The cytokine genes of Oncorhynchus masou formosanus include a defective interleukin-4/13A gene.

Oncorhynchus masou formosanus (Formosa landlocked salmon) is a critically endangered salmonid fish endemic to Taiwan. To begin to understand how its drastic change in lifestyle from anadromous to exclusively river-dwelling is reflected in its immune genes, we characterized the genes encoding six cytokines (IL-2A, IL-2B, IL-4/13A, IL-4/13B1, IL-4/13B2, and IL-17A/F2a) important for T cell responses as no genomic data is available for this fish. Interestingly, all genes appeared homozygous indicative of a genetic bottleneck. The IL2 and IL17A/F2a genes and their products are highly similar to their characterized homologs in Oncorhynchus mykiss (rainbow trout) and other salmonid fish. Two notable differences were observed in IL4/13 family important for type 2 immune responses. First, O. m. formosanus carries not only one but two genes encoding IL-4/13B1 proteins and expansions of these genes are present in other salmonid fish. Second, the OmfoIL4/13A gene carries a 228 bp deletion that results in a premature stop codon and hence a non-functional IL-4/13A cytokine. This suggests a reduced ability for T cell responses against parasitic infections in this species.

Quality assessment of cryopreserved Portuguese oyster (Crassostrea angulata) sperm through ultrastructural and flow cytometry analysis.

We investigated the effects of cryopreservation on the quality of Portuguese oyster (Crassostrea angulata) sperm, which were examined before and after freezing; sperm motility, fertilizing capacity, and ultrastructural morphology were analyzed. The motility percentage and fertilizing capacity of the cryopreserved sperm (mean ± standard error) were 16% ± 1% and 17% ± 8%, respectively. In the pre-freezing sperm, these were 58% ± 2% and 76% ± 4%, respectively. The sperm sustained substantial morphological and ultrastructural damage during cryopreservation. The morphological changes varied considerably in nature and extent, ranging from no apparent damage to virtual disintegration. Sperm were stained with fluorescent dyes to assess viability, plasma membrane integrity, mitochondrial activity, acrosomal membrane integrity, oxidation level, and DNA fragmentation and examined through flow cytometry. The methods used for the flow cytometry assays were slightly modified from those used for evaluating the semen quality of livestock. Relative to the pre-freezing sperm, the frozen-thawed sperm exhibited lower acrosomal membrane integrity (acrosomal damage, 59.86 ± 5.29; P < 0.05) and substantially higher oxidation levels (free radicals, 60.06 ± 0.82; P < 0.003). Oxidation level was found to be the most sensitive indicator of cryodamage. Along with ultrastructural analysis, we used flow cytometry to measure the qualitative and quantitative characteristics of Portuguese oyster sperm before and after cryopreservation rapidly, objectively, and accurately. This is the first study to assess the quality of Portuguese oyster sperm through these methods.

Sperm ultrastructure of Ruditapes variegata and Tapes literatus (Mollusca, Bivalvia, Veneridae, Tapetinae) from Pescadores, Taiwan.

In the present study, we have investigated the ultrastructures of the mature gonadal spermatozoa of R. variegata and T. literatus and presented comparisons with the Manila clam, R. philippinarum, sperm ultrastructure examined. Spermatozoa of R. variegata consist of (in anterior to posterior sequence): an elongate conical, deeply invaginated, acrosomal vesicle (length 1.58 ± 0.06 µm; width 0.99 ± 0.07 µm; invagination occupied by a granular subacrosomal material); a barrel-shaped nucleus (length 1.82 ± 0.06 µm; width 1.50 ± 0.03 µm); a midpiece consisting of two orthogonally arranged centrioles, surrounded by four spherical mitochondria; nine satellite fibers connecting the distal centriole to the plasma membrane; and a flagellum originating from the distal centriole. Contents of the acrosomal vesicle of R. variegata are differentiated into a very electron-dense basal ring and a less electron-dense zone (with seven dense transverse layers structure) on the anterior region of the acrosome. Spermatozoa of T. literatus differ from those of R. variegata and are characterized by a rounded-conical invaginated, acrosomal vesicle (length 0.88 ± 0.08 µm; width 0.77 ± 0.06 µm), with a basal ring; and an anteriorly-tapered, barrel-shaped nucleus (length 1.57 ± 0.04 µm; width 1.60 ± 0.09 µm); a midpiece composed of four mitochondria. Centriolar and flagellar details are essential as for R. variegata. Sperm morphology separating R. variegate, R. philippinarum, and T. literatus in different clades. The anterior region of the acrosomal vesicle in R. variegata sperm had the transverse bands structure whereas the apex of the acrosomal vesicle of T. literatus sperm had no such structure. This difference advocated that acrosomal feature could be an important character for taxonomic distinction. Our data supported the previous studies that the ultrastructure of bivalve sperm is species-specific. This advocates that the phyletic relationships of Tapetinae, commonly based on shell morphology, should also add additional and newer approaches.

Ultrastructure of sperm and complete mitochondrial genome in Meretrix sp. (Bivalvia: Veneridae) from Taiwan.

Spermatozoan ultrastructure and complete mitochondrial genome in the marine bivalve mollusk Meretrix sp. (Taiwan) from Taiwan are described and contrasted with other bivalves, especially within Meretrix. We have examined the features of the mature gonadal spermatozoa of Meretrix sp. (Taiwan) and provided comparisons with the other four Meretrix species (M. petechialis, M. meretrix, M. lyrata, and M. lamarckii). The morphological characteristics of these spermatozoa are diagnostic for each of the species studied here. The most marked interspecific difference was found in the acrosome. Meretrix sp. (Taiwan) is genetically distinct and is a different species from M. petechialis and M. lusoria (Japan) based on complete mitochondrial genome data. Sperm data for Meretrix are limited but show remarkable congruence with the molecular results. We suggest use Meretrix formosa Gwo and Hsu as the scientific name for Taiwanese hard clams, Meretrix sp. (Taiwan). Additional species, particularly the Japanese hard clam (M. lusoria) require examination before this tentative conclusion can be verified.

Genetic diversity and stock identification of small abalone (Haliotis diversicolor) in Taiwan and Japan.

Small abalone (Haliotis diversicolor) is a commercially valuable species for both fisheries and aquaculture. The production of annual farmed small abalone in Taiwan, once the highest in the world, has dramatically decreased in the past 15 years, and currently, the industry is close to collapse. Understanding the genetic diversity of small abalone and developing stock identification methods will be useful for genetic breeding, restoring collapsed stocks, managing stocks, and preventing illegal trade. We investigated 307 cultured and wild individuals from Taiwan, Japan, and Bali Island (Indonesia) by using the mitochondrial cytochrome c oxidase subunit I (COI) gene. Network analysis of mtDNA COI gene sequences revealed that the individuals collected from Taiwan, Japan, and Indonesia could be identified, and showed significant genetic divergence. In addition, the Indonesian population (Haliotis diversicolor squamata) was significantly different from the other populations and might need to be considered a separate species. We discovered a single nucleotide polymorphism marker in the mtDNA COI gene that can be used to distinguish the Taiwan population from the Japan population. We also developed a polymerase chain reaction-restriction fragment length polymorphism method for rapid detection. Furthermore, we could identify the cultured stocks, wild population, and hybrid stocks by using 6 microsatellites and amplified fragment length polymorphism. This study contributes useful tools for stock identification and the production of high-disease resistant small abalone strains (Japan × Taiwan or Taiwan × Japan). Efforts should be made to avoid unintentional random genetic mixing of the Taiwan population with the Japan population and subsequent breakdown of population differentiation, which impair local adaptation of the Taiwan wild population. Molecular markers revealed a split between the Taiwan and Japan populations, and the existence of a possible barrier to the free dispersal of small abalone is discussed.

Distribution of feminizing compounds in the aquatic environment and bioaccumulation in wild tilapia tissues.

This study sampled six times of river water, sediment, and tilapia (Oreochromis niloticus) in the Dan-Shui River, Taipei, Taiwan; 10 feminizing compounds were analyzed using ultra-performance liquid chromatography-tandem mass spectrometry. Bisphenol A (508 ± 634 ng/L, geometric mean (GM) 303 ng/L) and nonylphenol (491 ± 570 ng/L, GM 328 ng/L) were the most abundant among analytes in the river water. Nonylphenol (770 ± 602 ng/g wet weight, GM 617 ng/g wet weight) was also the highest in sediment. Fish may uptake nonylphenol and nonylphenol ethoxylates from river water and sediment because there were significant correlations between the concentrations in these matrixes and those in fish tissues (r s ranged from 0.21 to 0.49, p < 0.05). The bioaccumulation of nonylphenol, nonylphenol ethoxylates and bisphenol A in gonad, eggs, and liver was much higher than that in muscle (e.g. mean bioaccumulation factors of nonylphenol were 27,287, 20,971, 9,576 and 967, respectively) and might result in low liver fractions in fish body weights (0.66 % ± 0.39 %, GM 0.55 %) and the skewed sex ratio of fish (male to female = 0.52). This innovative study linked the environmental and internal doses statistically in the globally distributed wild fish by analyzing feminizing compounds in water, sediment, and four fish tissues including gonad and eggs.

DNA barcoding reveals cryptic diversity in the peanut worm Sipunculus nudus.

Peanut worm (Sipunculus nudus) is a cosmopolitan species mainly distributed in tropical and subtropical coastal waters. Analysis of the mitochondrial cytochrome c oxidase subunit I (COI) gene sequences among S. nudus from GenBank revealed high genetic variation (p-distance, 0.115-0.235; k2p, 0.128-0.297) and paraphyletic relationships. These indicated misidentification and/or cryptic diversity may be present in the genus Sipunculus. To understand the genetic diversity and to manage the recourse of S. nudus, we collected specimens from coastal waters of southern China and Taiwan. In the phylogenetic topology, specimens can be separated into four distinct clades; three of these clades (clade A, B and C) were only represented from this region (southern China and Taiwan), but the clade D grouped with individuals from Central America (Atlantic coast). Furthermore, individuals of clades A and D were collected at the same location, which does not support the hypothesis that this genetic break reflects contemporary geographical isolation. The four distinct clades observed among coastal waters of southern China and Taiwan indicated underestimated diversity. It is noteworthy that the cryptic diversity is vulnerable under high pressure of human activity.

Rapid sex identification of papaya (Carica papaya) using multiplex loop-mediated isothermal amplification (mLAMP).

Papaya (Carica papaya L.) is established as a cash crop throughout the tropical and subtropical regions due to its easy adaptation to diverse agricultural conditions, high yields, and prompt returns. The sex types of papaya plants are hermaphrodite, male, and female. Among them, hermaphroditic plants are the major type in papaya production, because the fruit has commercial advantages over that of the other sexes. Sex inheritance in papaya is determined by the M and M(h) dominant alleles in males and hermaphrodites, respectively, and a recessive m allele in females. Currently, all hermaphrodite seeds are not available due to the lethality of dominant homozygosity. Therefore, in this study, six male-hermaphrodite-specific markers were developed for a rapid sex identification using multiplex loop-mediated isothermal amplification (mLAMP) to efficiently and precisely select hermaphroditic individuals in the seedling or early growth stage. The LM1-LAMP assay consisted of two sex-LAMP reactions for amplifying two male-specific markers (T12 and Cpsm90) in one reaction, and showed several advantages in terms of a rapid reaction time (<1 h), isothermal conditions (less equipment required), a high efficiency (0.5 ng of DNA required in the reaction mixture), and an economical reaction system (5 µl in volume). The established method can be easily performed in the field by visual inspection and facilitates the selection of all hermaphroditic individuals in papaya production.

Ultra-high performance liquid chromatography/tandem mass spectrometry determination of feminizing chemicals in river water, sediment and tissue pretreated using disk-type solid-phase extraction and matrix solid-phase dispersion.

This study developed and validated a method of measuring the feminizing chemicals 4-tert-octylphenol, 4-nonylphenol, nonylphenol monoethoxycarboxylate (NP(1)EC), nonylphenol monoethoxylate (NP(1)EO), nonylphenol diethoxylate (NP(2)EO), estrone, 17ß-estradiol, estriol, 17α-ethinyl estradiol and bisphenol A in river water, sediment, and tissue using ultra-high performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) and isotope-dilution techniques. Water samples were pretreated using disk-type automated solid-phase extraction (SPE). Solid samples of sediment, fish, and clams were treated with matrix solid-phase dispersion (MSPD) using C(8) adsorbent. Eluents were directly passed following alumina cartridges for cleanup. The signal intensity of analytes on electrospray ionization (ESI) was compared with that of atmospheric pressure photoionization (APPI). The analytes were separated on a UHPLC C(18) column with aqueous 10-mM ammonium acetate for NPEOs and aqueous 10-mM N-methylmorpholine for the other compounds. On-line cleanup was evaluated using two-dimensional liquid chromatography (2-D LC). ESI could provide satisfactory response for all of the analytes. Though APPI did not offer suitable response for NP(1)EO, NP(2)EO and NP(1)EC, it provided better signal intensities for the steroid estrogens (1.0-2.4 times) and the phenols (3.2-4.4 times) than ESI. UHPLC shortened chromatographic time to less than 10 min. Disk-type automated SPE and MSPD dramatically increased the throughput of sample preparation. The extraction efficiency on surface water samples ranged from 10% to 91%. The extraction efficiency of MSPD on sediment, fish, and clams was 51-101%, 36-109%, and 30-111%, respectively. Acidic alumina cleanup was essential for the analysis of the tissue sample, and reduced matrix effects better than 2-D LC on-line cleanup. The limits of detection (LODs) in water ranged from 0.81 ng/L to 89.9 ng/L. The LODs in sediment and tissue ranged from tens of pg/g wet weight to only a few ng/g wet weight. This method proved to be accurate and reproducible, as both quantitative biases and relative deviations remained smaller than 20% at three spiked levels.

Testosterone improves the transition of primary oocytes in artificial maturation eels (Anguilla japonica) by altering ovarian PTEN expression.

In mammals, androgens appear to enhance the development of primary ovarian follicles, but PI3K (phosphoinositide 3-kinases) pathway is well recognized as one of the critical pathways in early follicular development. Roles of the PI3K were revealed by deletion of PTEN (phosphatase and tensin homolog on chromosome 10). PTEN is demonstrated to play an important role in the early stage of follicle development. In the Japanese eel, two forms of PTEN have been cloned, but what their functions on the development of early ovarian follicles are still not clear. The natural blockage and inducible of ovarian development was a benefit to address this question in the eel. Testosterone (T) shows to ameliorate the early ovarian development in the eel. The aims of this study were to elucidate the two forms of PTEN by cellular and physiological criteria and to study the effects of T on the ovarian PTEN production in the exogenous pituitary extracts-stimulated eel. Our results suggested that two forms of PTEN are existing in the Japanese eel, and eel ovarian development corresponded to the decrease in ovarian PTEN expression, vice versa. In addition, the supplement of T on eel early ovarian development can be attributed to its PTEN inhibitor role.

Species and sex identification of Formosa landlocked salmon using loop-mediated isothermal amplification.

Species and sex identification are among the most important parameters for conservation management. However, it is extremely difficult to perform such identification in Formosa landlocked salmon (Oncorhynchus masou formosanus). Both sexual dimorphism in landlocked dwarf form Formosa landlocked salmon and morphological difference among cherry salmon complex are minimal. We developed a simple, rapid and noninvasive method for identifying sex and species of this critically endangered species using a loop-mediated isothermal amplification (LAMP) technique. The LAMP assay showed the advantage of simple detection (evaluated by visual inspection), rapid reaction time (< 1 h), isothermal condition (less equipment required) and high efficiency (only 0.5-5 pg of DNA was required in the reaction mixture). Therefore, the method is more economical and practical than PCR. The LAMP assay can be easily performed in the field and is a valuable tool for detecting sex ratios in wild populations and identifying species in commercial imports. This is the first application of LAMP in identifying species and sex of salmonids as far as we know and clearly shows the potential application of LAMP in molecular ecology and conservation efforts.

A novel growth hormone 1 gene-derived probe for Oncorhynchus masou formosanus distinguished from the Oncorhynchus subspecies.

Traditional mitochondrial 16S rRNA is commonly used in many species identification studies. However, it is difficult to apply to the phylogenetic studies among the Oncorhynchus subspecies, which is a crucial need for management purposes for Oncorhynchus masou formosanus, Taiwan salmon. In this study, we have developed an improved species identification method for Taiwan salmon distinguished with other Oncorhynchus subspecies tested by exploiting PCR for growth hormone (GH) 1 gene. By comparing DNA sequences for GH1 from 11 species of Oncorhynchus subspecies we designed novel PCR primers that exploit differences between Taiwan salmon and other Oncorhynchus subspecies. Therefore, the technique is an important tool in the management of populations of the endangered land-locked Taiwan salmon preventing from their possible hybrids with other Oncorhynchus subspecies once tested.

Cryopreservation of a marine microalga, Nannochloropsis oculata (Eustigmatophyceae).

The cryopreservation of algae could prevent genetic drift and minimize labor costs compared to the current method of maintenance and subculturing. Clear, simple protocols for cryopreservation of marine microalga, Nannochloropsis oculata were developed and cryoprotectant choice and concentration optimized. The viability of the microalga was assessed directly after thawing, and algal concentration was measured after 2-30 days of growth. Five cryoprotectants (dimethyl sulphoxide, Me2SO; ethylene glycol, EG; glycerol, Gly; methanol, MeOH; and propylene glycol, PG) at five concentrations (10, 20, 30, 40, and 50%; v/v) were evaluated to determine the toxicity of various cryoprotectants to N. oculata. The toxicity of cryoprotectant (Me2SO, EG, MeOH, and PG) was observed only at higher concentrations of CPAs: > 20% for EG, > 30% for Me2SO and methanol, and > 40% for PG. Direct freezing of algae in liquid nitrogen resulted in a severe loss of viability and a modified cryopreservation protocol proved to be more appropriate for the preservation of N. oculata. Cryopreservation protocols developed and tested in the present study might be applied to cryopreserving other strains, or species, in this genus.

Evaluation of damage in Pacific oyster (Crassostrea gigas) spermatozoa before and after cryopreservation using comet assay.

We examined the applicability of the comet assay (single cell gel electrophoresis assay) to estimate the quality of frozen-thawed Pacific oyster (Crassostrea gigas) spermatozoa. Comet assay was performed on semen before and after cryopreservation followed by fluorescent staining with propidium iodide to assess DNA integrity. After cryopreservation, the percentage of spermatozoa with damaged DNA significantly increased, while only about half of the cells displayed intact DNA, even when protected with 10 percent DMSO. All the considered parameters (head length, head area, head intensity, total length, total area, total intensity, tail length percent, tail area percent, and tail intensity percent) were higher than the oyster sperm protected with 10 percent DMSO-artificial sea water after freezing and thawing. Only tail length percent, tail area percent, and tail intensity percent were increased significantly after cryopreservation. The tail length percent was found to be the most sensitive indicator of the cryopreservation-induced DNA damage. Our freeze-thawing procedure significantly affected oyster sperm DNA, as indicated by the reduced fertilization rate when frozen-thawed oyster sperm are used. Irreversible alteration of the genome may prevent fertilization or alter normal embryonic development. This study is the first to demonstrate that the comet assay is an inexpensive, rapid and sensitive method for determining DNA damage in Pacific oyster sperm quality assessments.

Semen cryopreservation of small abalone (Haliotis diversicolor supertexa).

Methods for cryopreserving spermatozoa and maximizing fertilization rate in Taiwan small abalone, Haliotis diversicolor supertexa, were developed. The gametes (spermatozoa and eggs) of small abalone were viable 3 h post-spawning, with fertilization, and development rate decreasing with time. A minimum of 10(2) cell/ml sperm concentration and a contact time of 2 min between gametes is recommended for artificial insemination of small abalone eggs. Eight cryoprotectants, dimethyl sulfoxide (DMSO), dimethyl acetamide (DMA), ethylene glycol (EG), propylene glycol (PG), butylene glycol (BG), polyethylene glycol, glycerol and methanol, were tested at concentrations between 5 and 25% to evaluate their effect on motility of spermatozoa exposed to cryoprotectant for up to 60 min at 25 degrees C before freezing. The least toxic cryoprotectant, 10% DMSO, was added to artificial seawater (ASW) to formulate the extender for freezing. Semen was diluted 1:1 with the extender, inserted into 1.5 ml microtubes and frozen using a cooling rate between -3.5 and -20 degrees C/min to various transition temperatures (0, -30, -60, -90 and -120 degrees C), followed by transfer and storage in liquid nitrogen (-196 degrees C). The microtubes were thawed from +45 to +145 degrees C/min. Spermatozoa, cooled to -90 degrees C at a cooling rate of -12 or -15 degrees C/min and then immersed in liquid nitrogen, had the best post-thaw motility. Post-thaw sperm motility was markedly reduced compared to fresh sperm. More frozen-thawed spermatozoa are required to achieve fertilization rates comparable to those achieved using fresh spermatozoa.

Ultrastructure of the spermatozoon of the teleost fish Acanthopagrus schlegeli (Perciformes: Sparidae).

Ultrastructurally the spermatozoon of Acanthopagrus schlegeli (Sparidae) has a spherical, homogeneously electron-dense nucleus with a deep axial nuclear fossa, and an unusual notch, shaped like a bowtie, in the nuclear region. The short midpiece contains four spherical mitochondria and encircles the basal body of the flagellum. It is concluded that the spermatozoon is of a primitive type, although it is characterized by several unique features which may provide useful systematic characters. © 1993 Wiley-Liss, Inc.