Interactions between marine megafauna and plastic pollution in Southeast Asia

Southeast (SE) Asia is a highly biodiverse region, yet it is also estimated to cumulatively contribute a third of the total global marine plastic pollution. This threat is known to have adverse impacts on marine megafauna, however, understanding of its impacts has recently been highlighted as a priority for research in the region. To address this knowledge gap, a structured literature review was conducted for species of cartilaginous fishes, marine mammals, marine reptiles, and seabirds present in SE Asia, collating cases on a global scale to allow for comparison, coupled with a regional expert elicitation to gather additional published and grey literature cases which would have been omitted during the structured literature review. Of the 380 marine megafauna species present in SE Asia, but also studied elsewhere, we found that 9.1 % and 4.5 % of all publications documenting plastic entanglement (n = 55) and ingestion (n = 291) were conducted in SE Asian countries. At the species level, published cases of entanglement from SE Asian countries were available for 10 % or less of species within each taxonomic group. Additionally, published ingestion cases were available primarily for marine mammals and were lacking entirely for seabirds in the region. The regional expert elicitation led to entanglement and ingestion cases from SE Asian countries being documented in 10 and 15 additional species respectively, highlighting the utility of a broader approach to data synthesis. While the scale of the plastic pollution in SE Asia is of particular concern for marine ecosystems, knowledge of its interactions and impacts on marine megafauna lags behind other areas of the world, even after the inclusion of a regional expert elicitation. Additional funding to help collate baseline data are critically needed to inform policy and solutions towards limiting the interactions of marine megafauna and plastic pollution in SE Asia

Identification of Oil Body Proteins in Pine Megagametophytes and Microgametophytes

Unique proteins including steroleosin, caleosin, oleosin-L, and oleosin-H have been identified in oil bodies of angisoperm species. However, the oil body proteins in gymnosperm species are rarely studied. In this study, we intend to investigate the oil-body proteins in pine species. Mature pine (Pinus massoniana) megagametophytes oil bodies were stably isolated and found to comprise mostly triacylglycerols as examined by thin layer chromatography analysis and confirmed by both Nile red and BODIPY stainings. Fatty acids released from the triacylglycerols of pine oil bodies were mainly unsaturated, including linoleic acid (60%), adrenic acid (12.3%) and vaccenic acid (9.7%). Complete cDNA fragments encoding these four pine oil-body proteins, tentatively named caleosin, oleosin-L and oleosin-G and steroleosin were obtained by PCR cloning and further confirmed by mass spectrometric analysis. The pine seed caleosin was found evolutionarily close to the caleosin in oil bodies of cycad megagametophytes. The pine oleosin-L was found evolutionarily close to oleosin-L isoforms in oil bodies of angiosperm seeds. The pine oleosin-G seemed to represent a new class of seed oleosin isoform evolutionarily close to the pollen oleosin isoform, oleoisn-P. The pine steroleosin (41 kDa) was found evolutionarily more closely-related to steroleosin-B than steroleosin-A found in angiosperm seed oil bodies. The sterol binding domain of steroleoisn was demonstrated to be responsible for the sterol binding capability of this oil-body protein. Artificial oil bodies constituted with recombinant steroleosin as well as native pine oil bodies were shown to possess sterol-coupling dehydrogenase activity. Oleosin and caleosin have been identified in oil bodies of angiosperm pollen. Whether the same or different oil-body proteins are present in oil bodies of gymnosperm pollen has not been addressed so far. In this study, the pollen oil bodies also investigated to observe the presence of oil-body proteins in gymnosperm species. Mature pine pollen grains with wing-like bladders were collected from pine (Pinus elliottii). Ultrastructural studies showed that oil bodies were present in pollen grains, but not in the attached bladders. Stable oil bodies were successfully purified from pine pollen grains, and analyzed to be mainly composed of triacylglycerols. One caleosin and one oleosin were identified in oil bodies of pine pollen. Pine pollen caleosin (27 kDa) was found evolutionarily close to pine seed caleosin. The pine pollen oleosin (15 kDa) was found evolutionarily close to pine seed oleosin-G and lily pollen oleosin-P. A comprehensive analysis of oil-body proteins in gymnosperm seed and pollen should scientifically advance our knowledge for the understanding of oil-body proteins in evolution.TABLE OF CONTENTS ABSTRACT ………….…………………………………………….. ii TABLE OF CONTENTS …………………………………………….. iv ABBREVIATION …………………………..................... vi LIST OF TABLES ………………………………………………… viii LIST OF FIGURES …………………………………………………. ix CHAPTER 1. RESEARCH BACKGROUND …………………………….. 1 1. Plant Oil bodies ..……………………………………….. 2 2. Pines ……………….………………………………….. 3 3. Reference ……………………………................. 5 CHAPTER 2. IDENTIFICATION OF OIL BODIES PROTEINS IN PINE MEGAGAMETOPHYTE OIL BODIES………………... 8 1. Introduction …………………………............. 9 2. Material and Methods …...…………………………….. 12 3. Result …………………………………….……………. 21 4. Discussion ……………………………………………... 28 5. Reference …………………………………............ 52 CHAPTER 3. IDENTIFICATION OF CALEOSIN AND OLEOSIN IN PINE POLLEN OIL BODIES….............................................. 57 1. Introduction ……………………………............ 58 2. Material and Methods ……………………………….... 60 3. Result …………………………………………………. 65 4. Discussion …………………………………......... 69 5. Reference ………………………................. 82 CONCLUSION ……………………………………………………. 8

Nitrogen-deprivation elevates lipid levels in Symbiodinium spp. by lipid droplet accumulation: morphological and compositional analyses.

Stable cnidarian-dinoflagellate (genus Symbiodinium) endosymbioses depend on the regulation of nutrient transport between Symbiodinium populations and their hosts. It has been previously shown that the host cytosol is a nitrogen-deficient environment for the intracellular Symbiodinium and may act to limit growth rates of symbionts during the symbiotic association. This study aimed to investigate the cell proliferation, as well as ultrastructural and lipid compositional changes, in free-living Symbiodinium spp. (clade B) upon nitrogen (N)-deprivation. The cell proliferation of the N-deprived cells decreased significantly. Furthermore, staining with a fluorescent probe, boron dipyrromethane 493/503 (BODIPY 493/503), indicated that lipid contents progressively accumulated in the N-deprived cells. Lipid analyses further showed that both triacylglycerol (TAG) and cholesterol ester (CE) were drastically enriched, with polyunsaturated fatty acids (PUFA; i.e., docosahexaenoic acid, heneicosapentaenoic acid, and oleic acid) became more abundant. Ultrastructural examinations showed that the increase in concentration of these lipid species was due to the accumulation of lipid droplets (LDs), a cellular feature that have previously shown to be pivotal in the maintenance of intact endosymbioses. Integrity of these stable LDs was maintained via electronegative repulsion and steric hindrance possibly provided by their surface proteins. Proteomic analyses of these LDs identified proteins putatively involved in lipid metabolism, signaling, stress response and energy metabolism. These results suggest that LDs production may be an adaptive response that enables Symbiodinium to maintain sufficient cellular energy stores for survival under the N-deprived conditions in the host cytoplasm

The change of fatty acid compositions in TAGs of <i>Symbiodinium</i> after five days of nitrogen deprivation.

<p>Relative amounts (%) of fatty acid compositions in purified TAGs from total <i>Symbiodinium</i> were determined (see the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087416#s2" target="_blank">Materials and methods</a>” section). The data represents mean ± SD (n = 3).</p

Effect of nitrogen-deprivation on the cell proliferation and lipid accumulation in <i>Symbiodinium</i>.

<p>(A) Growth of <i>Symbiodinium</i> cells cultivated in control versus nitrogen-deprivation media. The data represents mean ± SD (n = 3). (B) The visualization of neutral lipid accumulation using BODIPY 493/503 in control vs. nitrogen-deprived cultures. Scale bar, 10 µm.</p

The TLC analysis of lipids extracted from <i>Symbiodinium</i> spp. cells.

<p>The comparison between control and nitrogen-deprivation treated cells (A). The lipid content of the purified LDs from <i>Symbiodinium</i> after five days of nitrogen deprivation is shown in (B).</p

Identification of lipid droplet proteins in <i>Symbiodinium</i> spp.

<p>a)MS/mps(p): Mowse score/number of total matched peptides (numbers of different matched peptides).</p

The ultrastructural examination of morphological changes and LD formation in <i>Symbiodinium</i> after nitrogen deprivation.

<p>Transmission electron micrographs of <i>Symbiodinium</i> in control (A, B) and nitrogen-deprivation media (five days: C–D; seven days: E–F). Insets in A, C and D were magnified as B, D and F, respectively. Arrows in A, C, and E indicated cell walls, while arrowheads in F indicated the OsO₄-negative “inclusion bodies”. Abbreviations: LD, lipid droplet; Ch, chloroplast; S, starch granule; P, pyrenoids; N, nucleolus.</p

Light microscopy of the LDs purified from <i>Symbiodinium</i> cells after different treatments.

<p>The LDs were suspended in the (A) pH 7.5 grinding buffer, (B) pH 6.5 grinding buffer or (C) treated by the trypsin digestion. (D) Phospholipid analyses by TLC showing the presence of phospholipids (PLs) in purified LDs (the top layer during the centrifugation) but not lower layer fractions after detergent (0.1% Triton X-100) washing.</p

LDs purification and protein analyses.

<p>(A) SDS-PAGE analyses of isolated LDs fraction and the LD purity assessment by RuBisCO western blotting. <i>Symbiodinium</i> spp. cells harvested after five days of nitrogen deprivation were homogenized and fractionated to purify LDs as shown in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087416#s2" target="_blank">Materials and methods</a>” section. The purity of LDs was examined based on the absence of RuBisCO contamination by western blotting. Proteins bands 1 to 5 were excised for mass spectrometric analysis. (B) BODIPY 493/503 staining of isolated LDs displayed the abundance of neutral lipid.</p