Adenosine 5'-monophosphate-activated protein kinase (AMPK) negatively regulates the immunity and resistance to Vibrio alginolyticus of white shrimp, Penaeus vannamei.

Shrimp immunology is vital in establishing prophylactic and therapeutic strategies for controlling pathological problems that threaten shrimp production. Apart from dietary treatments, the adenosine 5'-monophosphate-activated protein kinase (AMPK), an important regulatory enzyme that restores cellular energy balance during metabolic and physiological stress, is known to have therapeutic potential to improve shrimp's defense mechanism. Despite this, studies targeting the AMPK pathway in shrimp exposed to stressful conditions are vastly limited. In this study, AMPK was knocked down to assess the immunological changes and white shrimp, Penaeus vannamei resistance to Vibrio alginolyticus infection. Shrimps were injected individually and simultaneously with dsRNA targeting specific genes such as AMPK, Rheb, and TOR, after which the hepatopancreas was analyzed for the different gene expressions. The gene expressions of AMPK, Rheb, and TOR were effectively suppressed after being treated with dsRNAs. The Western blot analysis further confirmed a reduction in the protein concentration of AMPK and Rheb in the hepatopancreas. The suppression of AMPK gene led to a robust increase in the shrimp's resistance to V. alginolyticus, whereas the activation of AMPK by metformin decreased the shrimp's disease resistance. Among the mTOR downstream targets, the HIF-1α expression in shrimp treated with dsAMPK significantly increased at 48 h but returned to normal levels when shrimp were treated with dsAMPK and either dsRheb or dsTOR. Immune responses such as respiratory burst, lysozyme activity, and phagocytic activity increased, while superoxide dismutase activity decreased following the knockdown of the AMPK gene compared to the control group. However, co-injection with dsAMPK and dsTOR or dsRheb restored immune responses to normal levels. Collectively, these results demonstrate that the inactivation of AMPK may ameliorate shrimp's innate immune response to recognize and defend against pathogens via the AMPK/mTOR1 pathway.

Dietary administration of a postbiotic, heat-killed Pediococcus pentosaceus PP4012 enhances growth performance, immune response and modulates intestinal microbiota of white shrimp, Penaeus vannamei.

The efficacy of postbiotics on the immune-related gene expression and gut microbiota of white shrimp, Penaeus vannamei remains unexplored. A commercial heat-killed postbiotic Pediococcus pentosaceus PP4012 was used to evaluate the growth performance, intestinal morphology, immunological status, and microbial community of white shrimp after dietary administration in this study. White shrimp (0.040 ± 0.003 g) were divided into three treatments; a control, inanimate P. pentosaceus (105 CFU g feed-1) at low concentration (IPL) and inanimate P. pentosaceus (106 CFU g feed-1) at high concentrations (IPH). The diets of IPL and IPH significantly increased final weight, specific growth rate and production compared to the control group. Shrimp fed with IPL and IPH significantly utilized feed more efficiently than those fed the control diet. The IPH treatment significantly lowered the cumulative mortality rate compared to the control and IPL diet following Vibrio parahaemolyticus infection. No significant difference was observed for Vibrio-like and lactic acid bacteria in intestine of shrimp fed with the control diet and the experimental diets. Adding inanimate P. pentosaceus significantly improved immune responses such as lysozyme and phagocytic activity compared to the control group. However, the total hemocyte count, phenoloxidase activity, respiratory burst, and superoxide dismutase activity were not significantly different among treatments. The immune-related genes alf, pen3a, and pen4 expression were significantly higher in shrimp fed IPL diet compared with control and IPH. Taxonomic identification of bacterial genera in all dietary groups belonged to two predominant phyla, Proteobacteria and Bacteroidota. An abundance of Photobacterium, Motilimonas, Litorilituus, and Firmicutes bacterium ZOR0006 were identified in the intestine of shrimp fed postbiotic diets. Unique microbes such as Cohaesibacter was discovered in the shrimp fed IPL while Candidatus Campbellbacteria, uncultured Verrucomicrobium DEV114 and Paenalcaligenes were discovered in the intestines of shrimp fed IPH diet. Collectively, these data suggest that including heat-killed P. pentosaceus, particularly IPH, can enhance growth performance, promote microbial diversity, elevate immune responses, and increase shrimp's resistance to V. parahaemolyticus.

Enhancement of immunity and disease resistance in the white shrimp, Litopenaeus vannamei, by the probiotic, Bacillus subtilis E20.

Effects of Bacillus subtilis E20 isolated from fermented soybean on immune parameters and the disease resistance of the white shrimp (Litopenaeus vannamei) after 98 days of B. subtilis E20 feeding were evaluated in this study. Shrimp fed B. subtilis E20-containing diets at concentrations of 10(6) (E206), 10(7) (E207), and 10(8) (E208)cfu kg(-1), respectively, had significantly increased survival rates of 13.3%, 16.7%, and 20%, compared to the control (fed no probiotic) after being challenged with Vibrio alginolyticus. There were no significant differences in the total hemocyte count, respiratory burst, or superoxide dismutase glutathione peroxidase among all treatments. Shrimp fed a higher concentration of the probiotic (E208) exhibited significant increases in phenoloxidase activity, phagocytic activity, and clearance efficiency compared to control shrimp. In addition, B. subtilis E20 showed a weaker inhibitory effect against the growth of Aeromona hydrophila with around a 0.3-cm inhibitory zone, but showed no inhibitory effects against other selected pathogens, such as white shrimp pathogens: V. alginolyticus and Vibrio vulnificus. These results suggest that the increased resistance of shrimp after B. subtilis E20 consumption occurs through immune modifications, such as increases in phenoloxidase activity, phagocytic activity, and clearance efficiency against V. alginolyticus.

Environmental and cortisol-mediated control of Ca(2+) uptake in tilapia (Oreochromis mossambicus).

Ca(2+) is a vital element for many physiological processes in vertebrates, including teleosts, which live in aquatic environments and acquire Ca(2+) from their surroundings. Ionocytes within the adult gills or larval skin are critical sites for transcellular Ca(2+) uptake in teleosts. The ionocytes of zebrafish were found to contain transcellular Ca(2+) transporters, epithelial Ca(2+) channel (ECaC), plasma membrane Ca(2+)-ATPase 2 (PMCA2), and Na(+)/Ca(2+) exchanger 1b (NCX1b), providing information about the molecular mechanism of transcellular Ca(2+) transports mediated by ionocytes in fish. However, more evidence is required to establish whether or not a similar mechanism of transcellular Ca(2+) transport also exists in others teleosts. In the present study, ecac, pmca2, and ncx1 were found to be expressed in the branchial ionocytes of tilapia, thereby providing further support for the mechanism of transcellular Ca(2+) transport through ionocytes previously proposed for zebrafish. In addition, we also reveal that low Ca(2+) water treatment of tilapia stimulates Ca(2+) uptake and expression of ecac and cyp11b (the latter encodes a cortisol-synthesis enzyme). Treatment of tilapia with exogenous cortisol (20 mg/l) enhanced both Ca(2+) influx and ecac expression. Therefore, increased cyp11b expression is suggested to enhance Ca(2+) uptake capacity in tilapia exposed to low Ca(2+) water. Furthermore, the application of cortisol receptor antagonists revealed that cortisol may regulate Ca(2+) uptake through glucocorticoid and/or mineralocorticoid receptor (GR and/or MR) in tilapia. Taken together, the data suggest that cortisol may activate GR and/or MR to execute its hypercalcemic action by stimulating ecac expression in tilapia.

The complete mitochondrial genome sequence of Onychostoma alticorpus (Cypriniformes, Cyprinidae).

We determined the complete mitochondrial genome (mitogenome) sequence of Onychostoma alticorpus, which is known as an endemic freshwater species in Taiwan, by using long polymerase chain reaction method. The total length of O. alticorpus mitogenome is 16,680 bp, consisting of 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNAs genes and a noncoding control region. The overall base composition of O. alticorpus is 30.88% for A, 23.57% for T, 16.56% for G and 28.99% for C, with a slight AT bias of 54.45%. Gene location and specific usage of distinct termination codon types characterize typically the vertebrate mitochondrial genome. The determination of O. alticorpus mitogenome would play an important role not only in the delineation of phylogeographic history and population genetic structure, but reflection of conservation efforts on the genetic diversity as well as population vitality.

Complete mitochondrial genome of Onychostoma barbata (Cypriniformes, Cyprinidae).

In this study, the complete mitogenome sequence of Onychostoma barbata has been determined using long polymerase chain reaction method. The mitogenome, consisting of 16,592 base pairs (bp), had the typical vertebrate mitochondrial gene arrangement, including 13 protein-coding, 22 transfer RNAs, 2 ribosomal RNAs genes, and a noncoding control region (CR). CR of 937 bp lengths long is located between tRNA(Pro) and tRNA(Phe). The overall base composition of O. barbata is 24.49% for T, 28.04% for C, 31.54% for A, and 15.94% for G, with a slight AT bias of 56.03%.

Action of vitamin D and the receptor, VDRa, in calcium handling in zebrafish (Danio rerio).

The purpose of the present study was to use zebrafish as a model to investigate how vitamin D and its receptors interact to control Ca(2+) uptake function. Low-Ca(2+) fresh water stimulated Ca(2+) influx and expressions of epithelial calcium channel (ecac), vitamin D-25-hydroxylase (cyp2r1), vitamin D receptor a (vdra), and vdrb in zebrafish. Exogenous vitamin D increased Ca(2+) influx and expressions of ecac and 25-hydroxyvitamin D(3)-24-hydroxylase (cyp24a1), but downregulated 1α-OHase (cyp27b1) with no effects on other Ca(2+) transporters. Morpholino oligonucleotide knockdown of VDRa, but not VDRb, was found as a consequence of calcium uptake inhibition by knockdown of ecac, and ossification of vertebrae is impaired. Taken together, vitamin D-VDRa signaling may stimulate Ca(2+) uptake by upregulating ECaC in zebrafish, thereby clarifying the Ca(2+)-handling function of only a VDR in teleosts. Zebrafish may be useful as a model to explore the function of vitamin D-VDR signaling in Ca(2+) homeostasis and the related physiological processes in vertebrates.

Reverse effect of mammalian hypocalcemic cortisol in fish: cortisol stimulates Ca2+ uptake via glucocorticoid receptor-mediated vitamin D3 metabolism.

Cortisol was reported to downregulate body-fluid Ca(2+) levels in mammals but was proposed to show hypercalcemic effects in teleostean fish. Fish, unlike terrestrial vertebrates, obtain Ca(2+) from the environment mainly via the gills and skin rather than by dietary means, and have to regulate the Ca(2+) uptake functions to cope with fluctuating Ca(2+) levels in aquatic environments. Cortisol was previously found to regulate Ca(2+) uptake in fish; however, the molecular mechanism behind this is largely unclear. Zebrafish were used as a model to explore this issue. Acclimation to low-Ca(2+) fresh water stimulated Ca(2+) influx and expression of epithelial calcium channel (ecac), 11ß-hydroxylase and the glucocorticoid receptor (gr). Exogenous cortisol increased Ca(2+) influx and the expressions of ecac and hydroxysteroid 11-beta dehydrogenase 2 (hsd11b2), but downregulated 11ß-hydroxylase and the gr with no effects on other Ca(2+) transporters or the mineralocorticoid receptor (mr). Morpholino knockdown of the GR, but not the MR, was found to impair zebrafish Ca(2+) uptake function by inhibiting the ecac expression. To further explore the regulatory mechanism of cortisol in Ca(2+) uptake, the involvement of vitamin D(3) was analyzed. Cortisol stimulated expressions of vitamin D-25hydroxylase (cyp27a1), cyp27a1 like (cyp27a1l), 1α-OHase (cyp27b1) at 3 dpf through GR, the first time to demonstrate the relationship between cortisol and vitamin D(3) in fish. In conclusion, cortisol stimulates ecac expression to enhance Ca(2+) uptake functions, and this control pathway is suggested to be mediated by the GR. Lastly, cortisol also could mediate vitamin D(3) signaling to stimulate Ca(2+) uptake in zebrafish.

Effects of stanniocalcin 1 on calcium uptake in zebrafish (Danio rerio) embryo.

Stanniocalcin (STC) formerly called hypocalcin or teleocalcin, is a 50-kDa disulfide-linked homodimeric glycoprotein that was originally identified in fish and secreted from the corpuscles of Stannius (CS). One of the main functions of STC-1 is Ca(2+) uptake inhibition; however, the mechanisms remain unknown. In the present study, we provide molecular evidence to elucidate how zebrafish STC-1 regulates Ca(2+) uptake in zebrafish embryos. In a wide variety of tissues including the kidney, brain, gill, muscle, and skin, zstc-1 was expressed. Incubating zebrafish embryos in low-Ca(2+) (0.02 mM) freshwater stimulated whole body Ca(2+) influx and zebrafish epithelial Ca(2+) channel (zECaC) mRNA expression, while downregulated zstc-1 expression. A morpholino microinjection approach was used to knockdown the zSTC-1 protein, and the results showed that the Ca(2+) content, Ca(2+) influx, and zECaC mRNA expression all increased in morphants. These data suggest that zSTC-1 negatively regulates ECaC gene expression to reduce Ca(2+) uptake in zebrafish embryos.

Molecular cloning and characterisation of prophenoloxidase cDNA from haemocytes of the giant freshwater prawn, Macrobrachium rosenbergii, and its transcription in relation with the moult stage.

Expression of prophenoloxidase (proPO) cDNA was determined from haemocytes of the giant freshwater prawn Macrobrachium rosenbergii by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA using oligonucleotide primers based on the proPO sequence of tiger shrimp Penaeus monodon, freshwater crayfish Pacifastacus leniusculus, green tiger shrimp Penaeus semisulcatus, kuruma shrimp Marsupenaeus japonicus, and white shrimp Litopenaeus vannamei. The proPO of M. rosenbergii was constitutively expressed. The 2,547-bp cDNA contained an open reading frame (ORF) of 2,013 bp, a 96-bp 5'-untranslated region, and a 438-bp 3'-untranslated region containing the poly A tail. The molecular mass of the deduced amino acid (aa) sequence (671 aa) was 76.7 kDa with an estimated pI of 7.05. It contained putative copper-binding sites, a complement-like motif (GCGWPRHM), a proteolytic activation site, and a conserved C-terminal region common to all known proPOs. However, no signal peptide sequence was detected in giant freshwater prawn proPO. Comparison of amino acid sequences showed that prawn proPO is similar to the proPO of penaeid, crayfish and lobster. Prawn proPO was only synthesised in haemocytes. The proPO transcript was significantly increased in the A stage and achieved the highest level in the B stage, and then declined sharply in the C stage and reached the lowest level in the D(2)/D(3) stage.

Molecular cloning and characterisation of peroxinectin, a cell adhesion molecule, from the giant freshwater prawn Macrobrachium rosenbergii.

Expression of peroxinectin cDNA was determined from haemocytes of giant freshwater prawn Macrobrachium rosenbergii using oligonucleotide primers and reverse transcription polymerase chain reaction (RT-PCR) based on the peroxinectin sequence of white shrimp Litopenaeus vannamei, tiger shrimp Penaeus monodon, and freshwater crayfish Pacifastacus leniusculus. The peroxinectin of M. rosenbergii was constitutively expressed. Analysis of the nucleotide sequence revealed that the cDNA clone has an open reading frame of 2,403 bp encoding a protein of 801 amino acids including a 20 amino acid signal peptide. The calculated molecular mass of the mature protein (781 amino acids) was 88.7 kDa with an estimated pI of 6.8. A putative peroxidase domain and a putative integrin-binding motif, KGD (Lys-Gly-Asp) were observed in prawn peroxinectin at the C-terminal. Sequence comparison showed that peroxinectin deduced amino acid of M. rosenbergii had an overall similarity of 62%, 64%, and 66% to that of P. leniusculus, P. monodon, and L. vannamei, respectively. Quantitative real-time PCR analysis showed that peroxinectin transcript in haemocyte of M. rosenbergii decreased significantly after 3, 6 and 12h injection with Lactococcus garvieae.