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The antennal glands (AnGs) are recognized as an important organ that functions in ion regulation and excretion in decapods. Previously, many studies had explored this organ at the biochemical, physiological, and ultrastructural levels but had few molecular resources. In this study, the transcriptomes of the male and female AnGs of Portunus trituberculatus were sequenced using RNA sequencing (RNA-Seq) technology. Genes involved in osmoregulation and organic/inorganic solute transport were identified. This suggests that AnGs might be involved in these physiological functions as versatile organs. A total of 469 differentially expressed genes (DEGs) were further identified between male and female transcriptomes and found to be male-biased. Enrichment analysis showed that females were enriched in amino acid metabolism and males were enriched in nucleic acid metabolism. These results suggested differences in possible metabolic patterns between males and females. Furthermore, two transcription factors related to reproduction, namely AF4/FMR2 family members Lilli (Lilli) and Virilizer (Vir), were identified in DEGs. Lilli was found to be specifically expressed in the male AnGs, whereas Vir showed high expression levels in the female AnGs. The expression of up-regulated metabolism and sexual development-related genes in three males and six females was verified by qRT-PCR and the pattern was found to be consistent with the transcriptome expression pattern. Our results suggest that although the AnG is a unified somatic tissue composed of individual cells, it still demonstrates distinct sex-specific expression patterns. These results provide foundational knowledge of the function and differences between male and female AnGs in P. trituberculatus.

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Panulirus argus virus 1 (PaV1) (Family Mininucleoviridae) causes chronic and systemic infection in wild juvenile spiny lobsters Panulirus argus (Latreille, 1804), ending in death by starvation and metabolic wasting. In marine decapods, the antennal gland is involved in osmoregulation and excretion. In this compact organ, fluid is filtered from the hemolymph, and ions are reabsorbed to produce a hypotonic urine. Although PaV1 is released with the urine in infected individuals, little is known regarding the metabolic effect of PaV1 in the antennal gland. The objective of this study was to perform a comparative evaluation of the metabolic profile of the antennal gland of clinically PaV1-infected lobsters versus those with no clinical signs of infection, using proton nuclear magnetic resonance analysis. Overall, 48 compounds were identified, and the most represented metabolites were those involved in carbohydrate, amino acid, energy, and nucleotide metabolism. Most of the metabolites that were down-regulated in the infected group were essential and non-essential amino acids. Some metabolites involved in the urea cycle and carbohydrate metabolism were also altered. This study represents a first approach to the metabolic evaluation of the antennal gland. We broadly discuss alterations in the content of several proteinogenic and non-proteinogenic amino acids and other key metabolites involved in energetic and nucleotide metabolism.

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Brachyurans inhabit a variety of habitats and have evolved diverse osmoregulatory patterns. Gills, antennal glands and a lung-like structure are important organs of crabs that maintain their homeostasis in different habitats. Species use different processes to regulate ions in the antennal gland, especially those with high terrestriality such as Grapsoidea and Ocypodoidea. Our phylogenetic generalized least square (PGLS) result also suggested that there is a correlation between antennal gland NKA activity and urine-hemolymph ratio for Na+ concentration in hypo-osmotic environments among crabs. Species with higher antennal gland NKA activity showed a lower urine-hemolymph ratio for Na+ concentration under hypo-osmotic stress. These phenomenon may correlate to the structural and functional differences in gills and lung-like structure among crabs. However, a limited number of studies have focused on the structural and functional differences in the antennal gland among brachyurans. Integrative and systemic methods like next generation sequencing and proteomics method can be useful for investigating the differences in multi-gene expression and sequences among species. These perspectives can be combined to further elucidate the phylogenetic history of crab antennal glands.

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Within-host distributions of parasites can have relevance to parasite competition, parasite mating, transmission, and host health. We examined the within-host distribution of the adult trematode Alloglossidium renale infecting the paired antennal glands of grass shrimp. There are 4 possible parasite distributions for infections of paired organs: random, uniform, biased aggregation to 1 particular organ (e.g., left vs. right), or inconsistently biased (aggregated, but does not favor 1 side). Previous work has shown that morphological asymmetries in hosts can lead to biased infections of paired organs. Apparent symmetry between the antennal glands of grass shrimp leads to the prediction that there would be no bias for 1 particular organ. However, an alternative prediction stems from the fact that A. renale is hermaphroditic: aggregation between glands would increase outcrossing opportunities and thus, avoid inbreeding via self-mating. Existing methods to test for an overall pattern did not apply to the A. renale system because of low-intensity infections as well as many 0 values for abundance per unit of the antennal gland. Hence, we used Monte Carlo simulations to determine if the observed overall patterns differed from those expected by randomly allocating parasites into groups of 2. We found that in 3 of 4 data sets, A. renale infections did not deviate from random distributions. The fourth data set had a more uniform pattern than expected by chance. As there was no aggregation between glands and the proportion of worms in single gland infections did not differ from that expected by chance alone, we found no evidence of inbreeding avoidance as might be manifested via a within-host distribution. Given the large proportion of worms in single infections, we predict as a major evolutionary outcome that populations of A. renale will be largely inbred.

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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.

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Gills and the antennal gland are ion-regulatory organs in crabs. Previous studies have suggested that the differences in the morphology and ion regulation of gills and accessory respiratory organs between ocypodid and grapsid species are related to their distinct evolutionary transition to land habitats. In addition, Na+, K+-ATPase (NKA) activity and Na+ and NH4+ regulation in the antennal gland differ between ocypodid and grapsid species, which had different terrestrial adaptation trajectories. This study used five Ocypodoidea species and three Grapsoidea species from the intertidal and supratidal zones to further investigate the differences in ion regulation and NKA activity in the antennal gland between these crab families in different habitats. Crabs were transferred to 5 practical salinity unit (PSU) water, and osmolality, Na+ and Cl- concentrations in the urine and hemolymph, and NKA activity in the antennal gland were examined. Phylogenetic ANOVA results showed that the NKA activity in the antennal gland was higher in the ocypodid than grapsid groups, and Moran's I autocorrelation analysis also indicated that NKA activity in the antennal gland was phylogenetically correlated among crabs. K-means clustering showed a difference among the crabs in the crabs' Na+ and Cl- concentrations in the urine/hemolymph, NKA activities in the antennal gland and gill 6, and number of pairs of gills. Crabs with relatively high antennal gland NKA activity were found not only in the Ocypode species, which are better adapted to terrestrial environments, but also in two intertidal species of Gelasiminae. In conclusion, part of the Ocypodidae lineage may have a) the ability to reabsorb Na+ and b) higher NKA activity in the antennal gland than other families, and this phenomenon is phylogenetically correlated in Ocypodoidea and Grapsoidea. The physiological diversity in osmoregulation among intertidal and costal species provides a base to further investigate their ecological niches and guilds.

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Mikrocytids are a widespread but rather neglected group of parasites of aquatic invertebrates. One such parasite is Paramikrocytos canceri-discovered to infect the antennal gland of the juvenile edible crab, Cancer pagurus, taken from several intertidal sites across the United Kingdom. To determine if this parasite is also present in other species of decapod crustaceans, we surveyed crabs (n = 330) across two contrasting sites in Pembrokeshire (UK). Using a histopathological approach, P. canceri infection was confirmed in variable numbers of edible crabs from both survey sites, 7%-44%. No measurable signs of infection were encountered in four other co-located species, including European shore crabs (Carcinus maenas), Montagu's crabs (Xantho hydrophilus), velvet swimming crabs (Necora puber) and broad-clawed porcelain crabs (Porcellana platycheles). These data imply that P. canceri has a more limited host range than suggested by molecular diagnosis alone.

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Two experimental modules with different stocking densities (M1 = 70 and M2 = 120 shrimp m-2) were examined weekly during 72-day culture cycle at low-salinity water (1.9 g L-1) and zero-water exchange to examine the effects of water quality deterioration on the antennal gland (AG) of shrimp. Results showed survival rates of 87.7% and 11.9% in M1 and M2, respectively. Water temperature, pH, dissolved oxygen, and chlorophyll a were not significantly different between modules but the concentrations of the nitrogen compounds were significantly different between modules with the exception of nitrite-N, showing a higher histological alteration index in M2 (32 ± 10) than M1 (22 ± 0) with a strong correlation with the nitrogen compounds. During the last weeks was evidenced in M1 inflammation and hemocytic and hemolymph infiltration, while in M2, melanization, hemocytic melanized nodules and cells with kariorrexis.

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BACKGROUND: Understanding the genomic basis of osmoregulation (candidate genes and/or molecular mechanisms controlling the phenotype) addresses one of the fundamental questions in evolutionary ecology. Species distributions and adaptive radiations are thought to be controlled by environmental salinity levels, and efficient osmoregulatory (ionic balance) ability is the main mechanism to overcome the problems related to environmental salinity gradients. METHODS: To better understand how osmoregulatory performance in freshwater (FW) crustaceans allow individuals to acclimate and adapt to raised salinity conditions, here we (i), reviewed the literature on genes that have been identified to be associated with osmoregulation in FW crustaceans, and (ii), performed a transcriptomic analysis using cDNA libraries developed from mRNA isolated from three important osmoregulatory tissues (gill, antennal gland, hepatopancreas) and total mRNA from post larvae taken from the freshwater prawn, Macrobrachium australiense using Illumina deep sequencing technology. This species was targeted because it can complete its life cycle totally in freshwater but, like many Macrobrachium sp., can also tolerate brackish water conditions and hence should have genes associated with tolerance of both FW and saline conditions. RESULTS: We obtained between 55.4 and 65.2 million Illumina read pairs from four cDNA libraries. Overall, paired end sequences assembled into a total of 125,196 non-redundant contigs (≥200 bp) with an N50 length of 2,282 bp and an average contig length of 968 bp. Transcriptomic analysis of M. australiense identified 32 different gene families that were potentially involved with osmoregulatory capacity. A total of 32,597 transcripts were specified with gene ontology (GO) terms identified on the basis of GO categories. Abundance estimation of expressed genes based on TPM (transcript per million) ≥20 showed 1625 transcripts commonly expressed in all four libraries. Among the top 10 genes expressed in four tissue libraries associated with osmoregulation, arginine kinase and Na+/K+- ATPase showed the highest transcript copy number with 7098 and 660, respectively in gill which is considered to be the most important organ involved in osmoregulation. DISCUSSION: The current study provides the first broad transcriptome from M. australiense using next generation sequencing and identifies potential candidate genes involved in salinity tolerance and osmoregulation that can provide a foundation for investigating osmoregulatory capacity in a wide variety of freshwater crustaceans.

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The euryhaline shrimp Palaemonetes argentinus exemplifies an evolutionary transition from brackish to freshwater habitats that requires adequate osmoregulatory capacities. Hyperosmoregulation is functional at hatching and it likely begins during the embryonic phase allowing this species to develop entirely in fresh water. Here, we investigated the Na(+)/K(+)-ATPase α-subunit gene (nka-α) expression using quantitative real-time PCR and localized Na(+)/K(+)-ATPase (NKA) in ion-transporting epithelia through immunofluorescence microscopy. We reared shrimps from spawning to juvenile stages at two salinities (1, 15 ‰) and maintained adults for 3 weeks at three salinity treatments (1, 15, 25 ‰). nka-α gene expression was measured in: (1) embryos at an early (SI), intermediate (SII) and late (SIII) stage of embryonic development; (2) newly hatched larvae (Zoea I, ZI); and (3) isolated gill tissue of adults. The nka-α expression was low in SI and SII embryos and reached maximum levels prior to hatching (SIII), which were similar to expression levels detected in the ZI. The nka-α expression in SIII and ZI was highest at 15 ‰, whereas salinity did not affect expression in earlier embryos. In SIII, in ZI and in a later zoeal stage ZIV, NKA was localized in epithelial cells of pleurae, in the inner-side epithelium of branchiostegite and in the antennal glands. Gills appeared in the ZIV but NKA immunolabeling of the cells of the gill shaft occurred in a subsequent developmental larval stage, the decapodid. Extrabranchial organs constitute the main site of osmoregulation in early ontogenetic stages of this freshwater shrimp.

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The American lobster (Homarus americanus) fishery is the most economically significant fishery in Canada; although comparatively little is known about the lobsters' response to pathogenic challenge. This is the first study to investigate the expression of immune genes in tissues outside of the lobster hepatopancreas in response to challenges by the Gram-positive bacteria, Aerococcus viridans var. homari or the scuticociliate parasite, Anophryoides haemophila. The hepatopancreas has been regarded as the major humoral immune organ in crustaceans, but the contribution of other organs and tissues to the molecular immune response has largely been overlooked. This study used RT-qPCR to monitor the gene expression of several immune genes including three anti-lipopolysaccharide isoforms (ALF) Homame ALF-B1, Homame ALF-C1 and ALFHa-1, acute phase serum amyloid protein A (SAA), as well as thioredoxin and hexokinase, in antennal gland and gill tissues. Our findings indicate that the gene expression of the SAA and all ALF isoforms in the antennal gland and gill tissues increased in response to pathogenic challenge. However, there was differential expression of individual ALF isoforms that were dependent on both the tissue, and the pathogen used in the challenge. The gene expression changes of several immune genes were found to be higher in the antennal gland than have been previously reported for the hepatopancreas. This study demonstrates that increased immune gene expression from the gill and antennal gland over the course of pathogen induced disease contributes to the immune response of H. americanus.

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Brachyuran crabs from diverse habitats show great differences in their osmoregulatory processes, especially in terms of the structural and physiological characteristics of the osmoregulatory organs. In crustaceans, the antennal glands are known to be important in osmoregulation, and they play a functional role analogous to that of the vertebrate kidney. Nevertheless, the detailed structure and function of the antennal glands in different species have rarely been described. The aim of this study is to investigate the role of the antennal gland in ion regulation by examining the ultrastructure of the cells and the distribution of the ion regulatory proteins in each cell type in the antennal gland of a semi-terrestrial crab. The results showed that Na(+), K(+)-ATPase activity significantly increased in the antennal gland after a 4-day acclimation in dilute seawater and returned to its original (day 0) level after 7 days. Three major types of cells were identified in the antennal gland, including coelomic cells (COEs), labyrinthine cells (LBRs) and end-labyrinthine cells (ELBRs). The proximal tubular region (PT) and distal tubular region (DT) of the antennal gland consist of LBRs and COEs, whereas the end tubular region (ET) consists of all three types of cells, with fewer COEs and more ELBRs. We found a non-uniform distribution of NKA immunoreactivity, with increasing intensity from the proximal to the distal regions of the antennal gland. We summarise our study with a proposed model for the urine reprocessing pathway and the role of each cell type or segment of the antennal gland.

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The objective of our study was to describe by histologic methods the structural organization of the antennal gland of M. potiuna as well as the location through histochemical methods of the neutral and sulfated polissacharides that exist in these regions. This study showed that the gland is composed of the a non-secretive tubular portion and a secretive portion which is formed by a cubic simple epithelium, sub-epithelium layer and cellular chains.