Ultrastructural study of neuronal cells and localization of ghrelin-like peptide and its receptor in the ganglia of the golden apple snail (Pomacea canaliculata).

Pomacea canaliculata is an invasive snail species causing major problems in agriculture. The snail biology was then investigated. The main objective of the present study was to investigate the nervous system of the snail. The nervous system comprises pairs of cerebral, buccal, pedal, pleural, parietal ganglia and an unpaired visceral ganglion. Most neurons were concentrated at the periphery of the ganglia. The neurons were classified into four types: NR1, NR2, NR3, and NR4. The percentages of the NR3 and NR4 in the pleural and pedal ganglia were significantly higher than those of other ganglia. Ultrastructural study revealed that nuclei of all neuronal types exhibited mostly euchromatins. Many organelles including ribosomes and endoplasmic reticulum were found in their cytoplasm. However, various mitochondria were found in the NR2 and NR3. The immunohistochemistry revealed immunoreactivity of ghrelin-like peptide in the neurons of the cerebral, pleural and pedal ganglia. However, immunoreactivity of GHS-R1a-like peptide existed only in the neurons of the pleural and pedal ganglia. The present study is the first to demonstrate the existence of ghrelin-like peptide and its receptor in P. canaliculata nervous system.

Ultrastructure of cells constituting lymphoid tubules and circulating hemocytes in Penaeus monodon.

The two main groups of cells in the lymphoid tubule wall of Penaeus monodon are fixed cells and migrating hemocytes. Fixed cells include endothelial, stromal, and capsular cells. Together, they form the scaffold that defines the structure of the lymphoid tubule and provide physical support as well as a niche for transmigrating hemocytes. The luminal surface of lymphoid tubule was lined by elongated, spindle-shaped endothelial cells with a centrally located nucleus and rather thick plasma membrane. Stromal cells were the smallest type of fixed cell. They are stellate cells located between the inner endothelial and outer capsular cells. These cells formed a cyto-reticular network for migrating hemocytes. Capsular cells have a flattened and irregular shape with a ruffled border with long filamentous microvilli. The nucleus is centrally located within a small mass of cytoplasm. Together they form the outermost layer of the lymphoid tubular wall. Transmigrating hemocytes within the lymphoid tubules, as opposed to circulating hemocytes, were classified into hyaline (HH), small granular (SGH) and large granular (LGH) hemocytes. The HH have very few granules and a few cytoplasmic organelles, reflecting low synthetic activity. The granular hemocytes (SGH and LGH), despite being different in size, have similar ultrastructural characteristics. They contain high amounts of rough endoplasmic reticulum, ribosomes, mitochondria, and three types of granules. These characteristics implicate their higher synthetic as well as immunologic activities. Based on these characteristics we believe that all the hemocytes belong to a single line of cell differentiation.

Direct STR typing from human bones.

Identification by STR analysis of bones is time-consuming, mainly due to the lengthy decalcification required and complex DNA extraction process. To streamline this process, we developed a direct STR typing protocol from bone samples. We optimized bone sample amounts using femur and tibia and two commercial PCR kits (Identifiler™ Plus and IDplex Plus kits). Optimally, 100 mg of bone powder in 300 µL PBS buffer was heated at 98 °C for three minutes to produce a supernatant for DNA amplification. IDplex Plus performed better than Identifiler™ Plus in terms of allele recovery and peak height. Fifteen samples of each of seven bone elements (1st distal phalange of hand, capitate, femur, metacarpal 4, patella, talus, and tibia; N = 105) were then subjected to direct STR typing with the optimized protocol, and 94.3% were high partial to full profiles. The performance of the developed protocol was similar for all bone elements. Median peak heights were significantly better in profiles of cancellous bone than compact bone (p = 0.033) and significantly different across the bone elements (p < 0.001). Ten casework samples from various conditions and up to 7-year-PMI were subjected to both direct STR and conventional STR typing. No significant difference in the number of alleles was seen (95% HDI of -13.5 to 5.15). As well as being rapid, convenient, and safe, the protocol could help improve STR typing from bones.

Changes in the histological organization and spheroid formation in lymphoid organ of Penaeus monodon infected with yellow head virus.

The changes in the histological and three dimensional organizations of lymphoid organ and characteristics of lymphoid cells after chronic infection with yellow head virus (YHV) in Penaeus monodon were investigated. The vascular cast of infected lymphoid organ showed less branching and dramatically shortened terminal capillaries that formed the lymphoid tubules, and only large stumps of these lymphoid tubules remained. This might occur because the terminal ends of the tubules were damaged from YHV infection, as stromal cells and hemocytes in the LT wall were infected and formed foci that could give rise to lymphoid spheroids that broke away from the original lymphoid tubules. Histologically, there was a decrease of PAS-stained connective tissue materials in lymphoid spheroids as well as a decrease of stromal cells as marked by anti-vimentin antibody. This indicated that stromal cells together with type 1 fibers and associated extracellular matrix degenerated in lymphoid spheroids, while type 2 or reticular fibers proliferated and encapsulated the spheroids.

Histological and three dimensional organizations of lymphoid tubules in normal lymphoid organ of Penaeus monodon.

The normal lymphoid organ of Penaeus monodon (which tested negative for WSSV and YHV) was composed of two parts: lymphoid tubules and interstitial spaces, which were permeated with haemal sinuses filled with large numbers of haemocytes. There were three permanent types of cells present in the wall of lymphoid tubules: endothelial, stromal and capsular cells. Haemocytes penetrated the endothelium of the lymphoid tubule's wall to reside among the fixed cells. The outermost layer of the lymphoid tubule was covered by a network of fibers embedded in a PAS-positive extracellular matrix, which corresponded to a basket-like network that covered all the lymphoid tubules as visualized by a scanning electron microscope (SEM). Argyrophilic reticular fibers surrounded haemal sinuses and lymphoid tubules. Together they formed the scaffold that supported the lymphoid tubule. Using vascular cast and SEM, the three dimensional structure of the subgastric artery that supplies each lobe of the lymphoid organ was reconstructed. This artery branched into highly convoluted and blind-ending terminal capillaries, each forming the lumen of a lymphoid tubule around which haemocytes and other cells aggregated to form a cuff-like wall. Stromal cells which form part of the tubular scaffold were immunostained for vimentin. Examination of the whole-mounted lymphoid organ, immunostained for vimentin, by confocal microscopy exhibited the highly branching and convoluted lymphoid tubules matching the pattern of the vascular cast observed in SEM.