Resolution of the Acanthopagrus black seabream complex based on mitochondrial and amplified fragment-length polymorphism analyses.

In this study, DNA analyses were employed to verify the identity of six morphologically similar species that occur in the coastal waters of Taiwan: the black seabream complex (Acanthopagrus latus, Acanthopagrus schlegelii, Acanthopagrus sivicolus, Acanthopagrus taiwanensis, Acanthopagrus chinshira and Acanthopagrus pacificus). Amplified fragment-length polymorphism (AFLP) analyses clearly distinguished the same six species that are morphologically diagnosable based on subtle differences in scale counts and anal-fin colouration. In contrast, mitochondrial DNA analyses based on cytochrome b gene sequences did not distinguish individuals of A. schlegelii and A. sivicolus, reflecting either historical introgression or recent speciation and incomplete sorting of their mitochondrial lineages. Phylogenetic relationships among these six north-west Pacific Ocean species of Acanthopagrus analysed using AFLP data were consistent with scale rows above the lateral line (TRac), sperm ultrastructure and geographical distribution. The study provides molecular tools for future research relevant to improved management of these resources, and an increased understanding of the evolutionary history of this radiation.

Two spinefoot colour morphs: mottled spinefoot Siganus fuscescens and white-spotted spinefoot Siganus canaliculatus are synonyms.

Mottled spinefoot Siganus fuscescens and white-spotted spinefoot Siganus canaliculatus are two similar species that differ subtly in colouration and morphology. Three major mtDNA clades were identified for these species, but individuals were clustered by amplified fragment length polymorphism (AFLP) according to geography rather than morphology, suggesting that the colour morphs are interbreeding.

Ultrastructure of the sperm of blue sprat, Spratelloides gracilis; Teleostei, Clupeiformes, Clupeidae.

The general sperm structure of the investigated clupeoids possess an oliviform head with a distinct deep nuclear fossa, a midpiece with one mitochondrion and a posterior flagellum. They are characterized by two apomorphies: an annular or C-shaped mitochondrion and ITDs (intratubular differentiation) in the A tubules of the axonemal doublets. The fine structure of the spermatozoa of blue sprat, Spratelloides gracilis, was investigated to see if resulting data conformed to the current hypotheses of the taxonomy of the Clupeidae. The mature sperm is characterized by the following features: (1) the nucleus is oliviform; its prominent deep nuclear fossa encloses the initial portion of flagellum, (2) a proximal centriole has not been identified, (3) a single spherical mitochondrion is located laterally in relation to the flagellum and (4) no cytoplasmic canal is present. Our blue sprat-spermatozoan morphology data suggest that the blue sprat has Clupeomorpha affinities, while indicating no close affinity with Elopomorpha. Clupeoid sperm exhibit morphological variations with the nucleus and mitochondrion being particularly variable. This study provides useful systematic characters to the existing knowledge of comparative spermatology and may provide additional clue to Euteleost phylogeny.

Spermatozoal ultrastructure of four Sparidae fishes: Acanthopagrus berda, Acanthopagrus australis, Lagodon rhomboids and Archosargus probatocephus.

The mature spermatozoa of two Taiwan protandrous hermaphrodite Sparidae Acanthopagrus berda and Acanthopagrus australis are investigated and compared with those of other two Sparidae (Lagodon rhomboids and Archosargus probatocephus) from the Western hemisphere. Ultrastructurally the spermatozoon of these four species has a spherical, homogeneously electron-dense nucleus with an axial nuclear fossa. The midpiece contains one to four spherical mitochondria and encircles the basal body of the flagellum. The mature spermatozoa of the four species are of the primitive or ect-aquasperm form and conform to the teleostean type I spermatozoon with the flagellar axis inserts perpendicular and medial to the nuclear fossa. Variation in the depths of the nuclear fossa and mitochondria number is substantial in these four Sparidae species. This study provide useful systematic characters to the existing knowledge of comparative spermatology of Sparidae.

Ultrastructure of Pagrus major and Rhabdosargus sarba spermatozoa (Perciformes: Sparidae: Sparinae).

Transmission and scanning electron microscopy were used to investigate the ultrastructure of spermatozoa in two Sparinae species Pagrus major and Rhabdosargus sarba. Ultrastructurally, the spermatozoa of P. major and R. sarba both consist of a spherical, homogeneously electron-dense nucleus with a deep axial nuclear fossa, and an unusual notch, in the nuclear region. The midpiece contains two spherical mitochondria in R. sarba and one in P. major. The comparison of spermatozoal ultrastructure of these two species of Sparidae shows that they closely resemble one another and suggests that they are closely related. Variation in the geometry and dimensions of the mitochondrion and nucleus is substantial in these two Sparidae species. It is concluded that the spermatozoa of both species are of primitive type, and they are distinguished by several unique features which may provide useful systematic characteristics.

Spermatozoal ultrastructures of two marine perciform teleost fishes, the goatfish, Paraupeneus spilurus (Mullidae) and the rabbitfish, Siganus fuscescens (Siganidae) from Taiwan.

Mature spermatozoa of two perciform teleost fishes, Paraupeneus spilurus (Mullidae) and Siganus fuscescens (Siganidae) from Taiwan were examined using transmission and scanning electron microscopy. Despite the fact that spermatozoa of both species are of the primitive type, the results of the present study highlight the potential application of spermatozoal morphology in studies of fish phylogenetic relationships. To our knowledge, the flattened nucleus observed in P. spilurus spermatozoa is reported for the first time. Several features common to Sigandae spermatozoa-the unusual almost parallel situation of the centrioles, the arrangement of mitochondria and the near absence of shallow nuclear fossa-are significantly different from other common teleost sperm types. These unique features may be synapomorphies for the Siganidae and Mullidae and evidently contribute to the study of phylogenetic relationships in teleosts.

The ultrastructure of the mature spermatozoon of the bivalve Gafrarium tumidum (Bivalvia, Heterodonta, Veneridae, Circinae).

Using transmission and scanning electron microscopy, we have described the mature spermatozoon of the bivalve Gafrarium tumidum (Heterodonta, Veneridae, Circinae) for the first time. The spermatozoon of G. tumidum is the ect-aquasperm type. The head contains a slightly curved nucleus. At its apex is a short cone-shaped acrosome. The structure of the acrosome is typical of heterodont bivalves and two major components of the acrosome vesicle material can be distinguished. The midpiece is an annular band of five mitochondria which surround the centriole complex. Sperm ultrastructure of G. tumidum provides additional information for the existing Veneridae phylogeny.

Spermatozoan morphology of four species of bivalve (Heterodonta, Veneridae) from Taiwan.

Using transmission and scanning electron microscopy, the mature spermatozoa of four bivalves of the family Veneridae--Gafrarium tumidum and Circe scripta (Circinae), Pitar sulfureum (Pitarinae) and Gomphina aequilatera (Tapetinae)--are described for the first time and compared with those of other bivalves, particularly other heterodonts. As our observations show, the spermatozoa of these four species are of the primitive type or ect-aquasperm form. The head contains a slightly curved nucleus with a short cone-shaped acrosome. The structure of the acrosome is typical of heterodont bivalves and two major components of the acrosomal vesicle material can be distinguished. The midpiece exhibits four or five mitochondria which surround the proximal and the distal centrioles. Variation in the shape and dimensions of the acrosomal vesicle and nucleus is substantial in these four Veneroidea species.

Cryopreservation of sperm from the endangered formosan landlocked salmon (Oncorhynchus masou formosanus).

The Formosan landlocked salmon (Oncorhynchus masou formosanus) are at a high risk of extinction, and the sustained maintenance of the population will soon depend on aquaculture systems, which use cryopreservation of spermatozoa to increase genetic diversity. We investigated the effectiveness of dimethyl sulfoxide (DMSO), dimethyl-acetamide (DMA), and methanol as cryoprotectants in combination with 300 mM glucose as extender on the freezing of Formosan landlocked salmon spermatozoa. We also evaluated the morphological changes of Formosan landlocked salmon spermatozoa after their immediate dilution in the 300 mM glucose-DMSO extender and after freeze-thawing. The spermatozoa frozen with DMSO as a cryoprotectant showed significantly higher post-thaw motility and fertility than spermatozoa frozen with DMA or methanol. The fertilization capacity of frozen-thawed Formosan landlocked salmon was comparable to that of fresh spermatozoa. Intersubspecies fertilization trials between cryopreserved Formosan landlocked salmon spermatozoa and Amago salmon eggs showed high fertilization rates. Based on the findings, the potential value of using sperm bank to safeguard this endangered species is discussed.

Preliminary experiments on the cryopreservation of penaeid shrimp (Penaeus japonicus) embryos, nauplii and zoea.

To improve availability of penaeid seedstock during periods of high demand, experiments were conducted to determine the feasibility of stockpiling embryos by freezing them. Embryos were screened for developmental stage; cryoprotectants, chilling effects, and freezing regimens were likewise evaluated. Juvenile forms (embryos, nauplii and zoea) of Penaeus japonicus were exposed to various cryoprotectants, including dimethyl sulfoxide, glycerol, methanol, ethylene glycerol and polyethylene glycol 300 under ambient temperature (25 degrees C). Following this bioassay, maximum safe concentrations of each cryoprotectant were tested on the juveniles under chilling to 0 degree C and with 42 freezing regimens. Methanol was found to be relatively nontoxic. Early developmental stages were the most sensitive to chilling. Initial attempts to freeze P. japonicus juveniles were reported. The survival rate of nauplii and zoea treated with 10% methanol in natural sea water (35 ppt salinity) and frozen to -15 degrees C was 85%, and some nauplii and zoea survived freezing to -25 and -196 degrees C. However, no treatment yielded normal nauplii or zoea after freezing.

Ultrastructural study of osmolality effect on spermatozoa of three marine teleosts.

With particular emphasis on mitochondria that may provide endogenous energy for spermatozoan motility, the morphological changes of the spermatozoa of three marine teleosts, black porgy (Acanthopagrus schlegelli), black grouper (Epinephelus malabaricus), and Atlantic croaker (Micropogonias undulatus), were compared either after activation in artificial sea water or when immersed in various osmotic pressure media. The midpieces of these three teleosts spermatozoa are composed of mitochondria surrounding the flagellum. Each mitochondrion is enclosed by distinct outer and inner membranes. The inner membrane separates the organelle's volume into two phases: the matrix and the intermembrane space. The inner membrane displays numerous infolding cristae that vary in number and shape and extend into the matrix. Following activation with artificial sea water, spermatozoa became motile and both the size and number of mitochondria decrease and then totally disappear. The present study strongly suggests that an energy source(s), responsible for motility, is located within the mitochondria in the midpiece of these three marine teleost spermatozoa.

Changes in mechanical tolerance and chilling sensitivity of red drum (Sciaenopus ocellatus) embryos during development.

The effects of mechanical shock and thermal stress on red drum (Sciaenopus ocellatus) embryos at specific developmental stages were studied. The tolerance of red drum embryos to mechanical and chilling shocks depended on the embryo's stage of development. Gastrulae stage was more susceptible to mechanical shock than the other developmental stages. However, a different ontogenetic tolerance pattern for chilling shock was observed. It is suggested that the embryonic developmental stage needs be considered in future studies on the cryopreservation of fish embryos.

Cryopreservation of oyster (Crassostrea gigas) embryos.

Several critical variables associated with successful cryopreservation of oyster embryos (Crassostrea gigas) were examined. These were 1) embryo developmental stage, 2) kind and concentration of cryoprotectant, 3) equilibration time, and 4) freezing rate. The percentage of survival was scored as the number of recovered embryos that swam actively 12 h after thawing and had developed into veliger stage. The oyster embryos became increasingly susceptible to the cryoprotectants as the concentration was increased and the equilibration time was lengthened. The stage of development appears to be a critical factor for survival of oyster embryos, with trochophore stage embryos more resistant than morula and gastrula stages embryos to cryoprotectant exposure and having better surviving after freezing. The optimum cryoprotectant concentration for the trochophore embryos differed markedly from the morula stage. Cryopreservation of fertilized eggs (2 to 8 cells) was unsuccessful. Varying degrees of success were achieved using gastrula- and trochophore-stage embryos. Maximum survival was obtained when trochophore embryos incubated in 10% propylene glycerol-artificial sea water were cooled at -2.5 degrees C/min to -30 degrees C and were then directly placed into liquid nitrogen. The results showed a clear effect of the stage of development on survival.

Cryopreservation of yellowfin seabream (Acanthopagrus latus) spermatozoa (Teleost, Perciformes, Sparidae).

The effects of both osmolality and cation in the initiation of sperm motility were examined in yellowfin seabream, Acanthopagrus latus. Various factors involved in the cryopreservation of yellowfin seabream spermatozoa on motility are discussed. Extender containing only glucose proved to be a suitable medium for freezing yellowfin seabream spermatozoa to -196 degrees C. Glycerol seems to have a direct osmotic effect on yellowfin seabream sperm cells, and it induced sperm motility before freezing and during thawing. However, this exhausted the energy needed for sperm motility for fertilization. Dimethyl sulfoxide (DMSO) proved superior to ethylene glycerol, propylene glycerol, glycerol and methanol as a cryoprotectant. Prolonged equilibration time had a detrimental effect on both prefreezing and post-thawing sperm motility. The estimated optimum freezing rate was in the range of -20 to -154 degrees C/min. More frozen-thawed than fresh spermatozoa are required to achieve comparable fertilization rates.

Spermatogenesis in the black porgy, Acanthopagrus schlegeli (Teleostei: Perciformes: Sparidae).

The ultrastructure of spermatogenesis and of the spermatozoon of Acanthopagrus schlegeli (Sparidae) are described. The testis is of the unrestricted type. Germ cells are surrounded by cyst cells. Spermiogenesis involves conspicuous modifications such as intracellular movements (diplosome and mitochondria migration, nuclear rotation, and depression) and structural changes (chromatin condensation, shape of mitochondria, and loss of cytoplasm). The mature spermatozoon has a spherical nucleus with a deep, axial nuclear fossa, and an unusual notch, shaped like a bow tie. The short midpiece contains four spherical mitochondria and encircles the basal body of the flagellum. It is concluded that the A. schlegeli spermatozoon is of a primitive type, but that it is characterized by a unique feature which may provide a useful systematic character.

Induced ovulation in Atlantic croaker (Sciaenidae) using hCG and an LHRH analog: A preliminary study.

A single dose of LHRHa (D-Ala(6), des Gly(10)- LHRH-ethylamide; 100 mug/kg body weight); administered by intramuscular injection, effectively induced ovulation in the Atlantic croaker (Micropogonias undulatus ); 37 of 40 (92%) females were successfully hand-stripped and there was no mortality. A single dose of human chorionic gonadotropin (hCG; 500 IU/kg body weight) was successful in inducing oocyte hydration. However, only 16 of 30 (53%) females ovulated successfully and could be hand-stripped. Another 8 females became extremely bloated and died. Thus LHRHa was shown to be the more reliable method of inducing ovulation in the Atlantic croaker. The response interval between hormone treatment and ovulation at different water temperatures was also determined.

Cryopreservation of Atlantic croaker spermatozoa: evaluation of morphological changes.

The spermatozoon of the Atlantic croaker (Micropogonias undulatus) is a primitive type in that it lacks an acrosome. The kidney-shaped head has a diameter of about 1.5 microns and is occupied by a granular and electron-dense nucleus. The short midpiece contains 3 spherical mitochondria and encircles the basal body of the flagellum but is separated from it. The flagellum consists of the typical 9 + 2 axoneme and surrounding plasma membrane but lacks a lateral ridge. Spermatozoa of Atlantic croaker diluted in either NaCl or sodium citrate solutions with or without DMSO were examined with the electron microscope before freezing in liquid nitrogen and after thawing. Damage following cryopreservation appeared to be greater to the mitochondria, plasma membrane, and 9 + 2 axoneme than to the nucleus. The incidence of postthaw damage in spermatozoa diluted with NaCl solutions containing DMSO was remarkably lower than that with either pure NaCl solutions, pure sodium citrate solutions, or sodium citrate solutions containing DMSO.