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Antene, ki so pomemben senzorični organ kopenskih enakonožcev, so pogosto na udaru plenilcev in tudi pripadnikov vrste, zato prihaja do amputacij. Njihova regeneracija je pri enakonožcih povezana z levitvijo. V nalogi smo želeli ugotoviti: I) ali amputacija antene vpliva na dolžino levitvenega cikla II) ali se regenerirane antene dimenzijsko razlikujejo od izvornih III) ali se število senzoričnih elementov na regeneriranih antenah razlikuje od izvornih. Odraslim enakonožcem Porcellio scaber smo dan po levitvi anteno amputirali in skozi levitveni cikel spremljali spremembe na ostanku okončine. Po uspešni regeneraciji smo regenerat ponovno amputirali. Tako smo pridobili izvorno anteno in dva regenerata istega osebka. Za dimenzijsko primerjavo zadnjih treh členov anten smo analizirali fotografije narejene s svetlobnim mikroskopom. Na SEM mikrografijah smo analizirali dimenzijo, obliko in število senzoričnih struktur izvornih anten in regeneratov. Amputaciji antene na tretjem členu je sledila samoamputacija med prvim in drugim členom. Do levitve je regeneracija potekala v notranjosti štrclja, ki ga je zapirala pigmentirana krasta. Dolžina levitvenega cikla se ni spremenila. Regenerirana antena je bila vedno krajša in ožja od izvorne. Število estetaskov je bilo na regeneratu praviloma manjše, prav tako je bila pogosto zmanjšana tudi gostota trikornov, medtem ko se dolžina apikalnega organa in drugih senzil praviloma ni razlikovala. Antennae, an important sensory organ of terrestrial isopods, are often attacked and amputated by predators or members of the same species. The regeneration of amputated limbs is associated with molting. The aims of our work were to find out: I) whether amputation of the antenna affects the length of the moult cycle II) whether the regenerated antennas differ dimensionally from the original III) whether the number of sensory elements on the regenerated antennae differs from the original. One day after molting the antenna of adult isopods Porcellio scaber was amputated and changes of the stump were monitored throughout the molting cycle. After successful regeneration, the regenerate was amputated again. Thus we obtained the original antenna and two regenerates of the same specimen. Light microscope photographes of antennae were analysed for a dimensional comparison of the last three articles. On SEM micrographs we analysed the size, shape and number of sensory structures of the original and regenerated antennae. Amputation of the antenna on the third article was followed by self-amputation between the first and second article. Until molting, regeneration took place inside the stoup closed with a pigmented scab. The length of the molting cycle has not changed. The regenerated antenna was always shorter and narrower than the original one. The number of aesthetascs was generally smaller on the regenerate, and the density of tricorns was also often reduced, while the length of the apical organ and other sensilla generally did not differ.

Subterranean termite control methods using chitin synthesis inhibitors (CSIs) aim at eliminating colonies that feed upon a bait formulation. Several benzoylurea active ingredient formulations are currently commercially available as alternative termite management strategies to liquid termiticides. Individual workers need to molt on a regular basis and CSIs interfere with such molting process, allowing sufficient time for the acquisition of a colony-wide lethal dose prior to widespread mortality. As workers progressively die, the colony eventually collapses, leaving only soldiers and primary reproductives that starve to death. One common observation is that young workers often die early owing to their relatively short molting cycle. However, the absence of brood in dying colonies raises questions about the potential fate of eggs laid by the queen. This study aims to determine if CSI baits also terminate the ability of a colony to produce a new cohort of workers by disabling the ongoing brood development. Incipient termite colonies were used to test the impact of noviflumuron on the queen’s ability to lay eggs and on the eggs' ability to hatch. Our results showed that queens in colonies exposed to CSI not only initially laid less eggs than the control queens, but eggs also did not develop and were progressively cannibalized, eventually leading to colony establishment failure. This result implies that queens of mature colonies exposed to CSI would lose the ability to lay viable eggs as the colony collapses, leading to an absence of worker replacement, aiding in colony elimination.

Ecdysone plays a crucial role in the molting process, is synthesized by neverland (nvd) and regulates the transcription of early response genes, HR3 and E75. However, their roles in the development and reproduction of small crustaceans are still poorly understood at the molecular level. Thus, this study aimed to identify and characterize the nvd, HR3, and E75 genes in the brackish water flea Diaphanosomal celebensis, and to investigate their transcriptional modulation upon exposure to bisphenol (BP) analogs. Three genes found in the local D. celebensis transcriptome database were sequenced and identified using BLAST X and conserved domain search. The relative expression patterns of these genes at different ages and under exposure to BP analogs were investigated by real-time reverse transcription polymerase chain reaction. Sequencing analysis showed that nvd, HR3, and E75 had conserved domains. The mRNA expression patterns of Dc-nvd were highly upregulated at day 5 and day 7 of D. celebensis life cycle. The E75 and HR3 mRNA expression patterns differed according to age. The BP analogue exposure test showed that the three genes were significantly modulated with different patterns. The nvd, HR3, and E75 had conserved domains, suggesting that they have conserved functions in D. celebensis. Age-dependent expression of these three genes implies their involvement in the molting cycle. Our findings also suggest that BPS, BPF, and BPA may disrupt the edcysteroid signaling pathway in this species by different mechanisms.

Significance The ever-growing human population faces problems providing sufficient animal proteins. For shellfish, wild catch is supplemented by aquaculture to meet this increasing global demand. However, aquaculture faces serious problems with infectious diseases. One of the main problems for oriented disease control is lack of information on pathogen entry in the host. The present study fully describes the anatomy of the shrimp’s excretory organ, the antennal gland, identifying the organ as a major portal candidate. Additional findings show that pathogens may indeed enter through this organ naturally, infecting shrimp. We also demonstrate involvement in molting. These insights into the molting process and pathogen entry open doors in fundamental biology and the potential development of disease control measures., Viruses, such as white spot syndrome virus, and bacteria, such as Vibrio species, wreak havoc in shrimp aquaculture [C. M. Escobedo-Bonilla et al., J. Fish. Dis. 31, 1–18 (2008)]. As the main portal of entry for shrimp-related pathogens remain unclear, infectious diseases are difficult to prevent and control. Because the cuticle is a strong pathogen barrier, regions lacking cuticular lining, such as the shrimp’s excretory organ, “the antennal gland,” are major candidate entry portals [M. Corteel et al., Vet. Microbiol. 137, 209–216 (2009)]. The antennal gland, up until now morphologically underexplored, is studied using several imaging techniques. Using histology-based three-dimensional technology, we demonstrate that the antennal gland resembles a kidney, connected to a urinary bladder with a nephropore (exit opening) and a complex of diverticula, spread throughout the cephalothorax. Micromagnetic resonance imaging of live shrimp not only confirms the histology-based model, but also indicates that the filling of the diverticula is linked to the molting cycle and possibly involved therein. Based on function and complexity, we propose to rename the antennal gland as the “nephrocomplex.” By an intrabladder inoculation, we showed high susceptibility of this nephrocomplex to both white spot syndrome virus and Vibrio infection compared to peroral inoculation. An induced drop in salinity allowed the virus to enter the nephrocomplex in a natural way and caused a general infection followed by death; fluorescent beads were used to demonstrate that particles may indeed enter through the nephropore. These findings pave the way for oriented disease control in shrimp.

Spiders molt periodically before reaching full maturity, but several spiders continue to molt after sexual maturity. This post-maturity molting (PMM) behavior has been observed in the barn spider Araneus cavaticus (Araneae: Araneidae) among the orb-web spiders. In this study, we investigated molt-related changes in the ampulla and tail regions of the major ampullate gland during the PMM sequences (intermolt, pre-molt, ecdysis, and post-molt). The results showed that all gland units consist of a monolayer of epithelial cells surrounding a large central lumen, and two types of secretory granules (Type-M and Type-S). During the molting period, most cells showed fine structural modification in their organelles, and conspicuous tissue swelling was detected at the glandular epithelium. Following the molting cycle, the amount of Type-M granules continues to increase in the cell with a corresponding swelling, but Type-S granules gradually disappeared during the process of ecdysis. This suggests that the molt-related changes in spider silk production originates from the periodic production of Type-S secretory granules in the ampulla region. As Type-M granules flow toward the funnel, it is coated with viscous liquid secretion of Type-S granules in order to produce dragline silk fibers. We provide fine structural evidence for Type-S granules of hexagonal crystalline substructures representing glycoprotein substances to maintain high level of water content.

This study aimed to determine the effect of rearing water temperature on survival, growth and molting cycle of the mud crab, S. paramamosain from Megalopa up to 1st crablet instar (Crablet 1) and from Crablet 1 to 2nd crablet instar (Crablet 2). Different water temperature regimes were implemented i.e. 20 °C, 24 °C, 28 °C, 32 °C, 36 °C and ambient temperature. The observed parameters were: successful molting from Megalopa to Crablet 1 and from Crablet 1 to Crablet 2, survival in Crablet 1 and Crablet 2, growth (carapace increment of crablets), Specific Growth Rate of carapace width (SGRCW) and, molting interval (in days) from Megalopa to Crablet 1 and from Crablet 1 to Crablet 2. The results showed that the highest survival rate from Megalopa to Crablet 1 was found at 24 and 28 °C where both could reach up to 100%. At water temperature of 32 °C and ambient temperature, the survival rate were 96.66%. The lowest survival rate was found at 20 °C and 36 °C treatments (p 0.05), but there were significant differences in molting intervals (p

The effect of temperature (20, 25, 30 and 35 oC), salinity ( 0, 5, 10 and 15 ppt), pH (6, 7, 8 and 9) and light (24h L:00hD, 12hL:12hD and 00hL:24hD) on molting cycle, growth and survival of larvae of the freshwater prawn Macrobrachium rosenbergii were investigated independently under laboratory conditions. Temperature, salinity and pH significantly (P 0.05). The best levels of temperature, salinity and pH, which gave the fast molt with the highest growth increment in M. rosenbergii were 30oC, 5 ppt, and pH 8, respectively. Outside these levels, more molting did not produce more growth. These results have direct implications in the aquaculture production of Macrobrachium rosenbergii and accelerate the growth of prawns in conditions of intensive culturing with a reduction in production costs.

Micro- and nano- polystyrene particles have been widely detected in environment, posing potential threats to human health. This study was designed to evaluate the neurodevelopmental toxicity of polystyrene nanoparticles (NPs) in Caenorhabditis elegans (C. elegans), to screen crucial genes and investigate the underlying mechanism. In wild-type C. elegans, polystyrene NPs (diameter 50 nm) could concentration-dependently induce significant inhibition in body length, survival rate, head thrashes, and body bending, accompanying with increase of reactive oxygen species (ROS) production, lipofuscin accumulation, and apoptosis and decrease of dopamine (DA) contents. Moreover, pink-1 mutant was demonstrated to alleviate the locomotion disorders and oxidative damage induced by polystyrene NPs, indicating involvement of pink-1 in the polystyrene NPs-induced neurotoxicity. RNA sequencing results revealed 89 up-regulated and 56 down-regulated differently expressed genes (DEGs) response to polystyrene NPs (100 μg/L) exposure. Gene Ontology (GO) enrichment analysis revealed that predominant enriched DEGs were correlated with biological function of cuticle development and molting cycle. Furthermore, mutant strains test showed that the neurodevelopmental toxicity and oxidative stress responses induced by 50 nm polystyrene NPs were regulated by dpy-5 and rol-6. In general, polystyrene NPs induced obvious neurodevelopmental toxicity in C. elegans through oxidative damage and dopamine reduction. Crucial genes dpy-5 and rol-6 might participate in polystyrene NPs-induced neurodevelopmental toxicity through regulation on synthesis and deposition of cuticle collagen.

Mayflies (order Ephemeroptera), insects favored as food by freshwater fish and as models for the artificial lures of fly fishers, live double lives. During their immature (nymphal) stages, they pursue an underwater career for 1 y or 2 y, molting their cuticular exoskeleton on numerous occasions as they grow. As in other insects, their future wings grow only slowly, but, as maturity approaches, the organs of flight grow rapidly, and the adult insect emerges from the water in a completely different form than the nymph. All winged hexapods (Pteryogota) undergo a metamorphosis of more or less this kind, but mayflies are unique among living insects in having more than one winged developmental stage (1), the final imago or adult instar being preceded by a short-lived subimago (called by anglers a “dun”) (Fig. 1). A paper in PNAS by Kamsoi et al. (2) now presents a molecular analysis of the hormonal and cellular control of metamorphosis in a model ephemeropteran species, Cloeon dipterum , casting fresh light on the evolutionary significance of the mayfly subimaginal stage. Fig. 1. Subimago of the mayfly C. dipterum . Image credit: Isabel Almudi (Centro Andaluz de Biologia del Desarrollo, Seville, Spain). It has long been known that the mayfly’s two different winged instars are distinct developmental stages, separated by a highly compressed but otherwise normal molting cycle (3). In this, mayflies appear intermediate between all other winged insects, which cease to shed their skins on achieving the adult condition, and wingless insects from the orders Archeognatha (jumping bristletails) and Zygentoma (silverfish and firebrats), which undertake multiple molts as adults while they continue to reproduce (4). The paper by Kamsoi et al. (2) unequivocally shows that the molt of the last nymphal stage of C. dipterum to the subimago makes use of the same hormones, receptors, and … [↵][1]1Email: S.E.Reynolds{at}bath.ac.uk. [1]: #xref-corresp-1-1

Eyestalk ablation allows the removal of neurohormones that inhibit early reproduction and decreases the time to complete the molting cycle. The present study evaluated the impact of unilateral eyestalk ablation on the behavior of blue crab breeders Callinectes arcuatus in the short term under laboratory conditions. Immature male and female crabs and individuals in the intermolt stage were collected in Ohuira Bay, Ahome, Sinaloa, Mexico. Three bioassays were performed: males, females, and male-females each with three treatments; control + xylocaine (50 μL, 5%), squash ablation + xylocaine (50 μL, 5%), ablation by string ligation + xylocaine (50 μL, 5%), each with three replicas and three periods of evaluation after the ablation procedure (10 min, 2 h, and 24 h). Kruskal-Wallis and Chi-square (χ2) tests were used to evaluate significances (P < 0.05) in the behavior of C. arcuatus. The sizes of blue crab C. arcuatus were 9.72 ± 1.0 cm of carapace width. The feeding behavior of the male-females bioassay was significant at 2 h (P < 0.05). The results obtained were satisfactory using two techniques, unilateral eyestalk ablation by squash and ligation in which pain and aggressiveness indicators were mitigated. Disorientation and aggressiveness in males were significant at 2 h (P < 0.05), and in females after 10 min, and 2 h after ablation (P < 0.05). In the male-females bioassay, aggressiveness was significant at 2 h (P < 0.05). Courtship and copulation were recorded in those crabs subjected to unilateral eyestalk squash ablation (13 and 8, respectively).

Termites feed on a carbon-rich but nitrogen-poor diet and evolved efficient nitrogen conservation strategies. It was previously suggested that during time of low access to resources (i.e., starvation), subterranean termites (Rhinotermitidae) colonies would adopt an energy conservation strategy by cannibalizing soldiers, a dependent caste. However, such hypothesis was tested with relatively small groups of foragers, which may not have reflected how resources are reallocated in whole colonies when food (carbon) is scarce. The current study subjected 2-year-old colonies (≈ 3000 termites with all castes and instars) to starvation, in order to reexamine if cannibalism is part of an active energy conservation strategy. Within 12 days of starvation, eggs and larvae were all cannibalized, followed by young workers. Soldiers then died marginally faster than old workers. By 22 days, cannibalism was no longer observed, and the king and queen were among the last individuals to die. Termites that engaged in the process of ecdysis with no energetic resources, failed to molt, died in the process, and the subsequent cannibalism was a passive mortality-driven process. Therefore, cannibalism was primarily the result of the inherent termite behavior for recycling nitrogen resources. The hemimetabolous developmental pathway, the difference of timing in the molting cycle of termite instars and the relatively rapid exhaustion of soldiers, indirectly determined the sequence of starvation-induced mortality and subsequent cannibalism. Although termites have evolved efficient nitrogen conservation strategies, they have not evolved an efficient carbon conservation strategy, which is essentially limited to keeping the primary reproductives alive as long as possible.

Bisphenol A (BPA) is a representative endocrine disrupting chemical (EDC) that has estrogenic effects in aquatic animals. In recent years, due to the continuing usage of BPA, its analogues have been developed as alternative substances to replace its use. The molting process is a pivotal point in the development and reproduction of crustaceans. However, studies of the effects of EDCs on molting in crustaceans at the molecular level are scarce. In the present study, we examined the acute toxicity of BPA and its analogues bisphenol F (BPF) and S (BPS) to the brackish water flea Diaphanosoma celebensis. We further identified four ecdysteroid pathway - related genes (cyp314a1, EcRA, EcRB, and USP) in D. celebensis, and investigated the transcriptional modulation of these genes during molting and after exposure to BPA and its analogues for 48 h. Sequencing and phylogenetic analyses revealed that these four genes are highly conserved among arthropods and may be involved in development and reproduction in the adult stage. The mRNA expression patterns of cyp314a1, EcRA and USP were matched with the molting cycle, suggesting that these genes play a role in the molting process in the adult stage in cladocerans. Following relative real-time polymerase chain reaction (RT-PCR) analyses, BPA and its analogues were found to modulate the expression of each of these four genes differently, indicating that these compounds can disrupt the normal endocrine system function of D. celebensis. This study improves our understanding of the molecular mode of action of BPA and its analogues in D. celebensis.

Crustacean growth is characterized by molting, whereby the old exoskeleton is shed and replaced by a new and larger version. The cellular events that lead to molting are driven by steroid hormones (ecdysteroids) secreted by paired endocrine glands (Y-organs). Between molts, ecdysteroid production is suppressed by a polypeptide molt-inhibiting hormone (MIH) released from neurosecretory cells in the eyestalks. Although a decrease in the MIH titer precedes the upsurge in ecdysteroidogenesis, it is hypothesized that a positive regulatory signal is also required for full activation of Y-organs. Existing data point to an intracellular Ca2+ signal. Ca2+ signaling is dependent on a tightly regulated Ca2+ gradient, achieved through membrane transport proteins. One such protein, the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), pumps Ca2+ from cytosol to the lumen of the ER. We have recently cloned from Y-organs of the blue crab (Callinectes sapidus) a cDNA encoding a putative Cas-SERCA protein. In studies reported here, quantitative PCR (QPCR) was used to quantify Cas-SERCA transcript abundance in Y-organs during a molting cycle, and radioimmunoassay was used to quantify ecdysteroids in hemolymph. The abundance of the Cas-SERCA transcript in Y-organs increased gradually during pre-molt. Similarly, the level of ecdysteroids in hemolymph increased during pre-molt. The results are consistent with the hypothesis that Cas-SERCA functions to maintain Ca2+ homeostasis in Y-organs. Cas-SERCA transcript abundance also changed in several non-ecdysteroidogenic tissues during a molting cycle. The pattern of change differed among tissues suggesting a functional role for SERCA in each.

The insulin-like androgenic gland hormone (IAG) plays a key role in male sexual differentiation and spermatogenesis in crustaceans. The expression of IAG is usually negatively regulated by neuropeptide hormones through the “eyestalk-AG” endocrine axis. However, the underlying mechanism is still largely unknown. In the present study, we performed a comparative transcriptome analysis on the androgenic gland (AG), the main secretory organ of IAG, of L. vannamei before and after unilateral eyestalk ablation. A total of 67 differentially expressed genes (DEGs) were identified, including some putative genes involved in sexual development. Interestingly, several genes related to molting and endocrine processes were found differentially expressed between the two treatments. Further investigation on the expression profiles of these genes and Lv-IAG showed that their expression patterns were closely correlated throughout the molting cycle. In addition, injection of 20-hydroxyecdysone (20E) could inhibit the expression of Lv-IAG at different molting stages in a dosage-dependent manner. The data indicated that ecdysteroids played important roles in regulating the Lv-IAG expression and might bridge the endocrine axis between eyestalk and AG. The present study provided new insights into understanding the regulation of sexual development in male crustacean.

This study was carried out to determine the physiological changes (survival, growth, molting cycle, sex differentiation, and gill condition) of mud crab, Scylla paramamosain crablet at different water temperatures of 24, 28 and 32 °C, and ambient temperature of 27 to 30 °C. Thermoregulatory behavior, represented by preferred temperature (29.83 ± SD 2.47 °C), critical thermal minimum (17.33 ± SD 0.58 °C), critical thermal maximum (40 ± SD 0.00 °C), and thermal tolerance interval (22.67 ± SD 0.58 °C), were checked for Crablet 1 stage only (with ambient temperature as acclimation temperature).Both low (24 °C) and high (32 °C) temperatures were associated with lower growth performance, and survival rate (p &lt, 0.05), in comparison with both 28 °C and ambient temperature treatments.Male ratio at low temperaturetreatment (24 °C) was higher (80.09 ± SD 18.86%) than for other treatments (p &lt, 0.05), observed as 44.81 ± D 10.50%, 41.94 ± SD 19.44%, and 76.30 ± SD 5.13% for 28 °C, 32 °C and ambient temperature treatments, respectively. However, there was no significant difference observed between 24 °C, 28 °C, and ambient temperature treatments. Anatomical alterations of gill lamellae of S. paramamosain crablet for both 32 °C, and 24 °C treatments, appeared thinner and paler than at both 28 °C, and ambient temperature treatments. Based on this study, temperature of 28 to 30 °C was recommended as the optimal temperature for the long-term nursery phase of S. paramamosain.

Background: Molting is an inherent cyclic process that occurs in all crustaceans and is essential for growth, metamorphosis, and reproduction. Recent studies have shown that many organs and systems are involved in molting regulation, including the hepatopancreas, the immune system, and production of ecdysteroids by Y-organs. However, the molecular mechanisms underlying the dynamic process of crab molting remain poorly understood at the whole-individual level. Therefore, we investigated global expression changes in the transcriptomes of the mud crab Scylla paramamosain, the most cultured crab species worldwide. We revealed a cascade of sequential expression events of molting-related genes involved in various aspects of the molting process by using whole-body sequencing of juvenile crabs. Results: RNA-sequencing (RNA-seq) produced 139.49 Gb of clean reads and 20,436 differentially expressed genes (DEGs) among different molting stages. The expression patterns for genes involved in several molecular events critical for molting, such as cuticle reconstruction, cytoskeletal structure remodeling, hormone regulation, immune responses, and metabolism, were characterized and considered as mechanisms underlying molting in S. paramamosain. Among these genes, we identified 10,695 DEGs in adjacent molting stages. In further GO and KEGG analyses, the significantly enriched pathways included structural constituents of cuticle, binding and chitin metabolic processes, steroid hormone biosynthesis, insulin resistance, and amino sugar metabolic processes. The expression profiles of 12 functional genes detected via RNA-Seq were corroborated via real-time RT-PCR assays. The results indicated gene expression profiles across the molting cycle and identified possible activation pathways for future investigation of the underlying molecular mechanisms. Conclusions: This study establishes a comprehensive transcriptomic repertoire for the molting process in S. paramamosain. The data generated in this study will serve as an important transcriptomic resource for the crab research community to facilitate gene and genome annotation and provide fundamental support for future research on molecular mechanisms in this species. Furthermore, the comprehensive analysis results will improve our understanding of morphological variation in the molting cycle and serve as a potential blueprint for future research on molting in crustaceans and other animals.

International audience; In the grow-out ponds of the blue shrimp (Litopenaeus stylirostris) farms of New Caledonia, animals with orange gills (OG) have been observed over the last ten years, with up to 70% of the shrimp in a given pond being affected. During the processing and marketing of the product, this coloring leads to reduced quality and selling prices, resulting in lower income for the producers concerned. Individual observations and transfer experiments led us to conclude that gill coloration intensity varies according to the intermolt stages, ranging from white in the postmolt stage to a deep orange in the premolt stage, which then disappeared after molting. Histological, biochemical studies and a semiquantitative analysis by scanning electronic microscopy (SEM + EDX) showed that the coloration is due to layers of iron which settle on the gill tissue surface in a heterogeneous way. Because Cr and Co showed an increase in their concentrations on the whole orange gills but not at their surface, it is possible that elements were mobilized and transported (translocated) from the exoskeleton to the tissue. Animals kept out of contact with sediment show a decrease in OG intensity, suggesting a link with the sediment biogeochemistry. In grow-out ponds, orange gills first appeared in shrimp populations between 10 and 13 weeks after stocking and reached a maximum after 15 weeks. These findings are discussed with a view to identifying the environmental processes that lead to metal accumulation on gills.

The salmon louse, Lepeophtheirus salmonis, is an ectoparasitic crustacean that annually inflicts substantial losses to the aquaculture industry in the northern hemisphere and poses a threat to the wild populations of salmonids. The salmon louse life cycle consists of eight developmental stages each separated by a molt. Fushi Tarazu Factor-1 (FTZ-F1) is an ecdysteroid-regulated gene that encodes a member of the NR5A family of nuclear receptors that is shown to play a crucial regulatory role in molting in insects and nematodes. Characterization of an FTZ-F1 orthologue in the salmon louse gave two isoforms named αFTZ-F1 and βFTZ-F1, which are identical except for the presence of a unique N-terminal domain (A/B domain). A comparison suggest conservation of the FTZ-F1 gene structure among ecdysozoans, with the exception of nematodes, to produce isoforms with unique N-terminal domains through alternative transcription start and splicing. The two isoforms of the salmon louse FTZ-F1 were expressed in different amounts in the same tissues and showed a distinct cyclical expression pattern through the molting cycle with βFTZ-F1 being the highest expressed isoform. While RNA interference knockdown of βFTZ-F1 in nauplius larvae and in pre-adult males lead to molting arrest, knockdown of βFTZ-F1 in pre-adult II female lice caused disruption of oocyte maturation at the vitellogenic stage. No apparent phenotype could be observed in αFTZ-F1 knockdown larvae, or in their development to adults, and no genes were found to be differentially expressed in the nauplii larvae following αFTZ-F1 knockdown. βFTZ-F1 knockdown in nauplii larvae caused both down and upregulation of genes associated with proteolysis and chitin binding and affected a large number of genes which are in normal salmon louse development expressed in a cyclical pattern. This is the first description of FTZ-F1 gene function in copepod crustaceans and provides a foundation to expand the understanding of the molecular mechanisms of molting in the salmon louse and other copepods.

The arteriovenous fistula (AVF) is the first choice for vascular access for hemodialysis of renal failure patients. Venous remodeling after exposure to high fistula flow is important for AVF to mature but the mechanism underlying remodeling is still unknown. The objective of this study is to identify the molecular mechanisms that contribute to venous remodeling after AVF. To screen and identify the differentially expressed genes (DEGs) that may involve venous remodeling after AVF, we used bioinformatics to download the public microarray data (GSE39488) from the Gene Expression Omnibus (GEO) and screen for DEGs. We then performed gene ontology (GO) function analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and gene set enrichment analysis (GSEA) for the functional annotation of DEGs. The protein-protein interaction (PPI) network was constructed and the hub genes were carried out. Finally, we harvested 12 normal vein samples and 12 AVF vein samples which were used to confirm the expressions of the hub genes by immunohistochemistry. A total of 45 DEGs were detected, including 32 upregulated and 13 downregulated DEGs. The biological process (BP) of the GO analysis were enriched in the extrinsic apoptotic signaling pathway, cGMP-mediated pathway signaling, and molting cycle. The KEGG pathway analysis showed that the upregulated DEGs were enriched in glycosaminoglycan biosynthesis and purine metabolism, while the downregulated DEGs were mainly enriched in pathways of glycosaminoglycan biosynthesis, antifolate resistance, and ABC transporters. The GSEA analysis result showed that the top three involved pathways were oxidative phosphorylation, TNFA signaling via NF-K B, and the inflammatory response. The PPI was constructed and the hub genes found through the method of DMNC showed that INHBA and NR4A2 might play an important role in venous remodeling after AVF. The integrated optical density (DOI) examined by immunohistochemistry staining showed that the expression of both INHBA and NR4A2 increased in AVF compared to the control group. Our research contributes to the understanding of the molecular mechanism of venous remodeling after exposure to high fistula flow, which may be useful in treating AVF failure.

Chitin deacetylases are essential enzymes in the chitin-modifying process and play vital roles in arthropod molting. In this study, we identified and characterized a chitin deacetylase-like (EsCDA-l) gene in the Chinese mitten crab, Eriocheir sinensis. The open reading frame of EsCDA-l was 2555 bp and encoded 554 amino acid residues that contained typical domain structure of carbohydrate esterase family 4. Phylogenetic analysis reveal that EsCDA-l belongs to the group I chitin deacetylase family. Quantitative real-time PCR analyses showed that EsCDA-l was highly expressed in exoskeletal tissues and megalopa stages. During the molting cycle, EsCDA-l was up-regulated periodically in the post-molt stage. Knockdown of EsCDA-l resulted in the abnormal ultrastructure of cuticle, prevented molting to high mortality suggesting EsCDA-l is indispensable for molting. The characterization and function analysis of the EsCDA-l should provide useful reference for further research on the utility of key genes involved in the chitin metabolic pathway in the molting process of the Chinese mitten crab as well as other crustaceans.

Background Osteoarthritis (OA) is the most common arthritic disease in humans, affecting the majority of individuals over 65 years of age. The aim of this study is to identify the gene expression profile specific to subchondral bone in OA by comparing the different expression profiles in experimental and sham-operation groups. Methods Gene expression profile GSE30322 was downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were obtained by limma package. And Database for Annotation, Visualization and Integrated Discovery (DAVID) databases were further used to identify the potential gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Furthermore, a protein–protein interaction (PPI) network was constructed and significant modules were extracted. Results Totally, 588 DEGs were identified including 199 upregulated DEGs and 389 downregulated DEGs screened in OA and sham-operation. GO showed that DEGs were significantly enhanced for ribosomal subunit export from nucleus and molting cycle. KEGG pathway analysis revealed that target genes were enriched in thiamine metabolism. Conclusion These key candidate DEGs that affect the progression of OA, and these genes might serve as potential therapeutic targets for OA.

The transforming growth factor beta superfamily encompasses a large family of ligands that are well conserved across many organisms. They are regulators of a number of physiological and pathological processes. The model nematode Caenorhabditis elegans has been instrumental in identifying key components of the transforming growth factor beta (TGF&beta, ) pathway. In C. elegans, the TGF&beta, homolog DAF-7 signals through the DAF-1 Type I and DAF-4 Type II receptors to phosphorylate downstream R-SMADs DAF-8 and DAF-14. These R-SMADs translocate into the nucleus to inhibit Co-SMAD DAF-3. Many of the roles of the canonical DAF-7 pathway, involving both DAF-1 and DAF-3, have been identified using targeted genetic studies. Few have assessed the global transcriptomic changes in response to these genes, especially in adult animals. In this study, we performed RNA sequencing on wild type, daf-1, and daf-1, daf-3 adult hermaphrodites. To assess the overall trends of the data, we identified differentially expressed genes (DEGs) and performed gene ontology analysis to identify the types of downstream genes that are differentially expressed. Hierarchical clustering showed that the daf-1, daf-3 double mutants are transcriptionally more similar to wild type than daf-1 mutants. Analysis of the DEGs showed a disproportionally high number of genes whose expression is increased in daf-1 mutants, suggesting that DAF-1 acts as a general repressor of gene expression in wild type animals. Gene ontology analysis of the DEGs produced many significantly enriched terms, including Molting Cycle, Response to Topologically Incorrect Protein, and Response to Biotic Stimulus. Understanding the direct and indirect targets of the DAF-7 TGF&beta, pathway through this RNA-seq dataset can provide insight into novel roles of the multifunctional signaling pathway, as well as identify novel genes that may participate in previously reported functions of TGF&beta, signaling.

Juvenile hormones (JHs) are sesquiterpenoids synthesized by the corpora allata (CA). They play critical roles during insect development and reproduction. The first JH was described in 1934 as a "metamorphosis inhibitory hormone" in Rhodnius prolixus by Sir Vincent B. Wigglesworth. Remarkably, in spite of the importance of R. prolixus as vectors of Chagas disease and model organisms in insect physiology, the original JH that Wigglesworth described for the kissing-bug R. prolixus remained unidentified. We employed liquid chromatography mass spectrometry to search for the JH homologs present in the hemolymph of fourth instar nymphs of R. prolixus. Wigglesworth's original JH is the JH III skipped bisepoxide (JHSB3), a homolog identified in other heteropteran species. Changes in the titer of JHSB3 were studied during the 10-day long molting cycle of 4th instar nymph, between a blood meal and the ecdysis to 5th instar. In addition we measured the changes of mRNA levels in the CA for the 13 enzymes of the JH biosynthetic pathway during the molting cycle of 4th instar. Almost 90 years after the first descriptions of the role of JH in insects, this study finally reveals that the specific JH homolog responsible for Wigglesworth's original observations is JHSB3., Facultad de Ciencias Naturales y Museo, Consejo Nacional de Investigaciones Científicas y Técnicas

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.

Made available in DSpace on 2020-12-10T19:50:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-02-07 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The environmental quality of a Ramsar wetland site located at the Cananeia-Iguape-Peruibe Protected Area (CIP-PA), in Sao Paulo, Brazil, was assessed by geochemical analyses and biomarker assessments (GPx, GST, GSH, GST, MT, LPO, DNA damage) performed in swimming crab Callinectes danae Smith, 1869 organs (posterior and anterior gills and hepatopancreas) to estimate sediment contaminant bioavailability. The results indicated that two sampling stations, PT and PM, exhibited the worst environmental conditions, as sediments collected at both points contained metal contamination, while crabs exhibited significant responses for GPx, GST, and LPO (mostly during winter). Sediment contamination tended to be associated to fine sediments (both seasons) and organic matter (winter). During the summer survey, Pb concentrations in sediments of station PT exceeded the Brazilian Sediment Quality Guidelines (SQGs) and the Canadian Interim Marine Sediment Quality Guidelines. Metal concentrations in sediments sampled in winter were higher compared with summer, with Co, Ni, and Pb exceeding SQGs levels at PT, whereas Co, Ni, Hg, Zn, and Pb exceeded SQGs at PM. Biomarker induction during summer appeared to be caused by natural variables (water salinity and temperature, and molting cycle), whereas oxidative stress and tissue damage during winter appeared to be more clearly linked to metal contamination. Anterior gills presented the clearest signs of seasonal variability, being more responsive to sediment contamination. The results suggest that metals originated from the upper Ribeira de Iguape River are transported toward the estuarine system, causing effects on C. danae individuals. Additionally, seasonality is a strong factor concerning CIP-PA toxicity, since the rainfall regime significantly modifies the freshwater flow and, consequently, estuarine water salinity, suspended particle and metal inputs, as well as the location of depositional areas. Thus, efforts to mitigate CIP-PA contamination should be based on the control of upstream pollution sources. Univ Sao Paulo, Inst Oceanog, Sao Paulo, SP, Brazil Univ Estadual Paulista Julio de Mesquita Filho UN, NEPEA, Campus Litoral Paulista, Sao Vicente, Brazil IPEN Nucl & Energy Res Inst, Res Reactor Ctr, Sao Paulo, Brazil Pontificia Univ Catolica Rio de Janeiro, Dept Chem QUI, Rio De Janeiro, Brazil Fundacao Oswaldo Cruz, Lab Avaliacao & Promocao Saude Ambiental, Inst Oswaldo Cruz, Rio De Janeiro, Brazil Univ Fed Rio de Janeiro, Inst Quim, Rio De Janeiro, RJ, Brazil Univ Fed Fluminense, Programa Posgrad Geoquim, Dept Geoquim, Niteroi, RJ, Brazil Univ Estadual Paulista Julio de Mesquita Filho UN, NEPEA, Campus Litoral Paulista, Sao Vicente, Brazil FAPESP: 09/52762-6 CNPq: 479899/2013-4 CNPq: 303620/2008-0 CNPq: 311609/2014-7 CNPq: 311820/2017-4 CNPq: 308649/2011

Lower termites, as well as their sister group, the subsocial wood-feeding cockroach Cryptocercus, rely on flagellated eukaryotic symbionts in the hindgut to cooperatively digest their wood diet. In Cryptocercus these flagellates undergo encystment cycles tightly coordinated with the molting cycle of their host, yet the resultant cysts play no demonstrated role in their transmission to neonates; the trophozoite stage of the flagellates is passed directly from parents to offspring via hindgut fluids (proctodeal trophallaxis). This pattern suggests that encystment is a vestige from a gregarious cockroach ancestor, when the flagellates had a functional, two-stage life cycle and the cysts were horizontally transmitted among hosts via coprophagy. The strong integration between flagellate encystment cycles and host developmental physiology in Cryptocercus indicates that the relationship of the flagellates with their proposed gregarious cockroach ancestor was not commensal, but parasitic, with flagellates likely obtaining benefits by taking advantage of host gut metabolites and ingested plant debris. When vertical transmission evolved the parasites were ‘captured,’ and their fitness became inescapably embedded in the fitness of their host. The vertical transmission of gut flagellates and the origin of host subsociality via proctodeal trophallaxis can be considered two sides of the same coin. From the host point of view proctodeal trophallaxis marks the origination of parental care by provisioning neonates with nourishment, metabolites and beneficial microbiota. From the flagellate point of view, proctodeal trophallaxis was a shift from horizontal to vertical transmission, pushing them from the parasitic to the mutualistic end of the symbiotic spectrum, arguably making this host behavioral change the most critical juncture in the evolutionary trajectory of the termite lineage.

Molting enables growth and development across ecdysozoa. The molting process is strictly controlled by hormones - ecdysteroids. Ecdysteroidogenesis occurs in theprothoracic glands and stimulated by prothoracicotropic hormone in insects, while it ensues in the Y-organ and regulated by the molt inhibiting hormone in crustaceans. A peak in ecdysteroids in the hemolymph induces a cascade of multiple neuropeptides including Ecdysis Triggering Hormone (ETH) and Corazonin. The role of ETH is well defined in controlling the molt process in insects, but it is yet to be defined in crustaceans. In this study, we investigated the behavioral response of intermolt crayfish to ETH and Corazonin injections as well as the impact of ETH on the molt period using in vivo assays. Injection of Corazonin and ETH resulted in a clear and immediate eye twitching response to these two neuropeptides. The Corazonin injection induced eye twitching in slow and asynchronous manner, while ETH injection caused eye twitching in a relatively fast and synchronous way. A single injection of ETH to crayfish resulted in a remarkable prolong molt period, at twice the normal molting cycle, suggesting that ETH plays a key role in controlling the molt cycle in decapod crustaceans. Given the key significance of ETH in molt regulation and its plausible application in pest control, we characterized ETH across the pancrustacean orders. Bioinformatic analysis shows the mature ETH sequence is identical in all studied decapod species. ETH can be classified into specific groups based on the associated motif in each insect order and shows an insect motif -KxxPRx to be conserved in crustaceans.

The present study was conducted to characterize the full-length cDNA of c-Jun N-terminal kinase (JNK) in Procambarus clarkii (Pcjnk) and evaluate its potential function under different molt cycle. The full-length cDNA of Pcjnk covered 2937 bp with an open reading frame of 1320 bp, encoding 439 amino acids. A typical conserved TPY motif (118Thr-Pro-120Tyr) was found in Pcjnk. Quantitative real-time PCR (qRT-PCR) analysis revealed a constitutive expression of Pcjnk in the tested tissue, with the highest expression occurring in the hepatopancreas. Additionally, the present study initially revealed that relative mRNA expression of Pcjnk and apoptosis level were significantly higher in the premolt stage (D1/D2 and D3/D4 stage) as compared to other molt stages. In contrast to the levels of superoxide dismutase (SOD) and malondialdehyde (MDA), catalase (CAT) and glutathione peroxidase (GPX) level decreased significantly from the intermolt stage (C stage) to the premolt stage (D1/D2 and D3/D4 stage), then increased from the premolt stage to the postmolt stage (A and B stage). The results obtained in the present study indicated that molt could cause apoptosis induced by oxidative stress through the activation of JNK in Procambarus clarkii.

The crustacean exoskeleton is composed primarily of chitin, proteins and various inorganic compounds. It is the inorganic compounds, such as calcium and magnesium, that underlie the exoskeletal mineralization process following ecdysis. Little is known about the hormonal mechanism for this process in crustaceans. Carbonic anhydrase (CA) in the epidermis has been suggested to aid in deposition of calcium and magnesium carbonates to the exoskeleton in crustaceans by generating bicarbonate ions, resulting in mineralization. Given a similar pattern of fluctuation in prevalence between epidermal CA and ecdysteroids during the crab molting cycle, it has been proposed in a previous study that post-ecdysial epidermal CA and subsequent metal deposition to the exoskeleton are controlled by the ecdysteroid molting hormones in the blue crab, Callinectes sapidus. This study sought to acquire evidence to support such a proposition. Early postmolt and early intermolt blue crabs were used to quantify epidermal CA mRNA expression when exposed in vitro to three physiologically relevant concentrations of 10 nM, 100 nM and 1 μM 20-hydroxyecdysone (20-HE), using the epidermis-with-exoskeleton (EWE) tissue assay method. It was found that 100 nM 20-HE significantly induced the mRNA of CasCAg, a CA isoenzyme, in epidermal tissues of early intermolt crabs and that injection of 20-HE at a dose of 15 ng/g significantly elevated epidermal CA activity in vivo in early intermolt crabs. These two lines of evidence clearly show that post-ecdysial epidermal CA is influenced by the molting hormone. Interestingly, exoskeletal calcium content was significantly lower in the 20-HE treated crabs than in the control, whereas magnesium content was unchanged. The inhibition of calcification in the post-ecdysial exoskeleton by the exogenous molting hormone may implicate that sclerotization and mineralization of the new shell must be coordinated and that enhanced deposition of carbonate salts as a result of increased epidermal CA activity following the administration of exogenous molting hormone would be inhibited to avoid the formation of a structurally disrupted exoskeleton.

Causal factors leading to epizootic shell disease (ESD) lesions in American lobster, Homarus americanus H. Milne-Edwards, 1837, are not well understood. We explore the structural and physiological bases for development of ESD from preclinical stages invisible to unaided eye to early visible stages. We present a lobster shell model, which develops structural functional vulnerability and suggests plausible routes to ESD. Medallions of carapace cuticle were obtained from carapace fixed with protocols to minimize movement of mineral and macromolecular components. Rapid processing of medallions was used to encourage large sample sizes compatible with environmental surveys. One- and two-dimensional analytic maps of polished sections of the cuticle, obtained with an electron microprobe, described the composite mineral and polymeric structures. Micro-Raman spectroscopy was used to identify bond properties of phosphates and carbonates, as well as signatures of organic structures. The frequency and properties of structures identified can be monitored through the lobster molting cycle using a high throughput application of micro-computed tomography (μCT). We observed density differences in the calcite layer, exocuticle, and endocuticle, and the frequency and structure of CaCO3 structures in the endocuticle and membranous layer of carapace cuticle during chosen stages of the molting cycle. The correlative microscopy and μCT of shell structures provides improved understanding of the lobster cuticle structure. Detailed structural differences quantified through development and under different environmental conditions can provide insight into causes and vulnerabilities associated with ESD.

The hepatopancreas is the key organ involved in energy storage, immune response, and metabolism during crustacean molting, yet the underlying molecular mechanisms in the hepatopancreas that regulate molting remain unknown. In the present study, we conducted a comprehensive proteomic analysis in the hepatopancreas and quantified 1527 proteins, of which 193 changed significantly in abundance among three molting stages (pre-molt: PrM, post-molt: PoM, and inter-molt: InM) of Scylla paramamosain using iTRAQ-coupled LC-MS/MS. Ten exoskeleton and cuticle reconstruction proteins, such as chitinase, cuticle protein and myosin heavy chain, were found change significantly in abundance between PoM and PrM. Six energy metabolism proteins such as mitochondrial cytochrome c oxidase, cytochrome b-c1 and cAMP-dependent protein kinase with positive loadings showed a higher abundance in InM than PoM. In addition, all differentially abundance proteins (DAPs) were annotated for GO function and KEGG pathway analysis. GO analysis demonstrated function subcategories mainly including thiamine metabolism, complement and coagulation cascades, endocrine, shigellosis, salmonella infection, and other factor-regulated calcium reabsorption. The KEGG pathway enrichment analysis indicated that the DAPs were mainly involved in reconstruction of the exoskeleton and cuticle, energy reserves, metabolism, and immune response during the molting process. The results for the proteins and key pathways involved in the molting process provide fundamental molecular evidence that will improve our understanding of morphological and metabolism variation in the molting cycle and will serve as a potential blueprint for future study on molecular mechanism of molting in crustaceans.

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.

Aeglids are unique freshwater decapods whose habitats are being impacted by metallic compounds, such as copper (Cu). Thus, we investigated the effects of acute Cu exposure on ionic regulation of Aegla castro. For this, male specimens in intermolt were collected from a reference stream and acclimated for 5 days in laboratory. After which, crabs were exposed to 11 μg L−1 Cu (Cu11) or only to water (CTR) for 24 h. Hemolymph samples were withdrawn for the determination of Na+, K+, Ca2+, and Mg2+ concentrations and the posterior gills removed for the analysis of Na+/K+-ATPase, Ca2+-ATPase, H+-ATPase, and carbonic anhydrase (CA) activities. Increased Ca2+ and Mg2+ hemolymph concentrations were observed in animals from Cu11, when compared with CTR group. In addition, decreased activity of CA was observed in animals exposed to Cu. In the current study, alterations in Ca2+ and Mg2+concentrations probably indicate that animals activated exoskeleton reabsorption mechanisms, characteristic of the premolt. Therefore, increased Ca2+ and Mg2+ concentrations in hemolymph may indicate that a biochemical signal associated with the molting cycle was triggered by Cu exposure. Despite the known harmful effects of Cu on osmoregulatory enzymes, here we observed decreased activity only in CA. However, decreased activity of CA could trigger both acid-base imbalance and ionic disruption, since CA provides H+ and HCO3− for intracellular pH maintenance, and underpins Na+ and Cl− for ionic regulation. Therefore, understanding how aeglids respond to metal contamination in laboratory conditions is crucial to assess their potential as an alternative biological model for aquatic ecotoxicology.