Morphological characterization of the tunic in the edible ascidian, Halocynthia roretzi (Drasche), with remarks on 'soft tunic syndrome' in aquaculture

'Soft tunic syndrome' is a serious problem in the aquaculture of the edible ascidian, Halocynthia roretzi (Drasche), and often leads to mass mortality. Here, we describe the tunic morphology of intact and diseased ascidians to reveal structural differences between them. Morphologically, diseased tunics are not very different from intact tunics, although the former are thinner and softer than the latter. While several types of cells are distributed in the tunic, the cell types and their cytomorphologies were almost identical in both groups. As bacterial/protozoan cells were not found in either intact or diseased tunics, they are not the direct cause of soft tunic syndrome. The most remarkable difference was in the bundles of tunic fibres that compose the tunic matrix; in intact tunics, the thick bundles interlace to form a firm matrix, whereas in soft tunics, the tunic fibres do not form thick bundles. Furthermore, areas of low fibre density were found in diseased tunics. Therefore, soft tunic syndrome probably causes inhibition of bundle formation and degradation of tunic bundles, creating areas of low fibre density, although the causes remain unknown.     

In vitro and in vivo efficacy of drugs against the protozoan parasite Azumiobodo hoyamushi that causes soft tunic syndrome in the edible ascidian Halocynthia roretzi (Drasche)

It was discovered recently that infection by a protozoan parasite, Azumiobodo hoyamushi, is the most probable cause for soft tunic syndrome in an edible ascidian, Halocynthia roretzi (Drasche). In an attempt to develop measures to eradicate the causative parasite, various drugs were tested for efficacy in vitro and in vivo. Of the 20 antiprotozoal drugs having different action mechanisms, five were found potent (24‐h EC₅₀ < 10 mg L^?1) in their parasite‐killing effects: formalin, H₂O₂, bithionol, ClO₂ and bronopol. Moderately potent drugs (10 < 24‐h EC₅₀ < 100 mg L^?1) were quinine, fumagillin, amphotericin B, ketoconazole, povidone‐iodine, chloramine‐T and benzalkonium chloride. Seven compounds, metronidazole, albendazole, paromomycin, nalidixic acid, sulfamonomethoxine, KMnO₄, potassium monopersulphate and citric acid, exhibited EC₅₀ > 100 mg L^?1. When ascidians were artificially infected with A. hoyamushi, treated using 40 mg L^?1 formalin, bronopol, ClO₂, or H₂O₂ for 1 h and then monitored for 24 h, very low mortality was observed. However, the number of surviving parasite cells in the ascidian tunic tissues was significantly reduced by treating with 40 mg L^?1 formalin or ClO₂ for 1 h. The data suggest that we might be able to develop a disinfection measure using a treatment regimen involving commonly available drugs.     

Growth performance and the soft body composition of juvenile abalone,Haliotis discus,Reeve 1846, fed the extruded pellets substituting fish meal and macroalgae with tunic meal of sea squirt,Halocynthia roretzi

Growth performance and the soft body composition of juvenile abalone fed the extruded pellets (EPs) substituting fish meal (FM) and macroalgae (MA) with tunic meal of sea squirt (SS) was investigated. A total of 1,260 abalone were distributed into 18 containers. Six experimental diets were prepared in triplicate. Five diets were pelletized by an extruder pelleter. The 140 g/kg FM and 250 g/kg mixture of MA were included into the control (Con) diet. Five hundred and 1,000 g/kg of each FM and MA were substituted with an equal amount of tunic meal of SS, referred to as the FM50, FM0, MA50 and MA0 diets, respectively. Finally, dry Undaria pinnatifida was prepared. Weight gain and specific growth rate of abalone fed all EPs were greater than those fed U. pinnatifida. Weight gain of abalone fed MA50 and FM50 diets was greater than Con and FM0 diets, but not different from MA0 diet. Higher crude protein and lipid contents were observed in soft body of abalone fed all EPs compared to U. pinnatifida. In conclusion, FM and MA up to 500 and 1,000 g/kg, respectively, could be replaced with tunic meal of SS in EPs without retardation in growth of abalone.     

Substitution effect of sea tangle (ST) (Laminaria japonica) with tunic of sea squirt (SS) (Halocynthia roretzi) in diet on growth and carcass composition of juvenile abalone (Haliotis discus,Reeve 1846)

Substitution effect of sea tangle (ST) with tunic of sea squirt (SS) in diet on growth and carcass composition of juvenile abalone was determined. One thousand four hundred and seventy abalones were distributed into 21 containers. Six formulated diets in triplicate were prepared. A 200 g/kg ST was included into the ST0 diet. The 200, 400, 600, 800 and 1000 g/kg of ST were substituted with the same amount of tunic of SS, referred to as the ST200, ST400, ST600, ST800 and ST1000 diets, respectively. Finally, Undaria was prepared to compare effect of the formulated diets on performance of abalone. The experimental diets were fed to abalone for 16 weeks. Weight gain of abalone fed the ST400 diet was higher than that of abalone fed the ST0, ST600, ST800 and ST1000 diets and Undaria. Weight gain of abalone fed the formulated diets was higher than that of abalone fed the Undaria. The chemical composition of the carcass of abalone was affected by dietary substitution of ST with tunic of SS. In conclusion, ST could be completely substituted with tunic of SS without retardation in performance of abalone. Abalone fed the ST400 diet substituting 400 g/kg ST with tunic of SS achieved the best growth.     

A review on the morphology, molecular characterization, morphogenesis and pathogenesis of white spot syndrome virus

Since it first appeared in 1992, white spot syndrome virus (WSSV) has become the most threatening infectious agent in shrimp aquaculture. Within a decade, this pathogen has spread to all the main shrimp farming areas and has caused enormous economic losses amounting to more than seven billion US dollars. At present, biosecurity methods used to exclude pathogens in shrimp farms include disinfecting ponds and water, preventing the entrance of animals that may carry infectious agents and stocking ponds with specific pathogen-free post-larvae. The combination of these practices increases biosecurity in shrimp farming facilities and may contribute to reduce the risk of a WSSV outbreak. Although several control methods have shown some efficacy against WSSV under experimental conditions, no therapeutic products or strategies are available to effectively control WSSV in the field. Furthermore, differences in virulence and clinical outcome of WSSV infections have been reported. The sequencing and characterization of different strains of WSSV has begun to determine aspects of its biology, virulence and pathogenesis. Knowledge on these aspects is critical for developing effective control methods. The aim of this review is to present an update of the knowledge generated so far on different aspects of WSSV organization, morphogenesis, pathology and pathogenesis.     

Studies on pathogenicity and prevalence of white spot syndrome virus in mud crab, Scylla serrata (Forskal), in Zhejiang Province, China

Mud crab, Scylla serrata (Forskal), is the most commercially important marine crab species in China. In recent years, serious diseases have occurred in major mud crab culture regions in SE China. PCR detection of white spot syndrome virus (WSSV) in diseased mud crabs collected from Zhejiang Province during 2006–2008 showed a prevalence of 34.82%. To study the pathogenicity of WSSV to mud crab, healthy mud crabs were injected intramuscularly with serial 10‐fold dilutions of a WSSV inoculum. The cumulative mortalities in groups challenged with 10^?1, 10^?2, 10^?3 and 10^?4 dilutions were 100%, 100%, 66.7% and 38.9% at 10 days post‐injection, respectively. All moribund and dead mud crabs except the control group were positive for WSSV by PCR. Based on the viral load of the WSSV inoculum by quantitative real‐time PCR, the median lethal dose (LD50) of WSSV in S. serrata was calculated as 1.10 × 10⁶ virus copies/crab, or 7.34 × 10³ virus copies g^?1 crab weight. The phenoloxidase, peroxidase and superoxide dismutase activities in haemolymph of WSSV‐infected moribund crabs, were significantly lower than the control group, whereas alkaline phosphatase, glutamate‐pyruvate transaminase and glutamic‐oxaloacetic transaminase were higher than in the control group. WSSV was mainly distributed in gills, subcuticular epithelia, heart, intestine and stomach as shown by immunohistochemical analysis with Mabs against WSSV. The epithelial cells of infected gill showed hypertrophied nuclei with basophilic inclusions. Numerous bacilliform virus particles were observed in nuclei of infected gill cells by transmission electron microscopy. It is concluded that WSSV is a major pathogen of mud crab with high pathogenicity.     

Nucleotide sequence variations of the major structural proteins (VP15, VP19, VP26 and VP28) of white spot syndrome virus (WSSV), a pathogen of cultured Litopenaeus vannamei in Mexico

White spot syndrome virus (WSSV) was first reported in farmed Litopenaeus vannamei stocks in Sinaloa and Sonora, Mexico during 1999 and continues to cause severe shrimp losses. WSSV genes encoding nucleocapsid (VP26 and VP15) and envelope proteins (VP19 and VP28) of a Mexican isolate were cloned in the pMosBlue vector. The nucleotide sequences of these genes were compared with WSSV isolates in GenBank. VP15 is highly conserved, and VP26 showed 99% homology to a Chinese isolate. The VP28 fragment demonstrated 100% homology to the majority of the isolates analysed (UniProt accession no. Q91CB7), differing from two Indian WSSV and one Chinese WSSV isolates by two non-conserved and one conserved replacements, respectively. Because of their highly conserved nature, these three structural proteins are good candidates for the development of antibody-based WSSV diagnostic tools or for the production of recombinant protein vaccines to stimulate the quasi-immune response of shrimp. In contrast, VP19 of the Mexican isolate was distinguishable from almost all isolates tested, including an American strain of WSSV (US98/South Carolina, GenBank accession no. AAP14086). Although homology was found with isolates from Taiwan (GenBank accession no. AAL89341) and India (GenBank accession no. AAW67477), VP19 may have application as a genetic marker.     

Evidence for the presence of white spot syndrome virus (WSSV) and monodon baculovirus (MBV) in wild Penaeus monodon (Fabricius) broodstock,in the southeast coast of India

A survey on the presence of the viruses of two economically significant diseases, white spot syndrome virus (WSSV) and monodon baculovirus (MBV) in wild‐collected Penaeus monodon broodstock, was conducted during different seasons of the year in two major coastal areas of southeast India. The broodstock were collected along the coast of Tamil Nadu and Andhra Pradesh during summer, premonsoon, monsoon and post‐monsoon seasons for three consecutive years. A total of 7905 samples were collected and subjected to MBV screening, and 6709 samples that were screened as MBV negative were diagnosed for WSSV. MBV was detected using rapid malachite green staining and WSSV by nested polymerase chain reaction. Prevalence data of the viruses were analysed using the EpiCalc 2000 program at 95% confidence interval. Samples collected from the Andhra Pradesh coast displayed a slightly higher prevalence of WSSV and MBV infection than those collected from Tamil Nadu, although this difference was not statistically significant (P > 005). In addition, it was found that the prevalence of both WSSV and MBV infections fluctuated according to season. Data on prevalence of these viruses in broodstock would be useful to develop strategies for shrimp health management along the southeast coast of India.