Genotoxicity and alteration of the Gene Regulatory Network expression during Paracentrotus lividus development in the presence of carbon nanoparticles

Nanoparticles are a newly emerging class of pollutants with eco-toxicological impacts on marine ecosystems; they are characterized by nano-scale size which improves their physical, chemical and biological properties. To better understand the mechanisms of embryotoxicity of carbon-based nanoparticles, the genotoxicity and the perturbation of the Gene Regulatory Network (GRN) expression have been investigated during the development of the sea urchin Paracentrotus lividus from fertilization to early pluteus stage. Increasing quantities of carbon nanoparticles (C-NPs), 0.5, 2.5 and 25 × 1013 C-NPs/500 cm3 of filtered seawater were administered during fertilization and the development was monitored up to the early pluteus stage (48 h). DNA damage and gene expression were assayed by Comet assay and Real-Time PCR, respectively. Taken together, our results indicate that embryo malformations taking place in the presence of C-NPs are due to altered regulation of the GNR and to a progressive accumulation of DNA single-strand breaks.

Towards a scientific-based farming of sea urchins: First steps in the cultivation of Diadema setosum, Diadema savignyi and Mesocentrotus nudus

FISHING OR BREEDING. This question arose relatively recently, but in the last decade, mankind will have to lean more towards the second. Sea reserves of useful species are exhausted. One possible solution to this problem is marine farming. We proposed to investigate the larval development of three sea urchin species: Diadema setosum (Leske, 1778), D. savignyi (Audouin, 1829) (South China Sea), and Mesocentrotus nudus (A. Agassiz, 1864) (Japan Sea). The larvae of Diadema setosum and D. savignyi were very similar, and some differences could only be observed at the late pluteus stage. These sea urchins were developed through the modified pluteus, which only had two pairs of larval arms. The arms were very long - in D. setosum above 2 mm, and in D. savignyi about 5.5 mm. Larval development took about 45 days in D. setosum and 47–50 days in D. savignyi. In contrast, Mesocentrotus nudus (A. Agassiz, 1864) was developed through the pluteus larvae, which had some differences from the pluteus of the genus Strongylocentrotus. Their dimensions did not reach one millimetre. The larval development of Mesocentrotus nudus lasted about 30 days. Analysis of material and time costs has led to the conclusion that Mesocentrotus nudus is the most convenient for obtaining seed material. However, this species cannot be used for the tropical zone. The results of D. savignyi and D. setosum can be used to increase the number of cultivated species.

DYNAMICS OF SPATIAL ABUNDANCE OF ZOOPLANKTON IN MORELLA COASTAL WATERS, CENTRAL MALUKU

The dynamics abundance of zooplankton in Morella coastal waters is poorly known.The purpose of this study was to detemine the abundance of zooplankton in Morella coastal waters during Southeast Monsoon. This research was conducted in August 2011. Plankton samples were collected from five stations, by NORPAC net that was vertically hauled from 10 meter depth up to the surface (except in Station 5, less than 10 m). The result showed that composition of zooplankton in Morella consisted of 43 taxa of zooplankton (33 taxa of holoplankton and 10 taxa of meroplankton). Total abundance of zooplankton was between 752 and 1050 ind/m3 (average 890±128 ind/m3), which was dominated by Copepods (53.70%). The abundace of the Copepods was between 368 and 742 ind/m3 (average 481±153 ind/m3). Copepods that typically found in coastal water may have higher tolerant on oceanographic factors than one that typically found in oceanic water. Sagitta enflata was identified as the most abundant, followed by Oncaea sp and Acrocalanus gibber. Echinoderms larvae were abundant in the pluteus stage (8 arms) indicated that spawning process occurred in August. The Bray-Curtis clustering analyses showed that in station 4 there were six different species zooplankton indentified in this location. Keywords: structure community, copepods, meroplankton, chaetoghnata, Morella

DYNAMICS OF SPATIAL ABUNDANCE OF ZOOPLANKTON IN MORELLA COASTAL WATERS, CENTRAL MALUKU

<p><em>T</em><em>he dynamics abundance of zooplankton in Morella coastal waters is poorly known</em><em>.</em><em>The purpose of this study was to detemine the abundance of zooplankton in Morella coastal waters during Southeast Monsoon</em>. <em>This research was conducted in August 2011. Plankton samples were collected from five stations, by NORPAC net that was vertically hauled from 10 meter depth up to the surface (except in Station 5, less than 10 m). The result showed that composition of zooplankton in Morella </em><em>consisted of</em><em> 43 taxa of zooplankton (33 taxa </em><em>of</em><em> holoplankton</em><em> and</em><em> 10 taxa </em><em>of </em><em>meroplankton). Total abundance of zooplankton </em><em>was </em><em>between 752</em><em> and </em><em>1050 ind/m³(average 890</em>±<em>128</em> <em>ind/m³</em>), <em>which was dominated by Copepods (53.70%)</em>. <em>The abundace of the Copepods </em><em>was </em><em>between</em><em> </em><em>368</em><em> and </em><em>742 ind/m³ (average 481±153 ind/m³). Copepods </em><em>that typically found in </em><em>coastal </em><em>water </em><em>may </em><em>have higher </em><em>tolerant </em><em>on</em><em> oceanograph</em><em>ic</em><em> factors</em><em> than one that typically found in oceanic water</em><em>. <span style="text-decoration: underline;">Sagitta</span> <span style="text-decoration: underline;">enflata</span> was identified as the most abundant, followed by <span style="text-decoration: underline;">Oncaea</span> <span style="text-decoration: underline;">sp</span> </em>and <em><span style="text-decoration: underline;">Acrocalanus</span> <span style="text-decoration: underline;">gibber</span></em>.<em> Echinoderms larvae </em><em>we</em><em>re abundant in the pluteus stage (8 arms)</em><em> indicated</em><em> that spawning process occur</em><em>red</em><em> in </em><em>August</em><em>. </em><em>The Bray-Curtis clustering</em><em> analys</em><em>es showed that in station 4 there were six different species zooplankton indentified in this location.</em></p> <p> </p> <strong><em>Keywords: </em></strong><em>structure community, copepods, meroplankton, chaetoghnata, Morella</em>

Left-right positioning of the adult rudiment in sea urchin larvae is directed by the right side

Indirect-developing sea urchins eventually form an adult rudiment on the left side through differential left-right development in the late larval stages. Components of the adult rudiment, such as the hydropore canal, the hydrocoel and the primary vestibule, all develop on the left side alone, and are the initial morphological traits that exhibit left-right differences. Although it has previously been shown that partial embryos dissected in cleavage stages correctly determine the normal left-right placement of the adult rudiment, the timing and the mechanism that determine left-right polarity during normal development remain unknown. In order to determine these, we have carried out a series of regional operations in two indirect-developing sea urchin species. We excised all or a part of tissue on the left or right side of the embryos during the early gastrula stage and the two-armed pluteus stage, and examined the left-right position of the adult rudiment, and of its components. Excisions of tissues on the left side of the embryos, regardless of stage, resulted in formation of a left adult rudiment, as in normal development. By contrast, excisions on the right side of the embryos resulted in three different types of impairment in the left-right placement of the adult rudiment in a stage-dependent manner. Generally, when the adult rudiment was definitively formed only on the right side of the larvae, no trace of basic development of the components of the adult rudiment was found on the left side, indicating that a right adult rudiment results from reversal of the initial left-right polarity but not from a later inhibitory effect on the development of an adult rudiment. Thus, we suggest that determination of the left-right placement of the adult rudiment depends on a process, which is directed by the right side, of polarity establishment during the gastrula and the prism stages; however, but commitment of the cell fate to initiate formation of the adult rudiment occurs later than the two-armed pluteus stage.

Regulative potential to form an amniotic cavity in mesomeres of a direct developing echinoid, Peronella japonica

Peronella japonica, a direct developer, exhibits certain peculiar features during development, particularly heterochrony, a change in the relative timing of expression among tissues and organs. One of the important heterochronical changes in the species was found in the development of the amniotic cavity, a component of an adult rudiment. In indirect developers the amniotic cavity is formed on the left side of the larval body in the late pluteus stage. In P. japonica the organ is formed at the gastrula stage in the region located on the midline of the larval body.In the present study, the ability of partial embryos isolated from 8- or 16-cell stage embryos of P. japonica to differentiate an amniotic cavity was investigated to assess the regulative potential of a direct developer.The embryos were dissected at 8-cell stage with a glass needle to obtain half embryos. Some of the half embryos were further divided into four blastomeres to obtain mesomere pairs. Each half embryo and blastomere that did not form micromeres but divided equally during the next cleavage was identified as an animal cap and presumptive mesomere pair. Isolated animal caps and mesomere pairs were cultured, and differentiation of the amniotic cavity was examined at 24 and 48 h after fertilisation, when the organ in the normal embryos had already completed differentiation.

Distribution of apoptosis-like cells in sea urchin early embryogenesis

It has been reported that the number of cells per embryo increases from the cleavage stage to the pluteus stage. Also, it has been reported that the number of cells per embryo from the early gastrula stage to the mid-gastrula stage increases very slightly (Mizoguchi, 1999). A detailed analysis of cell proliferation during this period would thus seem to be necessary.On the other hand, Roccheri et al. (1997) reported spontaneous apoptosis at the early pluteus stage, especially in the regions of arm and intestine. However, it is unknown whether apoptosis occurs before the pluteus stage.Using the Tumor, Neuro and/or Cardio TACS in situ apoptosis detection kit (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling: TUNEL method, Trevigen, USA), the Comet assay kit (Trevigen, USA) and DAPI (4,6-diamidino-2-phenylinodole dihydrochloride) staining, we investigated the distribution of apoptosis-like cells in sea urchins during early embryogenesis to clarify the relationship between cell death and morphogenesis.The embryos of the sea urchin Hemicentrotus pulcherrimus were used in the present study. Three methods of detection of apoptosis signals in the embryos were performed: (1) defects in chromosomes and punctured nuclear envelopes of cells were detected by DAPI staining; (2) apoptosis-like cells were stained a brown colour by the TUNEL method; (3) some clear tails which detected the fragmentation of DNA were found by the Comet assay. These signals indicate apoptosis-like cells.

Inversion of left–right asymmetry in the formation of the adult rudiment in sea urchin larvae: removal of a part of embryos at the gastrula stage

At the late pluteus stage, sea urchin larvae form an adult rudiment left–right (LR) asymmetrically, on only the left side. Little is known about how the LR asymmetry of the adult rudiment is established in earlier stages during which the larval body is basically LR symmetric. To investigate how the different regions of the embryo function to establish LR asymmetry, we removed different regions at the gastrula stage and assessed the effects of the operation on the establishment of LR asymmetry of the adult rudiment.Surgery was performed on mid- to late-gastrula embryos of two indirect developing species: Scaphechinus mirabilis and Hemicentrotus pulcherrimus. The embryos were placed under a dissecting microscope (SMZ8, Leica) and ectodermal epithelium together with underlying mesenchyme cells was dissected out with a glass needle. The region along the midline of the embryo at the width of the archenteron was designated the ‘midline region’, and the region lateral to the ‘midline region’ was designated the ‘lateral region’. When the left and/or the right side was excised, the whole lateral region was precisely removed on the animal side, but the plane of incision deviated more laterally on the vegetal side to avoid the ventro-lateral cluster of primary mesenchyme cells, so that a part of the defined ‘lateral region’ was left on the vegetal side. The vertical excision was made by cutting mid-gastrula embryos vertically to the archenteron at a level just superior to the tip of the archenteron. In the sham operation, embryos were pressed with the needle as in the vertical excision, but the incision was stopped slightly before the animal and vegetal halves of the embryos were completely divided from each other. The operated embryos were examined through an optical microscope (Optiphoto, Nikon) to confirm that the excision was correct, and they were then cultured with a food supply (diatoms, Chaetoceros gracilis). The handedness of the adult rudiment was examined at the six-armed or eight-armed pluteus stage through an optical microscope (Table 1).

Embryonic and post-embryonic utilization and subcellular localization of the nuclear receptor SpSHR2 in the sea urchin

SpSHR2 (Strongylocentrotus purpuratus steroid hormone receptor 2) is a nuclear receptor, encoded by a maternal RNA in the sea urchin embryo. These maternal SpSHR2 transcripts, which are present in all cells, persist until the blastula stage and then are rapidly turned over. A small fraction of the embryonic SpSHR2 protein is maternal, but the majority of this nuclear receptor in the embryo is the product of new synthesis, presumably from the maternal RNA after fertilization. In agreement with the mRNA distribution, the SpSHR2 protein is also detected in all embryonic cells. Contrary to the RNA though, the SpSHR2 protein persists throughout embryonic development to the pluteus stage, long after the mRNA is depleted. Following fertilization and as soon as the 2-cell stage, the cytoplasmic SpSHR2 protein enters rapidly into the embryonic nuclei where it appears in the form of speckles. During subsequent stages (from fourth cleavage onward), SpSHR2 resides in speckled form in both the nucleus and the cytoplasm of the embryonic cells. The cytoplasmic localization of SpSHR2 differs between polarized and non-polarized cells, maintaining an apical position in the ectoderm and endoderm versus a uniform distribution in mesenchyme cells. Following the end of embryonic development (pluteus stage), the SpSHR2 protein is depleted from all tissues. During the ensuing four weeks of larval development, the SpSHR2 is not detected in either the larval or the rudiment cells which will give rise to the adult. Just prior to metamorphosis, at about 35 days post-fertilization, the protein is detected again but in contrast to the uniform distribution in the early embryo, the larval SpSHR2 is specifically expressed in cells of the mouth epithelium and the epaulettes. In adult ovaries and testes, SpSHR2 is specifically detected in the myoepithelial cells surrounding the ovarioles and the testicular acini. Nuclear SpSHR2 in blastula extracts binds to the C1R hormone response element in the upstream promoter region of the CyIIIb actin gene indicating that the latter may be a target of this nuclear receptor in the sea urchin embryo.