Wei-Tung Huang

Acute Changes in Gill Na+‐K+‐ATPase and Creatine Kinase in Response to Salinity Changes in the Euryhaline Teleost, Tilapia (Oreochromis mossambicus)

Some freshwater (FW) teleosts are capable of acclimating to seawater (SW) when challenged; however, the related energetic and physiological consequences are still unclear. This study was conducted to examine the changes in expression of gill Na+‐K+‐ATPase and creatine kinase (CK) in tilapia (Oreochromis mossambicus) as the acute responses to transfer from FW to SW. After 24 h in 25 ppt SW, gill Na+‐K+‐ATPase activities were higher than those of fish in FW. Fish in 35 ppt SW did not increase gill Na+‐K+‐ATPase activities until 1.5 h after transfer, and then the activities were not significantly different from those of fish in 25 ppt SW. Compared to FW, the gill CK activities in 35 ppt SW declined within 1.5 h and afterward dramatically elevated at 2 h, as in 25 ppt SW, but the levels in 35 ppt SW were lower than those in 25 ppt SW. The Western blot of muscle‐type CK (MM form) was in high association with the salinity change, showing a pattern of changes similar to that in CK activity; however, levels in 35 ppt SW were higher than those in 25 ppt SW. The activity of Na+‐K+‐ATPase highly correlated with that of CK in fish gill after transfer from FW to SW, suggesting that phosphocreatine acts as an energy source to meet the osmoregulatory demand during acute transfer.

A 3D Model of the Membrane Protein Complex Formed by the White Spot Syndrome Virus Structural Proteins

Background Outbreaks of white spot disease have had a large negative economic impact on cultured shrimp worldwide. However, the pathogenesis of the causative virus, WSSV (whit spot syndrome virus), is not yet well understood. WSSV is a large enveloped virus. The WSSV virion has three structural layers surrounding its core DNA: an outer envelope, a tegument and a nucleocapsid. In this study, we investigated the protein-protein interactions of the major WSSV structural proteins, including several envelope and tegument proteins that are known to be involved in the infection process. Principal Findings In the present report, we used coimmunoprecipitation and yeast two-hybrid assays to elucidate and/or confirm all the interactions that occur among the WSSV structural (envelope and tegument) proteins VP51A, VP19, VP24, VP26 and VP28. We found that VP51A interacted directly not only with VP26 but also with VP19 and VP24. VP51A, VP19 and VP24 were also shown to have an affinity for self-interaction. Chemical cross-linking assays showed that these three self-interacting proteins could occur as dimers. Conclusions From our present results in conjunction with other previously established interactions we construct a 3D model in which VP24 acts as a core protein that directly associates with VP26, VP28, VP38A, VP51A and WSV010 to form a membrane-associated protein complex. VP19 and VP37 are attached to this complex via association with VP51A and VP28, respectively. Through the VP26-VP51C interaction this envelope complex is anchored to the nucleocapsid, which is made of layers of rings formed by VP664. A 3D model of the nucleocapsid and the surrounding outer membrane is presented.

Penaeus monodon TATA Box-Binding Protein Interacts with the White Spot Syndrome Virus Transactivator IE1 and Promotes Its Transcriptional Activity

ABSTRACT We show here that the white spot syndrome virus (WSSV) immediate-early protein IE1 interacts with the Penaeus monodon TATA box-binding protein (PmTBP) and that this protein-protein interaction occurs in the absence of any other viral or cellular proteins or nucleic acids, both in vitro and in vivo. Mapping studies using enhanced green fluorescent protein (EGFP) fusion proteins containing truncations of IE1 and PmTBP delimited the interacting regions to amino acids (aa) 81 to 180 in IE1 and, except for aa 171 to 230, to aa 111 to 300 in PmTBP. A WSSV IE1 transactivation assay showed that large quantities (>800 ng) of the GAL4-IE1 plasmid caused “squelching” of the GAL4-IE1 activity and that this squelching effect was alleviated by the overexpression of PmTBP. Gene silencing of WSSV ie1 and PmTBP by pretreatment with double-stranded RNAs (dsRNAs) prior to WSSV challenge showed that the expression of these two target genes was specifically inhibited by their corresponding dsRNAs 72 and 96 h after dsRNA treatment. dsRNA silencing of ie1 and PmTBP expression also significantly reduced WSSV replication and the expression of the viral early gene dnapol (DNA polymerase gene). These results suggest that WSSV IE1 and PmTBP work cooperatively with each other during transcription initiation and, furthermore, that PmTBP is an important target for WSSV IE1s transactivation activity that can enhance viral gene expression and help in virus replication.

Expression and in vitro regulation of pituitary adenylate cyclase-activating polypeptide (pacap38) and its type I receptor (pac1-r) in the gonads of tilapia (Oreochromis mossambicus)

Pituitary adenylate cyclase-activating polypeptide (PACAP), a pleiotropic neuropeptide, has diverse functions in mammals. However, studies of the expression and function of PACAP and its receptor in fish, particularly in the reproductive system, are still limited. In this report, semi-quantitative RT-PCR and immunohistochemical staining were performed to identify expression domains of commercially important tilapia (Oreochromis mossambicus). PACAP (tpacap(38)) and its type I receptor (tpac(1)-r). Transcripts were detected in the brain, gallbladder, gill, heart, intestine, kidney, muscles, pancreas, spleen, stomach, testes, and ovaries, but not in the liver. Expression of tpacap(38) and tpac(1)-r mRNA in brain tissue was significantly higher in both sexes compared with other tissues. Addition of exogenous ovine PACAP(38) (0.25-5 nM), cAMP analog (dibutyryl-cAMP, 0.25-1.5 mM) or forskolin (adenylate cyclase activator, 1-10 microM) significantly upregulated tpacap(38) in the gonads via a dose- and time-dependent fashion. This effect reached a maximal level at 2 h after induction, and then decreased with prolonged culture for up to 4 or 8 h. Additionally, the expression levels of tpac(1)-r were not significantly affected by ovine PACAP(38) or dibutyryl-cAMP in either sex. Forskolin had a slightly inductive effect and its function could be suppressed with the addition of protein kinase A (PKA) inhibitor, H89 (10 microM), indicating involvement of the cAMP-PKA signaling pathway in the regulation of tpacap(38). Expression of tpacap(38) and tpac(1)-r in the gonads of tilapia suggests that PACAP may mediate gonadotropin action via paracrine/autocrine mechanisms in this bony fish.

Application of RNAi Technology to the Inhibition of Zebrafish GtHα, FSHβ, and LHβ Expression and to Functional Analyses

Abstract Zebrafish (Danio rerio) were used as a model fish, and the technique of RNA interference (RNAi) was employed to knockdown three subunits of the gonadotropin alpha (GtHα, common α), follicle-stimulating hormone beta (FSHβ), and luteinizing hormone beta (LHβ) genes. Three short-hairpin RNA (shRNA) expression vectors and three mismatched shRNA expression vectors as controls for each subunit gene were constructed, and the depression efficiency was tested in vivo by microinjection; the RNA or protein expression levels of the GtH genes were monitored by RT-PCR, Southern blotting, and green fluorescent protein (GFP) analyses. Expression of GtH mRNA was obviously and more efficiently depressed by GtHα RNAi expression compared with the other two subunits. A GtHα morpholino analysis showed that the GtHα morpholino led to suppression of embryonic development and the production of embryonic mutants as a result of an injection of GtHα -shRNA. Taken together, these results show that GtHα-shRNA, which more efficiently targets RNAi, may have an essential role in the further development of sterility technology of transgenic fish for biosafety purposes.

Bioenergetics of Adaptation to a Salinity Transition in Euryhaline Teleost (Oreochromis mossambicus) Brain

Freshwater (FW) teleosts are capable of acclimating to seawater (SW) following such a transfer from FW. However, their osmo-regulating mechanisms are still unclear, particularly those in the brain. The present study was conducted to examine acute changes that occur in brain Na+-K+-ATPase activity, creatine kinase (CK) activity, creatine, creatinine contents, and ATP levels of tilapia (Oreochromis mossambicus) in response to this transition. After transfer to SW (25 ppt), the Na+-K+-ATPase activity was maintained for 8 hr at higher levels than that in FW. In contrast, in 35 ppt SW, Na+-K+-ATPase was maintained at a even higher level than in FW for the first 2 hr. Brain Na+-K+-ATPase contents in both the 25 and 35 ppt SW groups were significantly elevated within 1 and 0.5 hr after transfer from FW, respectively. Interestingly, brain CK activities and content (homodimer of the B subunit [BB] form) in both the 25 and 35 ppt SW groups were significantly elevated within 1 hr after transfer from FW. The ATP contents in 35 ppt SW increased abruptly within 0.5 hr, and then gradually decreased during the next 2 hr. Unlike the 35 ppt group that declined in ATP contents, the 25 ppt group leveled off within 24 hr. The elevations in CK activity and creatine levels after transfer from FW to SW imply that abrupt salinity changes alter phosphocreatine/CK ratio. Such changes are needed to satisfy the increases in the energetic requirement of the cotransport mechanisms mediating osmoregulation

The Hormonal Regulation of Color Changes in the Sexually Dichromatic Frog Buergeria robusta

During the breeding season, dynamic changes in body coloration are regularly observed in the male brown tree frog Buergeria robusta. This study investigated the hypothesis that this sexual dichromatism in male B. robusta is mediated through hormonal regulation. Frogs were exogenously injected with testosterone (T) or estradiol (E2). This manipulation revealed that the body coloration (hue, brightness, and saturation) of the male frog increased significantly (i.e., the brilliant yellow color developed) in response to T but not in response to E2. Concurrently, the levels of expression of brain-derived neurotrophic factor (BDNF) and pituitary adenylate cyclase-activating polypeptide (PACAP) in the pituitary gland were reduced in frogs whose coloration was pale brown on a yellow background. In particular, the weakest expressions of BDNF, PACAP, and PACAP type II receptors (VPAC-1R) were found in male frogs with a brilliant yellow body color during the breeding season regardless of background color. These changes may decrease α-melanocyte-stimulating hormone production associated with the PACAP receptors (VPAC-1R), resulting in the aggregation of black pigment in melanophores and the production of a brilliant yellow body color. The effects of hormones on skin coloration were further examined in isolated skin in vitro. The results of this investigation showed that the dispersion of xanthophores was stimulated by T or prolactin (PRL) and that the melanophores were aggregated by melatonin (MEL) but not by E2. Furthermore, yellow pigments in the xanthophores were significantly dispersed following the PRL+T treatment. In the T+MEL, PRL+MEL, and T+PRL+MEL treatments, xanthophores were dispersed, and melanophores were aggregated and subsequently moved to the low spongiosum layer of the dorsal skin, causing the increase in yellow coloration. These results reveal that multiple hormones play major roles in the regulation of the brilliant yellow coloration of male B. robusta by high plasma T during the breeding season.

Ovine thyroid stimulating hormone (TSH) heterologously stimulates production of thyroid hormones from Chinese soft-shell turtle (Pelodiscus sinensis) and bullfrog (Rana catesbeiana and Rana rugulosa) thyroids in vitro.

Thyroid hormones are important for regulating a variety of developmental processes in vertebrates, including growth, differentiation, metamorphosis, and oxidative metabolism. In particular, this study focused on the in vitro production of thyroxine (T(4)) and triiodothyronine (T(3)) from thyroids in American bullfrogs (Rana catesbeiana), Chinese bullfrogs (Rana rugulosa Wiegmann), and Chinese soft-shell turtles (Pelodiscus sinensis) treated with ovine thyroid stimulating hormone (TSH) at different culture intervals (2, 4, 8, and 12 h) and dosages (1, 10, 50 or 100 ng). The levels of T(4) and T(3) in the tested animals were elevated upon stimulation in a time- and dose-dependent manner, indicating de novo synthesis of T(4) and T(3). Significantly higher hormone levels were observed in the Chinese bullfrog compared to the other two species, for both the time-course and dose-response experiments. Although the bullfrog secreted significantly higher levels of T(4) and T(3), a higher T(4)-conversion capacity was found in the Chinese soft-shell turtle. The highest ratios of T(3) to T(4) were observed in the American bullfrog and Chinese soft-shell turtle for the time-course and dose-response experiments, respectively. These findings suggest that the Chinese soft-shell turtle and bullfrog thyroids can accept ovine TSH for T(4)- and T(3)-formation in a time- and dose-dependent manner, supporting the hypothesis that the binding interactions between TSHs and thyroidal receptors are conserved in vertebrates.

Roles of hepatocyte nuclear factors (HNF) in the regulation of reproduction in teleosts

Hepatocyte nuclear factor (HNF) families are composed of liver-enriched transcription factors and upstream regulators of many liver-specific genes. HNF are involved in liver-specific gene expression, metabolism, development, cell growth and many cellular functions in the body. HNF genes can be activated or influenced by several hormones and insulin-like growth factors (IGF), and different combinations of the four HNF factors form a network in controlling the expression of liver-specific or liver-enriched genes. The functions of these factors and their interactions within the gonads of bony fishes, however, are not well understood, and the related literature is scant. Recently, several members of the HNF families have been detected in teleost gonads together with their downstream genes (IGF-I and IGF-II), suggesting that these HNF could be upregulated in vitro by steroid hormones. Thus, the hormone-HNF-IGF-gonad interaction may be an alternative axis in the reproductive mechanism that acts in concert with the conventional hypothalamus-pituitary-gonad pathway. This may help the early development and maturation of the gonad or gamete, sexual maturity or reversion and spawning-regulating mechanisms among fishes to be understood.