Morphometric characteristic of sex cells and seasonal changes of female gonads of Cucumaria japonica (Semper, 1868) (Echinodermata: Holothuroidea) from Peter the Great Bay, Sea of Japan

The oocytes at different stages of development were characterized morphometrically, and seasonal changes in female gonads of the Far-eastern edible holothurian Cucumaria japonica (Semper, 1868) from the Peter the Great Bay were described. Groups of oocytes and categories of sexual tubules corresponding to different stages of development of gonads were distinguished. Free laying oocytes were divided into several groups, i.e., oocytes 3, 4 and 5. The greatest size of C. japonica oocytes was 460 μm. In various seasons of the year there was greater growth of oocytes in the gonads, which reached its maximum volume during reproduction of the holothurians. The gonads developed asynchronously. All the stages of development were observable simultaneously in the gonads of each individual of this species during the reproductive cycle, and the stage of fast growth prevailed. C. japonica spawned twice a year, in May–June and from July to October. Unreleased oocytes were subjected to resorption.     

The composition and seasonal dynamics of the hemocyte cell population in the clams <Emphasis Type="Italic">Corbicula japonica</Emphasis> Prime (1864) of the Kievka River (the basin of the Sea of Japan)

The parameters of a hemocyte cell population were investigated in the bivalve mollusk Corbicula japonica of the Kievka River (the basin of the Sea of Japan) at four seasonal points corresponding to different stages of the reproductive cycle: (1) March (sexual rest); (2) May (gametogenesis); (3) July (spawning); and (4) November (the end of reproductive activity). Using light microscopy, we identified four cell morphotypes in the hemolymph: small and large hyalinocytes, as well as basophilic and eosinophilic granulocytes. Flow cytometry confirmed the structural heterogeneity of the cell population and allowed us to estimate the seasonal variability of the cell composition in C. japonica hemolymph. The total cell count in the mollusk hemolymph did not change significantly during the annual cycle and did not correlate with the average cell size of hemocytes. In March, May, and November, granulocytes dominated in the cell population (74.3 ± 2.9, 77.5 ± 3.5, and 86.7 ± 2.6%, respectively), while in July their relative content was reduced dramatically (37.2 ± 5.1%) causing a significant decrease of both the average cell size and granularity in circulation. Most likely, this is connected with the summer migration of mature eosinophilic granulocytes from the hemolymph to spawning gonads for resorption of the unspent sexual products, as well as to other internal organs for participation in digestion and to provide the immune defense against pathogens.     

Reinvestigation of epithelial lining of the genital coelomic sinus in asteroids. An ultrastructural study

Ultrastructural study of gonadal muscles in sea star, Asterina pectinifera, showed that myoepithelial cells were located only in the epithelial lining of the genital coelomic sinus. No myoepithelial cells were found in the visceral peritoneal epithelium or within connective tissue layer of the outer sac. Morphology of the myoepithelial cells in gonads of A. pectinifera varies during the reproductive cycle. During the gametogenic phase of the reproductive cycle, the myoepithelial cells get an elongated, spindle-like shape having a length of 20–30 μm. In prespawning gonads, many of the myoepithelial cells form cytoplasmic extensions of 3–5 μm in length, filled with myofilaments and penetrating into the underlying connective tissue of the outer sac or haemal sinus. Besides, myoepithelial cells, simultaneously anchored in the inner and outer sacs, were also observed. These changes result in development of more elaborated musculature and increase in contractility of the gonadal wall in prespawning gonads as compared to that during other stages of the reproductive cycle.     

Rapid recovery of the intertidal seagrass Zostera japonica following intense Manila clam (Ruditapes philippinarum) harvesting activity in Korea

Although the Manila clam (Ruditapes philippinarum) culture grounds are occasionally located in Zostera japonica beds along the coasts of Korea, plant responses to the clamming activity have not been reported for this seagrass species. Intense Manila clam harvesting activity took place in the intertidal Z. japonica bed during April 2004. The Z. japonica bed at the study site has been monitored since January 2003. Thus, this study provided a unique opportunity to compare the structure of the Z. japonica population before and after the clamming activity, which was conducted for approximately 1 week in April 2004. All Z. japonica shoots were removed and buried in the sediment immediately after the clamming activity. However, a few shoots were found at the disturbed area in July 2004, 3 months after the clamming activity. By September 2004, 5 months after the disturbance, shoot density and biomass were almost recovered to the levels reported before the clamming activity. No Z. japonica seedlings were observed when the shoot density rapidly increased in August and September 2004, 4-5 months after the disturbance, because revegetation of the disturbed seagrass bed has occurred before the seed germination time which is typically winter or early spring in this area. Thus, the initial rapid revegetation of the disturbed area occurred via asexual reproduction through new shoot formation from the buried below-ground tissues. The reproductive shoot density and reproductive efforts of Z. japonica were significantly higher after the disturbance relative to the levels recorded before the disturbance, and the duration of the fertile period was approximately three times longer following the clamming activity. The belowground biomass after the disturbance was also significantly higher than that before the disturbance. These results suggest that Z. japonica allocated more energy to sexual reproduction, as well as the maintenance of belowground tissues, to persist their population under unstable environmental conditions.     

Occupational strategy of runner reed (Phragmites japonica): Change of growth patterns with developmental aging

The characteristics of P. japonica stands growing along ten transect in an AFP were assessed. Especially those of the reproductive organs in terms of developmental stages were focused. Four consecutive stages were observed based on the existence of reproductive organs (flowers and stolons). Plants in the ‘stolon’ and ‘flower and stolon’ stages played a role as a colonizer by stolonization (the longest length of stolon was more than 9 m) while those in the ‘flower’ stage played a role as an occupant via forming tall and dense shoots on tussocks (more than 2 m and about 20 shoots per tussock including dead standing shoots). Consecutive developmental stages according to changes in growth patterns, particularly in the conversion of reproductive organ from asexual (stolon) to sexual (flower), was the critical strategy of P. japonica for efficient monotypic occupation. This occupational strategy of P. japonica also resulted in a sharp decrease of the plant species richness (two to three including P. japonica) growing in the same area with P. japonica in the mature (‘flower’) stage.     

The state of reproductive glands of the pike Esox lucius from water bodies of the Chernobyl trail in 1999–2004

The state of the reproductive system of the pike Esox lucius, descendants of individuals exposed to X-ray irradiation due to the catastrophe at the Chernobyl Nuclear Power Plant in 1986, was studied. The material was collected in water bodies of Ukraine contaminated with radionuclides—Kiev Reservoir, the Teterev River, and Lake Glubokoe in the postcatastrophe period 1999–2004. It was shown that the total number and the extent of disturbances in the pike gonads are positively correlated with the levels of contamination of the water bodies: in the cleanest water body, the Teterev River, the proportion of fish with gonads without considerable deviations was 70%; in the Kiev Reservoir, it was 45%; no individuals with normal gonads were recorded in Lake Glubokoe. The great number of anomalies in sexual cells and gonads has led to a decrease in the reproductive capacity of the pike populations studied. A unique case was revealed of synchronous hermaphroditism in a pike from the Kiev Reservoir that belonged to the fourth generation (F₄) of fish that underwent emergency loads. Among postemergency pike generations, the maximum number of disturbances in sexual cells and gonads was recorded in F_2–4, which is possibly accounted for by the phenomenon of “prolonged mutagenesis.”     

COMPARISONS OF LIFE-HISTORY TRAITS AND SEXUAL DIMORPHISM BETWEEN ASSIMINEA JAPONICA AND ANGUSTASSIMINEA CASTANEA (GASTROPODA: ASSIMINEIDAE)

Life histories of two sympatric assimineid gastropods, Assimineajaponica and Angustassiminea castanea, were investigated ata reed marsh in northern Japan. Size frequency distributionsrevealed that growth curves of females and males differed inboth species resulting in sexual dimorphisms in shell size.In A. japonica, the shell height of females was larger thanthat of males, whereas in A. castanea males attained a biggershell than females. Mating behaviour of A. japonica was observedmainly from February to June, while that of A. castanea wasobserved from May to August. The diameter of deposited eggsof A. japonica was 200 to 250 mm, whereas that of A. castaneawas 125 to 150 mm. The length of the veliger larva of A. japonicaat hatching was approximately 200 mm, whereas that of A. castaneawas approximately 125 mm. Longevity, age at sexual maturityand opportunities for reproduction during life time were estimatedin the field. For A. japonica, sexual maturity was establishedby the age of 17 months and longevity was about three years.Opportunities for reproduction of A. japonica occurred twiceduring a lifetime. For A. castanea, sexual maturity was establishedby the age of 10 months and longevity was almost five years.Opportunities for reproduction of A. castanea occurred fivetimes during a lifetime. The differences of life-history traitsand sexual dimorphism between species and possible explanationsof evolutionary factors are discussed. To whom correspondence should be addressed. (Received 21 January 1999; accepted 8 September 1999)     

Activation of the Hedgehog pathway in the mouse fetal ovary leads to ectopic appearance of fetal Leydig cells and female pseudohermaphroditism

Proper cell fate determination in mammalian gonads is critical for the establishment of sexual identity. The Hedgehog (Hh) pathway has been implicated in cell fate decision for various organs, including gonads. Desert Hedgehog (Dhh), one of the three mammalian Hh genes, has been implicated with other genes in the establishment of mouse fetal Leydig cells. To investigate whether Hh alone is sufficient to induce fetal Leydig cell differentiation, we ectopically activated the Hh pathway in Steroidogenic factor 1 (SF1)-positive somatic cell precursors of fetal ovaries. Hh activation transformed SF1-positive somatic ovarian cells into functional fetal Leydig cells. These ectopic fetal Leydig cells produced androgens and insulin-like growth factor 3 (INLS3) that cause virilization of female embryos and ovarian descent. However, the female reproductive system remained intact, indicating a typical example of female pseudohermaphroditism. The appearance of fetal Leydig cells was a direct consequence of Hh activation as evident by the absence of other testicular components in the affected ovary. This study provides not only insights into mechanisms of cell lineage specification in gonads, but also a model to understand defects in sexual differentiation.     

Allocation of nutrients during the reproductive cycle of Ophidiaster ophidianus (Echinodermata: Asteroidea)

The reproductive cycle of Ophidiaster ophidianus (strictly protected status) from São Miguel Island, in the Azorean Archipelago was studied. The reproductive strategy; the energy allocation of each sex during the reproductive cycle and the nutritional condition of the population were analyzed. Gonadal index (GI) showed a clear seasonal pattern with spawning between August and October but histological examination revealed that gamete release can occur throughout the entire year. The pyloric caeca index (PCI) showed little annual variation but with an inverse relationship with the GI. Allocation of energy to the gonads and to the pyloric caeca reflected the seasonal reproductive strategy of this species. Individuals were able to simultaneously develop gonads, pyloric caeca, and quickly regenerate lost arms. There was a major expenditure of energy by females compared to males but, sexual size dimorphism was not observed. The reproductive pattern observed in O. ophidianus combining rich food availability and seawater temperatures characteristic of a temperate zone may be the key to the success of this species in the Azorean oceanic Island.     

Relationship between burrowing activity of the polychaetous annelid, Neanthes japonica (Izuka) and nitrification-denitrification processes in the sediments

The influence of the benthic organism, Neanthes japonica (Izuka) on nitrification-denitrification processes has been studied in experimental aquaria. The aquaria were filled with diluted sea water with or without nitrate, and contained or did not contain N. japonica. In the series supplied with diluted sea water without nitrate, extremely high concentrations of nitrite + nitrate nitrogen were found in the inner layer of the burrow wall, and denitrification activity in the surface layer (0–0.5 cm) containing N. japonica was three times that of control. In the series supplied with nitrate, there was no significant difference in the activity between surface layers with and without N. japonica. The influence of bioturbation of sediments by N. japonica on nitrification-denitrification processes and its mechanisms have been discussed.     

Histology of the reproductive tissue of the clam Ruditapes decussatus from the gulf of Gabès (Tunisia)

Summary

The gametogenic cycle of the clam Ruditapes decussatus was investigated during one year. Samples were collected each month in two different sites located at the gulf of Gabès area. Gametogenic activity was characterized on the basis of histological analysis of the gonads. A classification and a scoring system were then proposed. According to our study, Ruditapes decussatus gametogenesis can be divided in four major steps: (1) a sexual rest (December- February), (2) starting of gametogenesis (February-March), (3) development (March-May), (4) ripe (from April onwards). This study revealed that the gonad of the clam Ruditapes decussatus was surrounded by muscular tissue which could represent the principal storage tissue. Two kinds of hermaphroditism appeared since maturity of gonads: simultaneous one showing gonadal tubules of both males and females in regionally distinct and separate zones and a “superposed” one showing a gonad consisting essentially of mature male gametes and few residual oocytes.     

Intersex Occurrence in Rainbow Trout (Oncorhynchus mykiss) Male Fry Chronically Exposed to Ethynylestradiol

This study aimed to investigate the male-to-female morphological and physiological transdifferentiation process in rainbow trout (Oncorhynchus mykiss) exposed to exogenous estrogens. The first objective was to elucidate whether trout develop intersex gonads under exposure to low levels of estrogen. To this end, the gonads of an all-male population of fry exposed chronically (from 60 to 136 days post fertilization – dpf) to several doses (from environmentally relevant 0.01 µg/L to supra-environmental levels: 0.1, 1 and 10 µg/L) of the potent synthetic estrogen ethynylestradiol (EE2) were examined histologically. The morphological evaluations were underpinned by the analysis of gonad steroid (testosterone, estradiol and 11-ketotestosterone) levels and of brain and gonad gene expression, including estrogen-responsive genes and genes involved in sex differentiation in (gonads: cyp19a1a, ER isoforms, vtg, dmrt1, sox9a2; sdY; cyp11b; brain: cyp19a1b, ER isoforms). Intersex gonads were observed from the first concentration used (0.01 µg EE2/L) and sexual inversion could be detected from 0.1 µg EE2/L. This was accompanied by a linear decrease in 11-KT levels, whereas no effect on E2 and T levels was observed. Q-PCR results from the gonads showed downregulation of testicular markers (dmrt1, sox9a2; sdY; cyp11b) with increasing EE2 exposure concentrations, and upregulation of the female vtg gene. No evidence was found for a direct involvement of aromatase in the sex conversion process. The results from this study provide evidence that gonads of male trout respond to estrogen exposure by intersex formation and, with increasing concentration, by morphological and physiological conversion to phenotypic ovaries. However, supra-environmental estrogen concentrations are needed to induce these changes.     

The Maestro (Mro) Gene Is Dispensable for Normal Sexual Development and Fertility in Mice

The mammalian gonad arises as a bipotential primordium from which a testis or ovary develops depending on the chromosomal sex of the individual. We have previously used DNA microarrays to screen for novel genes controlling the developmental fate of the indifferent embryonic mouse gonad. Maestro (Mro), which encodes a HEAT-repeat protein, was originally identified as a gene exhibiting sexually dimorphic expression during mouse gonad development. Wholemount in situ hybridisation analysis revealed Mro to be expressed in the embryonic male gonad from approximately 11.5 days post coitum, prior to overt sexual differentiation. No significant expression was detected in female gonads at the same developmental stage. In order to address its physiological function, we have generated mice lacking Maestro using gene targeting. Male and female mice homozygous for a Mro null allele are viable and fertile. We examined gonad development in homozygous male embryos in detail and observed no differences when compared to wild-type controls. Immunohistochemical analysis of homozygous mutant testes of adult mice revealed no overt abnormalities. Expression profiling using DNA microarrays also indicated no significant differences between homozygote embryonic male gonads and controls. We conclude that Maestro is dispensable for normal male sexual development and fertility in laboratory mice; however, the Mro locus itself does have utility as a site for insertion of transgenes for future studies in the fields of sexual development and Sertoli cell function.     

Discovery and evaluation of candidate sex-determining genes and xenobiotics in the gonads of lake sturgeon (Acipenser fulvescens)

Modern pyrosequencing has the potential to uncover many interesting aspects of genome evolution, even in lineages where genomic resources are scarce. In particular, 454 pyrosequencing of nonmodel species has been used to characterize expressed sequence tags, xenobiotics, gene ontologies, and relative levels of gene expression. Herein, we use pyrosequencing to study the evolution of genes expressed in the gonads of a polyploid fish, the lake sturgeon (Acipenser fulvescens). Using 454 pyrosequencing of transcribed genes, we produced more than 125 MB of sequence data from 473,577 high-quality sequencing reads. Sequences that passed stringent quality control thresholds were assembled into 12,791 male contigs and 32,629 female contigs. Average depth of coverage was 4.2 × for the male assembly and 5.5× for the female assembly. Analytical rarefaction indicates that our assemblies include most of the genes expressed in lake sturgeon gonads. Over 86,700 sequencing reads were assigned gene ontologies, many to general housekeeping genes like protein, RNA, and ion binding genes. We searched specifically for sex determining genes and documented significant sex differences in the expression of two genes involved in animal sex determination, DMRT1 and TRA-1. DMRT1 is the master sex determining gene in birds and in medaka (Oryzias latipes) whereas TRA-1 helps direct sexual differentiation in nematodes. We also searched the lake sturgeon assembly for evidence of xenobiotic organisms that may exist as endosymbionts. Our results suggest that exogenous parasites (trematodes) and pathogens (protozoans) apparently have infected lake sturgeon gonads, and the trematodes have horizontally transferred some genes to the lake sturgeon genome.     

Ecological responses and remediation ability of water fern (Azolla japonica) to water pollution

The ability of the water fern Azolla japonica to remediate phosphorus (P), nitrogen (N), and iron (Fe) contamination, and its physiological responses to three common sources of water pollution (landfill leachate, swine lagoon sewage, and fish farm sewage) and standard solution were investigated. The biomass, water content, and chlorophyll content of Azolla japonica in each solution were measured, and the concentrations and accumulation rates of polluting elements in the solutions were determined. A. japonica showed over eight-fold increase in biomass within only 20 d in every solution except in swine lagoon sewage, extremely high in N concentration. Consistent chlorophyll and water contents of the plant in most solutions showed that A. japonica can adapt to highly concentrated solutions. N, P, and Fe concentrations of the solutions decreased significantly within the 20 d. In most treatments, A. japonica showed high N accumulation and also showed total uptake of P and Fe from the solutions. In reference to this result, using this species as a phytoremediator plant would have additional benefits of helping maintaining endangered populations of A. japonica. Therefore, the plant??s fast growth, good element remediation efficiency, and conservation needs makes A. japonica a suitable plant species for pollution remediation.     

An iridescent virus fromPopillia japonica (Col.: Scarabaeidae)

A very low incidence (<0.01%) of a blue iridovirus (IV) was found in larvae of the Japanese beetle,Popillia japonica Newman, that were sampled over a two year period on Terceira Island (Azores, Portugal). In the most heavily infected larvae, a deep blue iridescence was observed, particularly in the fat body. Transmission electron microscopy revealed the characteristic crystalline arrays of the hexagonal virus particles in the cytoplasm of fat body cells, tracheal matrix, muscle, hypodermis and blood cells. Crystals of the virus particles were also observed freely circulating in the hemolymph. The average diameter of negatively stained purified virus particles was 157 nm. Similarities and differences with other IVs found in the Scarabaeidae are discussed. Considering the broad host range of some of the iridescent viruses, the relatively recent invasion of Terceira byP. japonica, and the rarity of the virus in the beetle, it is probable that the infection was the result of transmission from another species of soil-inhabiting arthropod. Its value as a potential biological control agent ofP. japonica is negligible.     

Photosynthesis-nitrogen relationship in a Hinoki cypress (Chamaecyparis obtusa) canopy: a comparison with Japanese cedar (Cryptomeria japonica)

The relationship between light-saturated photosynthetic capacity (P _max) and leaf nitrogen (N) content was investigated for one year in a 15-year-old Chamaecyparis obtusa canopy and was compared with a Cryptomeria japonica canopy previously described. The linear regression between P _max and leaf N content tended to converge toward a single line segment from July to January and in May for C. obtusa. The slope of the linear regression between P _max and leaf N content of C. obtusa was gentler than that of C. japonica. The smaller regression coefficient of C. obtusa may reflect species differences in nitrogen nutrition requirements between C. obtusa and C. japonica. A pronounced decrease in the slope of the linear regression lines due to low temperature was observed in February and March. During this period, P _max of C. obtusa declined more than that of C. japonica suggesting that C. obtusa is less tolerant to low temperatures than C. japonica.     

Life cycle stages of the benthic palytoxin-producing dinoflagellate Ostreopsis cf. ovata (Dinophyceae)

The asexual and sexual reproduction of Ostreopsis cf. ovata was studied in the field and in cultures isolated from two locations in the Mediterranean Sea. Asexual division took place in the motile stage by the sharing of theca (desmoschisis). High cell-size variability and differences in division capability were detected in the cultures. Thecal analyses and nuclear division patterns allowed characterization of the different phases of dividing cells obtained during an in situ cell-cycle sampling performed off Llavaneres beach (Northeast Spain). During the 45-h cycle, binucleated cells accounted for 2.6% of the population. Division was initiated with the onset of dusk and reached a maximum 3–4 h before dawn. No dividing cells were detected after 09:00 AM. Sexuality occurred both in cultures and in natural populations of O. cf. ovata. Mating gamete pairs were the only sexual stages that could be distinguished from vegetative stages. The differences between these pairs and dividing cells are described herein. None of the individually isolated gamete pairs underwent fusion nor encystment, instead, in most of them one member of the gamete pair divided. Rather than plasmogamy, there was evidence of nuclear migration from one gamete to the other. Pellicle cysts, thin-walled cysts, and thecate cysts were also identified and studied. These cysts, embedded in abundant mucilage in a bloom-derived incubated sample, were able to germinate for as long as 6 months after their formation. Our results suggest that they constitute the overwintering population that causes recurrent blooms of O. cf. ovata in some areas of the Mediterranean Sea.