Cryopreservation of sperm from Atlantic halibut (Hippoglossus hippoglossus, L.) for commercial application

Development of Atlantic halibut (Hippoglossus hippoglossus) aquaculture will be enhanced with cryopreservation of halibut sperm by ensuring a reliable supply of sperm of desired quality and quantity. To assist in its commercial application, the cryopreservation of large volumes of halibut sperm was investigated. Three cryoprotectants were compared: dimethyl sulfoxide (DMSO), polyethylene glycol (PG) and glycerol (GLY) at two concentrations (10% or 15%). Two salt solutions, Hanks’ balanced salt solution (HBSS) and 0.1 M KHCO₃ with 0.125 M sucrose solution (KS) were tested as diluents. Both factors were examined in 1.6 mL volumes. A cryopreservation volume of 4 mL and a low dilution ratio (1:1) were examined separately. Based on motility and fertilization rate, 10% and 15% DMSO diluted with HBSS or KS solution proved to be effective extenders with mean fertilization rates ranging from 52.2 ± 27.2% to 65.8 ± 26.1%; none of which were significantly different from that of the control. Four other extenders, 10% PG or 10% GLY with HBSS or KS, resulted in significantly lower fertilization rates. Use of a 4 mL cryopreservation volume did not exhibit a significant effect on fertilization rate or motility of post-thawed sperm compared to a 1.6 mL volume (P > 0.05); while the use of a dilution ratio of one part sperm with three parts cryopreservation solution (1:3 v/v with sperm concentration of 0.51 ± 0.11 × 10¹⁰ cells/ml) had a significantly better preservation effect than using a ratio of 1:1 with sperm concentration of 1.02 ± 0.21 × 10¹⁰ cells/ml (P < 0.05). From these results, an optimized protocol for the cryopreservation of Atlantic halibut sperm using a volume as large as 4 mL has been established.     

Detection and basic properties of carbamoyl phosphate synthetase III during teleost ontogeny: a case study in the Atlantic halibut (Hippoglossus hippoglossus L.)

The presence of carbamoyl phosphate synthetase III (CPSase III), catalyzing the first step of the urea cycle in fish, in Atlantic halibut (Hippoglossus hippoglossus L.) yolk-sac larvae and adult white muscle has been established using gel filtration chromatography to separate the CPSase III from the pyrimidine-pathway related CPSase II. The results are consistent with the hypothesis that teleostean fish express urea cycle enzymes during early development and with recent observations of low levels of CPSase III in muscle tissue. The presence of CPSase III in crude extracts could not be established using sensitive assay conditions to discriminate between CPSase III and CPSase II. However, kinetic characterization after chromatographic separation identified each as typical CPSase II and CPSase III activities, respectively. The CPSase III was less sensitive to activation by N-acetyl- -glutamate and had a higher K_m for ammonia than CPSase III found in other species. These results suggest that precise quantitation of low levels of CPSase III in the presence of CPSase II by assaying crude extracts may be difficult unless the enzymes are first separated and the kinetic properties of CPSase III are determined; the results indicate that assaying larval extracts of Atlantic halibut in the presence of uridine triphosphate results in CPSase activity that reflects mostly CPSase III and can, therefore, be used to measure changes in CPSase III activity.     

Histomorphology of the early yolk-sac larvae of the Atlantic halibut (Hippoglossus hippoglossus L.)—an indication of the timing of functionality

Halibut larvae hatch at a very immature stage, and the duration of the yolk-sac period is very long (up to 50 days). This paper describes the histomorphological development of organs of the yolk-sac larvae (6° C) by use of light and electron microscopy. Rudimentary branchial cavities were open from 2 days after hatching. Kidneys seemed functional 16 days after hatching and onwards, and primitive lamellae on the gill arches were beginning to form at this age. Pancreatic zymogen granules were first observed 20daysafter hatching. The liver was segmented into lobes between 20 and 23 days after hatching, and the gall bladder seemed functional from day 23. The hindgut became extensively folded from day 26, and branchial capillaries were first observed at this stage. The larvae were able to catch food particles 24 days after hatching. Judging from ultrastructural observations, it seemed that halibut larvae were able to digest food particles between day 24 and 26 after hatching (around 150 daydegrees and 50% yolk absorption).     

Ciliated epithelium in the gut of larval Atlantic halibut, Hippoglossus hippoglossus

The presence of cilia was documented in the hindgut of larval Atlantic halibut ( Hippoglossus hippoglossus ) with the use of electron microscopy. Cilia were shown to be present as late as 8 and 9 day post hatch. These observations were compared to similar observations in more primitive teleosts.     

Immune- and enzyme histochemical characterisation of leukocyte populations within lymphoid and mucosal tissues of Atlantic halibut (Hippoglossus hippoglossus)

Leukocyte populations within the kidney, spleen, posterior intestine and gills of Atlantic halibut were investigated using a panel of histological, enzyme- and immunohistochemical methods. In the kidney and spleen, a diverse population of leukocytes was associated with the extensive network of sinusoids and larger blood vessels present in these tissues. IgM+ cells (B-cells, plasma cells and IgM-bearing macrophages) and large mononuclear cells showing reactivity for non-specific esterase (NSE) and acid phosphatase (ACP), representing macrophage populations, were often associated with vessel walls that were also the site of trapping of fluorescent microspheres. In the kidney, trapping of 0.1 and 0.5 microm diameter microspheres occurred at these sites but in the spleen, the 0.1 microm diameter microspheres were retained in ellipsoids. The lymphoid tissues of the kidney and spleen possessed a spread population of 5'-nucleotidase+ (5'N+) cells but compartmentalisation of the splenic white pulp was suggested by an absence of these 5'N+ reticular cells in areas associated with melanomacrophage accumulations and in areas rich in IgM+ cells. A striking feature of the mucosal tissues was the diversity of leukocyte populations within the epithelium particularly of the posterior intestine, including IgM+ cells and NSE+, ACP+ and 5'N+ mononuclear cells. Although limited in numbers in the posterior intestine, IgM+ cells were more common in the epithelium than in the lamina propria. In the gills, leukocytes as detected by enzymatic reactivity were scarce, but IgM+ cells were very abundant in the stratified epithelium of the gill arch and filaments. The difference in distribution of these leukocyte populations between the intestines and gills suggested a compartmentalisation of the mucosal immune system and the need to assess the immunological competence of mucosal tissues in Atlantic halibut.     

Development of 10 polymorphic microsatellite loci primers for Codonopsis pilosula Nannf. (Campanulaceae)

Codonopsis pilosula Nannf., as an important traditional Chinese medicinal plant species, has been used to treat fatigue, thirst and loss of appetite. In this study, we developed 10 new microsatellite loci primers from the genome of this species using the combined biotin capture method. The polymorphism of each locus was further assessed through 27 individuals from four geographically distant populations. The number of alleles per locus ranged from 6 to 11. The observed and expected heterozygosity ranged from 0.15 to 0.24 and 0.27 to 0.40, respectively. We further performed cross-priming tests of these loci in the other three congeneric species. These microsatellite markers will be useful for investigating genetic diversity within and between populations of these species.     

Isolation and characterization of eight polymorphic microsatellite loci for the critically endangered Arenaria nevadensis (Caryophyllaceae)

We report on the isolation and characterization of eight microsatellite markers from enriched libraries for Arenaria nevadensis, one of the most critically endangered plant species in the Iberian Peninsula. These are the first microsatellite loci reported for Arenaria species. The number of alleles ranged from two to eight, and the expected heterozygosity from 0.067 to 0.873. These markers will be useful for characterizing the genetic diversity in A. nevadensis and understanding its population structure, and will provide important genetic data for the conservation and recovery of this species.     

A study of the mechanism of algal uptake in yolk-sac larvae of Atlantic halibut (Hippoglossus hippoglossus)

The drinking rate of water, and the ingestion- and assimilation rates of algae in yolk-sac larvae of Atlantic halibut were examined by use of³H-dextran and¹⁴C-labelled algae. The drinking rate throughout the yolk-sac stage was in the range of 7–160 nl per larva h⁻¹, with a slight increase towards the end of the period. The ingestion rate of algae ( Tetraselmis sp.) was very low before day 30 and showed a peak between day 43 and 48 at 5° C. The assimilation efficiency of the algae was in the range of 1-5%. The clearance rate of algae was 100–1000 times higher than the drinking rate, and was correlated to the distance between the gill arches. Bigger cells were more effectively retained than smaller ones. The results suggested that the larvae acted as filter feeders from day 30 up to the point when they are expected to start feeding on bigger prey.     

Variation in size and location of the Ag-NOR in the Atlantic halibut (<Emphasis Type="Italic">Hippoglossus hippoglossus</Emphasis>)

The distribution of differentially stained chromatin was studied in the Atlantic halibut (Hippoglossus hippoglossus) chromosomes (2n = 48). Four pairs of homologous chromosomes were identified using a combination of traditional cytogenetic staining techniques (Giemsa/DAPI/CMA3/Ag-NO3). Chromosome 1 showed a length polymorphism (1^S-short, 1^L-long isoforms of the chromosome 1) which was related to the variation of the size of the Ag-NORs. In one specimen the Ag-NOR was translocated from chromosome 1 into the telomeric region on the q-arm of the chromosome 2 forming a derivative chromosome der(2)t(1^S;2)(q?;q?). Four Ag-NOR genotypes have been shown: 1^S1^S, 1^S1^L, 1^L1^L and 1^S der(2)t(1^S;2)(q?;q?). The chromosome rearrangements did not leave any interstitially located telomeric sequences and the telomeres were confined to the ends of the chromosomes. A single chromosomal location of 5S rDNA clusters was found using the PRINS technique. In the extended metaphase spreads two adjacent clusters of 5S rDNA could be seen on one chromosome while condensed chromatin gave a single hybridization signal. Double 5S rDNA signals on the same chromosome arm suggested paracentric inversion of the minor rDNA site. 5S rDNA clusters were not co-localized with Ag-NORs. Although female and male karyotypes were compared no sex related cytogenetic markers were found.     

Energy and ration requirements of juvenile Pacific halibut (Hippoglossus stenolepis) based on energy consumption and growth rates

Growth of captive juvenile Pacific halibut was linearly related to energy consumption (J g⁻¹ day⁻¹) at 4°C by the following equation: growth (% body weight (b.w.) day⁻¹)=0–007 (consumption J g⁻¹ day⁻¹)– 0.192; r² =0.81. Weight gain was independent of size for fish between 9 and 7000 g when growth was expressed as a function of consumption in J g⁻¹ day⁻¹. Maintenance ration determined in feeding–growth experiments averaged 27.4 J g⁻¹ day⁻¹ at 4–0°C. Small halibut ate significantly more food than large fish. Single meals following 2 day fasts averaged 4.1% b.w. for halibut under 100 g, 1.72% b.w. for 1.2 kg fish and 1.1% B.W. for 6.8 kg fish. Both large and small size categories of halibut tended to evacuate their meal in about 3 days even though small fish ate relatively larger meals. Minimum estimates for daily ration to achieve growth rates observed in the Gulf of Alaska were approximately 0.5 to 2.4% b.w. day⁻¹ depending on fish size and whether northern shrimp or yellowfin sole were their prey.     

Expressed sequence tags analysis of Atlantic halibut (Hippoglossus hippoglossus) liver, kidney and spleen tissues following vaccination against Vibrio anguillarum and Aeromonas salmonicida

To investigate the response of Atlantic halibut to vaccination and pathogen exposure, a cDNA library was constructed from liver, kidney and spleen mRNA collected following vaccination against Vibrio anguillarum and Aeromonas salmonicida. After sequencing 1114 clones 1072 (96.23%) readable sequences were obtained of which 106 sequences are the first reported from the fish. Of these, 182 clones (16.98%) contained cell/organism defence genes including immunoglobulin light chain, MHC class I and II, interferon consensus sequence binding protein, B-cell receptor-associated protein, early B-cell factor, 10 complement components, heat shock protein 70 and 90, antimicrobial peptides hepcidin type 1 and 2, and CC chemokine (macrophage inflammatory protein-1 beta-like chemokine, MIP-1beta). Expression of MIP-1beta-like was elevated in the kidney and spleen at 1, 2, 7 and 14 days post vaccination. Functional genes involved in cellular processes of hematopoietic tissues were also identified. These results indicate that this cDNA library contains many important genes involved in the immune response, making it an important resource for studying the response of Atlantic halibut to vaccination or pathogen exposure.     

Quantitative investigation of antigen and immune response in nervous and lymphoid tissues of Atlantic halibut (Hippoglossus hippoglossus) challenged with nodavirus

The present study reports the quantitative analysis of the spatio-temporal development of nodavirus infection and corresponding immune response in juvenile Atlantic halibut (Hippoglossus hippoglossus) challenged by intramuscular injection of nodavirus. Novel quantitative real-time RT-PCR protocols were applied to evaluate the absolute copy numbers of nodavirus RNA2 (RNA2) and secretory-IgM mRNA (sec-igmicro) in the eye, brain, mid/posterior kidney and spleen sampled over a period of 81 days. In the eye and brain, levels of both RNA2 and sec-igmicro increased significantly early in the infection. In the spleen and mid/posterior kidney, both RNA2 and sec-igmicro were detected but the levels remained unchanged during the experimental period. The levels of RNA2 and sec-igmicro in the eye and brain were strongly correlated (P<0.0001). Nodavirus antigen was demonstrated by immunohistochemistry (IHC) in the retina of eyes from a relatively few fish from day 34 post challenge (brain not examined), but not at any time in the spleen and anterior kidney. By IHC, IgM+ cells were observed in conjunction with nodavirus positive IHC labelling in the retina. In both the spleen and anterior kidney, the number of IgM+ cells increased from day 3 post challenge. By conventional real-time RT-PCR, RNA2 was only sporadically demonstrated in the posterior intestine, heart and gills. ELISA analysis revealed a nodavirus specific antibody response in serum that was significant from day 18 post challenge. No clinical signs or mortality related to nodavirus infection were observed in the challenged halibut. The results suggest that the nodavirus infection induced a significant antibody response through activation of B-cells in the kidney and spleen, and involved a substantial migration of antibody-secreting cells to infected peripheral tissues.     

Isolation and characterization of microsatellite loci in the Japanese pipistrelle (Pipistrellus abramus)

We describe the first set of ten microsatellite markers isolated in Pipistrellus abramus. The number of alleles per locus ranged from 7 to 13. The observed and expected heterozygosities values ranged from 0.486 to 0.971 and from 0.752 to 0.876, respectively. Three loci revealed significant departure from Hardy–Weinberg equilibrium and no linkage disequilibrium was found between loci pairs. These informative microsatellite markers will be a powerful molecular tool for studying the population genetic structure of P. abramus, as well as other species of this genus.     

Twelve novel polymorphic microsatellite loci for the Yellow grouper (Epinephelus awoara) and cross-species amplifications

Yellow grouper (Epinephelus awoara) is a commercially important marine fish species. A dinucleotide-enriched genomic library of E. awoara was constructed using the method of FIASCO. Twelve loci were polymorphic in a test population with alleles per locus ranging from two to eight, and observed and expected heterozygosities per locus from 0.13 to 1.00 and from 0.20 to 0.86, respectively. Five loci significantly deviated from Hardy–Weinberg equilibrium and no significant linkage disequilibrium was found between pairs of loci. Cross-species amplification of these polymorphic microsatellite loci was performed in additional two related species. These polymorphic microsatellite loci would be useful for investigating genetic diversity of E. awoara and related species. L. Zhao and C. Shao have contributed equally to this work.     

Isolation and characterization of polymorphic microsatellite loci from a dinucleotide-enriched genomic library of starry flounder (Platichthys stellatus) and cross-species amplification

Starry flounder (Platichthys stellatus) is a rare fish species in China. Here, we reported 12 polymorphic microsatellite loci isolated from a dinucleotide-enriched genomic library of starry flounder (P. stellatus). The number of alleles, observed and expected heterozygosity per locus in 30 individuals ranged from two to six, from 0.2500 to 1.0000 and from 0.4512 to 0.7667, respectively. One locus significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction and no significant linkage disequilibrium between pairs of loci was found. Cross-species amplification of these microsatellite loci in additional three fish species was performed. These polymorphic microsatellite loci would be useful for investigating genetic population structure and construction of genetic linkage map in P. stellatus. Guidong Miao and Changwei Shao have contributed equally.     

Isolation and characterization of microsatellite loci in Ostryopsis davidiana (Betulaceae)

Ostryopsis (Betulaceae) is a samll genus endemic to China with only two species. Both of them play an important role in restoring the local ecosystems. The distribution of genetic diversity between and within populations in each species are important to further utilize the wild genetic resources and explore the interspecific divergence. In this study, we developed 10 microsatellite loci from O. davidiana by the combining biotin capture method for the first time. A total of 27 microsatellite sequences were recovered through screening the library and 10 of them are polymorphic. The number of alleles per locus in 18 sampled individuals ranged from 3 to 6, expected heterozygosity and observed heterozygosity ranged from 0.2958 to 0.4767 and from 0.1591 to 0.2997, respectively. In addition, all markers have been crossly checked in the other congeneric species. These microsatellite markers would together provide a useful tool for investigating the genetic diversity and structure of both species and speciation mechanism between them.     

Activity and swimming speed at time of first feeding of halibut (Hippoglossus hippoglossus) larvae

Atlantic halibut larvae show an increase in activity, together with a decrease in swimming speed during active periods, occurring from day 26 onwards, which corresponds approximately to 50% yolk absorption.     

Characterization of novel microsatellite loci in the great leaf-nosed bat, Hipposideros armiger and cross-amplification in other related species

Eight microsatellite loci were isolated from an enriched genomic library of the great leaf-nosed bat, Hipposideros armiger. The polymorphism of these loci was tested on a population of 48 individuals from Anhui Province, China. All loci revealed the polymorphism ranging from three to 12 alleles. The observed heterozygosity values were from 0.213 to 0.875 and expected heterozygosity from 0.232 to 0.820. No significant linkage disequilibrium was detected. Two loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction. In addition, successful cross-amplification also suggested that these microsatellite markers will facilitate research on the population genetics and gene flow of H. armiger and other related species.     

Eight polymorphic microsatellite loci for the Chinese medicinal plant Artemisia annua L. (Asteraceae)

Artemisia annua is an important medicinal plant from which Artemisinin was extracted to cure malaria effectively. We developed eight microsatellite markers from the genome of A. annua using the FIASCO protocol. Polymorphism of each locus was assessed in 54 individuals from two Chinese populations. The average allele number of these microsatellites was 3.1 per locus, ranging from 2 to 6. The observed (H _O) and expected (H _E) heterozygosity were from 0.019 to 0.907 and from 0.055 to 0.793, respectively. These microsatellite markers would provide a useful tool for genetic studies of A. annua. H.-R. Huang and G. Zhou have contributed equally to this work.