Effects of cadmium on respiratory burst,intracellular Ca2+ and DNA damage in the white shrimp Litopenaeus vannamei

Acute effects of heavy metal ions on shrimp have been an area of intense study worldwide. However, the molecular mechanism by which cadmium-induced injury occurs remains largely unclear, and methods for mitigating toxicity in vivo have rarely been reported. In this study, the changes in respiratory burst and intracellular free calcium in haemocytes of pacific white shrimp, Litopenaeus vannamei, after exposure to Cd²⁺ (CdCl₂) were examined using flow cytometry. Meanwhile, DNA damage and repair in haemocytes and hepatopancreas cells were studied using the comet assay. Respiratory burst generation, intracellular Ca²⁺ concentration ([Ca²⁺]i) and DNA damage in haemocytes and hepatopancreas cells all exhibited a dose-dependent increase and a time-dependent change after treatment with Cd²⁺ compared with controls. These results indicate that Cd can induce oxidative stress and DNA damage in the shrimp L. vannamei. Moreover, the results also demonstrate that these parameters can be used as sensitive indicators of exposure to this genotoxicant.     

The immune cellular effectors of terrestrial isopod Armadillidium vulgare: meeting with their invaders, Wolbachia

Background

Most of crustacean immune responses are well described for the aquatic forms whereas almost nothing is known for the isopods that evolved a terrestrial lifestyle. The latter are also infected at a high prevalence with Wolbachia, an endosymbiotic bacterium which affects the host immune system, possibly to improve its transmission. In contrast with insect models, the isopod Armadillidium vulgare is known to harbor Wolbachia inside the haemocytes.

Methodology/Principal Findings

In A. vulgare we characterized three haemocyte types (TEM, flow cytometry): the hyaline and semi-granular haemocytes were phagocytes, while semi-granular and granular haemocytes performed encapsulation. They were produced in the haematopoietic organs, from central stem cells, maturing as they moved toward the edge (TEM). In infected individuals, live Wolbachia (FISH) colonized 38% of the haemocytes but with low, variable densities (6.45±0.46 Wolbachia on average). So far they were not found in hyaline haemocytes (TEM). The haematopoietic organs contained 7.6±0.7×10³ Wolbachia, both in stem cells and differentiating cells (FISH). While infected and uninfected one-year-old individuals had the same haemocyte density, in infected animals the proportion of granular haemocytes in particular decreased by one third (flow cytometry, Pearson''s test = 12 822.98, df = 2, p<0.001).

Conclusions/Significance

The characteristics of the isopod immune system fell within the range of those known from aquatic crustaceans. The colonization of the haemocytes by Wolbachia seemed to stand from the haematopoietic organs, which may act as a reservoir to discharge Wolbachia in the haemolymph, a known route for horizontal transfer. Wolbachia infection did not affect the haemocyte density, but the quantity of granular haemocytes decreased by one third. This may account for the reduced prophenoloxidase activity observed previously in these animals.     

Molecular cloning and expression of a C-type lectin gene from Venerupis philippinarum

C-type lectins have been demonstrated to play important roles in invertebrate innate immunity by mediating the recognition of pathogens and clearing the micro-invaders. In the present study, a C-type lectin gene (denoted as VpCTL) was identified from Venerupis philippinarum by expressed sequence tag and rapid amplification of cDNA ends approaches. The full-length cDNA of VpCTL consists of 904 nucleotides with an open-reading frame of 456 bp encoding a peptide of 151 amino acids. The deduced amino acid sequence of VpCTL shared high similarity with C-type lectins from other species. The C-type lectin domain and the characteristic EPN and WND motifs were found in VpCTL. The VpCTL mRNA was dominantly expressed in the haemocytes of the V. philippinarum. After Listonella anguillarum challenge, the temporal expression of VpCTL mRNA in haemocytes was increased by 97- and 84-fold at 48 and 96 h, respectively. With high expression level in haemocytes and hepatopancreas, and the up-regulated expression in haemocytes indicted that VpCTL was perhaps involved in the immune responses to L. anguillarum challenge.     

Effects of infection of EGFP-expressing Escherichia coli on haemocytes in Ciona intestinalis

The effects of infection of EGFP-expressing Escherichia coli on the haemocytes of the ascidian Ciona intestinalis were investigated. The results showed that THC of the infected individuals changed significantly. Hyaline amoebocytes phagocytosed E. coli in 5 min and excreted lysosome particles that attached to the surface of the bacteria. Granular amoebocytes released lots of particles for humoral immunity while stem-cell-like haemocytes remained intact. With the increase in THC, the stem-cell-like haemocytes showed division and proliferation. A small portion of hyaline amoebocytes was at early apoptosis stage 1 h after infection and typical apoptosis bodies emerged in granular amoebocytes. A few of the infected haemocytes showed DNA damage using SCGE assay. Flow cytometry analysis revealed an obvious apoptosis peak in infected haemocytes. In conclusion, apoptosis was found to be an important immune response of ascidian haemocytes response to bacterial infection. To our best knowledge, this is the first report of the occurrence of apoptosis of haemocytes in ascidians.     

Apoptosis of tiger shrimp (Penaeus monodon) haemocytes induced by Escherichia coli lipopolysaccharide

This study was aimed at investigating the toxicity mechanism of lipopolysaccharide (LPS) on Penaeus monodon haemocytes at a cellular level. Reactive oxygen species (ROS) production, nitric oxide (NO) production, non-specific esterase activity, cytoplasmic free-Ca^2 + (CF-Ca^2 +) concentration, DNA damaged cell ratio and apoptotic cell ratio of in vitro LPS-treated haemocytes were measured by flow cytometry. Two concentrations of Escherichia coli LPS (5 and 10 μg mL^? 1) were used. Results showed that ROS production, NO production and CF-Ca^2 + concentration were significantly induced in the LPS-treated haemocytes. Ratio of DNA damaged cell and apoptotic cell increased caused by LPS, while esterase activity increased at the initial 60 min and dropped later. The initial increase in esterase activity suggested that LPS activated the release of esterase, and the later decrease might result from apoptosis. These results indicated that LPS would induce oxidative stress on shrimp haemocytes, and cause Ca^2 + release, DNA damage and subsequently cell apoptosis. This process of ROS/RNS-induced Ca^2 +-mediated apoptosis might be one of the toxicity mechanisms of LPS on shrimp haemocytes.     

Identification and characterization of an intracellular Cu,Zn-superoxide dismutase (icCu/Zn-SOD) gene from clam Venerupis philippinarum

Superoxide dismutase (SOD, EC 1.15.1.1) represents one kind of enzyme involved in scavenging the high level of reactive oxygen species (ROS) into molecular oxygen and hydrogen peroxide. In the present study, the intracellular Cu/Zn-SOD gene (icCu/Zn-SOD) of Venerupis philippinarum (denoted as VpSOD) was identified from haemocytes by homology cloning and RACR approaches. The full-length cDNA of VpSOD consisted of 910 nucleotides with a canonical polyadenylation signal sequence AATAAA, a polyA tail, and an open-reading frame of 465 bp encoding 154 amino acids. The deduced amino acid of VpSOD shared high similarity with the icCu/Zn-SODs from other species, indicating that VpSOD should be a new member of icCu/Zn-SOD family. Several highly conserved motifs including Cu, Zn binding sites (H⁴⁶, H⁴⁸, H⁶³, H¹²⁰ for Cu binding, and H⁶³, H⁷¹, H⁸⁰, D⁸³ for Zn binding), intracellular disulfide bond and two Cu, Zn SOD signatures were also identified in VpSOD. The temporal expression of VpSOD in haemocytes after Vibrio anguillarum challenge was recorded by quantitative real-time RT-PCR. The relative expression level of VpSOD mRNA was up-regulated rapidly at 6 h post-infection and reached 18-fold of the control group. After a drastic decrease at 12 h, the expression level increased again and reached 22-fold to that in the control group at 96 h post-infection. All these results indicated that VpSOD was an acute-phase protein involved in the immune responses of V. philippinarum.     

Species composition and distribution of free-living marine nematodes in Vostok Bay, Sea of Japan

The qualitative and quantitative composition of free-living marine nematodes have been studied in Vostok Bay (Peter the Great Bay, Sea of Japan). It is found that the population density of nematodes in the bottom sediments of Vostok Bay shows an uneven distribution. The mean population density equaled 56800 ± 23400 specimens/m². A correlation has been revealed between the population density of nematodes and the substrate type. Altogether, 85 species of nematodes have been found; they were dominated by Sabatieria palmaris, Rhabdodemania orientalis, Araeolaimus parvibulbosus, Oncholaimium paraolium, Doryolaimopsis peculiaris, and Metachromadora itoi. Six taxocenoses of nematodes were distinguished, taking into account the species dominating in the population density and using cluster analysis of the obtained data. The dominating trophic assemblage of nematodes was “scrapers.” In general, the species composition of nematodes in Vostok Bay is characterized by the relatively great similarity with that in other areas of Peter the Great Bay (Sea of Japan).     

Variation in nitrogen isotopic composition in the Selenga river watershed, Mongolia

The stable nitrogen (N) isotope ratio (??¹⁵N) has been used to examine the anthropogenic N input (i.e., septic water, wastewater, and manure) to aquatic ecosystems, because anthropogenic N generally has a ??¹⁵N signature distinct from that found in nature. Aquatic organisms and the derived organic matter such as sediments are reported to become increasingly enriched in ¹⁵N as the human population density increases in watersheds. However, little is known about the relationship in steppe ecosystems, where the livestock population is greater than that of humans. Here, we conducted a preliminary study in the Selenga river mainstream watershed in Mongolia, which covers an area of approximately 300,000?km². A multiple regression analysis revealed that the ??¹⁵N of the riverine sediment was significantly affected by the human population density and more significantly by livestock population density. The population density, including both humans and livestock, significantly influenced ??¹⁵N of the macrophytic Potamogeton spp. The results showed that ??¹⁵N of riverine organic matter can be an indicator of the human and livestock population density, which is likely associated with the status of N cycles in livestock-dominated watersheds.     

Bursicon-induced adenyl cyclase activity in the haemocytes of the American cockroach

Partially purified preparations of bursicon were injected, between ecdyses, into American cockroaches (Periplaneta americana) held at 4°C. After short periods of time, the haemocytes were removed and found to contain appreciable amounts of cyclic adenosine 5′-monophosphate (cAMP). Identical experiments using animals reared at 23°C did not result in the accumulation of cAMP. These and additional data suggest that phosphodiesterase is not active during the initial stages of bursicon-stimulated adenyl cyclase.Incubation of haemocyte soluble proteins in the presence of [³H]cAMP, followed by molecular sieve chromatography on P-60 polyacrylamide gel, shows the binding of cAMP to large molecular weight proteins. Similar experiments, but with phosphodiesterase inhibition revealed a reduced level of radioactive bound protein. From these studies one can assume that the haemocytes contain a soluble cAMP-dependent protein kinase.If either [¹⁴C]-leucine or [¹⁴C]-tyrosine is incubated with whole blood taken from animals between ecdyses, little or no uptake by the haemocytes can be seen. Identical studies, but with the addition of cAMP, results in a dramatic enhancement of amino acid uptake. It appears that bursicon stimulates blood-cell adenyl cyclase, which in turn produces cAMP. The cAMP subsequently activates a specific protein kinase that phosphorylates potential membrane proteins. The membrane phosphoprotein may be instrumental in the enhanced uptake of amino acids.     

Subcellular distribution of glycoside hydrolase and polysaccharide depolymerase enzymes in the rumen entodiniomorphid ciliatePolyplastron multivesiculatum

The distribution of 12 acid hydrolase and two polysaccharide depolymerase enzymes in the rumen entodiniomorphid ciliatePolyplastron multivesiculatum, isolated from the ovine rumen 2 h after feeding, was examined by differential and density-gradient centrifugation. Approximately 60%–70% of the recovered activity was sedimentable in fractions prepared by centrifugation at 10³ g for 10 min (F1) and 10⁴ g for 10 min (F2) with 25%–35% of the acid hydrolases and 15%–20% of acid phosphatase and the polysaccharidases remaining nonsedimentable (in fraction F5) after centrifugation at 10⁵ g for 60 min. Approximately 60% of the sedimentable activity was located in fraction F1. Latency of the hydrolase activity was demonstrated. After isopycnic centrifugation in sucrose density gradients, the hydrolytic enzymes cosedimented in acid phosphatase-containing, membrane-bound, pleomorphic lysosomelike vesicles 0.1–1.0 m in size, with a mean equilibrium density of 1.17 (1.15–1.19) g/ml.     

Susceptibility of Larvae of Galleria mellonella to Infection by Aspergillus fumigatus is Dependent upon Stage of Conidial Germination

The ability of conidia of the human pathogenic fungus Aspergillus fumigatus to kill larvae of the insect Galleria mellonella was investigated. Conidia at different stages of the germination process displayed variations in their virulence as measured using the Galleria infection model. Non-germinating (‘resting’) conidia were avirulent except when an inoculation density of 1 × 10⁷ conidia per insect was used. Conidia that had been induced to commence the germination process by pre-culturing in growth medium for 3 h were capable of killing larvae at densities of 1 × 10⁶ and 1 × 10⁷ per insect. An inoculation density of 1 × 10⁵ conidia per insect remained avirulent. Conidia in the outgrowth phase of germination (characterised as the formation of a germ tube) were the most virulent and were capable of killing 100% of larvae after 5 or 24 h when 1 × 10⁷ or 1 × 10⁶ conidia, that had been allowed to germinate for 24 h, were used. Examination of the response of insect haemocytes to conidia at different stages of the germination process established that haemocytes could engulf non-germinating conidia and those in the early stages of the germination process but that conidia, which had reached the outgrowth stages of germination were not phagocytosed. The results presented here indicate that haemocytes of G. mellonella are capable of phagocytosing A. fumigatus conidia less than 3.0 μm in diameter but that conidia greater than this are too large to be engulfed. The virulence of A. fumigatus in G. mellonella larvae can be ascertained within 60–90 h if infection densities of 1 × 10⁶ or 1 × 10⁷ activated conidia (pre-incubated for 2–3 h) per insect are employed.     

Cloning and expression of an actin gene in the haemocytes of pearl oyster (Pinctada fucata, Gould 1850)

An actin gene (designated pfact1) of pearl oyster, Pinctada fucata, was cloned from haemocytes by the techniques of homological cloning and rapid amplification of cDNA ends (RACE). The full length of Pfact1 cDNA was 1608 bp in length, having a 5′ untranslated region (UTR) of 82 bp, a 3′ UTR of 395 bp, and an open reading frame (ORF) of 1131 bp encoding a polypeptide of 376 amino acids with a predicted molecular weight of 41.76 kDa and an estimated isoelectric point of 5.29. Sequence analysis revealed that Pfact1 shared high similarity with other actins and was more closely related to vertebrate cytoplastic actins than muscle types. Phylogenetic analysis indicated that molluscan actins could also be generally grouped into two classes: muscle type and cytoplasmic type, although both are similar to vertebrate cytoplastic actins. Fluorescent real-time quantitative RT-PCR was used to examine the expression level of Pfact1 in haemocytes of P. fucata after the challenge of Vibrio alginolyticus, and results showed that Pfact1 exhibited stable expression in all time points, indicating that Pfact1 could be a suitable internal control for gene expression analysis in haemocytes of P. fucata.     

Intracellular dynamics of alkaline phosphatase-containing granules in electropermeabilized human neutrophils

 Human neutrophils possess alkaline phosphatase-containing intracellular granules which are upregulated to the cell surface upon stimulation. The mechanism that governs the intracellular dynamics of these granules is, however, poorly understood. The aim of the present study was to investigate the possible participation of GTP-binding proteins in the reorganization and exocytosis of the alkaline phosphatase-containing granules using electropermeabilized cells. Biochemical assays using intact neutrophils showed that the alkaline phosphatase activity was upregulated and exocytosed into the extracellular space upon stimulation with AlF₄ ^– and N-formyl peptide. This upregulation was inhibited by treatment of cells with pertussis toxin and botulinum toxin. Alkaline phosphatase activity was also upregulated in electropermeabilized cells stimulated with guanosine 5′-O-(3-thiotriphosphate) (GTPγS), but not with guanosine 5′-O-(2-thiodiphosphate) (GDPβS). Cytochemically, alkaline phosphatase-containing granules were dispersed throughout the cytoplasm in unstimulated electropermeabilized neutrophils. Upon stimulation with GTPγS, but not with GDPβS, these granules fused to form elongated tubular structures which eventually became associated with the plasma membrane. Nocodazole disturbed the reorganization of the alkaline phosphatase-containing granules in cells stimulated with GTPγS. The results from this study indicate that GTP-binding proteins participate in the reorganization and exocytosis of alkaline phosphatase-containing granules associated with the microtubules in electropermeabilized human neutrophils. Accepted: 31 March 1998     

Growth-promoting effect of haemocytes on insect epidermis in vitro

The epidermis of 21-day-old leg regenerates of cockroaches (Leucophaea maderae) was cultivated in vitro. Outgrowth of the epidermis only occurred in connexion with haemocytes.Haemocytes contaminating the epidermal explants show strong adherence to epidermal cells. The epidermal cells adhering to moving haemocytes are stretched out to long projections or completely pulled out of the epithelium. When more haemocytes are present, they can form an uninterrupted line at the margin of the epidermis. By the adhesion of marginal cells of the epidermis to the moving haemocytes, the epithelium is apparently pulled out into broad tongues. In these tongues the epidermal cells become highly flattened, especially at the front, and soon begin to divide. Outgrowth in the tongues continues only as long as there are haemocytes at the front. When they have disappeared, outgrowth stops, the flattened epidermal cells detach from the glass surface, round up, and the outgrown tissue may withdraw again.For further analysis of the interactions of haemocytes and epidermal cells the epidermis is placed on a monolayer of haemocytes. The epidermis rapidly grows out on such a monolayer. The epidermal cells either move over or under the haemocytes indicating that there are substances on both sides of the haemocytes which are attractive to the epidermal cells and cause their flattening and outgrowth. Similar outgrowth occurs on fixed monolayers of haemocytes. There is no outgrowth on areas where the monolayer has been scraped away. No principal differences can be found between monolayers consisting almost exclusively of either plasmatocytes or granular haemocytes.The similarities of the observed interactions of haemocytes and epidermal cells to encapsulation and wound healing are pointed out. A hypothesis is presented which assumes that the haemocytes during wound healing not only serve as a mechanical support but also as a chemical guide by which the closure of the wound by epidermal cells is enhanced.