Spermatogenesis and distinctive mature sperm in the giant freshwater prawn, Macrobrachium rosenbergii (De Man, 1879)

The structures of differentiating male germ cells in the testis of the giant freshwater prawn, Macrobrachium rosenbergii, were studied by light and electron microscopy. Based on ultrastructural characteristics, the developing male germ cells are classified into 12 stages, including spermatogonia, six phases of primary spermatocytes (leptotene, zygotene, pachytene, diplotene, diakinesis and metaphase), secondary spermatocyte, three stages of spermatids and mature sperm. During spermatogenesis, the differentiating germ cells have characteristics similar to those of other invertebrates, but they exhibit some unique characteristics during spermiogenesis. In particular, an early spermatid has a round nucleus with highly condensed heterochromatin, appearing as thick interconnecting cords throughout the nucleus. In contrast to most invertebrates and vertebrates, the chromatin begins to decondense in one-half of the nucleus at the mid spermatid stage. In the late spermatid, the chromatin becomes almost entirely decondensed with only a small crescent-shaped heterochromatin patch remaining at the anterior pole of the nucleus. Mature sperm possess an everted umbrella-shaped plate with a spike covering the anterior pole of the nucleus, whose chromatin is totally decondensed as only small traces of histones H3 and H2B remain. The acrosome appears at the ruffled border of the spike plate as small sac-like structures. Few mitochondria remain in the cytoplasm at the posterior pole.     

Cells producing insulin-like androgenic gland hormone of the giant freshwater prawn, Macrobrachium rosenbergii, proliferate following bilateral eyestalk-ablation

We found that the androgenic gland (AG) of Macrobrachium rosenbergii possesses three cell types. Type I cells are small polygonal shaped-cells (13.4 μm in diameter), stain strongly with hematoxylin-eosin (H&;E), have abundant multilayered rough endoplasmic reticulum (rER), and nuclei containing mostly heterochromatin. Type II cells are slightly larger (18.6 μm in diameter), stain lightly with H&;E, have rER with dilated cisternae, and nuclei containing mostly euchromatin. Type III cells (previously undescribed) are similar in size and shape to type I cells, but the cytoplasm is unstained and they have a high amount of smooth endoplasmic reticulum (sER) and mitochondria with tubular cristae. Bilateral eyestalk-ablation resulted in AG hypertrophy with a proliferation and predominance of type I cells as determined by bromodeoxyuridine (BrdU) assays. Expression of insulin-like androgenic gland hormone (Mr-IAG), determined by immunohistochemistry, was weak in type I cells, strong in type II cells of both the intact and eyestalk-ablated, and negative in type III cells. It was also detected in spermatogonia, nurse cells, and epithelium lining of the spermatic duct. The function of Mr-IAG in these tissues is yet to be elucidated but the distribution implies a strong role in male reproduction.     

Fischoederius cobboldi: A scanning electron microscopy investigation of surface morphology of adult rumen fluke

Adults Fischoederius cobboldi are conical-shaped, concave ventrally and convex dorsally, measures about 8-10mm in length and 4-6mm in width across the mid section. Scanning electron microscopy (SEM) of entire body showed that the tegumental surface exhibits highly corrugation and transverse folds alternating with grooves and without spines. At higher magnification, the surface of each fold is further increased with a meshwork of ridges separated by irregular-sized pits. The ventral surface has more complex corrugations and invaginations than those of the dorsal surface of the body. Both anterior and posterior suckers have thick edges covered with transverse folds and appear spineless. The genital pore is located at the anterior one-third of the body. There are two types of sensory papillae on the surface: type 1 is bulbous in shape and nipple-like tips, measuring 10-15 μm in diameter at the base, and also type 2 is a similar shape and has short cilia on tips. These sensory papillae occur in large clusters, each having between 7 and 25 units depending on the region of the body. Clusters of papillae on the ventral surface and around the anterior suckers tend to be more abundant and larger in size. The dorsal side of the body exhibit similar surface features, but papillae appear less numerous and are smaller. Corrugations and invaginations of the dorsal aspect are also less extensive than those on the ventral surface of the body.     

Distribution of dopamine and octopamine in the central nervous system and ovary during the ovarian maturation cycle of the giant freshwater prawn, Macrobrachium rosenbergii

Dopamine (DA), octopamine (OA) and serotonin (5-HT) are the key neurotransmitters that control gonadal development in decapod crustaceans. 5-HT stimulates, while DA and OA delay gonadal development in Macrobrachium rosenbergii. In the present study, we have further investigated the distribution patterns of DA and OA in the central nervous system (CNS) and ovary during various stages of the ovarian maturation cycle of this giant freshwater prawn. DA- and OA-immunoreactive neurons and fibers were distributed extensively in several regions of the brain, subesophageal ganglion (SEG), thoracic ganglia and abdominal ganglia. In the brain, the two neurotransmitters were present in neurons of clusters 6, 7, 11, 17, and nearby neuropil regions. In the SEG, thoracic ganglia and abdominal ganglia, immunoreactive neurons and fibers were found along the midline and in several neuronal clusters around each neuropil region. Staining for DA and OA was more intense in the thoracic ganglia than in other parts of the CNS. In the ovary, DA- and OA-immunoreactivities were present at high intensity in early oocytes. The presence of DA- and OA-immunoreactivities in neural ganglia as well as ovary suggests that DA and OA may also be involved in the reproductive process, particularly ovarian development and differentiation of oocytes in this species.     

Fasciola gigantica: Production and characterization of a monoclonal antibody against recombinant cathepsin B3

A number of monoclonal antibodies (MoAbs) against a recombinant cathepsin B3 (rCatB3) of Fasciola gigantica were produced in BALB/c mice. Reactivity and specificity of these MoAbs were assessed by indirect ELISA and immunoblotting techniques. Six stable clones, namely 1C4, 1E9, 2E5, 2F9, 5B4, 5D7 were obtained. All MoAbs reacted with rCatB3 at molecular weight (MW) 37 kDa as well as the glycosylated peptide at 55–75 kDa and with the native CatB3 at MW 37 kDa in WB extracts of metacercariae (Met) and newly excysted juveniles (NEJ). It was found to be IgG₁ and λ light chain isotypes. Immunolocalization of CatB3 in metacercariae, NEJ, 4-week-old juvenile and adult F. gigantica performed by immunoperoxidase technique by using these MoAbs as probes indicated that CatB3 was present in high concentration in the caecal epithelium and caecal lumen of the Met and NEJ, but not in the 4-week-old juvenile and adult fluke. The MoAbs show no cross-reactions with antigens of other parasites including Gigantocotyl explanatum, Eurytrema pancreaticum, Paramphistomum cervi, Schistosoma spindale, S. mansoni, Haemonchus placei and Setaria labiato-papillosa. Thus, it is possible that these MoAbs could be a good candidate for immunodiagnosis of fasciolosis.     

Distribution and changes of serotonin and dopamine levels in the central nervous system and ovary of the Pacific white shrimp, <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>, during ovarian maturation cycle

We investigated changes in serotonin (5-HT) and dopamine (DA) levels and in their distribution patterns in the central nervous system (CNS) and ovary during the ovarian maturation cycle in the Pacific white shrimp, Litopenaeus vannamei. The concentrations of these two neurotransmitters were determined by using high performance liquid chromatography with electrochemical detection. The 5-HT concentration exhibited a gradual increase in the brain and thoracic ganglia during early ovarian stages I, II, and III, reaching a maximum at the mature ovarian stage IV, whereas DA showed its highest concentration at ovarian stage II in the brain and thoracic ganglia and then declined to its lowest concentration at ovarian stage IV. In the ovaries, 5-HT was lowest at ovarian stage I and gradually increased to a peak at ovarian stage IV. Conversely, the concentration of DA was highest at ovarian stages I and II and lowest at ovarian stage IV. In the brain, 5-HT immunoreactivity (−ir) from stage IV and DA-ir from stage II were distributed extensively in neurons of clusters 6, 11, and 17, in fibers, and in the anterior and posterior medial protocerebral, olfactory, antenna II, and tegumentary neuropils. In the circumesophageal, subesophageal, thoracic, and abdominal ganglia, both 5-HT-ir and DA-ir were detected in neuropils and surrounding neurons and fibers. 5-HT-ir and DA-ir were more intense in the thoracic ganglia than in other parts of the CNS. In the ovary, 5-HT-ir exhibited high intensity in late oocytes, whereas DA-ir was more intense in early oocytes. Thus, opposing changes occur in the levels of these two neurotransmitters and in their specific localizations in the CNS and ovary during ovarian maturation, indicating their important involvement in female reproduction.     

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.     

Microbial degradation of halogenated aromatics: molecular mechanisms and enzymatic reactions

Halogenated aromatics are used widely in various industrial, agricultural and household applications. However, due to their stability, most of these compounds persist for a long time, leading to accumulation in the environment. Biological degradation of halogenated aromatics provides sustainable, low-cost and environmentally friendly technologies for removing these toxicants from the environment. This minireview discusses the molecular mechanisms of the enzymatic reactions for degrading halogenated aromatics which naturally occur in various microorganisms. In general, the biodegradation process (especially for aerobic degradation) can be divided into three main steps: upper, middle and lower metabolic pathways which successively convert the toxic halogenated aromatics to common metabolites in cells. The most difficult step in the degradation of halogenated aromatics is the dehalogenation step in the middle pathway. Although a variety of enzymes are involved in the degradation of halogenated aromatics, these various pathways all share the common feature of eventually generating metabolites for utilizing in the energy-producing metabolic pathways in cells. An in-depth understanding of how microbes employ various enzymes in biodegradation can lead to the development of new biotechnologies via enzyme/cell/metabolic engineering or synthetic biology for sustainable biodegradation processes.     

Existence and distribution of gonadotropin-releasing hormone-like peptides in the central nervous system and ovary of the Pacific white shrimp, <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

We used antibodies against octopus gonadotropin-releasing hormone (octGnRH) and tunicate GnRH (tGnRH-I) in order to investigate the existence and distribution of GnRH-like peptides in the central nervous system (CNS) and in the ovary during various stages of the ovarian cycle of the white shrimp, Litopenaeus vannamei. OctGnRH-immunoreactive and tGnRH-I-immunoreactive neurons and fibers were present in several regions of the supraesophageal ganglion (brain), subesophageal ganglion (SEG), thoracic ganglia, and abdominal ganglia. In the brain, both octGnRH immunoreactivity (ir) and tGnRH-I-ir were detected in neurons of clusters 6, 11, 17, and associated fibers, and the anterior medial protocerebral, posterior medial protocerebral, olfactory, and tegumentary neuropils. In the SEG and thoracic ganglia, octGnRH-immunoreactive and tGnRH-I-immunoreactive neurons and fibers were present in dorsolateral and ventromedial cell clusters and in surrounding fibers. Only immunoreactive fibers were detected in the abdominal ganglia. In the ovary, both octGnRH and tGnRH-I were detected at medium intensity in the cytoplasm of early step oocytes (Oc2) and, at high intensity, in Oc3. Furthermore, octGnRH-ir and tGnRH-I-ir were intense in follicular cells surrounding Oc2 and Oc3. The presence of GnRH-ir in the CNS and ovary indicates that GnRH-like peptides occur in the white shrimp, and that GnRHs are involved in the reproductive process, especially ovarian maturation and the differentiation of oocytes, as reported in other species.     

Expression of the male reproduction-related gene (Mar-Mrr) in the spermatic duct of the giant freshwater prawn, Macrobrachium rosenbergii

Phosphorylated sperm proteins are crucial for sperm maturation and capacitation as a priori to their fertilization with eggs. In the freshwater prawn, Macrobrachium rosenbergii, a male reproduction-related protein (Mar-Mrr) was known to be expressed only in the spermatic ducts as a protein with putative phosphorylation and may be involved in sperm capacitation in this species. We investigated further the temporal and spatial expression of the Mar-Mrr gene using RT-PCR and in situ hybridization and the characteristics and fate of the protein using immunblotting and immunocytochemistry. The Mar-Mrr gene was first expressed in 4-week-old post larvae and the protein was produced in epithelial cells lining the spermatic ducts, at the highest level in the proximal region and decreased in the middle and distal parts. The native protein had a MW of 17 kDa and a high degree of serine/threonine phosphorylation. It was transferred from the epithelial cells to become a major protein at the anterior region of the sperm. We suggest that it is involved in sperm capacitation and fertilization in this open thelycal species and this is being investigated.