Acute toxicity of mixture of acetaminophen and ibuprofen to Green Neon Shrimp, Neocaridina denticulate.

In recent years, numerous studies have indicated that various long-term use drugs, such as antibiotics or analgesics, not only cannot be completely decomposed via sewage treatment but also exhibit biological toxicity if they enter the environment; thus, the release of these drugs into the environment can damage ecological systems. This study sought to investigate the acute toxicity of two commonly utilized analgesics, ibuprofen (IBU) and acetaminophen (APAP), to aquatic organisms after these drugs have entered the water. To address this objective, the acute toxicity (median lethal concentration, LC50, for a 96-h exposure) of IBU alone, APAP alone, and mixtures containing different ratios of IBU and APAP in green neon shrimp (Neocaridina denticulata) were measured. The results of four tests revealed that the 96-h LC50 values for IBU and APAP alone were 6.07 mg/L and 6.60 mg/L, respectively. The 96-h LC50 for a 1:1 mixture of IBU and APAP was 6.23 mg/L, and the toxicity of this mixture did not significantly differ from the toxicity of either drug alone (p<0.05). The experimental results for mixtures containing unequal ratios of IBU and APAP indicated that mixtures with high APAP concentrations and low IBU concentrations exhibited markedly greater toxicity in N. denticulata (LC50=4.78 mg/L) than APAP or IBU alone. However, mixtures with high IBU concentrations and low APAP concentrations exhibited lower toxicity in N. denticulata (LC50=6.78 mg/L) than IBU or APAP alone. This study demonstrated that different mixtures of IBU and APAP were associated with different toxic effects in green neon shrimp.

Function of an anti-lipopolysaccharide factor (ALF) isoform isolated from the hemocytes of the giant freshwater prawn Macrobrachium rosenbergii in protecting against bacterial infection.

In this study, a 780-bp full-length cDNA encoding Macrobrachium rosenbergii anti-lipopolysaccharide factor (MrALF) from hemocytes was cloned and identified. The ALF isoform exhibited immune activities, and its concentration in hemolymph was determined. An in vivo expression study showed that the ALF mRNA level of hemocytes could be induced by lipopolysaccharides (LPSs) in an exposure time-dependent manner. Purified recombinant MrALF (rMrALF) expressed in the yeast Pichia pastoris SMD1168 eukaryotic protein expression system demonstrated antibacterial activity against the Gram-negative prawn pathogen Aeromonas hydrophila (minimum inhibitory concentration (MIC)=0.806µM, minimum bactericidal concentration (MBC)=1.606µM) but not the Gram-positive pathogen Lactococcus garvieae exposed to 25.696µM of rALF. However, rMrALF can bind to Gram-negative and -positive bacteria. An in vivo expression study demonstrated that the ALF concentrations in prawn hemocytes and plasma were 0.176µM and 0.168µM, respectively; following LPS treatment for 6h, the corresponding concentrations were 0.133µM in hemocytes and 0.272µM in plasma. Furthermore, the percentage of hemocytes phagocytosing bacteria cells was higher in hemoyctes previously treated with MrALF than those treated with sterile medium. These results suggest that in the innate immune response of M. rosenbergii, the MrALF from hemocytes may play an opsonin during a bacterial invasion.

Differential immune responses of the green neon shrimp (Neocaridina denticulate) to dipropyl phthalate.

This study set out to understand the sublethal effect of xenobiotic phthalate esters (PAEs) on the relationship between the susceptibility of shrimp to bacterial infection and the immune response of the shrimp. Neocaridina denticulate were exposed to different concentrations of the PAE dipropyl phthalate (DPrP), and mortality and six immune parameters were measured on days 1, 3, 5, and 10 after exposure. On days 1 and 3 after exposure, shrimp exposed to 0, 1, 5, 10, and 50 mg/L of DPrP and challenged with Aeromonas veronii experienced 14% and 16%, 16% and 16%, 18% and 18%, 34% and 24%, and 38% and 26% mortality, respectively. On day 1, five immune parameters (acid phosphatase, AcP; ß-glucuronidase, ß-Glu; phenoloxidase, PO; superoxide dismutase, SOD; and haemocyanin mRNA) were significantly altered in the all of the groups treated with DPrP compared to the untreated shrimp and were elevated in the 10 mg/L- and 50 mg/L-treated groups. Beta-Glu activity and haemocyanin mRNA levels were significantly increased in a dose-dependent manner. These results suggest that the increased susceptibility of N. denticulate exposed to DPrP is short-term and may be related to the increased expression of DPrP-induced immune mediators.

Genes are differentially expressed at transcriptional level of Neocaridina denticulata following short-term exposure to nonylphenol.

To assess the toxicity of nonylphenol towards aquatic crustaceans, Neocaridina denticulata were exposed short-term to sublethal concentration (0.001-0.5 mg/L). Following treatment, differentially expressed genes were identified using suppression subtractive hybridization on samples prepared from whole specimens. There were 20 differentially expressed sequence tags that corresponded to known genes and could be divided into six functional classes: defence, translation, metabolism, ribosomal gene expression, respiration, and genes involved in the stress response. Using semi-quantitative RT-PCR, we found that 14 of the differentially expressed sequence tags significantly responded to nonylphenol, including six at a nominal concentration of 0.01 mg/L; among them, 12 genes were down-regulated. These results suggest that under non-lethal concentrations of nonylphenol, the polluted aquatic environment may still present a potential risk to N. denticulata.

Effect of nonylphenol on giant freshwater prawn (Macrobrachium rosenbergii) via oral treatment: toxicity and messenger RNA expression of hemocyte genes.

A previous in vitro study has indicated that two alkylphenol ethoxylates (APEs) could potentially damage hemocytes and influence cellular immunity of prawns (Macrobrachium rosenbergii). The aim of this study was to investigate the effects of nonylphenol (NP) on susceptibility to a pathogen and on the mRNA expression of hemocyte genes, including four immune-related genes. NP at different concentrations was fed continuously to prawn (M. rosenbergii) for 1, 3, 6, and 9 days. Challenging prawns with Lactobacillus garvieae resulted in 44-50%, 20-24% and 10-12% mortality were detected after prawns were fed with 100, 10 and 1microNP/prawn for 6 days, respectively. In comparison with control prawns fed with phosphate-buffered solution (PBS), the increase of mRNA levels of four immune-related genes, alpha-2 microglobulin (alpha-2m), antimicrobial peptides (amp), peroxinectin (pon), and prophenoloxidase (propo), was detected on days 1, 3 and 6 after feeding with 100microg/prawn; on day 9, only the mRNA level of amp of the NP-treated group was significantly increased, while that of the remaining groups was not different from that of the control. In addition, two other hemocyte genes were also studied, including a respiration-related gene, cytochrome oxidase subunit (cos), and an unknown gene, L12X3. The mRNA level of cos was elevated during the experimental period, but an increase of L12X3 expression was detected only on day 1 after treatment. Regarding sensitivity of these genes to NP, the results from NP-treated prawns on day 1 after treatment revealed (1) that mRNA expression of the six genes in the 100-microg-NP-treated group was significantly different from that of control group, (2) that the mRNA levels of three immune-related genes (amp, pon and propo) in 10-microg-treated group were significantly higher than that of control group, and (3) that a significant change of propo was detected in 1-microg-treated group. These results suggest that NP may enhance the immune response of prawns, but the effect created by a high concentration of NP may damage prawns, and then increase the susceptibility to pathogen.

Differentially enhanced gene expression in hemocytes from Macrobrachium rosenbergii challenged in vivo with lipopolysaccharide.

In order to better understand the immune response in prawns after treatment with the immunostimulant lipopolysaccharide (LPS), in this study, the differential gene expression of the hemocytes from LPS-injected versus non-injected prawns (Macrobrachium rosenbergii) were isolated and identified using suppression subtractive hybridization (SSH). The hemocytes were extracted after treatment for 1, 6, and 12h. The upregulated genes (i.e., where gene expression was elevated) were identified and could be divided into four classes on the basis of physiological function: genes concerning defense-related molecules, genes involved in energy-production (respiration), genes related to protein synthesis and folding, and genes with unknown function. The time-course for gene expression indicated that, except for expression of the gene anti-microbial peptide (amp), which was increased at 12h after LPS treatment, the expression of the other two immune-related genes was much earlier (at 1h), including alpha-2-macroglobulin (alpha2-M) and Mas-like protein (mas). These results suggest that in the early phase of LPS stimulation some immune reactions regulated by alpha-2M and Mas may be induced, such as the activation of prophenoloxidase activating system, opsonization, and anti-microbial activity. In addition, six unigenes with unproven function were particularly interesting and worthy of further study because their expression in LPS-treated hemocytes was clearly enhanced.

The effect of two CpG oligodeoxynucleotides with different sequences on haemocytic immune responses of giant freshwater prawn, Macrobrachium rosenbergii.

A previous study has demonstrated that the intrahaemocytic phenoloxidase (PO) activity of the prawn Macrobrachium rosenbergii was enhanced by CpG oligodeoxynucleotide (ODN) 2006, but not by os-ODN13. The study described herein determined the binding characteristics of the two ODNs to haemocytes. Treating haemocytes with FAM-ODN or FAM-ODN plus different concentrations of the same ODN revealed that both ODNs specifically bound to haemocytes. Results from haemocytes treated with FAM-ODN2006 plus ROX-os-ODN13 in a competitive assay indicated that about 91% of haemocytes were simultaneously bound by the two ODNs and that the remaining haemocytes were only bound by ODN2006; moreover, ODN2006 binding to haemocytes was stronger than that of os-ODN13. To clarify the interactive effect of the two ODNs on haemocytic function, mRNA levels of haemocytic genes from single or double ODN-injected prawns were determined by semi-quantitative RT-PCR. The expression of either the prophenoloxidase (proPO) or peroxinectin (pon) genes was elevated by ODN2006 but reduced by os-ODN13; furthermore, ODN2006-increased proPO expression was abated following treatment with os-ODN13. In comparison with ODN-injected prawns alone, the NF-kappaB inhibitor PDTC reduced the proPO mRNA levels induced by ODN2006, but it elevated proPO expression inhibited by os-ODN13. These results support the notion that os-ODN13 may be able to neutralise or negate the enhancing effect of ODN2006 on proPO expression via the NF-kappaB signalling pathway as well as an unknown signalling pathway.

Initiation of signal transduction of the respiratory burst of prawn haemocytes triggered by CpG oligodeoxynucleotides.

Phagocytosis is important in the immune system of the prawn and is believed to be a defence parameter. Previous studies have demonstrated that CpG oligonucleotides enhance the activation of the prophenoloxidase activating system of the prawn through either the G-protein/protein kinase C (PKC) or the cAMP pathway. This study investigated the influence of CpG ODN on the respiratory burst used as the indicator of phagocytic activity and on the initiation of the signal pathway in haemocytes of Macrobrachium rosenbergii. When haemocytes were treated in vitro with 50 microg ml(-1) of ODN2006 for 15 min, the increase of nitroblue-tetrazolium (NBT) reduction suggested that the respiratory burst of haemocytes can be enhanced by ODN2006 stimulation. In an attempt to determine which signal transduction pathway is involved in the enhancement effect, haemocytes were separately treated with activators or inhibitors of specific signalling components. The results showed that the NBT reduction of haemocytes increased after treatment with sodium fluoride (a G-protein activator) and decreased after treatment with GDP-beta-S (a G-protein inhibitor). When ODN2006-stimulated haemocytes were treated with GDP-beta-S, the inductive effect was significantly reduced. In haemocytes treated with 8-bromo-cAMP (a PKA activator), the NBT reduction was not significantly different from the control. The addition of phosphodiesterase-inhibiting caffeine, which inhibits the degradation of cAMP, decreased the NBT reduction of ODN2006-stimulated haemocytes; however, the addition of phenol-12-myristate-13-acetate (PMA) significantly increased the NBT reduction. When PMA-stimulated haemocytes were treated with chelerythrine (a PKC inhibitor), the induced NBT reduction was significantly reduced. Furthermore, the study of ODN2006-stimulated haemocytes treated with chelerythrine showed that the enhancement effect of ODN2006 on the NBT reduction was significantly decreased. All results suggest that the enhancement of the respiratory burst of prawn haemocytes is induced by ODN2006 via a PKC-activating signalling pathway, but negatively regulated via the cAMP pathway.

Cloning the prophenoloxidase cDNA and monitoring the expression of proPO mRNA in prawns (Macrobrachium rosenbergii) stimulated in vivo by CpG oligodeoxynucleotides.

The prophenoloxidase (proPO) system forms the basis of the non-specific defence system in crustaceans. The aim of this study was to develop an RT-PCR procedure to determine proPO gene expression. We used several degenerate primers designed from the conserved regions in amino acid sequences of proPOs from other species to clone the possible cDNA(s) from the haemocytes of the prawn Macrobrachium rosenbergii. One DNA fragment, 2016 bp long, was cloned by a combination of reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (3'/5'-RACE). This fragment could encode a putative polypeptide with 671 amino acids. Further analysis showed that this putative polypeptide contained six histidine residues and a thiol ester-like motif (GCGWPRHM) just like the structural features of proPOs from the shrimp Penaeus monodon. A partial fragment of the sequence with 934 bp containing three histidine residues and the thiol ester-like motif was used as a target to monitor the activation of the proPO gene by the semi-quantitative RT-PCR analysis. The result showed that the highest level of proPO mRNA was detected at 1h after in vivo injection with 5 microg of CpG oligodeoxynucleotide 2006 per prawn; in the same experiment, the highest PO activity was detected at 6 h after injection. In the control, a continuous and slow elevation of PO activity was observed during the experimental period, but such elevation of proPO mRNA was not observed. From our previous study and the time course of gene expression of proPO and enzymatic activity of PO in this study, it can be concluded that the enhancement effect was through its transcriptional level, its translational level and then its post-translational level sequentially. These results suggest that, both for reliability, sensitivity and for the timing of sampling, the change in proPO mRNA is more useful than the PO activity in monitoring the activation of the proPO non-specific defence system of prawns after treatment with stimulants.

The toxic effect of phthalate esters on immune responses of giant freshwater prawn (Macrobrachium rosenbergii) via oral treatment.

A previous in vitro study has indicated that four phthalate esters (PAEs) could damage hemocytes and decreases the cellular immunity of prawns [Sung, H.H., Kao, W.Y., Su, Y.J., 2003. Effects and toxicity of phthalate esters to hemocytes of giant freshwater prawn, Macrobranchium rosenbergii. Aquat. Toxicol. 64, 25-37]. The aim of this study was to investigate the in vivo effect of four PAEs, diethyl phthalate (DEP), dihexyl phthalate (DHP), dipropyl phthalate (DPrP) and diphenyl phthalate (DPP) on the defense system of the giant freshwater prawn, M. rosenbergii. PAE dissolved in corn oil was continuously fed to prawns for 8 days and five immune parameters (total hemocyte count, THC; ratio of granulocytes to hyalinocytes, G/H; intrahemocytic total phenoloxidase activity, PO(T); intracellular superoxide anion (O2-) production; transglutaminase (TGase) activity) were separately detected on days 1, 4 and 8. In addition, mortality was determined on days 4 and 8 after challenging the prawns with Lactobacillus garvieae. In comparison with untreated prawns, the results showed that DHP demonstrated the lowest toxicity in that it only influenced the PO activity and O2- production before 4 days after treatment and caused 6.6% mortality on day 8. DEP decreased G/H, PO(T) and TGase activity on day 1 and reduced THC, G/H and PO(T) and caused 16.6% mortality on day 4; however, on day 8, it increased O2- production and caused no mortality. In the DPrP-treated group, a reduction of all the immune reactions apart from TGase activity and 22.2% mortality were detected on day 4. As for the effect of DPP, results showed that it decreased all the immune parameters apart from THC on days 1 and 4, but caused no mortality on day 4; but on day 8, an increase of O2- production and 17.7% mortality were detected. These results indicated that the immune reactions of prawns were variable due to the different toxic effects of PAEs. In addition, it was found that, on day 8 after treatment, the three PAEs, DHP, DPrP and DPP increased O2- production and did not influence the other four reactions, but mortality was detected in these groups. These results suggest that other physiological responses may also be affected to increase the susceptibility of prawns to pathogens.

Signal transduction of the prophenoloxidase activating system of prawn haemocytes triggered by CpG oligodeoxynucleotides.

Intracellular phenoloxidase (PO) activity in haemocyte lysate supernatant (HLS) of giant freshwater prawn (Macrobrachium rosenbergii) was shown to be enhanced by CpG oligodeoxynucleotide (ODN) 2006, but not by so-ODN13. When haemocytes were treated in vitro with 50 microg/ml of ODN2006 for 30 min, the increases in both intra- and extracellular stimulated PO activity (POS) and extracellular total PO activity (POT) and the reduction of POT suggest that the PO activity of haemocytes is enhanced by ODN2006 stimulation, but new prophenoloxidase (proPO) is not synthesised. In an attempt to determine which signal transduction pathway is involved in the activation of the proPO system, haemocytes were separately treated with activators or inhibitors of specific signalling components. The results show that there was an increase in both intra- and extracellular POT of haemocytes treated with sodium fluoride (a G-protein activator); the addition of phosphokinase A (PKA)-activating 8-bromo-cAMP to haemocytes only increased intracellular POT, and the addition of either phorbol-12-myristate-13-acetate (PMA; a phosphokinase C (PKC) activator) or caffeine (a phosphodiesterase inhibitor) only increased extracellular POT. When PMA-stimulated haemocytes were treated with chelerythrine (a PKC inhibitor), the induced extracellular POT was significantly reduced. Furthermore, the study of ODN2006-stimulated haemocytes treated with chelerythrine or palmitoyl-DL-carnitine (a PKC inhibitor) showed that the enhancement effects of ODN2006 on the intra- and extracellular POS and extracellular POT were significantly decreased. ODN-stimulated haemocytes treated with genistein (an inhibitor of protein tyrosine kinase) showed a further increase in extracellular POT, but the other PO activities remained the same as those of the ODN-stimulated group. These results suggest that the activation of the proPO system of prawn haemocytes, including degranulation and PO activity, is induced by ODN2006 via a PKC-activating signalling pathway, but negatively regulated via the tyrosine kinase pathway.

Characterization of Cryptocaryon irritans, a parasite isolated from marine fishes in Taiwan.

The ciliated protozoan parasite Cryptocaryon irritans infecting marine fishes in Taiwan is described. Developmental characteristics and sequences of the ribosomal DNA regions such as part of 18 S, the entire first internal transcribed spacer, and part of 5.8 S of various Taiwan isolates of C. irritans were investigated. A total of 5 isolates was obtained from different fish-host species and localities, the majority from cultured fish species. C. irritans from Taiwan is able to shift its developmental characteristics, i.e. from non-adherent to adherent tomonts, from individualistic to aggregate-forming tomonts, from infection of the gills only to infection of the gills and body. Thus, it is not possible to classify strains of C. irritans on the basis of these parameters. Premature tomonts that developed from dead fishes were able to produce theronts that could infect fish host. Isolates from Pingtung and the USA had identical nucleotide sequences while an isolate from Malaysia was identical to an Israel isolate. Percentage variation among pairs of Taiwan isolates showed a higher degree of variation than isolate sequences listed in GenBank. Sequence analysis revealed highly aberrant isolates in Taiwan, and a phylogenetic tree distinguished a marine and a low-salinity variant. C. irritans from marine fishes in Taiwan, therefore, display some characteristics not previously reported. Since manipulation of salinity in brackishwater ponds and marine cage sites is not feasible, there is a need to develop new strategies for the control and prevention of cryptocaryoniasis.

Effects and toxicity of phthalate esters to hemocytes of giant freshwater prawn, Macrobrachium rosenbergii.

Phthalate esters (PAEs) have been considered as environmental pollutants and have been subject to control in the United States of America and Japan. The aim of this study was to investigate the effects and toxicity of eight PAEs to hemocytes and the defense functions of giant freshwater prawn (Macrobrachium rosenbergii), including hemocytic adhesion, pseudopodia formation, phenoloxidase (PO) activity, and superoxide anion (O(2)(-)) production, by means of in vitro exposure experiments. After hemocytes were treated separately with eight PAEs at concentrations of 100 microg/ml, the results showed that two PAEs (dipropyl phthalate, DPrP and diethyl phthalate, DEP) increased cells with pseudopodia formation, but decreased adhesive cells; reduction in the percentages of both pseudopodia formation and adhesive cells were detected in the dihexyl phthalate (DHP) and diphenyl phthalate (DPP) experiment groups; and di-(2-ethyl hexyl) phthalate (DEHP) decreased pseudopodia formation, but did not affect the adhesion. In addition, both PO activity and O(2)(-) production were decreased after hemocytes were treated with five PAEs (benzyl butyl phthalate (BBP), di-n-butyl phthalate (DBP), DEP, DHP and DPrP), respectively. At the same time, microscopy showed that both DPrP and DHP altered morphology of the cell nucleus and led to the presence of vacuoles in cytosol of hemocytes. Using the annexin assay, and after analysis of DNA fragmentation and transmission electron microscopy (TEM), it was found that hemocytes exposed to DHP and DPrP for more than 10 min would primarily die via apoptosis, the fatality correlates with increasing treatment time; and hemocytes treated with either BBP, dicyclohexyl phthalate (DCP), DEP or DPP would primarily die via necrosis. According to these results, we suggest that all eight PAEs examined could damage hemocytes and further influence the defense mechanism of prawns. This study reveals an important precaution for prawn cultivation.