Effect of antiprotozoal drugs on the proliferation of the bivalve parasite Perkinsus olseni

The protozoan parasite Perkinsus olseni causes severe losses among Ruditapes decussatus clams. The development of an in vitro culture of this parasite together with the use of a proliferation assay has provided the opportunity to screen for drug sensitivity of this parasite. Xenobiotics known for their antimalarial and antiprotozoal properties were tested against P. olseni. Only four of these drugs, namely cycloheximide, pyrimethamine, deferoxamine (DFO) and 2,2-bipyridyl (BIP), showed in vitro inhibitory effect on the parasite proliferation. Two in vivo experiments were designed to determine the effect of iron chelators on reducing P. olseni infection in clams. For this purpose, naturally infected clams from the Ria Formosa, Portugal, were exposed to DFO and BIP at various concentrations. In the first experiment, hemolymph samples were taken from each clam before and after treatment to determine the infection intensity and in the second experiment, clams were randomly distributed in groups of five and the parasite burden in treated and untreated groups was determined at the end of the experiment on the whole clam wet tissues. Only DFO was found to be effective in reducing in vivo P. olseni infections. In addition, acute toxicity of DFO and BIP has been determined and no mortality of Perkinsus-free clams was observed.     

Experimental challenges of juvenile and adult Manila clams with the protozoan Perkinsus olseni at different temperatures

Infection with Perkinsus species, primarily P. olseni, is thought to be a major cause of the decline of Manila clam populations in Japan since the 1980s. However, the pathogenicity of the infection has not been sufficiently evaluated to estimate the impact of infection on wild Manila clam populations. We experimentally challenged juvenile (3- to 6-mm shell length) and adult (18- to 22-mm shell length) Manila clams with P. olseni at 18, 23, 28, and 30 °C. Mortality was significantly higher in challenged groups than in control groups. The difference in mean mortality between the challenged and control groups (all life stages and temperatures) was only significant above a threshold of infection intensity ~10⁶ cells/g soft wet tissue (SWT). As temperature increased, the onset of mortality occurred more rapidly. The increase in mortality occurred earlier in juveniles than adults at 28 °C and lower. Our results suggest that the pathogenicity of P. olseni is higher in juveniles than in adults and at higher water temperatures. Given the infection intensities (ca. 10⁶ cells/g SWT) previously reported in wild Manila clams, the parasite likely has considerable impact on wild Manila clam populations, particularly juveniles during periods of high temperature.     

Application of enzyme-linked immunosorbent assay (ELISA) for the study of reproduction in the Manila clam Ruditapes philippinarum (Mollusca: Bivalvia): II. Impacts of Perkinsus olseni on clam reproduction

We investigated the effects of infection by the protozoan Perkinsus olseni on the reproduction of female Manila clams, Ruditapes philippinarum, from a population in Gomso Bay, Korea. The reproductive effort of the clams was assessed by ELISA using a clam egg-specific antibody and was expressed as a weight-based gonadosomatic index (GSI). The number of Perkinsus infecting each clam was estimated from the gills using Ray's fluid thioglycollate medium (RFTM) along with a NaOH digestion assay. We found that reproductive effort was negatively correlated with the intensity of the Perkinsus infection: more heavily infected clams produced fewer eggs during the spawning period from May to August. Frequency of spawning was also negatively correlated with the level of Perkinsus infection; heavily infected clams (HIC) exhibited a single spawning pulse in late July, whereas lightly infected clams (LIC) showed three spawning peaks in mid-May, late July, and late August. Egg production of HIC was only 30–75% of LIC during spawning. The level of total protein in LIC was also higher year round than in HIC. In conclusion, our investigation demonstrates that a high level of Perkinsus infection affects spawning frequency and reduces egg production, which may have long-term impacts on clam recruitment and population growth.     

Expression and localization of MCsialec, a sialic acid-specific lectin in the marine bivalve Manila clam, Ruditapes philppinarum

A novel sialic acid-specific lectin (MCsialec) was detected from an expressed sequenced tag (EST) sequence from Manila clam haemocytes infected with Perkinsus olseni. The cDNA of the lectin was cloned using gene-specific primers based on a previously determined EST and characterized. The full-length cDNA of MCsialec is 603 bp in length and encodes a polypeptide of 200 amino acids with a calculated molecular mass of 21.928 kDa. Sequence alignment and protein motif analyses showed that MCsialec shares identity with sialic acid-specific invertebrate lectins from Cepaea hortensis, Helix pomatia and Haliotis discus discus. The lectin was expressed in Escherichia coli M15 cells and purified using a Ni-NTA His-binding resin matrix for antibody production. The presence of the lectin in various tissues of Perkinsus-infected and uninfected Manila clams was analysed by both PCR and immunohistochemical localization assays. MCsialec was detected in each tissue of the clams; however, upon infection, the level of expression of the lectin increased in each tissue. Vibrio tapetis infection also induced high-level expression of MCsialec in the haemocytes. These data suggest that MCsialec plays a crucial role in the immune system of the Manila clam during pathogenic infection.     

Comparison of taste and odour characteristics of three mass‐produced aquaculture clams in China

Differences in taste and odour between three kinds of clam with the highest aquacultural production in China were investigated. Meretrix petechialis, Mactra veneriformis and Ruditapes philippinarum (fresh and dried product) were analysed firstly by electronic tongue and electronic nose. Fresh and dried clams could be easily distinguished, and there was little difference between uncooked fresh clams, while greater difference occurred between R. philippinarum and other fresh clams after cooking. The total free amino acid (FAA) content of uncooked clams increased after cooking but decreased in dried clam; the highest proportion was of sweet taste FAAs. Ala, Glu, Arg, 5′‐adenosine monophosphate and 5′‐inosine monophosphate were the most important active taste compounds. The umami intensity order was found to be fresh R. philippinarum, followed by M. petechialis and M. veneriformis, and finally dried R. philippinarum. The greatest number of volatile compounds was found in dried clam, while M. petechialis and M. veneriformis had the most compounds in common.     

The immune response in rohu,Labeo rohita (Actinopterygii: Cyprinidae) to Argulus siamensis (Branchiura: Argulidae) infection: kinetics of immune gene expression and innate immune response

Argulus siamensis is a major pathogen in freshwater aquaculture. The immune responses of Indian major carp, Labeo rohita to experimental infection of A. siamensis was evaluated by quantitation of immune‐relevant gene expression in head kidney and skin, and serum innate immune parameters through the course of infection. In skin of infected fish, antioxidant genes like natural killer cell enhancing factor (NKEF‐B) and superoxide dismutase (MnSOD) were significantly up‐regulated in addition to lysozyme G and β₂ microglobulin (β₂M). Both tumour necrosis factor α (TNFα) and toll‐like receptor 22 (TLR22) genes were significantly down‐regulated in skin during early phases of the infection. Most of the genes exhibited significant down‐regulation in head kidney; immunoglobulin (IgM) and β₂M genes being the exceptions which were significantly up‐regulated at 12 h and 3 days post infection. Most of the innate immune parameters like serum complement activity and ceruloplasmin levels showed significant reduction in infected fish. The observed results are indicative of A. siamensis modulating the immune response of rohu by down‐regulation of many immune factors which may explain the susceptibility of rohu to A. siamensis infection. The interaction of this parasite with the host need to be further explored to understand its pathogenesis.     

Effects of low salinity stress on immune response and evaluating indicators of the swimming crab Portunus trituberculatus

The purpose of the present study was to investigate the effects of salinity stress on immune responses and evaluating indicators in swimming crab Portunus trituberculatus. The crabs (150 ± 8.5 g in body weight) were exposed to different salinities as 21, 26 and 31‰ (control) for 6 days. The results showed the total haemocyte counts (THC) and prophenoloxidase (proPO) activity in the haemocytes decreased significantly in the treatment groups after 6 hr, and reached the lowest levels at 12 hr. The phenoloxidase (PO) activity in the plasma increased significantly and peaked at 12 hr, then recovered to control level after 24 hr. The phagocytic per cent of haemocyte, antibacterial and bacteriolytic activities in the plasma decreased significantly in the treatment groups, and reached the lowest level at 12 hr, then recovered to control level after 72 hr. The dopamine (DA) and 5‐hydroxytryptamine (5‐HT) contents in the plasma increased significantly and peaked at 12 hr, then recovered to control level, while the noradrenaline (NE) content in the plasma had no significant change throughout the duration of the experiment. The DA and 5‐HT receptors were significantly up‐regulated in the treatment groups. The highest value of mRNA expression of DA and 5‐HT receptors occurred at 12 hr and recovered to control level after 24 hr. In addition, the cAMP and protein kinase A (PKA) contents in the haemocytes increased significantly and peaked at 12 hr, then recovered to control level after 72 hr. The phospholipase C (PLC) and protein kinase C (PKC) contents in the haemocytes increased significantly and peaked at 12 hr, then resumed to control level after 24 hr. These results speculated that biogenic amine (DA and 5‐HT) is likely to play an important role in immune modulation via cAMP/PKA signalling pathway or PLC/PKC signalling pathway when P. trituberculatus is exposed to low salinity and these results will provide scientific data for immune evaluation.     

Synergistic osmoregulatory dysfunction during salmon lice (Lepeophtheirus salmonis) and infectious hematopoietic necrosis virus co‐infection in sockeye salmon (Oncorhynchus nerka) smolts

While co‐infections are common in both wild and cultured fish, knowledge of the interactive effects of multiple pathogens on host physiology, gene expression and immune response is limited. To evaluate the impact of co‐infection on host survival, physiology and gene expression, sockeye salmon Oncorhynchus nerka smolts were infected with the salmon louse Lepeophtheirus salmonis (V?/SL+), infectious hematopoietic necrosis virus (IHNV; V+/SL?), both (V+/SL+), or neither (V?/SL?). Survival in the V+/SL+ group was significantly lower than the V?/SL? and V?/SL+ groups (p = 0.024). Co‐infected salmon had elevated osmoregulatory indicators and lowered haematocrit values as compared to the uninfected control. Expression of 12 genes associated with the host immune response was analysed in anterior kidney and skin. The only evidence of L. salmonis‐induced modulation of the host antiviral response was down‐regulation of mhc I although the possibility of modulation cannot be ruled out for mx‐1 and rsad2. Co‐infection did not influence the expression of genes associated with the host response to L. salmonis. Therefore, we conclude that the reduced survival in co‐infected sockeye salmon resulted from the osmoregulatory consequences of the sea lice infections which were amplified due to infection with IHNV.     

Dietary fulvic acid effects on survival and expression of immune‐related genes in Litopenaeus vannamei challenged with Vibrio parahaemolyticus

The effects of fulvic acid (FA) on survival and immune‐related gene expression were investigated in Litopenaeus vannamei challenged with Vibrio parahaemolyticus by immersion. Shrimp were fed with different dietary FA concentrations (1, 2, 4 and 6 g/kg feed) for 20 days (first bioassay) or 8 days (second bioassay, 2 g/kg feed of FA added every 2 days) and then challenged with V. parahaemolyticus. In a third bioassay, the expression of three immune‐related genes (translationally controlled tumour protein [TCTP], superoxide dismutase [SOD] and heat‐shock protein 70 [HSP70]) in haemocytes or hepatopancreas of experimental shrimp was measured by real‐time quantitative PCR at 0, 6, 12, 24, 48, 72 and 96 hr after FA (2 g/kg feed) administration. Fulvic acid increased survival at a concentration of 2 g/kg feed supplied every two days. Interestingly, TCTP gene expression was upregulated, whereas gene expression of SOD and HSP70 was downregulated. In conclusion, dietary fulvic acid improves survival in white shrimp challenged with V. parahaemolyticus and modulates the immune response. Therefore, FA merits further evaluation as prophylactic treatment in commercial shrimp farms.     

Bromodeoxyuridine DNA labelling reveals host and parasite proliferation in a fish‐myxozoan model

Enteromyxum leei is a myxozoan parasite responsible for enteritis in gilthead sea bream (Sparus aurata). The parasite proliferates in the paracellular space of the intestinal epithelium and induces an inflammatory reaction. To assess intestinal cell turnover and parasite proliferation, fish were infected with the parasite by anal intubation; after 17 and 64 days, the cell proliferative marker bromodeoxyuridine (BrdU) was administered; and after 24 hr, tissue samples were taken for immunohistochemical detection. Parasite exposure induced increased epithelial and immune cell proliferation in all intestinal segments at all time points, even before parasite establishment. This increased turnover was triggered early after intubation and mainly at a local level, as shown by an increased proliferating cell nuclear antigen (pcna) gene expression only at the posterior intestine after 17 days (not found in lymphohaematopoietic organs). Incorporation of BrdU in parasite secondary and tertiary daughter cells indicated that parasite endogeny is not by schizogonial division, which uses de novo synthesis pathway of pyrimidines. Altogether, BrdU immunolabelling and pcna gene expression showed the rapid proliferative response of the fish intestines upon a myxozoan infection and how this response is effectively triggered even before the parasite reaches or establishes in the site.     

Identification of potential general markers of disease resistance in Exopalaemon carinicauda via three‐round challenge selection with an acute hepatopancreatic necrosis disease‐causing strain of Vibrio parahaemolyticus

Sixteen candidate disease‐resistant parameters were selected through which to evaluate the acute hepatopancreatic necrosis disease (AHPND)‐resistant capability of Exopalaemon carinicauda after three generations of selection for AHPND‐causing strain of Vibrio parahaemolyticus (VP_AHPND) resistance in our previous study. However, these parameters required further verification. In this study, another AHPND‐resistant E. carinicauda series was obtained through a short‐term selection procedure, consisting of three virulent challenge rounds of selection (about three‐week interval for each challenge) with VP_AHPND infection. After this selection, the survival rate at 144 hr post infection (hpi) increased from 23.33% to 37.78% and the observed 48‐hr LD₅₀ of VP_AHPND to shrimp increased from 10^5.5 cfu/ml to 10^6.5 cfu/ml. Then, the immune response of this AHPND‐resistant E. carinicauda was studied using the 16 candidate AHPND‐resistant parameters selected for in our previous study. The improved VP_AHPND clearance rate in hpi, increased total haemocyte counts, haemocyanin concentration, alkaline phosphatase activity and expressions of six immune‐related genes (Tollip and ALF in haemocytes and hepatopancreas; lysozyme, crustin and cathepsin B in hepatopancreas; and LGBP in haemocytes) at 24 hpi after the three‐round challenge selection suggest that these immune parameters may be reliable markers for the evaluation of the physiological status and potential AHPND‐resistant phenotypes in E. carinicauda.     

Evaluation of macroalgal detritus as food source for juvenile Manila clam,Ruditapes philippinarum: Effects on growth,amino acid content and fatty acid composition

Sufficient high‐quality microalgae are required for indoor nursery of juvenile Ruditapes philippinarum. However, culturing numerous microalgae to support clam feeding is a heavy burden on many hatcheries. The effects of detritus from the macroalgae Ulva pertusa, Chondrus ocellatus and Undaria pinnatifida on the growth, amino acid content and fatty acid profile of R. philippinarum were assessed as potential substitute diets. The green microalga Tetraselmis cordiformis served as comparative diet. Results revealed that the clams ingesting distinct diets presented no significant differences in growth of soft tissues, but the nutritional component of these clams differed dramatically. The clams fed with Undaria + Tetraselmis had the highest content of essential amino acids and proteins. In addition, the clams fed with single macroalgal diets and mixed macroalgal detritus and Tetraselmis showed significantly higher or statistically equal levels in n‐3/n‐6 ratio and docosahexaenoic acid (DHA)/eicosapentaenoic acid (EPA) ratio with respect to Tetraselmis diets. The relative percentages of EPA and DHA in clams fed with Undaria were 28% and 63% higher than those fed with Tetraselmis, and the arachidonic acid abundances in clams ingesting Undaria + Tetraselmis and Tetraselmis were significantly higher than those in clams ingesting other diets. Together, the diets containing single Undaria or mixed Undaria + Tetraselmis produced Manila clams with nutritional advantages in terms of essential amino acids and polyunsaturated fatty acids. Thus, the detritus of macroalgae, especially Undaria, is an appropriate substitute diet, at least partially, for culture of nutrition‐improved R. philippinarum.     

Molecular cloning and characterization of the cathepsin L gene in Pelodiscus sinensis and its expression in response to bacterial challenge

Cathepsin L is one of the most important lysosomal cysteine proteases for the initiation of protein degradation, and it is involved in the immune response in many vertebrates. However, its function in the innate immune system of turtles remains poorly understood. Here, we cloned the cathepsin L gene from the Chinese soft‐shelled turtle Pelodiscus sinensis. We then examined the mRNA expression in different tissues and at different time points after infection by Aeromonas hydrophila or Vibrio parahemolyticus. The full‐length cDNA sequence cloned from P. sinensis was 1,805 bp with a 1,071 bp open reading frame, which encodes a 40.39 kDa polypeptide of 356 amino acids. The deduced protein sequence contained an active triad of Cys, His and Asn, and conserved ERWNIN and GNFD motifs. Homology analysis showed that the deduced amino acid sequence shared 77%–96% identity with other known species. Sequence alignment, phylogenetic analysis and structural comparisons revealed that cathepsin L is a member of the cathepsin family. Real‐time PCR analyses showed that turtle cathepsin L mRNA is ubiquitously expressed in various tissues, and the expression level is higher in the liver than in other tissues. The expression level in the liver peaks 24 hr after A. hydrophila infection and at two times (12 and 72 hr) after V. parahemolyticus infection. These results suggest that the cathepsin L gene of P. sinensis is likely involved in the process of the antibacterial response to A. hydrophila and V. parahemolyticus. This study is of great importance for future work exploring the molecular mechanism of antibacterial immune responses in P. sinensis.     

Labeo rohita and Argulus siamensis infection: Host size,local inflammatory reaction and immunity modulate ectoparasite load on fish

Parasite species often show a heterogeneous, highly dispersed pattern of infestation within hosts. Varieties of factors including morphological, physiological, immunological and nutritional characteristics affect the infestation level of a specific parasite in homogenous pray. Limited attempts, however, have been made to explore such underlying drivers of infestation pattern. Here, three stages of Labeo rohita (fingerling, juvenile and pre‐adult) were challenged with ectoparasite, Argulus siamensis in same aquaria. The parasite load on individuals was determined at 5‐day interval for 1 month. The load was found to be highest in pre‐adult stage followed by juveniles and fingerlings. On day 20 post infection, the load of parasite on pre‐adult fish was high along with detectable skin damages. Skin tissues were collected for immune gene expression analysis and histopathology. Histological studies showed increased melanization in the dermis and mild inflammatory cellular reactions in pre‐adult fish whereas, massive subcutaneous myositis with engorged blood vessels were observed in fingerlings. The expression levels of various inflammation and innate immune‐related genes viz., interleukin (IL)‐8, IL‐10, IL‐11, IL‐15, natural killer enhancing factor, toll‐like receptor 4, apolipoprotein A–I and immunoglobulin Z were significantly high in skin samples of infected fingerlings as compared to other two growth stages or controls of each stage. On the other hand, the expression of immunoglobulin M was down‐regulated in all infected samples as compared to their respective controls. The results thus depict that local immuno‐inflammatory response plays a significant role in determining susceptibility of host in intra‐specific group, and has important implications for ecology and aquaculture.     

Immune responses of whiteleg shrimp,Litopenaeus vannamei (Boone, 1931), to bacterially expressed dsRNA specific to VP28 gene of white spot syndrome virus

In this study, dsRNA specific to VP28 gene of white spot syndrome virus (WSSV) of shrimp was synthesized in Escherichia coli in large scale and studied the immune response of shrimp to dsRNA‐VP28. The haematological parameters such as clotting time and total haemocytes counts, and immunological parameters such as prophenoloxidase (proPO), superoxide dismutase (SOD), superoxide anion (SOA) and malondialdehyde content, as well as the mRNA expression of ten immune‐related genes were examined to estimate the effect of dsRNA‐VP28 on the innate immunity of Litopenaeus vannamei. The activities of proPO, SOA and SOD significantly increased in haemocyte after dsRNA‐VP28 treatment, whereas MDA content did not change significantly. Among the ten immune‐related genes examined, only the mRNA expression of proPO, cMnSOD, haemocyanin, crustin, BGBP, lipopolysaccharides (LPs), lectin and lysozyme in haemocytes, gill and hepatopancreas of L. vannamei, was significantly upregulated at 12 h after dsRNA‐VP28 treatment, while no significant expression changes were observed in Toll receptor and tumour receptor genes. The increase of proPO and SOD activities, and SOA level and mRNA expression level of proPO, cMnSOD, haemocyanin, crustin, BGBP, LPs, lectin and lysozyme after dsRNA‐VP28 stimulation indicate that these immune‐related genes were involved in dsRNA‐VP28‐induced innate immunity in shrimp.     

Rainbow trout Oncorhynchus mykiss (Walbaum) type IV ice‐structuring protein LS‐12 in the acute‐phase response to Flavobacterium psychrophilum infection

A previous proteomic study examining the plasma acute‐phase response of rainbow trout to sterile inflammation highlighted an unidentified 9.5‐kDa spot using 2D‐PAGE, which was dramatically increased. The 15 amino acid sequence obtained from this protein spot allowed rapid amplification of cDNA ends PCR to generate a 443‐bp nucleotide sequence that was 98.6% similar to type‐4 ice‐structuring protein LS‐12 from Atlantic salmon Salmo salar Linnaeus. Quantitative reverse translation PCR and an ELISA were used to measure gene expression and plasma concentrations of LS‐12 following experimental intraperitoneal injection of rainbow trout with either 10⁶ or 10⁸ colony‐forming units (CFU) of Flavobacterium psychrophilum. There was no significant change in the plasma concentration of LS‐12 up to 15 days post‐infection in any group. Hepatic LS‐12 gene expression was significantly reduced at 3 and 6 days (p < 0.001) post‐infection in fish injected with 10⁸ CFU of F. psychrophilum relative to control fish, while branchial or head kidney expression was unchanged. Infected fish had significantly increased hepatic gene expression of serum amyloid A, confirming an acute‐phase response. Under the conditions used, LS‐12 is not a positive acute‐phase protein in rainbow trout.     

Comparison of immune responses and antioxidant status of different generations of growth‐selected Portunus trituberculatus families

The purpose of this study was to investigate the effects of inbreeding on the immune responses and antioxidant status of Portunus trituberculatus juveniles. Results showed that inbreeding affected the total haemocyte count, and phagocytic, pro‐phenoloxidase (propo), phenoloxidase and antibacterial activities decreased after the seventh generation. Antioxidant status showed a similar pattern: total antioxidant capacity, superoxide dismutase (SOD) activity and GSH/GSSG in the cell‐free haemolymph and hepatopancreas decreased, while catalase activity in the cell‐free haemolymph increased (P < 0.05). There were no significant differences in α₂‐macroglobulin and bacteriolytic activities in the cell‐free haemolymph and glutathione peroxidase activity in the cell‐free haemolymph and hepatopancreas among nine inbreeding generations. Gene expression levels of proPO and crustin in haemocytes and SOD in haemocytes and hepatopancreas also decreased significantly as the inbreeding generations increased. The results suggest that a high level of inbreeding could severely reduce the physiological health of P. trituberculatus. Our obtained data would be particularly useful for P. trituberculatus breeding programmes.