Efficacy of orally administered praziquantel against Zeuxapta seriolae and Benedenia seriolae (Monogenea) in yellowtail kingfish Seriola lalandi

We investigated the efficacy of praziquantel (PZQ) administered orally to yellowtail kingfish (Seriola lalandi in sea-cage aquaculture in South Australia) against the monogeneans Zeuxapta seriolae and Benedenia seriolae infesting gills and skin, respectively. PZQ was administered to fish by surface-coating feed pellets (Trial 1) or by direct intubation of the stomach (Trial 2). In both trials 4 daily doses were administered: 50 and 75 mg kg(-1) body weight (BW) d(-1) for 6 d, and 100 and 150 mg kg(-1) BW d(-1) for 3 d. Mean parasite intensity was compared between medicated fish and unmedicated control fish. In Trial 1, fish fed lower daily doses of PZQ for 6 d (50 and 75 mg kg(-1) BW d(-1)) had fewer Z. seriolae and B. seriolae than fish fed higher daily doses for 3 d (100 and 150 mg kg(-1) BW d(-1)). Fish rejected feed pellets surface-coated with PZQ, suggesting PZQ affected palatability of feed, and may explain differences in efficacy between treatments. In Trial 2, where PZQ was administered by intubation, there were fewer Z. seriolae and B. seriolae in medicated fish than control fish. Intubated PZQ was also effective against newly recruited Z. seriolae and B. seriolae. PZQ could be developed as a useful treatment for Z. seriolae and B. seriolae parasitising S. lalandi in sea-cage aquaculture if suspected palatability problems are resolved.     

Effects of temperature on fecundity in vitro, egg hatching and reproductive development of Benedenia seriolae and Zeuxapta seriolae (Monogenea) parasitic on yellowtail kingfish Seriola lalandi

Globally, aquaculture industries involved with commercial culture of kingfish (Seriola spp.) experience outbreaks of monogenean parasites, which can cause heavy stock losses. In Australia and New Zealand, aquaculturists of kingfish Seriola lalandi incur financial losses caused by two monogenean species: Benedenia seriolae and Zeuxapta seriolae which parasitise the skin and gills, respectively. This study provides information on some basic temperature-dependent life-cycle parameters of these problematic monogeneans on S. lalandi. Hatching times and age at maturity were inversely related to water temperature within the range experienced by wild kingfish in New Zealand (13-21 degrees C). Mature B. seriolae in vitro laid on average 37 eggs/day that hatched over approximately 4 days; peak hatching occurred 9, 11 and 22 days post-deposition at temperatures of 21, 17.5 and 13+/-1.0 degrees C, respectively. Z. seriolae in vitro laid on average 246 eggs/day that hatched over 2 days; peak hatching occurred 7, 9 and 15 days post-deposition at these respective temperatures. B. seriolae matured within 20, 25 and 48 days p.i. at 21, 18 and 13 degrees C. Z. seriolae matured within 25, 37 and >52 days p.i. at the same temperatures. This research describes stages in the reproductive development of B. seriolae and Z. seriolae and discusses the inclusion of basic parasitic life-cycle parameters into management strategies designed to maximise treatment efficacy and limit monogenean epizootics in sea-cage kingfish culture.     

Morphological and Molecular Characterization of Philometroides seriolae from Japanese Amberjack Seriola quinqueradiata caught in East Sea,Republic of Korea

The Japanese amberjack Seriolae quinqueradiata is one of the most consumed fish species among the Koreans. However, information regarding parasitic infection in Japanese amberjack is scarce. This study described the morphological and molecular characteristics of a species of philometrid nematode, Philometroides seriolae, which was recovered from Japanese amberjack. This fish was caught in the sea of Goseong-gun, Gangwon-do, Republic of Korea (Korea). Six P. seriolae (Nematoda: Philometridae) were recovered from 2 Japanese amberjacks. These parasites were subgravid female which were 325–420 mm long and 2.95–3.27 mm wide. Furthermore, they had typical papillae distributed on their body surface with 14 papillae at the apical view. Sequence analysis of the small subunits of ribosomal RNA (SSU rRNA) showed high sequence identity (99.8%, 1,607/1,611-bp) with that of P. seriolae (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"FJ155811","term_id":"205318602"}}FJ155811). This nematode species has been newly added to the Korean nematode fauna.     

Isolation and characterization of microsatellite loci from yellowtail Seriola quinqueradiata and cross‐species amplification within the genus Seriola

We developed five microsatellite primer pairs for the yellowtail Seriola quinqueradiata. The loci were highly polymorphic, with eight to 14 alleles per locus, and can be used to study kinship and/or population structure. Many of these primer pairs amplified polymorphic loci in cross‐species amplification tests for two other Seriola species (S. lalandi and S. dumerili).     

Ceratomyxa seriolae n. sp. and C. buri n. sp. (Myxozoa: Myxosporea) from the gall-bladder of cultured yellowtail Seriola quinqueradiata

Ceratomyxa seriolae n. sp. and C. buri n. sp. (Myxozoa: Myxosporea) were found in the gall-bladder of cultured yellowtail Seriola quinqueradiata Temminck & Schlegel (Carangidae) in Japan. Mature spores of C. seriolae n. sp. were elongate and 6.5 (6.0-7.5) m long and 33.7 (28.0-41.5) m thick. Disporous plasmodia of C. seriolae n. sp., 40-100 m in size, were amoeboid to spherical. C. buri n. sp. were elliptical with a flattened posterior end, 6.5 (5.5-7.5) m long and 14.3 (11.0-16.5) m thick. Spherical plasmodia of C. buri n. sp., 15-20 m in diameter, were disporous. In periodical sampling of yellowtail bile from August, 1999 to February, 2000, the two new species of Ceratomyxa, as well as Myxobolus spirosulcatus Maeno, Sorimachi, Ogawa & Kearn 1995, first appeared in October, and the prevalences were very variable (20-100%) during the study period.     

Cell-surface properties of Lactococcus garvieae strains and their immunogenicity in the yellowtail Seriola quinqueradiata

The cell-surface properties of strains of Lactococcus garvieae were examined. Two capsular types were found, one with a highly developed capsule (KG9408) and one with a micro-capsule (MS93003) carrying fimbriae-like components projecting from the cell surface. One strain (NSS9310) had neither cell capsular nor fimbriae-like structures on its cell surface. The strains with the highly developed capsule were more virulent to fish than either the micro-capsular or non-capsular strains. The KG9408, MS93003 and NSS9310 strains could be clearly differentiated by their susceptibility to bacteriophages. Protection against L. garvieae infection was induced in the yellowtail Seriola quinqueradiata by immunization with formalin-killed L. garvieae KG9408 and MS93003 cells. Although protection was also induced by immunization with NSS9310, the level of protection was significantly lower than that with KG9408 and MS93003 vaccines. Passive immunization with yellowtail immune sera raised against KG9408 and MS93003 conferred strong protection on yellowtail with rapid bacterial clearance after challenge with L. garvieae. Immunoblotting analysis of protein antigens extracted from L. garvieae strains using rabbit anti-KG9408 and anti-MS93003 sera and yellowtail anti-KG9408 and anti-MS93003 sera indicated that some bands in KG9408 and MS93003 strains were not detectable in NSS9310.     

Single and nested polymerase chain reaction assays for the detection of Microsporidium seriolae (Microspora), the causative agent of 'Beko' disease in yellowtail Seriola quinqueradiata.

Single and nested polymerase chain reaction (PCR) assays were developed for the detection of the microsporidian parasite Microsporidium seriolae, which is responsible for emaciation and even death in farmed Japanese yellowtail. Extremely high rDNA identities exist between this parasite and other members of the as yet unclassified genus, necessitating the design of generic, rather than species-specific primer sets. The nested PCR was several orders of magnitude more sensitive than the standard single PCRs, with visible target product amplified from as little as 0.01 pg of parasite DNA (equivalent to that extracted from a single spore). The specificity of the assays was tested against a range of potential host fishes and 6 other microsporidians infecting either fish or the musculature of their hosts. Single PCRs were found to be specific to the target genus, but the nested PCR replicated rDNA from several different microsporidian genera, limiting its utility. This study highlights problems associated with the use of the rRNA gene for PCR assays of certain microsporidians, but nevertheless provides a rapid and sensitive means for the detection of pre-spore forms not possible by current staining methods. Consequently, these assays may be employed for further studies on the portals of entry, migration to the musculature and transmission of this economically important pathogen.     

Quantitative Trait Loci (QTL) Associated with Resistance to a Monogenean Parasite (Benedenia seriolae) in Yellowtail (Seriola quinqueradiata) through Genome Wide Analysis

Benedenia infections caused by the monogenean fluke ectoparasite Benedenia seriolae seriously impact marine finfish aquaculture. Genetic variation has been inferred to play a significant role in determining the susceptibility to this parasitic disease. To evaluate the genetic basis of Benedenia disease resistance in yellowtail (Seriola quinqueradiata), a genome-wide and chromosome-wide linkage analyses were initiated using F₁ yellowtail families (n = 90 per family) based on a high-density linkage map with 860 microsatellite and 142 single nucleotide polymorphism (SNP) markers. Two major quantitative trait loci (QTL) regions on linkage groups Squ2 (BDR-1) and Squ20 (BDR-2) were identified. These QTL regions explained 32.9–35.5% of the phenotypic variance. On the other hand, we investigated the relationship between QTL for susceptibility to B. seriolae and QTL for fish body size. The QTL related to growth was found on another linkage group (Squ7). As a result, this is the first genetic evidence that contributes to detailing phenotypic resistance to Benedenia disease, and the results will help resolve the mechanism of resistance to this important parasitic infection of yellowtail.     

Cloning,sequence and variability analysis of expressed immunoglobulin light chain genes from yellowtail Seriola quinqueradiata

The cDNA clones encoding immunoglobulin (Ig) light (L) chain variable (V) region associated with constant (C) region were isolated from yellowtail (Seriola quinqueradiata) kidney by expressed sequence tag analysis (accession numbers: AB062619-AB062668, AB064322). The sequences of both VL and CL region contain well-conserved cysteine residues important for intra- and inter-domain interaction in mammals. Comparisons of the amino acid sequence of the CL domain with those of other species showed a high degree of similarity, with 88.3%, 59.8%, and 60.6% to those of wolf fish (Anarhichas lupus), rainbow trout IgL I isotype (Oncorhynchus mykiss) and channel catfish G isotype (Ictalurus punctatus), respectively. Multiple sequence alignments of the CL domain with those of higher vertebrates, however, did not readily allow it to be classified as kappa or lambda isotypes. Furthermore, the pI, hydrophobicity and variability of yellowtail VL regions were studied in 65 cDNA clones and the diversity was observed in CDR1, CDR2 and CDR3 regions.     

Effect of water temperature on reproductive development of Benedenia seriolae (Monogenea: Capsalidae) from Seriola lalandi in Australia

The monogenean Benedenia seriolae (Yamaguti, 1934) Meserve, 1938 is a major pathogen of farmed yellowtail kingfish Seriola lalandi in South Australia. To control parasite populations in commercial farms, an understanding of the effect of water temperature on parasite development is vital. This study investigated the effect of water temperature (14, 18, 22 and 26 +/- 0.5 degrees C) on development, growth rates and age at sexual maturity of B. seriolae. Five distinct developmental stages, defined by the progressive development of reproductive organs, were used to describe development of B. seriolae from recently invaded larvae to sexually mature adults. Parasite age at the first sign of sexual maturity (ability to lay eggs) was strongly influenced by water temperature and was attained at 41, 24, 16 and 14 d post infection (p.i.) at 14, 18, 22 and 26 +/- 0.5 degrees C respectively. Four parameters (parasite total length, maximum parasite width, accessory sclerite length and anterior hamulus length) were examined for suitability as an index of parasite age. Growth rates for each parameter increased with water temperature; however, mean anterior hamulus length was identified as the most reliable index of parasite age. Equations derived from these data can be used to estimate parasite age and time to sexual maturity at water temperatures ranging from 14 to 26 degrees C. The equations provide a simple tool to assist implementation of strategic treatment plans for B. seriolae infections in commercial kingfish farms, not only in South Australia but in other localities worldwide where S. lalandi is farmed.     

Production of donor-derived offspring by allogeneic transplantation of Spermatogonia in the yellowtail (Seriola quinqueradiata)

Although the yellowtail (Seriola quinqueradiata) is the fish most commonly farmed in Japan, breeding of this species has not yet started. This is primarily due to the lack of sufficiently sophisticated methods for manipulating gametogenesis, which makes it difficult to collect gametes from specific dams and sires. If it were possible to produce large numbers of surrogate fish by transplanting germ cells isolated from donor individuals harboring desirable genetic traits, then the probability of acquiring gametes carrying the donor-derived haplotype would increase, and breeding programs involving this species might increase as a result. As a first step, we established a method for the allogeneic transplantation of yellowtail spermatogonia and the production of donor-derived offspring. Donor cells were collected from immature (10-month-old) yellowtail males with testes containing abundant type A spermatogonia, labeled with PKH26 fluorescent dye, and transferred into the peritoneal cavities of 8-day-old larvae. Fluorescence observation at 28 days post-transplantation revealed that PKH26-labeled cells were incorporated into recipients' gonads. To assess whether donor-derived spermatogonia could differentiate into functional gametes in the allogeneic recipient gonads, gametes collected from nine male and four female adult recipients were fertilized with wild-type eggs and milt. Analysis of microsatellite DNA markers confirmed that some of the first filial (F(1)) offspring were derived from donor fish, with the average contribution of donor-derived F(1) offspring being 66% and the maximum reaching 99%. These findings confirmed that our method was effective for transplanting yellowtail spermatogonia into allogeneic larvae to produce donor-derived offspring.     

Genotypic characteristics of a Mycobacterium sp. isolated from yellowtail Seriola quinqueradiata and striped jack Pseudocaranx dentex in Japan

In Japan, a Mycobacterium marinum‐like mycobacterium was isolated from the yellowtail, Seriola quinqueradiata. The species was identified as M. marinum by a commercial mycobacterial DNA‐DNA hybridization kit. Nevertheless, PCR restriction analysis of the DNA of its RNA polymerase β‐subunit gene definitively showed that this Mycobacterium sp. was M. ulcerans. PCR analysis revealed the genotypic characteristics of M. ulcerans in the Mycobacterium sp., only the mup053 gene sequence being absent, as has been found previously in other piscine mycobacteria such as M. marinum strains DL240490 and DL045 and M. pseudoshottsii. With one exception, this Mycobacterium sp. and M. pseudoshottsii had identical 16S rRNA gene sequences, which is also probably true of M. marinum strains DL240490 and DL045. Similarly, according to comparisons of the 16S rRNA gene, ITS region, and hsp65 gene sequences, this Mycobacterium sp. is more closely related to M. pseudoshottsii than to M. ulcerans or M. marinum. A PCR product of approximately 2000 bp was amplified from region of difference 9 in the Mycobacterium sp. The nucleotide sequence revealed insertion of IS2404, the sequence of which is 1366 bp long. The novel single nucleotide polymorphisms identified in this region distinguished this Mycobacterium sp. from M. marinum strain DL240490 and M. pseudoshottsii. The present findings raise the possibility that these species have a common ancestor. Further studies are required to improve our understanding of the relationship between their geographical origin and genetic diversity.     

Effects of lethal levels of environmental hypercapnia on cardiovascular and blood-gas status in yellowtail,Seriola quinqueradiata

The cardiorespiratory responses were examined in yellowtail, Seriola quinqueradiata exposed to two levels of hypercapnia (seawater equilibrated with a gas mixture containing 1% CO(2) (water PCO(2) = 7 mmHg) or 5% CO(2) (38 mmHg)) for 72 hr at 20 degrees C. Mortality was 100% within 8 hr at 5% CO(2), while no fish died at 1% CO(2). No cardiovascular variables (cardiac output, Q; heart rate, HR; stroke volume, SV and arterial blood pressure, BP) significantly changed from pre-exposure values during exposure to 1% CO(2). Arterial CO(2) partial pressure (PaCO(2)) significantly increased (P < 0.05), reaching a new steady-state level after 3 hr. Arterial blood pH (pHa) decreased initially (P < 0.05), but was subsequently restored by elevation of plasma bicarbonate ([HCO(3)(-)]). Arterial O(2) partial pressure (PaO(2)), oxygen content (CaO(2)), and hematocrit (Hct) were maintained throughout the exposure period. In contrast, exposure to 5% CO(2) dramatically reduced Q (P < 0.05) through decreasing SV (P < 0.05), although HR did not change. BP was transiently elevated (P < 0.05), followed by a precipitous fall before death. The pHa was restored incompletely despite a significant increase in [HCO(3)(-)]. PaO(2) decreased only shortly before death, whereas CaO(2) kept elevated due to a large increase in Hct (P < 0.05). We tentatively conclude that cardiac failure is a primary physiological disorder that would lead to death of fish subjected to high environmental CO(2) pressures.     

Effects of exogenous cholecystokinin and gastrin on the secretion of trypsin and chymotrypsin from yellowtail (Seriola quinqueradiata) isolated pyloric caeca

The humoral control of secretion of the proteolytic enzymes trypsin and chymotrypsin was studied in yellowtail (Seriola quinqueradiata). In vitro trials were performed to investigate the effects of cholecystokinin (CCK) and two commercially available gastrin peptides. Isolated preparations of pyloric caeca/pancreas release trypsin and chymotrypsin when incubated with cholecystokinin (CCK) at 10 microM and gastrin I (G1) at 50 microM after 15 min of incubation. On the other hand, G1 at 10 microM and gastrin-related peptide (G2) did not enhance trypsin and chymotrypsin secretion. The studies concerning the CCK effects at different incubation temperatures have shown that trypsin and chymotrypsin secretion at 25 degrees C was stimulated by CCK after 15 min, while at 10, 15 and 20 degrees C the stimulatory effects of CCK were observed only after 30 min of incubation. The CCK effects were increased at higher incubation temperatures and longer incubation periods.     

Myxobolus spirosulcatus n. sp. (Myxosporea,Bivalvulida) infecting the bile duct of the yellowtail Seriola quinqueradiata from Japan

Myxobolus spirosulcatus n. sp. (Myxosporea: Bivalvulida), infecting the cultured yellowtail Seriola quinqueradiata, is described. M. spirosulcatus n. sp. was found in the bile duct of yellowtails collected in the southern part of Japan during June 1990. The plasmodium of the new species has various shapes and contains a vegetative form plus maturing and mature spores. Mature spores, on average, measure 8.9 m in length, 7.8 m in width and 6.7 m in thickness. The unique morphological characteristics of the new species are spiral furrows in the peripheral part of the spore valves.     

Observations on egg production in the monogenean Entobdella soleae

Egg production in the monogenean Entobdella soleae increases as adult parasites grow. Medium-sized adults (anterior hamulus length 550–600 μm; total body length approx. 5 mm) produced about 30 eggs per day at 12°C. Parasites with anterior hamuli exceeding 650 μm in length (total body length about 6 mm) may produce more than 60 eggs per day. In smaller adults, eggs tend to spend longer in the ootype after assembly of the shell and the ootype remains empty for longer periods of time. The time taken to assemble a single egg is relatively constant (4–6 min) in adults of all sizes and there is evidence of a small but significant increase in egg size as parasites increase in size. Some eggs were retained in the uterus for as long as 168 min but others spent less than 5 s in the uterus, indicating that the uterus is no more than a passageway for these eggs to the outside world. The egg cell (or zygote) precedes the vitelline cells as they progress along the ovo-vitelline duct to the ootype.     

Occurrence of ditaurobilirubin, bilirubin conjugated with two moles of taurine, in the gallbladder bile of yellowtail, Seriola quinqueradiata

An unknown bilirubin conjugate was detected by HPLC analysis in the bile of yellowtail, Seriola quinqueradiata. The retention time of this unknown conjugate was identical with that of authentic ditaurobilirubin, a bilirubin conjugated with 2 mol of taurine. The retention times of the ethyl anthranilate diazo derivatives of both bilirubin conjugates were the same. The azo derivatives of this unknown conjugate were purified by Amberlite XAD-2 treatment and preparative TLC and hydrolyzed with 6 N HCl at 105 degrees C for 12 h. The hydrolyzates obtained were analyzed with an amino acid analyzer. The ninhydrin-positive substance in the hydrolyzates was purified by column chromatography and analyzed by TLC and mass spectrometry. The results of these analyses indicated that this ninhydrin-positive substance was taurine. Therefore, this unknown bilirubin conjugate was identified as ditaurobilirubin.     

Nutrient control of release of pancreatic enzymes in yellowtail (Seriola quinqueradiata): involvement of CCK and PY in the regulatory loop

Cholecystokinin (CCK) and neuropeptide Y (NPY)-related peptides are key regulators of pancreatic enzyme secretion in vertebrates. CCK stimulates enzyme secretion whereas peptide Y (PY), a NPY-related peptide, plays an antagonistic role to that of CCK. In fish, very little is known about how different nutrients affect the synthesis of CCK and PY in the digestive tract, and the mechanism by which CCK and PY actually regulate digestive enzyme secretion is not well understood. In order to determine how different nutrients stimulate the synthesis of CCK and PY in yellowtail (Seriola quinqueradiata), CCK and PY mRNA levels in the digestive tract were measured after oral administration of a single bolus of either phosphate-buffered saline (PBS: control), starch (carbohydrate), casein (protein), oleic acid (fatty acid) or tri-olein (triglyceride). In addition, in order to confirm the synthesis and secretion of digestive enzymes, the mRNA levels and enzymatic activities of three digestive enzymes (lipase, trypsin and amylase) were also analyzed. Casein, oleic acid and tri-olein increased the synthesis of lipase, trypsin and amylase, while starch and PBS did not affect the activity of any of these enzymes. CCK mRNA levels rose, while PY mRNA levels were reduced in fish administered casein, oleic acid and tri-olein. These results suggest that in yellowtail, CCK and PY maintain antagonistic control of pancreatic enzyme secretion after intake of protein and/or fat.     

The efficacy and safety of an oil-based vaccine against Photobacterium damsela subsp. piscicida in yellowtail (Seriola quinqueradiata): a field study

Protection after intraperitoneal (i.p.) vaccination of yellowtail (Seriola quinqueradiata) against pasteurellosis was studied in a field trial. Yellowtail juveniles captured from the wild or artificially hatched were immunised with an oil-based vaccine against Photobacterium damsela subsp. piscicida, and the fish were observed for 15 weeks after vaccination. Outbreak of pasteurellosis was observed at all five sites (mortality in control group ranged from 7% to 77%), and significant (p<0.01) protection against pasteurellosis relative to non-vaccinated control groups was observed at all sites. The vaccinated fish showed an increased level of agglutinating antibodies against Ph. damsela subsp. piscicida with a peak around 3-4 weeks post vaccination, increased phagocytic activity and increased production of superoxide anions in isolated leucocytes compared to controls, both assessed at 36 and 66 days post vaccination. Transient reduction in fish weight was observed in vaccinated groups until 10 weeks after vaccination; however at 15 weeks, the weight of the vaccinated group was significantly higher than that of the control group. This coincided with the development of side-effect scores at the injection site that had started to wane by 10 weeks, and the downward trend continued up to the last collection time (41 weeks), although with some variation between sites. The study shows that the tested vaccine protects against pasteurellosis in yellowtail under field conditions and that it is safe for use in the target species.