Antennal Gland Research Articles

Bioaccumulation kinetics and organ distribution of cadmium and zinc in the freshwater decapod crustacean Macrobrachium australiense.

This study used the radioisotopes (109)Cd and (65)Zn to explore the uptake, retention and organ distribution of these nonessential and essential metals from solution by the freshwater decapod crustacean Macrobrachium australiense. Three treatments consisting of cadmium alone, zinc alone, and a mixture of cadmium and zinc were used to determine the differences in uptake and efflux rates of each metal individually and in the metal mixture over a three-week period, followed by depuration for 2 weeks in metal-free water using live-animal gamma-spectrometry. Following exposure, prawns were cryosectioned and the spatial distribution of radionuclides visualized using autoradiography. Metal uptake and efflux rates were the same in the individual and mixed-metal exposures, and efflux rates were close to zero. The majority of cadmium uptake was localized within the gills and hepatopancreas, while zinc accumulated in the antennal gland at concentrations orders of magnitude greater than in other organs. This suggested that M. australiense may process zinc much faster than cadmium by internally transporting the accumulated zinc to the antennal gland. The combination of uptake studies and autoradiography greatly increases our understanding of how metal transport kinetics and internal processing may influence the toxicity of essential and nonessential metals in the environment.

Discovery of a novel insulin-like peptide and insulin binding proteins in the Eastern rock lobster Sagmariasus verreauxi

This study reports, for the first time in any of the commercially important decapod species, the identification of an insulin-like peptide (ILP), distinct from the androgenic gland hormone. Bioinformatics analysis of the de novo assembled spiny lobster, (Sagmariasus verreauxi) transcriptome, allowed identification of Sv-ILP1 as well as eight binding proteins. Binding proteins were termed as Sv-IGFBP, due to homology with the vertebrate insulin-like growth-factor binding protein and Sv-SIBD1-7, single insulin-binding domain protein (SIBD), similar to those identified in other invertebrate species. Sv-ILP1 was found to be expressed in the eyestalk, gonads and antennal gland of both sexes and to a lesser extent in male muscle, androgenic gland and hepatopancreas. The expression profiles of each binding protein were found to vary across tissues, with Sv-SIBD5, 6 and 7 showing higher expression in the gonad, demonstrated by PCR and digital gene expression. Further spatial investigations, using in-situ hybridisation, found Sv-ILP1 to be expressed in the neurosecretory cells of the thoracic ganglia, in keeping with the tissue expression of Drosophila ILP7 (DILP7). This correlative tissue expression, considered with the phylogenetic clustering of Sv-ILP1 and DILP7, suggests Sv-ILP1 to be a DILP7 orthologue. The broad expression of Sv-ILP1 strongly suggests that ILPs have a role beyond that of masculinisation in decapods. The function of these novel peptides may have application in enhancing aquaculture practices in the commercially important decapod species.

Cultivation reveals physiological diversity among defensive 'Streptomyces philanthi' symbionts of beewolf digger wasps (Hymenoptera, Crabronidae).

Background‘Candidatus Streptomyces philanthi’ is a monophyletic clade of formerly uncultured bacterial symbionts in solitary digger wasps of the genera Philanthus, Philanthinus and Trachypus (Hymenoptera, Crabronidae). These bacteria grow in female-specific antennal reservoirs and – after transmission to the cocoon – produce antibiotics protecting the host larvae from fungal infection. However, the symbionts’ refractoriness to cultivation has thus far hampered detailed in vitro studies on their physiology and on the evolutionary changes in metabolic versatility in response to the host environment.ResultsHere we isolated in axenic culture 22 ‘Streptomyces philanthi’ biovars from different host species. Sequencing of gyrB revealed no heterogeneity among isolates within host individuals, suggesting low levels of (micro)diversity or even clonality of the symbionts in individual beewolf antennae. Surprisingly, however, isolates from different host species differed strongly in their physiology. All biovars from the Eurasian/African Philanthus and the South American Trachypus host species had high nutritional demands and were susceptible to most antibiotics tested, suggesting a tight association with the hosts. By contrast, biovars isolated from the genus Philanthinus and the monophyletic North American Philanthus clade were metabolically versatile and showed broad antibiotic resistance. Concordantly, recent horizontal symbiont transfer events – reflected in different symbiont strains infecting the same host species – have been described only among North American Philanthus species, altogether indicative of facultative symbionts potentially capable of a free-living lifestyle. Phylogenetic analyses reveal a strong correlation between symbiont metabolic versatility and host phylogeny, suggesting that the host environment differentially affects the symbionts’ evolutionary fate. Although opportunistic bacteria were occasionally isolated from the antennae of different host species, only filamentous Actinobacteria (genera Streptomyces, Amycolatopsis and Nocardia) could replace ‘S. philanthi’ in the antennal gland reservoirs.ConclusionOur results indicate that closely related bacteria from a monophyletic clade of symbionts can experience very different evolutionary trajectories in response to the symbiotic lifestyle, which is reflected in different degrees of metabolic versatility and host-dependency. We propose that the host-provided environment could be an important factor in shaping the degenerative metabolic evolution in the symbionts and deciding whether they evolve into obligate symbionts or remain facultative and capable of a host-independent lifestyle.

High salinity induced expression profiling of differentially expressed genes in shrimp (Penaeus monodon).

Four suppression subtractive hybridization (SSH) cDNA libraries were constructed to identify differentially expressed salinity stress responsive genes of black tiger shrimp, Penaeus monodon exposed to high (55 ppt) salinity conditions. One each of the forward and reverse SSH cDNA libraries were developed from the gill and gut tissues of shrimp and clones having inserts larger than 300 bp were unidirectionally sequenced. Based on the sequence homology search, the identified genes were categorized for their putative functions related to a wide range of biological roles, such as nucleic acid regulation and replication, immune response, energy and metabolism, signal transduction, cellular process, structural and membrane proteins, stress and osmoregulation. Gene expression levels in response to high salinity conditions at 2 weeks post salinity stress for some of the differentially expressed genes (Na(+)/K(+)-ATPase α-subunit, glutathione peroxidase, intracellular fatty acid binding protein, elongation factor 2, 14-3-3 like protein, penaeidin, translationally controlled tumor protein, transglutaminase and serine proteinase inhibitor B3) identified from SSH cDNA libraries were analysed by real-time RT-PCR. The highest gene expression levels was observed for Na(+)/K(+)-ATPase α-subunit in gill tissues (15.23-folds) and antennal glands (12.01-folds) and intracellular fatty acid binding protein in gut tissues (14.05-folds) respectively. The differential and significant levels of gene expression indicate the functional role of these genes in shrimp salinity stress adaptive mechanisms.

Morphology and histochemistry of the aesthetasc-associated epidermal glands in terrestrial hermit crabs of the genus Coenobita (Decapoda: Paguroidea).

Crustaceans have successfully adapted to a variety of environments including fresh- and saltwater as well as land. Transition from an aquatic to a terrestrial lifestyle required adaptations of the sensory equipment of an animal, particularly in olfaction, where the stimulus itself changes from hydrophilic to mainly hydrophobic, air-borne molecules. Hermit crabs Coenobita spp. (Anomura, Coenobitidae) have adapted to a fully terrestrial lifestyle as adults and have been shown to rely on olfaction in order to detect distant food items. We observed that the specialized olfactory sensilla in Coenobita, named aesthetascs, are immersed in a layer of mucous-like substance. We hypothesized that the mucous is produced by antennal glands and affects functioning of the aesthetascs.Using various microscopic and histochemical techniques we proved that the mucous is produced by aesthetasc-associated epidermal glands, which we consider to be modified rosette-type aesthetasc tegumental glands known from aquatic decapods. These epidermal glands in Coenobita are multicellular exocrine organs of the recto-canal type with tubulo-acinar arrangement of the secretory cells. Two distinct populations of secretory cells were clearly distinguishable with light and electron microscopy. At least part of the secretory cells contains specific enzymes, CUB-serine proteases, which are likely to be secreted on the surface of the aesthetasc pad and take part in antimicrobial defense. Proteomic analysis of the glandular tissue corroborates the idea that the secretions of the aesthetasc-associated epidermal glands are involved in immune responses.We propose that the mucous covering the aesthetascs in Coenobita takes part in antimicrobial defense and at the same time provides the moisture essential for odor perception in terrestrial hermit crabs. We conclude that the morphological modifications of the aesthetasc-associated epidermal glands as well as the functional characteristics of their secretions are important adaptations to a terrestrial lifestyle.

Functional anatomy and ion regulatory mechanisms of the antennal gland in a semi-terrestrial crab, Ocypode stimpsoni.

ABSTRACTBrachyuran 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.

Artificial infection of the native Korean freshwater prawn Macrobrachium nipponense (De Haan, 1849) (Decapoda, Palaemonidae) with white spot syndrome virus (WSSV)

White spot syndrome virus (WSSV), an important shrimp pathogen that can affect both salt- and freshwater shrimp, has a high mortality rate that can reach up to 100% in 3-10 days. Therefore, we assessed the susceptibility of Macrobrachium nipponense (De Haan, 1849), a freshwater prawn native to Korea, to artificial WSSV infection. Although rearing in water containing WSSV-infected carcasses did not result in infection, M. nipponense was infected with WSSV after being fed a WSSV-containing diet, per os infection, or injection with the purified virus. In the feeding test, the first mortality was observed on the 7th day and cumulative mortality reached 30% by 3 weeks post-infection. After per os infection, experimental animals started dying on the second day and within 3 weeks mortality had reached 90%. In the injection test, the mortality was found to be dose-dependent. Prawn injected with 2.5 × 104, 900 and 80 copies/g body weight exhibited the first mortality at 1, 2 and 4 days post inoculation, with 100, 100 and 50% final mortality at 2, 10 and 12 days post inoculation, respectively. Histological observation revealed inclusion bodies in the gills, antennal glands and other tissues of ectodermal origin. Real-time polymerase chain reaction revealed the increase of viral titers, indicating viral replication in the inoculated host. This forms the first report of the experimental susceptibility of M. nipponense to WSSV.

Histochemical Study of the Progenetic Trematode Alloglossidium renale.

A histochemical study of the progenetic trematode Alloglossidium renale has demonstrated the absence of any secreted material between the adult worm and the host (freshwater shrimp) antennal gland tubules. Host tissue is affected only by the compression, abrasion, and ingestion by the parasite, and host tubule cells near the worm have the same staining patterns as those distant from the parasite. The trematode sometimes dies within the host, leaving a necrotic mass whose histochemical staining differs significantly from the living organism. In the necrotic mass, the only recognizable features were the ova and the vitellarium, which atrophied and resulted in tyrosine-positive staining within the mass. A melanin reaction was not observed in the host using a specialized ferro-ferricyanide stain. The only apparent host response to infection was a layer of damaged squamous host cells adhering to the necrotic worm. The results confirm benign host-parasite effects and a highly evolved relationship between the host and parasite, perhaps bordering on commensalism.

Chronic toxicity of zinc sulphate in Procambarus clarkii

We evaluated the chronic toxicity of zinc sulphate in the crayfish Procambarus clarkii.Mature P.clarkii were exposed to 0,0.40,4.04,or 40.37 mg/L Zn2+ for 21 days.Each day,we randomly selected five crayfish in each group and collected samples of the hepatopancreas,gill,and muscle tissue from males and females and ovar-ian tissue from each female.The tissues were placed in Bouin's fixative for 24 h,sectioned in paraffin,and stained using H?E.We observed a number of brick red particles in the cytoplasm of cells from the hepatopancreas and antennal gland in animals exposed to Zn2+.The number of these particles was positively correlated with the con-centration of Zn2+ and exposure time.There was no obvious histopathology in the group exposed to 0.40 mg/L Zn2+.We observed bleeding and necrosis in hepatopancreas,antennal gland,and gills in the group exposed to 4.04 mg/L after 7 d.The proportion of necrotic cells increased as the concentration of Zn2+ and time of exposure in-creased.We observed spermatocyte necrosis after 14 d in exposure to 40.37 mg/L Zn2+.After 21 d,we observed bleeding of the spermary and ovary and necrosis of the ootid and spermatocyte cells.A large number of hepato-cytes exhibited necrosis and had dropped from the hepatic ducts.The connective tissue exhibited hyperplasia,with large numbers of cells blocking the hepatic ducts,resulting in macroscopical bulky grains in the hapatopancreas of the group exposed to 40.37 mg/L.Our results suggest that exposure to 4.04 mg/L Zn2+ for 21 d causes pathological changes in the hepatopancreas,antennal gland,gills,and gonad tissue of P.clarkii.Given that zinc sulphate inter-feres with the metabolic,respiratory,and reproductive systems,chronic exposure is likely to threaten the survival of P.clarkii populations.

Cloning and tissue expression of hemocyanin gene in Cherax quadricarinatus during white spot syndrome virus infection

The role of hemocyanin in terms of immune defense was explored in Cherax quadricarinatus infected by WSSV. The cDNA of a hemocyanin gene (CqHC) was cloned from the hepatopancreas of C. quadricarinatus (crayfish) by the Rapid Amplification Complementary DNA Ends (RACE) method. The full-length cDNA of CqHC consisted of 2167bp with a 2043bp open reading frame encoding a 680 amino acid protein with a predicted signal peptide of 19 amino acids. The deduced CqHC protein sequence was found to contain two conserved copper-binding sites and to share 74%, 73%, 72%, 71% and 71% similarity with Penaeus monodon, Marsupenaeus japonicus, Fenneropenaeus chinensis, Metacarcinus magister and Litopenaeus vannamei, respectively. Phylogenetic analysis revealed that CqHC and HC from F. chinensis, M. japonicus and P. monodon clustered in the same phylogenetic branch. Real-time PCR analysis showed that CqHC was widely distributed, with the highest level in hepatopancreas, at lower amounts in hemocytes, gills and antennal gland. HC levels in hepatopancreas, hemocytes and gills from C. quadricarinatus were also compared after white spot syndrome virus (WSSV) infection. After 24h of infection, CqHC levels rose significantly higher than that of the controls (P<0.05) but then sharply declined over the infection period. The HC gene expression in hemocytes and gills (but not hepatopancreas) of crayfish injected with polysaccharides before infection were higher than that of the non-polysaccharide injected infected groups, showing an immunoprotective rate of 51.86% at 7days after exposure, indicating that polysaccharide can act as immunopotentiator and could improve the antiviral ability of C. quadricarinatus against WSSV.

Infection of juvenile edible crabs, Cancer pagurus by a haplosporidian-like parasite

This study aimed to examine the pathobiology of a haplosporidian-like infection in juvenile (pre-recruit) edible crabs (Cancer pagurus) from two locations in South West Wales, UK. Infected crabs showed no external symptoms of the disease but dissection revealed an infected and hypertrophic antennal gland. Histological examination showed extensive parasitisation of the antennal gland overlying the hepatopancreas. Heavily infected crabs also showed the presence of parasites with morphological similarities to Haplosporidia in the labyrinth of the antennal gland and in the gills. The spread of the infection from the antennal gland to the gills suggests that these parasites are released into the haemolymph. Attempts to characterise the haplosporidian-like organism using several primers previously shown to amplify members of the phylum Haplosporidia failed. The prevalence of infection in juvenile edible crabs varied throughout the sampling period of November 2011 to July 2012 with the lowest level of ca. 15% in November peaking at 70% in March. This parasite may represent a threat to the sustainability of edible crab fisheries in this region if the damage observed in the antennal gland and gills results in host mortality. The identification of these parasites as members of the phylum Haplosporidia based on morphology alone must be seen as tentative in the absence of sequence data.

Maternal and Environmental Effects on Symbiont-Mediated Antimicrobial Defense

Bacteria produce a remarkable diversity of bioactive molecules with antimicrobial properties. Despite the importance of such compounds for human medicine, little is known about the factors influencing antibiotic production in natural environments. Recently, several insects have been found to benefit from symbiont-produced antimicrobial compounds for defense against pathogenic microbes. In the European beewolf, Philanthus triangulum (Hymenoptera, Crabronidae), bacteria of the genus Streptomyces provide protection against pathogens by producing antimicrobials on the larval cocoon during hibernation, thereby significantly enhancing the survival probability of the beewolf larva. To investigate the effects of abiotic and biotic factors on antibiotic production, we exposed beewolf cocoons to different environmental conditions and quantified the amount of Streptomyces-produced antibiotics by using gas chromatography/mass spectrometry (GC/MS). The results revealed no significant influence of temperature, humidity, or pathogen load on the antibiotic amount, indicating that antibiotic production is not affected by current environmental conditions but rather may be optimized to serve as a reliable long-term protection during the unpredictable phase of beewolf hibernation. However, the amount of antibiotics was positively correlated with the symbiont population size on the cocoon, which in turn is affected by the number of Streptomyces cells provided by the mother into the brood cell. Additionally, we found a positive correlation between the amount of hydrocarbons and the number and length of bacterial cells in the antennal gland secretion, suggesting that maternal investment affects symbiont growth and, thus, antibiotic production on the larval cocoon.

Procambarin: A glycine-rich peptide found in the haemocytes of red swamp crayfish Procambarus clarkii and its response to white spot syndrome virus challenge

We cloned a novel glycine-rich peptide, procambarin, from the haemocytes of unchallenged crayfish Procambarus clarkii. The mature peptide (155 residues) has a13.44 KDa molecular mass with a theoretical pI about 12.12. It is characterized by a high level of glycine (57.42%) and a threefold repeated motif GLKPNVGGGGGFGGG. Generally, it belongs to cationic glycine-rich peptide. The transcripts of this peptide were detected in many tissues. The haemocytes showed the highest expression of glycine-rich peptide mRNA, followed by the ovaries, antennal gland and intestine. The gill, hepatopancreas and heart showed little expression of this peptide and no expression was detected in the musculature. There is no intron in the ORF of it. The fluctuation of mRNA expression level of procambarin after WSSV challenge indicates that this peptide participates in the antiviral immune reaction.

Adaptation to freshwater in the palaemonid shrimp Macrobrachium amazonicum: comparative ontogeny of osmoregulatory organs

The ontogeny of osmoregulatory organs was studied in two geographically isolated populations of the palaemonid shrimp Macrobrachium amazonicum, one originating from the Amazon estuary (A) and the other from inland waters of the Pantanal (P) in northeastern and southwestern Brazil, respectively. A previous investigation had shown that the estuarine population is able to hypo-osmoregulate in seawater, whereas the hololimnetic inland population has lost this physiological function. In the present study, the structural development of the branchial chamber and excretory glands and the presence of Na(+)/K(+)-ATPase (NKA) were compared between populations and between larval and juvenile stages after exposure to two salinities representing hypo- and hypertonic environments. In the newly hatched zoea I stage of both populations, gills were absent and NKA was localized along the inner epithelium of the branchiostegite. In intermediate (zoea V) and late larval stages (decapodids), significant differences between the two populations were observed in gill development and NKA expression. In juveniles, NKA was detected in the gills and branchiostegite, with no differences between populations. At all developmental stages and in both populations, NKA was present in the antennal glands upon hatching. The strong hypo-osmoregulatory capacity of the early developmental stages in population A could be linked to ion transport along the inner side of the branchiostegite; this seemed to be absent or weak in population P. The presence of fully functional gills expressing NKA appears to be essential for efficient hyper-osmoregulation in late developmental stages during successful freshwater adaptation and colonization.

Salinity tolerance of cross-breed shrimp families: morphological and biochemical approaches

Marine invertebrates respond to fluctuations in their environment throughout development and growth. Homeostatic adaptation is mediated in part through the regulation of Na+-K+ ATPase enzymes and members of this class of enzymes are localized to the antennal gland of many crustaceans. In this study, we investigated the morphological and biochemical changes of the antennal glands in eight families of cross-breed post-larval shrimp subjected to hypotonic (10 ppt) and isotonic (20 ppt) conditions. The greatest changes in the antennal glands were detected in the hypotonic condition with families two and five cross-breed shrimp exhibiting the highest tolerance to this diluted salinity. The changes detected in hypotonic tolerant shrimp included enlargement of coelomosac and labyrinth spaces, the presence of many dense cytoplasmic globules in podocytes as well as highly wavy apical architecture of labyrinth cells. Several alterations were also observed at the subcellular level such as widening of the intercellular spaces between podocytes, organelle destruction, and decreased basal membrane in-folding in labyrinth cells. In addition, shrimp exposed to hypotonic conditions displayed decreased expression of both α and β subunits of Na+-K+ ATPase and decreased enzymatic activity. The changes observed in animals upon exposure to hypotonic stress are discussed.

Two plasmolipins from the black tiger shrimp, Penaeus monodon and their response to virus pathogens

Two isoforms of plasmolipin were initially identified from the black tiger shrimp (Penaeus monodon) EST database and completed using 5′ RACE to reveal complete cDNAs of 558bp (PmPLP1) and 537bp (PmPLP2) with 87% nucleotide sequence identity. The deduced amino acid sequences contained four-transmembrane domains and showed the highest amino acid identity (49% and 51%, respectively) to the honey bee (Apis mellifera) chemokine-like factor (CKLF), with a very similar hydrophobic pattern to other plasmolipins. Transcripts of PmPLP1 and PmPLP2 were observed in all tested shrimp tissues with the highest expression levels in the gill and epipodite for PmPLP1 and in the hemocytes and antennal gland for PmPLP2. PmPLP1 transcript levels were significantly upregulated in hemocytes at 24 and 72h post infection (hpi) with yellow head virus (YHV) (7.4- and 14.7- fold, respectively), but only after 72hpi by white spot syndrome virus (WSSV). In contrast, PmPLP2 was only slightly (but statistically significant) up-regulated with YHV and WSSV. Thus, PmPLPs have the potential to be a part of viral infection mechanism or defense response. This is the first characterization of a plasmolipin gene in crustaceans.

Additional Host, Distribution, and Prevalence Records for Alloglossidium caridicolum and Alloglossidium dolandi (Digenea: Macroderoididae), Parasites of Procambarid Crayfish Inhabiting the Coastal Plains of the Southeastern United States

Abstract Examination of antennal glands of 11 specimens of Procambarus zonangulus, from a single locality in Texas and those of 4 specimens of Procambarus blandingii, from a single locality in South Carolina, indicated infections with adult Alloglossidium spp. Prevalence of Alloglossidium caridicolum in P. zonangulus was 18.2%, and that of Alloglossidium dolandi in P. blandingii was 100%. Both collections represented new hosts and distribution records for both digene species.

Molecular cloning of heat shock protein 60 (PtHSP60) from Portunus trituberculatus and its expression response to salinity stress

Heat shock protein 60 (HSP60) is a highly conserved and multi-functional molecular chaperone that plays an essential role in both cellular metabolism and stress response. Portunus trituberculatus is an important marine fishery and aquaculture species, and water salinity condition influenced its artificial propagations significantly. In order to investigate the function of P. trituberculatus HSP60 against osmotic stress, P. trituberculatus HSP60 gene was firstly cloned. The full-length cDNA of PtHSP60 contains 1,743 nucleotides encoding 577 amino acids with a calculated molecular weight of 61.25 kDa. Multiple alignments indicated that the deduced amino acid sequences of PtHSP60 shared a high level of identity with invertebrate and vertebrate HSP60 sequence including shrimp, fruit fly, zebrafish, and human. The expression profiles of PtHSP60 at mRNA and protein levels under salinity treatment were investigated by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. It was found that the mRNA transcripts of PtHSP60 gene varied among different tissues under normal salinity conditions, and the antennal gland showed the highest expression level among the tissues tested. As for low salinity challenge, the mRNA expression of PtHSP60 gene was higher in the gill and appendicular muscle compared with other tissues, and gill and hypodermis represented the higher gene expressions during the hyperosmotic stress, which indicated that those tissues were salinity-sensitive tissues. In addition, salinity challenges significantly altered the expression of PtHSP60 at mRNA and protein level in a salinity- and time-dependent manner in P. trituberculatus gill tissue. The results indicate that PtHSP60 played important roles in mediating the salinity stress in P. trituberculatus.

Cloning of aquaporin-1 of the blue crab, Callinectes sapidus: its expression during the larval development in hyposalinity.

BackgroundOntogenetic variation in salinity adaptation has been noted for the blue crab, Callinectes sapidus, which uses the export strategy for larval development: females migrate from the estuaries to the coast to spawn, larvae develop in the ocean, and postlarvae (megalopae) colonize estuarine areas. We hypothesized that C. sapidus larvae may be stenohaline and have limited osmoregulatory capacity which compromises their ability to survive in lower salinity waters. We tested this hypothesis using hatchery-raised larvae that were traceable to specific life stages. In addition, we aimed to understand the possible involvement of AQP-1 in salinity adaptation during larval development and during exposure to hyposalinity.ResultsA full-length cDNA sequence of aquaporin (GenBank JQ970426) was isolated from the hypodermis of the blue crab, C. sapidus, using PCR with degenerate primers and 5′ and 3′ RACE. The open reading frame of CasAQP-1 consists of 238 amino acids containing six helical structures and two NPA motifs for the water pore. The expression pattern of CasAQP-1 was ubiquitous in cDNAs from all tissues examined, although higher in the hepatopancreas, thoracic ganglia, abdominal muscle, and hypodermis and lower in the antennal gland, heart, hemocytes, ovary, eyestalk, brain, hindgut, Y-organs, and gill. Callinectes larvae differed in their capacity to molt in hyposalinity, as those at earlier stages from Zoea (Z) 1 to Z4 had lower molting rates than those from Z5 onwards, as compared to controls kept in 30 ppt water. No difference was found in the survival of larvae held at 15 and 30 ppt. CasAQP-1 expression differed with ontogeny during larval development, with significantly higher expression at Z1-2, compared to other larval stages. The exposure to 15 ppt affected larval-stage dependent CasAQP-1 expression which was significantly higher in Z2- 6 stages than the other larval stages.ConclusionsWe report the ontogenetic variation in CasAQP-1 expression during the larval development of C. sapidus and the induction of its expression at early larval stages in the exposure of hyposalinity. However, it remains to be determined if the increase in CasAQP-1 expression at later larval stages may have a role in adaptation to hyposalinity.

Roles of crustacean hyperglycaemic hormone in ionic and metabolic homeostasis in the Christmas Island Blue crabDiscoplax celeste

There is a growing body of evidence implicating the involvement of crustacean hyperglycaemic hormone (CHH) in ionic homeostasis in decapod crustaceans. However, little is known regarding hormonally influenced osmoregulatory processes in terrestrial decapods. As many terrestrial decapods experience opposing seasonal demands upon ionoregulatory physiologies, we reasoned that these would make interesting models in which to study the effect of CHH upon these phenomena. In particular, those (tropical) species that also undergo seasonal migrations might be especially informative, as we know relatively little regarding the nature of CHHs in terrestrial decapods, and hormonally mediated responses to seasonal changes in metabolic demands might also be superimposed or otherwise integrated with those associated with ionic homeostasis. Using Discoplax celeste as a model crab that experiences seasonal extremes in water availability, and exhibits diurnal and migratory activity patterns, we identified two CHHs in the sinus gland. We biochemically characterised (cDNA cloning) one CHH and functionally characterised (in terms of dose-dependent hyperglycaemic responses and glucose-dependent negative feedback loops) both CHHs. Whole-animal in situ branchial chamber (22)NaCl perfusion experiments showed that injection of both CHHs increased gill Na(+) uptake in a seasonally dependent manner, and (51)Cr-EDTA clearance experiments demonstrated that CHH increased urine production by the antennal gland. Seasonal and salinity-dependent differences in haemolymph CHH titre further implicated CHH in osmoregulatory processes. Intriguingly, CHH appeared to have no effect on gill Na(+)/K(+)-ATPase or V-ATPase activity, suggesting unknown mechanisms of this hormone's action on Na(+) transport across gill epithelia.