The presence and distribution of gamma-aminobutyric acid and dopamine during the developmental stages of the sea cucumber, Holothuria scabra, with emphasis on settlement organs.

In the sea cucumber, Holothuria scabra, the competent larvae require main settlement organs (SOs), including the ciliary bands (CiBs), tentacles (Ts), podia (PDs), and cues from neurotransmitters, including gamma-aminobutyric acid (GABA) and dopamine (DA), for successful settlement. In the present study, we investigated the spatial distribution of GABA and DA in the developmental stages of H. scabra, with special emphasis on SOs by detecting immunoreactivity (-ir) against these two neurotransmitters. Strong GABA-ir and DA-ir cells and fibers were specifically detected in several SO structures, including CiBs, CiB cells (CiBCs), and long cilia (LCi), of H. scabra larvae. Additionally, we found intense GABA-ir and DA-ir cells in the epithelial lining of bud-papillae (BP) and mesothelium (Me) in the stem (S) region of Ts in larvae and juveniles. Intense GABA-ir and DA-ir were observed in the epineural nerve plexus (ENP) and hyponeural nerve plexus (HNP) of Ts in H. scabra pentactula and juvenile stages. Staining for these two neurotransmitters was particularly intense in the PDs and their nerve fibers. We also found significant changes in the numbers of GABA-ir and DA-ir-positive cells and intensities in the CiBs, Ts, and PDs during the developmental stages. Taken together, we are the first to report on the existence and distribution of GABAergic and dopaminergic systems in structures associated with the settlement. Our findings provide new and important insights into the possible functions of these two neurotransmitters in regulating the settlement of this sea cucumber species.

Saponins enriched in the epidermal layer of Holothuria leucospilota body wall.

Saponins are secondary metabolites that provide medicinal benefits in controlling body homeostasis and metabolic functions. Sea cucumber has been consumed in many Asian countries due to their health benefits. Active chemicals found in sea cucumber include natural source of saponins which are enriched in their tissues, including the Cuvierian tubules and the body wall. Tissue origin of the saponin biosynthesis and accumulation is limitedly known. The present study is to indicate major compositions and distributions of saponins in the body wall of Holothuria leucospilota. Structurally, their body wall consisted of the pigmented layer of the epidermis, the dermal connective tissues, and inner muscular layers. Interestingly, release of the pigmented granules from the epidermis was related to detection of epidermal saponins. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) revealed identical mass spectra in the saponin extracts and compared to the known compounds of holothurians. To investigate the release of epidermal saponins, the epidermis dissolved in either butanol or distilled water were analyzed and presented the saponin masses with two prominent masses of m/z 1,243.3 (holothurin A and scabraside B) and 1,259.3 (holothurin A3). MALDI-IMS also demonstrated strong signals of the known saponins which were only localized in the epidermis of the body wall. Taken together, this study shows that granule release from epidermal pigmented cells is somehow related to the amount of epidermal saponins released to surrounding seawater. Hence, the future research in the sea cucumber better focuses on epidermal cells that are the enriched site of saponins, although several active compounds require further investigation.

Transcriptomic discovery and comparative analysis of neuropeptide precursors in sea cucumbers (Holothuroidea).

Neuropeptides synthesized and released by neuronal cells play important roles in the regulation of many processes, e.g. growth, feeding, reproduction, and behavior. In the past decade, next-generation sequencing technologies have helped to facilitate the identification of multiple neuropeptide genes in a variety of taxa, including arthropods, molluscs and echinoderms. In this study, we extend these studies to Holothuria scabra, a sea cucumber species that is widely cultured for human consumption. In silico analysis of H. scabra neural and gonadal transcriptomes enabled the identification of 28 transcripts that encode a total of 26 bilaterian and echinoderm-specific neuropeptide precursors. Furthermore, publicly available sequence data from another sea cucumber, Holothuria glaberrima, allowed a more in-depth comparative investigation. Interestingly, two isoforms of a calcitonin-type peptide precursor (CTPP) were deduced from the H. scabra transcriptome - HscCTPP-long and HscCTPP-short, likely the result of alternative splicing. We also identified a sea cucumber relaxin-type peptide precursor, which is of interest because relaxin-type peptides have been shown to act as gonadotropic hormones in starfish. Two neuropeptides that appear to be holothurian-specific are GLRFA, and GN-19. In H. scabra, the expression of GLRFA was restricted to neural tissues, while GN-19 expression was additionally found in the longitudinal muscle and intestinal tissues. In conclusion, we have obtained new insights into the neuropeptide signaling systems of holothurians, which will facilitate physiological studies that may enable advances in the aquaculture of sea cucumbers.

Distribution and changes of serotonin and dopamine levels in the central nervous system and ovary of the Pacific white shrimp, Litopenaeus vannamei, 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.

Existence and distribution of gonadotropin-releasing hormone-like peptides in the central nervous system and ovary of the Pacific white shrimp, Litopenaeus vannamei.

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.

Morphological and biochemical alterations of abalone testicular germ cells and spawned sperm and their fertilizing ability.

In this study, we aimed to detect morphological and biochemical changes in developing germ cells (Gc), testicular sperm (Tsp), and spawned sperm (Ssp) using capacitation-associated characteristics. Gradual changes in the profiles of two membrane proteins, namely NaCl- and detergent-extractable proteins, were observed as compared Gc with Tsp and Tsp with Ssp. These membrane modifications were accomplished mostly through the introduction of new protein sets, both peripheral and integral, into Tsp and Ssp membranes. Activation of serine proteases, particularly in Ssp detergent-extracted proteins with the molecular masses of 38-130 kDa was evident and marked a major difference between Ssp and Tsp. An increase in the level of tyrosine phosphorylation of the proteins ranging from 15 to 20 kDa was noted in Tsp and remained constant in Ssp. Specifically, these three capacitation-associated characteristics could be detected in Ssp, possessing full fertilizing capacity. The lack of an activated proteolytic activity in Tsp resulted in a delayed fertilization, but not affected fertilizing ability. We believe that these characteristics should be advantageous in predicting abalone sperm fertilizing capability, particularly in cases when isolated germ cells or purified Tsp are used in place of spawned sperm in abalone aquaculture.

Basic nuclear protein pattern and chromatin condensation in the male germ cells of a tropical abalone, Haliotis asinina.

The basic nuclear proteins (BNPs) in spermatozoa of a tropical abalone, Haliotis asinina, were composed of a majority of protamine-like (PL) protein and a small amount of histones H1 and H4. Abalone H1 and PL proteins exhibited strong immunological cross reactivities among themselves as well as with chick H5 and calf thymus H1. Thus, all these proteins may belong to the same family. Immunolocalization by indirect immunofluorescence and immunoelectron microscopy indicated that H1 and H4 were present in all steps of the male germ cells, however, with decreasing amount in late stage cells, particularly spermatids and spermatozoa. On the other hand, PL was present in middle step cells (secondary spermatocytes) with increasing amount in spermatids and spermatozoa when the chromatin became tightly packed. Thus, PL may be involved in the condensation of chromatin in the spermatozoa of this species.