during oocyte development, in particular E and PoA. The present study demonstrates that changes in haemolymph ecdysteroid titres relate to changes in moulting status and not to changes in oocyte development. Also, the study indicates that the hepatopancreas is involved in the metabolism of ecdysteroids related to the moulting cycle but may also be involved in ecdysteroid metabolism during oocyte development. Furthermore, the pronounced increase in oocyte ecdysteroids during oocyte development during periods where haemolymph ecdysteroids titre is low, indicates that the oocytes are capable of de novo synthesis of the three ecdysteroids investigated.

The cationic composition and pH in the moulting fluid of Porcellio scaber (Crustacea, Isopoda) during calcium carbonate deposit formation and resorption

Before moulting, terrestrial isopods resorb calcium carbonate (CaCO(3)) from the posterior cuticle and store it in sternal deposits. These consist mainly of amorphous calcium carbonate (ACC) spherules that develop within the ecdysial space between the anterior sternal epithelium and the old cuticle. Ions that occur in the moulting fluid, including those required for mineral deposition, are transported from the hemolymph into the ecdysial space by the anterior sternal epithelial cells. The cationic composition of the moulting fluid probably affects mineral deposition and may provide information on the ion-transport activity of the sternal epithelial cells. This study presents the concentrations of inorganic cations within the moulting fluid of the anterior sternites during the late premoult and intramoult stages. The most abundant cation is Na(+) followed by Mg(2+), Ca(2+) and K(+). The concentrations of these ions do not change significantly between the stages whereas the mean pH changed from 8.2 to 6.9 units between mineral deposition in late premoult, and resorption in intramoult, respectively. Measurements of the transepithelial potential show that there is little driving force for passive movements of calcium across the anterior sternal epithelium. The results suggest a possible role of magnesium ions in ACC formation, and a contribution of pH changes to CaCO(3) precipitation and dissolution.

Differentiation of the epipod chloride cells in the limb of Artemia

Summary The epipod forms at instar V as a sac of epidermal cells extending from the dorsal edge of the thoracopod. Pillar processes from the basal surface of the cells connect opposing epithelia across the hemocoel of the flattened sac. Differentiation started in late premolt of instar VIII and by instar IX all the cells had increased in height, greatly enhanced the surface area of the plasma membrane, and increased the number of mitochondria. Chloride transport (silver staining) followed the same temporal pattern demonstrating that differentiation of chloride cell morphology and transport function was attained during a period of one instar. The presence of mitotic figures revealed that the chloride cells retained the ability to replicate after differentiation.

Dynamics of in Vivo Release of Molt-Inhibiting Hormone and Crustacean Hyperglycemic Hormone in the Shore Crab, Carcinus maenas

Very little is known regarding the release patterns or circulating titers of neuropeptides in crustaceans, in particular those concerned with regulation of molting hormone (ecdysteroid) synthesis, molt-inhibiting hormone (MIH), and crustacean hyperglycemic hormone (CHH), which is also an adaptive hormone, centrally important in carbohydrate metabolism. Furthermore, the currently accepted model of molt control is founded on an untested hypothesis suggesting that molting can proceed only after decline in MIH titer. Accordingly, we measured simultaneous circulating neuropeptide profiles for both MIH and CHH by RIA of purified hemolymph during the molt cycle at fine temporal scale during day/night cycles and seasonally. For CHH we additionally determined release patterns after physiologically relevant stress. Results show that both hormones are released exclusively and episodically, rather than continuously, with notably short half-lives in circulation, suggesting dynamic and short-lived variations in levels of both hormones. During the molt cycle, there are no overt changes in MIH titer, except a massive and unprecedented increase in MIH during late premolt, just before ecdysis. The function of this hormone surge is unknown. Treatment with various stressors (hypoxia, temperature shock) showed that CHH release occurs extremely rapidly, within minutes of stress. Release of CHH after stressful episodes during premolt (when gut endocrine cells synthesize large quantities of CHH) is exclusively from the sinus gland: CHH from the gut is never involved in the stress response. The results show a hitherto unsuspected dynamism in release of MIH and CHH and suggest that currently accepted models of molt control must be reconsidered.

In vivo and in vitro cadmium accumulation during the moult cycle of the male shore crab Carcinus maenas—interaction with calcium metabolism

The effect of moult stage on cadmium accumulation and distribution was investigated in vivo in male shore crabs Carcinus maenas exposed to 1 mg Cd l −1 for 7 days. The accumulation of cadmium in all tissues examined was markedly higher in postmoult (A 1–2 and B 1–2) compared to intermoult (C 1, C 3 and C 4) and premoult (D 0–3). In addition, elevated levels of cadmium were found in gills of late premoult (D 2–3) animals. The total amount of cadmium accumulated in the tissues (haemolymph, gills, midgut gland and muscle) increased from 43 μg Cd in early premoult (D 0–1) to 391 μg Cd in late postmoult (B 1–2). Gills and midgut gland were the primary cadmium accumulating tissues in C 4-intermoult and premoult (D 0–3); in early postmoult (A 1–2) haemolymph and midgut gland were the main cadmium containing tissues, while midgut gland dominated in late postmoult (B 1–2) and early intermoult (C 1 and C 3). A detailed account of calcium distribution in haemolymph, gills, midgut gland, muscle and exoskeleton during the moult cycle is presented. Mechanistic links between cadmium and calcium uptake in posterior gills of C 4-intermoult and early postmoult (A 1–2) crabs were explored using an in vitro gill perfusion technique. Calcium and cadmium influxes were markedly higher in postmoult compared to intermoult. No differences between intermoult and postmoult effluxes were found for either calcium or cadmium. From intermoult to postmoult net influx increased from 2.4 to 29 μmol Ca 2+ g −1 ww gill h −1 and from 0.24 to 25 nmol Cd 2+ g −1 ww gill h −1. The results indicate that the postmoult increase in cadmium influx is due to increased active transport of cadmium, at least partly, by accidental uptake via calcium transporting proteins. The in vitro net influx rates corresponded accurately to the observed in vivo accumulation of both cadmium and calcium. Although cadmium accumulation and distribution are clearly linked to changes in calcium requirements, cadmium did not interfere with calcium accumulation or distribution at any stage during the moult cycle.

Copper and zinc handling during the moult cycle of male and female shore crabs Carcinus maenas

Tissue concentrations and contents of copper and zinc were determined at 11 different stages of the moult cycle in male and female shore crabs Carcinus maenas. Metal concentrations in haemolymph, gills, midgut gland, muscle, and exoskeleton of males and haemolymph, gills, and midgut gland of females were determined, as were haemocyanin concentrations and haemolymph volumes (using 14C-inulin) in males. The changes in tissue Cu and Zn concentrations and contents that occur throughout the moult cycle can be attributed to muscle breakdown in late premoult, the period of starvation in late premoult and early postmoult, the resorption from and shedding of the old exoskeleton, and the dilution of the haemolymph caused by water uptake around the time of ecdysis. The present study demonstrates that whole-body Cu and Zn contents remain constant during a large part of the moult cycle of male and female C. maenas. This state of whole-body trace metal homeostasis is maintained in spite of major changes in tissue proportions and tissue Cu and Zn concentrations and contents. Previous studies have not carried out the necessary analysis to move from theoretical estimates to quantitative determination of the changes in tissue metal distribution associated with moulting in crustaceans; the data presented illustrate the necessity of measuring both tissue concentrations and contents of metals to avoid misinterpretation of either.