The dopamine effect on sea urchin larvae depends on their age.

Activation of the dopamine type-D2 receptor in late gastrula of sea urchins is known to decrease the growth rate of post-oral arms of larvae, and, as a result, the phenotype of these larvae mimics that of larvae developing in the abundance of food. Our data indicate that the effect of dopamine on sea urchin larvae is stage-dependent. In our experiment, the early four-armed plutei (96 hours post fertilization, hpf) of Strongylocentrotus intermedius had substantially shorter post-oral arms if they developed from the larvae treated with dopamine at the early pluteus stage (48 hpf), when they had already formed the first dopaminergic neurons, as compared to the plutei from the larvae treated with dopamine at the mid to late gastrula stage (24 hpf), when they did not have any neurons yet. The pre-treatment of larvae in 6-hydroxydopamine, a neurotoxic analog of dopamine that specifically disrupts activity of dopaminergic neurons, prevented the development of the short post-oral arms phenotype in larvae. These results confirm the assumption that dopaminergic neurons play an important role in the development of the short post-oral arms phenotype in sea urchin larvae. Another finding of our study is that the dopamine treatment also affects the growth of the body rods and the overall larval body growth. Based on these observations, we suggest researchers to carefully select the developmental stage, pharmacological agents, and incubation time for experimental manipulation of sea urchin larvae phenotypes through dopaminergic nervous system.

Are myoepithelial cells confined to genital coelomic sinus in the gonads of sea stars?

An ultrastructural study of the gonadal wall in 10 sea star species from the orders Forcipulatida, Paxillosida, Spinulosida, Valvatida and Velatida has shown variations in the presence of myoepithelial cells in the visceral peritoneal epithelium. These cells have only been found in the peritoneal epithelium of the gonads in Aphelasterias japonica (Forcipulatida: Asteriidae), Asterias amurensis (Forcipulatida: Asteriidae), Distolasterias nipon (Forcipulatida: Asteriidae), Diplopteraster multipes (Velatida: Pterasteridae), Luidia quinaria (Paxillosida: Ctenodiscidae), and Pteraster sp. (Velatida: Pterasteridae). Our results may shed light on the evolution of peritoneal epithelium of sea star gonads. It is probable that, initially sea stars had myoepithelial cells in visceral peritoneal epithelium of the gonads. The species from the orders Forcipulatida and Velatida have retained this plesiomorphic state, while many species from the orders Paxillosida, Spinulosida and Valvatida have lost myoepithelial cells from visceral peritoneal epithelium of their gonads.

Microscopic anatomy of gonadal area in the deep-sea clam Calyptogena pacifica (Bivalvia: Vesicomyidae) with emphasis on somatic cells.

Somatic cells in the gonadal area of male and female deep-sea clams, Calyptogena pacifica, were examined using light and transmission electron microscopy. Acini both at the pre-spawning stage and at the stage of active spermatogenesis were observed to be simultaneously present in sections through a male gonad. Oocytes of various degrees of maturity were simultaneously present in female acini. No storage tissue and cells similar to adipogranular cells or vesicular connective tissue of other mollusks were found in the gonadal area of C. pacifica. Instead, in both males and females, numerous hemocytes surround the acini. Among hemocytes, two types of granulocytes and erythrocytes were identified. Bundles of muscle cells were also found in the interacinar space. Male intraacinar accessory cells were rather large, glycogen-rich, with lipid inclusions, and phagosomes contained spermatogenic cells. Female accessory cells had well-developed endoplasmic reticulum, but they did not form any follicles around oocytes, being in their basal part, closer to basal lamina. Such a specific pattern of gonad organization can presumably be explained by both symbiosis with sulfide-oxidizing bacteria and phylogenetic aspects that should be further studied. Some evidence for continuous gametogenesis are discussed.

External dsRNA Downregulates Anthocyanin Biosynthesis-Related Genes and Affects Anthocyanin Accumulation in Arabidopsis thaliana.

Exogenous application of double-stranded RNAs (dsRNAs) and small-interfering RNAs (siRNAs) to plant surfaces has emerged as a promising method for regulation of essential genes in plant pathogens and for plant disease protection. Yet, regulation of plant endogenous genes via external RNA treatments has not been sufficiently investigated. In this study, we targeted the genes of chalcone synthase (CHS), the key enzyme in the flavonoid/anthocyanin biosynthesis pathway, and two transcriptional factors, MYBL2 and ANAC032, negatively regulating anthocyanin biosynthesis in Arabidopsis. Direct foliar application of AtCHS-specific dsRNAs and siRNAs resulted in an efficient downregulation of the AtCHS gene and suppressed anthocyanin accumulation in A. thaliana under anthocyanin biosynthesis-modulating conditions. Targeting the AtMYBL2 and AtANAC032 genes by foliar dsRNA treatments markedly reduced their mRNA levels and led to a pronounced upregulation of the AtCHS gene. The content of anthocyanins was increased after treatment with AtMYBL2-dsRNA. Laser scanning microscopy showed a passage of Cy3-labeled AtCHS-dsRNA into the A. thaliana leaf vessels, leaf parenchyma cells, and stomata, indicating the dsRNA uptake and spreading into leaf tissues and plant individual cells. Together, these data show that exogenous dsRNAs were capable of downregulating Arabidopsis genes and induced relevant biochemical changes, which may have applications in plant biotechnology and gene functional studies.

Ultrastructural aspects of spermatogenesis in Calyptogena pacifica Dall 1891 (Vesicomyidae; Bivalvia).

The process of spermatogenesis and spermatozoon morphology was characterized from a deep-sea bivalve, Calyptogena pacifica (Vesicomyidae, Pliocardiinae), a member of the superfamily Glossoidea, using light and electron microscopy. Spermatogenesis in C. pacifica is generally similar to that in shallow-water bivalves but, the development of spermatogenic cells in this species has also some distinguishing features. First proacrosomal vesicles are observed in early spermatocytes I. Although, early appearance of proacrosomal vesicles is well known for bivalves, in C. pacifica, these vesicles are associated with electron-dense material, which is located outside the limiting membrane of the proacrosomal vesicles and disappears in late spermatids. Another feature of spermatogenesis in C. pacifica is the localization of the axoneme and flagellum development. Early spermatogenic cells lack typical flagellum, while in spermatogonia, spermatocytes, and early spermatids, the axoneme is observed in the cytoplasm. In late spermatids, the axoneme is located along the nucleus, and the flagellum is oriented anteriorly. During sperm maturation, the bent flagellum is transformed into the typical posteriorly oriented tail. Spermatozoa of C. pacifica are of ect-aqua sperm type with a bullet-like head of about 5.8 µm in length and 1.8 µm in width, consisting of a well-developed dome-shaped acrosomal complex, an elongated barrel-shaped nucleus filled with granular chromatin, and a midpiece with mainly four rounded mitochondria. A comparative analysis has shown a number of common traits in C. pacifica and Neotrapezium sublaevigatum.

Effect of dopamine on early larvae of sea urchins, Mesocentrotus nudus and Strongylocentrotus intermedius.

Larvae of many echinoids are known to be phenotypically plastic and capable of changing the growth rate of their post-oral arms depending on the microalgae concentration in their habitat. As literature data show, developing larvae use chemosensation to detect algae in the environment and "adjust" the rate of growth of their post-oral arms through dopamine signaling. According to our results, dopamine has a significant effect on the post-oral arm growth in early larvae of two sea urchin species, Mesocentrotus nudus and Strongylocentrotus intermedius. The dopamine effect depends on concentration: the higher the dopamine concentration in the water, the shorter the post-oral arms. We suggest that the pattern of response to variation in dopamine concentration, manifested by early larvae of both species, is similar to that observed at different concentrations of microalgae.

Autophagy in nutrient storage cells of the Pacific oyster, Crassostrea gigas.

In oysters, nutrients are stored in a special type of cells referred to as vesicular-connective tissue cells (VCT-cells). These cells accumulate and provide nutrient to satisfy various needs of the organism, including gametogenesis. During the annual reproductive cycle, VCT-cells pass through a series of changes in their morphology associated with nutrients mobilization for developing germ cells. The results presented here show an approximately 33-35% increase in the number of autophagic vesicles in cytoplasm of VCT-cells in the gonadal area of C. gigas during the stage of active gametogenesis as compared to the resting stage of reproductive cycle. No destruction of VCT-cells due to autophagy or any other factors was observed, both in males and females. Our results indicate that autophagy does increase in VCT-cells of C. gigas and plays a certain role in nutrient mobilization from these cells.

Induction of Transgene Suppression in Plants via External Application of Synthetic dsRNA.

Recent investigations show that exogenously applied small interfering RNAs (siRNA) and long double-stranded RNA (dsRNA) precursors can be taken up and translocated in plants to induce RNA interference (RNAi) in the plant or in its fungal pathogen. The question of whether genes in the plant genome can undergo suppression as a result of exogenous RNA application on plant surface is almost unexplored. This study analyzed whether it is possible to influence transcript levels of transgenes, as more prone sequences to silencing, in Arabidopsis genome by direct exogenous application of target long dsRNAs. The data revealed that in vitro synthesized dsRNAs designed to target the gene coding regions of enhanced green fluorescent protein (EGFP) or neomycin phosphotransferase II (NPTII) suppressed their transcript levels in Arabidopsis. The fact that, simple exogenous application of polynucleotides can affect mRNA levels of plant transgenes, opens new opportunities for the development of new scientific techniques and crop improvement strategies.

Morphology of nutrient storage cells in the gonadal area of the Pacific oyster, Crassostrea gigas (Thunberg, 1793).

Successful gametogenesis in invertebrates is tightly associated with functioning of specific nutrient-storing cells. In oysters, cells of vesicular connective tissue (VCT-cells), also referred to as storage cells, which form a meshwork around gonadal acini, are the major population of cells that accumulate and provide nutrients for developing gametes. During the annual reproductive cycle, populations of developing germ cells and VCT-cells demonstrate the inversely proportional size dynamics: the larger the acini, the smaller the VCT-cells. In the present study, the morphology of VCT-cells in the Pacific oyster, Crassostrea gigas, at the active gametogenesis stage of reproductive cycle has been studied using light and transmission electron microscopy. At this stage, VCT-cells are big, irregularly shaped cells containing large nucleus with a single large nucleolus. The cytoplasm contains weakly developed endoplasmic reticulum, mitochondria in the perinuclear area and at periphery of the cell, numerous lipid droplets, and glycogen particles. Ultrastructure of VCT-cells is similar to the organization of brown adipocytes in mammals. The surface of cells has numerous cytoplasmic processes that are presumably associated with the transport function and provide close interaction with adjacent cells. The spatial relationship between VCT-cells and myoepithelial elements of the gonad area is demonstrated and discussed.

Macroautophagy is involved in residual bodies formation during spermatogenesis in sea urchins, Strongylocentrotus intermedius.

An ultrastructural study of developing spermatids in sea urchins, Strongylocentrotus intermedius, showed that macroautophagy is involved in formation of residual bodies and removal of excessive cytoplasm by spermatids during spermatogenesis in this species. During late stages of spermatogenesis spermatids sequester excessive cytoplasm into vesicles, surrounded by a double membrane. Subsequently, these vesicles fused to one another into larger vacuoles, up to 1.5 µm in diameter. Finally, the vacuoles transformed into residual bodies by condensing their content into finely granular material of varying electron density, separated from cytoplasm by a single membrane. An immunoelectron microscopic study of late spermatids with the antibodies, raised against microtubule-associated protein 1 A/1B-light chain 3 (LC3), which is a marker of autophagosomes, showed that residual bodies in late spermatids of S. intermedius were LC3-positive.

Microautophagy in nutritive phagocytes of sea urchins.

Two types of cells were observed in germinative epithelium of male and female sea urchins: germ cells and somatic accessory cells; the latter referred to as nutritive phagocytes. At the onset of gametogenesis, nutritive phagocytes accumulate nutrients and greatly increase in their size. As gametogenesis progresses, the accumulated nutrients are transferred from nutritive phagocytes into developing gametes, and size of the nutritive phagocytes decreases. An electron microscopic study of nutritive phagocytes in sea urchins, Strongylocentrotus intermedius, at different stages of annual reproductive cycle showed for the first time that both macro- and microautophagy take place in nutritive phagocytes. Both processes occur simultaneously and regulate size and composition of nutritive phagocytes in male and female sea urchins. Nutritive phagocytes consume redundant cytoplasm via macroautophagy. Microautophagy is probably involved in consumption of redundant membranes that appear within nutritive phagocytes due to destruction of nutrient-storing globules, macroautophagy, and phagocytosis of germ cells or their remnants.

An ultrastructural study of testes permeability in sea urchins, Strongylocentrotus intermedius.

Permeability of testes in sea urchins, Strongylocentrotus intermedius, was investigated by using an electron-opaque tracer, lanthanum nitrate. This tracer is able to enter the basal compartment of germinative epithelium, where developing germ cells are located. However, its ability to penetrate the gonadal lumen was reduced. An incomplete permeability barrier between the basal compartment and the gonadal lumen is supposed to exist in testes of S. intermedius.

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 20­30 m. In prespawning gonads, many of the myoepithelial cells form cytoplasmic extensions of 3­5 m 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.

A brief summary of neuroendocrine regulation of reproduction in sea stars.

Over than fifty years starfishes have been widely used as model for studying the mechanisms of cell cycle regulation, oocyte maturation and fertilization. Besides, significant work has been done to investigate the role of nervous system in the control of reproduction and spawning in these animals. Nowadays, sea stars represent one of the most thoroughly studied model for hormonal regulation of reproduction among invertebrates. However, while the general picture of neuroendocrine control of asteroid reproduction can be drawn easily, our knowledge concerning the details of this process still has some gaps. Filling these gaps is essential for studying the diversity of hormonal mechanisms involved in regulation of animal reproduction. The present paper aims to briefly summarize current data on hormonal regulation of reproduction in sea stars and to highlight existing gaps in our knowledge on the details of this process.

Selective resorption in nutritive phagocytes of the sea urchin Anthocidaris crassispina.

Phagocytic resorption during spermatogenesis was studied in the sea urchin Anthocidaris crassispina. Nutritive phagocytes in gonad absorbed both waste sperm cells and residual bodies discarded from maturing spermatids, and these materials were subsequently compartmented in heterophagosomes. Based on 180 heterophagosomes examined by transmission electron microscopy, over 99% of heterophagosomes contained either residual bodies or sperm cells only. Simultaneous resorption of sperm cells and residual bodies in a heterophagosome was uncommon, with only approximately 0.56% occurrence, suggesting that heterophagosomes have a selective resorption ability in nutritive phagocytes.