Jirawat Saetan

Distribution of serotonin and dopamine in the central nervous system of the female mud crab, Scylla olivacea (Herbst).

In crustaceans serotonin (5-HT) and dopamine (DA) are neurotransmitters that play roles in the modulation of numerous physiological functions, including reproduction. However, in the mud crab, Scylla olivacea, the distributions of 5-HT and DA in the CNS have not yet been investigated. The aim of our study was to map the distributions of these two neurotransmitters in the central nervous system (CNS) of the female of this crab during the late stage of ovarian development. We found 5-HT immunoreactivity (-ir) and DA-ir in many parts of the CNS, including the eyestalk, brain, and thoracic ganglia. In the eyestalk, 5-HT-ir was localized in the medulla terminalis (MT), hemi-ellipsoid body (HB), and protocerebral tract (PT), whereas DA-ir was present in neuronal cluster 1, the LG neuropils, and PT. In the brain, 5-HT-ir and DA-ir were detected in cells and fibers of neuronal clusters 6, 7, 8, 9, 10, 11, 14, and 15. In the ventral nerve cord, 5-HT-ir was present in neurons of the abdominal ganglia, whereas DA was only present in fibers. These spatial distributions of 5-HT and DA suggest that they may be involved in the neuromodulation of important physiological functions, including ovarian maturation, as shown in other non-crab decapods.

Presence of gonadotropin-releasing hormone-like peptide in the central nervous system and reproductive organs of the male blue swimming crab, Portunus pelagicus , and its effect on spermatogenesis

Our previous studies have demonstrated that lamprey gonadotropin-releasing hormone-III (lGnRH-III)-like peptide occurs in the central nervous system (CNS) of decapod crustaceans (Macrobrachium rosenbergii, Penaeus monodon, Portunus pelagicus), and that lGnRH-III is the most potent in stimulating ovarian maturation compared with other GnRH isoforms. In this study, we examined the localization of lGnRH-III-like peptide in the CNS and male reproductive organs of the blue swimming crab by using anti-lGnRH-III as a probe. In the brain, lGnRH-III immunoreactivity (−ir) was detected in neurons of clusters 6, 10, 11, 14/15, 16, and 17 and in many neuropils. In the subesophageal ganglion, lGnRH-III-ir was present in neurons of the dorso-lateral and ventro-medial clusters. In the thoracic ganglia, lGnRH-III-ir was observed in the large-sized neurons between the thoracic neuropils and in the ventromedial cluster of the abdominal ganglia. In the testis, lGnRH-III-ir was detected in nurse cells, hemocytes, spermatids 2, and the outer and inner zones of the acrosomes of spermatozoa. Bioassay showed that lGnRH-III significantly increased the testis-somatic index, the percentage of late stages of seminiferous tubules (stages VII–IX), the diameter of the seminiferous tubules, and the number of BrdU-labeled early germ cells compared with the control groups. Thus, lGnRH-III-like peptide exists in the male crab and possibly enhances germ cell proliferation and maturation in the testes, leading to increased sperm production.

Transcriptomic analysis of the autophagy machinery in crustaceans

BackgroundThe giant freshwater prawn, Macrobrachium rosenbergii, is a decapod crustacean that is commercially important as a food source. Farming of commercial crustaceans requires an efficient management strategy because the animals are easily subjected to stress and diseases during the culture. Autophagy, a stress response process, is well-documented and conserved in most animals, yet it is poorly studied in crustaceans.ResultsIn this study, we have performed an in silico search for transcripts encoding autophagy-related (Atg) proteins within various tissue transcriptomes of M. rosenbergii. Basic Local Alignment Search Tool (BLAST) search using previously known Atg proteins as queries revealed 41 transcripts encoding homologous M. rosenbergii Atg proteins. Among these Atg proteins, we selected commonly used autophagy markers, including Beclin 1, vacuolar protein sorting (Vps) 34, microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B), p62/sequestosome 1 (SQSTM1), and lysosomal-associated membrane protein 1 (Lamp-1) for further sequence analyses using comparative alignment and protein structural prediction. We found that crustacean autophagy marker proteins contain conserved motifs typical of other animal Atg proteins. Western blotting using commercial antibodies raised against human Atg marker proteins indicated their presence in various M. rosenbergii tissues, while immunohistochemistry localized Atg marker proteins within ovarian tissue, specifically late stage oocytes.ConclusionsThis study demonstrates that the molecular components of autophagic process are conserved in crustaceans, which is comparable to autophagic process in mammals. Furthermore, it provides a foundation for further studies of autophagy in crustaceans that may lead to more understanding of the reproduction- and stress-related autophagy, which will enable the efficient aquaculture practices.

Enhancement of β‐Globin Gene Expression in Thalassemic IVS2‐654 Induced Pluripotent Stem Cell‐Derived Erythroid Cells by Modified U7 snRNA

The therapeutic use of patient‐specific induced pluripotent stem cells (iPSCs) is emerging as a potential treatment of β‐thalassemia. Ideally, patient‐specific iPSCs would be genetically corrected by various approaches to treat β‐thalassemia including lentiviral gene transfer, lentivirus‐delivered shRNA, and gene editing. These corrected iPSCs would be subsequently differentiated into hematopoietic stem cells and transplanted back into the same patient. In this article, we present a proof of principle study for disease modeling and screening using iPSCs to test the potential use of the modified U7 small nuclear (sn) RNA to correct a splice defect in IVS2‐654 β‐thalassemia. In this case, the aberration results from a mutation in the human β‐globin intron 2 causing an aberrant splicing of β‐globin pre‐mRNA and preventing synthesis of functional β‐globin protein. The iPSCs (derived from mesenchymal stromal cells from a patient with IVS2‐654 β‐thalassemia/hemoglobin (Hb) E) were transduced with a lentivirus carrying a modified U7 snRNA targeting an IVS2‐654 β‐globin pre‐mRNA in order to restore the correct splicing. Erythroblasts differentiated from the transduced iPSCs expressed high level of correctly spliced β‐globin mRNA suggesting that the modified U7 snRNA was expressed and mediated splicing correction of IVS2‐654 β‐globin pre‐mRNA in these cells. Moreover, a less active apoptosis cascade process was observed in the corrected cells at transcription level. This study demonstrated the potential use of a genetically modified U7 snRNA with patient‐specific iPSCs for the partial restoration of the aberrant splicing process of β‐thalassemia. Stem Cells Translational Medicine 2017;6:1059–1069

Characterization and tissue distribution of neuropeptide F in the eyestalk and brain of the male giant freshwater prawn, Macrobrachium rosenbergii

We previously analyzed the central nervous system (CNS) transcriptome and found three isotypes of long neuropeptide F (MrNPF-I, −II, −III) and four isoforms of short NPF (sMrNPF) in the giant freshwater prawn, Macrobrachium rosenbergii. We now validate the complete sequences of the MrNPF-I and −II precursor proteins, which show high similarity (91–95 %) to NPFs of the penaeus shrimp (PsNPF). MrNPF-I and -II precursors share 71 % amino acid identity, whereas the mature 32-amino-acid MrNPF-I and 69-amino-acid MrNPF-II are identical, except for a 37-amino-acid insert within the middle part of the latter. Both mature MrNPFs are almost identical to PsNPF-I and −II except for four amino acids at the mid-region of the peptides. Reverse transcription plus the polymerase chain reaction revealed that transripts of MrNPF-I and -II were expressed in various parts of CNS including the eyestalk, brain and thoracic and abdominal ganglia, with the highest expression occurring in the brain and thoracic ganglia and with MrNPF-I showing five- to seven-fold higher expression than MrNPF-II. These peptides were also expressed in the midgut hindgut, and hepatopancreas, with MrNPF-I expression in the former two organs being at the same level as that in the brain and thoracic ganglia and about 4-fold higher than NPF-II. The expression of NPFs was also detected in the testes and spermatic duct but appeared much weaker in the latter. Other tissues that also expressed a considerable amount of NPF-I included the hematopoeitic tissue, heart and muscle. By immunohistochemistry, we detected MrNPFs in neurons of clusters 2, 3 and 4 and neuropils ME, MT and SG of the optic ganglia, neurons in cluster 6 and neuropils AMPN, PMPN, PT, PB and CB of the medial protocerebrum, neurons in clusters 9 and 11 and neurophils ON and OGTN of the deutocerebrum and neurons in clusters 14, 15 and 16 and neuropils TN and AnN of the tritocerebrum. Because of their high degree of conservation and strong and wide-spread expression in tissues other than CNS, we believe that, in addition to being a neuromodulator in controlling feeding, MrNPFs also play critical roles in tissue homeostasis. This should be further explored.

Spermatophore affects the egg-spawning and egg-carrying behavior in the female giant freshwater prawn, Macrobrachium rosenbergii

In crustaceans, mating occurs during the ecdysis after female molting. During this period, a male transfers its spermatophore into a female which, in some species, stores the spermatophore for a long period prior to spawning and fertilization. However, in some species including the giant freshwater prawn, Macrobrachium rosenbergii, the male deposits its spermataphore onto the external surface of the thoracic segment of the female which affects the spawning time and maternal behavior. This study investigated the spawning behavior of the M. rosenbergii females, which was divided into pre-spawning, spawning, and post-spawning phases. It was revealed that mated female prawns with attached spermatophore exhibited an earlier spawning than unmated individuals, leading to assessment of the factors that may elicit this phenomenon. Four groups of female prawns were allocated to groups including mating females with spermatophore still attached, mating females with the spermatophore removed, artificially inseminated females with spermatophores, and an unmated control. There was a significant reduction in the time of egg-spawning in the presence of spermatophores, and the mating activity was also a contributing factor. Furthermore, over 90% of the mated and artificially inseminated females in which spermatophores were deposited carried the eggs in the abdominal brood chamber until completion of embryonic development while others discarded the eggs within 2 days post-spawning. This study implies that the spermatophore may contain ovulation-inducing factors which stimulate an earlier spawning and fostering of brooding behavior.

Three-dimensional organization of the brain and distribution of serotonin in the brain and ovary, and its effects on ovarian steroidogenesis in the giant freshwater prawn, Macrobrachium rosenbergii

The giant freshwater prawn, Macrobrachium rosenbergii, is an economically important crustacean species which has also been extensively used as a model in neuroscience research. The crustacean central nervous system is a highly complex structure, especially the brain. However, little information is available on the brain structure, especially the three-dimensional organization. In this study, we demonstrated the three-dimensional structure and histology of the brain of M. rosenbergii together with the distribution of serotonin (5-HT) in the brain and ovary as well as its effects on ovarian steroidogenesis. The brain of M. rosenbergii consists of three parts: protocerebrum, deutocerebrum and tritocerebrum. Histologically, protocerebrum comprises of neuronal clusters 6–8 and prominent anterior and posterior medial protocerebral neuropils (AMPN/PMPN). The protocerebrum is connected posteriorly to the deutocerebrum which consists of neuronal clusters 9–13, medial antenna I neuropil, a paired lateral antenna I neuropils and olfactory neuropils (ON). Tritocerebrum comprises of neuronal clusters 14–17 with prominent pairs of antenna II (AnN), tegumentary and columnar neuropils (CN). All neuronal clusters are paired structures except numbers 7, 13 and 17 which are single clusters located at the median zone. These neuronal clusters and neuropils are clearly shown in three-dimensional structure of the brain. 5-HT immunoreactivity (-ir) was mostly detected in the medium-sized neurons and neuronal fibers of clusters 6/7, 8, 9, 10 and 14/15 and in many neuropils of the brain including anterior/posterior medial protocerebral neuropils (AMPN/PMPN), protocerebral tract, protocerebral bridge, central body, olfactory neuropil (ON), antennal II neuropil (Ann) and columnar neuropil (CN). In the ovary, the 5-HT-ir was light in the oocyte step 1(Oc1) and very intense in Oc2–Oc4. Using an in vitro assay of an explant of mature ovary, it was shown that 5-HT was able to enhance ovarian estradiol-17β (E2) and progesterone (P4) secretions. We suggest that 5-HT is specifically localized in specific brain areas and ovary of this prawn and it plays a pivotal role in ovarian maturation via the induction of female sex steroid secretions, in turn these steroids may enhance vitellogenesis resulting in oocyte growth and maturation.

Molecular cloning and distribution of oxytocin/vasopressin-like mRNA in the blue swimming crab, Portunus pelagicus, and its inhibitory effect on ovarian steroid release

This study was aimed to characterize the full length of mRNA of oxytocin/vasopressin (OT/VP)-like mRNA in female Portunus pelagicus (PpelOT/VP-like mRNA) using a partial PpelOT/VP-like sequence obtained previously in our transcriptome analysis (Saetan, 2014) to construct the primers. The PpelOT/VP-like mRNA was 626 bp long and it encoded the preprohormones containing 158 amino acids. This preprohormone consisted of a signal peptide, an active nonapeptide (CFITNCPPG) followed by the dibasic cleavage site (GKR), and the neurophysin domain. Sequence alignment of the PpelOT/VP-like peptide with those of other animals revealed strong molecular conservation. Phylogenetic analysis of encoded proteins revealed that the PpelOT/VP-like peptide was clustered within the group of crustacean OT/VP-like peptide. Analysis by RT-PCR revealed the expression of mRNA transcripts in the eyestalk, brain, ventral nerve cord (VNC), ovary, intestine and gill. The in situ hybridization demonstrated the cellular localizations of the transcripts in the central nervous system (CNS) and ovary tissues. In the eyestalk, the mRNA expression was observed in the neuronal clusters 1-5 but not in the sinus gland complex. In the brain and the VNC, the transcripts were detected in all neuronal clusters but not in the glial cell. In the ovary, the transcripts were found in all stages of oocytes (Oc1, Oc2, Oc3, and Oc4). In addition, synthetic PpelOT/VP-like peptide could inhibit steroid release from the ovary. The knowledge gained from this study will provide more understanding on neuro-endocrinological controls in this crab species.

The presence of abalone egg-laying hormone-like peptide in the central nervous system and ovary of the Spotted Babylon, Babylonia areolata

Recently, the neuronal classification of the ivory shell Spotted Babylon, Babylonia areolata, was readily demonstrated. Regarding its importance as marine economic molluscan species, the attempt to understand the neuroendocrine regulation is necessary. This study firstly demonstrated the neurosecretory cells as well as the existence and distribution of the egg-laying hormone (ELH)-like peptide in the central nervous system (CNS) and ovary of the B. areolata. The neurosecretory cell was characterized by the cytoplasmic purple dot-like structure as stained by the Gomoris paraldehyde fuchsin. Using the anti-abalone (a) ELH, we detected the aELH-like-peptide in neurons (Nr) and neurosecretory cells (Ns) of all ganglia including the cerebral, pleural, parietal, pedal and buccal ganglia. The aELH-like peptide was also present in the neuropil of each. It was noted that not all Ns presented the aELH-like peptide. In the ovary, the aELH-like peptide was slightly detected in early developing oocytes and strongly detected in late developing oocytes and follicular cells. This study firstly reported the evidence of ELH-like peptide in the CNS and ovary of the B. areolata. The molecular cloning as well as to investigate the function of ELH in this species is needed as it will be beneficial for future applications in aquaculture.