Gastrodermal lipid bodies (LBs) are organelles involved in the regulation of the mutualistic endosymbiosis between reef-building corals and their dinoflagellate endosymbionts (genus Symbiodinium). As their molecular composition remains poorly defined, we herein describe the first gastrodermal LB proteome and examine in situ morphology of LBs in order to provide insight into their structure and function. After tissue separation of the tentacles of the stony coral Euphyllia glabrescens, buoyant LBs of the gastroderm encompassing a variety of sizes (0.5-4 μm in diameter) were isolated after two cycles of subcellular fractionation via stepwise sucrose gradient ultracentrifugation and detergent washing. The purity of the isolated LBs was demonstrated by their high degree of lipid enrichment and as well as the absence of contaminating proteins of the host cell and Symbiodinium. LB-associated proteins were then purified, subjected to SDS-PAGE, and identified by MS using an LC-nano-ESI-MS/MS. A total of 42 proteins were identified within eight functional groups, including metabolism, intracellular trafficking, the stress response/molecular modification and development. Ultrastructural analyses of LBs in situ showed that they exhibit defined morphological characteristics, including a high-electron density resulting from a distinct lipid composition from that of the lipid droplets of mammalian cells. Coral LBs were also characterized by the presence of numerous electron-transparent inclusions of unknown origin and composition. Both proteomic and ultrastructural observations seem to suggest that both Symbiodinium and host organelles, such as the ER, are involved in LB biogenesis.
Symbiosomes are specific intracellular membrane-bound vacuoles containing microalgae in a mutualistic Cnidaria (host)-dinoflagellate (symbiont) association. The symbiosome membrane is originally derived from host plasma membranes during phagocytosis of the symbiont; however, its molecular components and functions are not clear. In order to investigate the protein components of the symbiosome membranes, homogenous symbiosomes were isolated from the sea anemone Aiptasia pulchella and their purities and membrane intactness examined by Western blot analysis for host contaminants and microscopic analysis using various fluorescent probes, respectively. Pure and intact symbiosomes were then subjected to biotinylation by a cell impermeant agent (Biotin-XX sulfosuccinimidyl ester) to label membrane surface proteins. The biotinylated proteins, both Triton X-100 soluble and insoluble fractions, were subjected to 2-D SDS-PAGE and identified by MS using an LC-nano-ESI-MS/MS. A total of 17 proteins were identified. Based on their different subcellular origins and functional categories, it indicates that symbiosome membranes serve as the interface for interaction between host and symbiont by fulfilling several crucial cellular functions such as those of membrane receptors/cell recognition, cytoskeletal remodeling, ATP synthesis/proton homeostasis, transporters, stress responses/chaperones, and anti-apoptosis. The results of proteomic analysis not only indicate the molecular identity of the symbiosome membrane, but also provide insight into the possible role of symbiosome membranes during the endosymbiotic association.
PCR and in situ hybridization analysis were used for detection of white spot syndrome virus (WSSV) in an infected, cultured shrimp population over a long period in the absence of disease outbreaks. The shrimp were derived from a single WSSV-carrier brooder and cultured first in a tank and then in outdoor ponds. Prior to harvest at 13 mo, no l-step PCR-positive specimens were found, even though most tested specimens were found to be 2-step PCR-positive. At 7 mo, 2-step PCR-positive tissues were found in 5 sampled shrimp. Heart, gill, integument, muscle and stomach tissues best supported viral replication At 13 mo several shrimp died, and l-step PCR-positive individuals were found for the first time Although superficially healthy, 10% of the surviving adults had tiny white spots on their carapace, and In sjtu hybridization analysis revealed WSSV-positive cells in 40% of the specimens examined. As before, most were found in the stomach, integument and gills, and only very few in the lymphoid organ and other organs. These observations contrasted to those for experimentally infected shrimp with gross signs of terminal WSSV infection, where strong positive signals were also observed in the lymphoid organ and in other organs of ectodermal or mesodermal origin. Our results showed clearly that whatever the source, WSSV was carried in the shrimp population at a low intensity (i.e. nested PCR was required for detection) for a very long time in the absence of massive mortality. We hypothesize that disease outbreaks do not occur if shrimp defense mechanisms manage to contain lowintensity viral infections under low-stress culture conditions. Conversely, outbreaks may occur under stressful conditions.
An open reading frame (ORF) that encodes a 715-amino-acid polypeptide was found in an 8421-bp EcoRI fragment of the shrimp white spot syndrome virus (WSSV) genome. The polypeptide shows significant homology to eukaryotic serine/threonine protein kinase (PK) and contains the major conserved subdomains for eukaryotic protein kinases. Coupled in vitro transcription and translation generated a protein having an apparent molecular mass of about 87 kDa according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. For transcriptional analysis of the pk gene, total RNA was isolated from WSSV-infected shrimp at different times after infection. Northern blot analysis with pk-specific riboprobe found a major and a minor transcript of 2.7 and 5.7 kb, respectively. Rapid amplification of the 5' cDNA ends of the major 2.7-kb pk transcript showed that there were two transcriptional initiation sites located at nucleotide residues -38(G) and -39(G) relative to the ATG translational start codon. Temporal expression analysis by RT-PCR indicated that the transcription of the pk gene started 2 h after infection and continued for at least 60 h. Phylogenetic analysis showed that WSSV protein kinase does not have any close relatives and does not fall into any of the major protein kinase groups.
We re-tested stored (frozen) DNA samples in 5 independent polymerase chain reaction (PCR) replicates and confirmed that equivocal test results from a previous study on white spot syndrome virus (WSSV) in brooders and their offspring arose because amounts of WSSV DNA in the test samples were near the sensitivity limits of the detection method. Since spawning stress may trigger WSSV replication, we also captured a fresh batch of 45 brooders for WSSV PCR testing before and after spawning. Replicates of their spawned egg batches were also WSSV PCR tested. For these 45 brooders, WSSV prevalence before spawning was 67 % (15/45 l-step PCR positive, 15/45 2-step PCR positive and 15/45 2-step PCR negative). Only 27 (60%) spawned successfully. Of the successful spawners, 56% were WSSV PCR positive before spawning and 74 % after. Brooders (15) that were heavily infected (i.e. l-step PCR positive) when captured mostly died within 1 to 4 d, but 3 (20%) did manage to spawn. All their egg batch sub-samples were l-step PCR positive and many failed to hatch. The remaining 30 shrimp were divided into a Lightly infected group (21) and a 2-step PCR negative group (9) based on replicate PCR tests. The spawning rates for these 2 groups were high (81 and 78%, respectively). None of the negative spawners (7) became WSSV positive after spawning and none gave egg samples positive for WSSV. In the lightly infected group (21), 6 brooders were 2-step WSSV PCR negative and 15 were 2-step WSSV PCR positive upon capture. However, all of them were WSSV PCR positive in replicate tests and after spawning or death. Four died without spawning. The remaining 17 spawned but only 2 gave egg samples PCR negative for WSSV. The other 15 gave PCR positive egg samples, but they could be divided into 2 spawner groups: those (7) that became heavily infected (i.e. l-step PCR positive) after spawning and those (8) that remained lightly infected (i.e. became or remained 2-step PCR positive only). Of the brooders that became heavily infected after spawning, almost all egg sample replicates (91%) tested 2-step PCR positive One brooder even gave heavily infected (i.e. l-step PCR positive) egg samples. For the brooders that remained lightly infected after spawning, only 27 % of the egg sample replicates were 2-step PCR positive. Based on these results, we recommend that to avoid false negatives in WSSV PCR brooder tests screening tests should be delayed until after spawning. We also recommend, with our PCR detection system, discarding all egg batches from brooders that are l-step PCR positive after spawning. On the other hand, it may be possible with appropriate monitoring to use eggs from 2-step PCR positive brooders for production of WSSV-free or lightly infected postlarvae. These may be used to stock shrimp ponds under low-stress rearing conditions.
In a survey of 27 Penaeus monodon culture ponds stocked with postlarvae (~PL10) at medium density (~40 shrimp m -2 ), single-step nested white spot syndrome virus (WSSV) PCR was used to measure the WSSV infection rates in the shrimp populations within 1 mo after stocking. Seven ponds were initially WSSV-free, and the shrimp in 5 of these were harvested successfully. In the ponds (n = 6) where detection rates were higher than 50%, mass mortality occurred during the growth period, and none of these ponds was harvested successfully. In a subsequent study, P. monodon brooders were classified into 3 groups according to their WSSV infection status before and after spawning: brooders that were WSSV-positive before spawning were assigned to group A; spawners that became WSSV-positive only after spawning were assigned to group B; and group C consisted of brooders that were still WSSV-negative after spawning. WSSV screening showed that 75, 44 and 14%, respectively, of group A, B and C brooders produced nauplii that were WSSV-positive. Most (57%; 16/28) of the brooders in group A produced nauplii in which the WSSV prevalence was high (> 50%).When a pond was stocked with high-prevalence nauplii from 1 of these group A brooders, an outbreak of white spot syndrome occurred within 3 wk and only ~20% of the initial population survived through to harvest (after 174 d). By contrast, 2 other ponds stocked with low-prevalence and WSSV-negative nauplii (derived respectively from 2 brooders in group B), both had much higher survival rates (70 to 80%) and yielded much larger (~3 × by weight) total harvests. We conclude that testing the nauplii is an effective and practical screening strategy for commercially cultured P. monodon.
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