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Hadwiger and Hajós conjectured that for every positive integer t , K t + 1 -minor free graphs and K t + 1 -topological minor free graphs are properly t -colorable, respectively. Clustered coloring version of these two conjectures which only require monochromatic components to have bounded size has been extensively studied. In this paper we consider the clustered coloring version of the immersion-variant of Hadwiger's and Hajós' conjecture proposed by Lescure and Meyniel and independently by Abu-Khzam and Langston. We determine the minimum number of required colors for H -immersion free graphs, for any fixed graph H , up to a small additive absolute constant. Our result is tight for infinitely many graphs H. A key machinery developed in this paper is a lemma that reduces a clustering coloring problem on graphs to the one on the torsos of their tree-cut decomposition or tree-decomposition. A byproduct of this machinery is a unified proof of a result of Alon, Ding, Oporowski and Vertigan and a result of the author and Oum about clustered coloring graphs of bounded maximum degree in minor-closed families. [ABSTRACT FROM AUTHOR]

Nash-Williams' Strong Immersion Conjecture states that graphs are well-quasi-ordered by the strong immersion relation. That is, given infinitely many graphs, one graph contains another graph as a strong immersion. In this paper we study the analogous problem for directed graphs. It is known that digraphs are not well-quasi-ordered by the strong immersion relation, but for all known such infinite antichains, paths that change direction arbitrarily many times can be found. This paper proves that the converse statement is true: for every positive integer k , the digraphs that do not contain a path that changes direction k times are well-quasi-ordered by the strong immersion relation, even when vertices are labeled by a well-quasi-order. This result is optimal for classes of digraphs closed under taking subgraphs since paths that change direction arbitrarily many times with vertex-labels form an infinite antichain with respect to the strong immersion relation. [ABSTRACT FROM AUTHOR]

A graph G contains another graph H as an immersion if H can be obtained from a subgraph of G by splitting off edges and removing isolated vertices. There is an obvious necessary degree condition for the immersion containment: if G contains H as an immersion, then for every integer k , the number of vertices of degree at least k in G is at least the number of vertices of degree at least k in H. In this paper, we prove that this obvious necessary condition is "nearly" sufficient for graphs with no edge-cut of order 3: for every graph H , every H -immersion free graph with no edge-cut of order 3 can be obtained by an edge-sum of graphs, where each of the summands is obtained from a graph violating the obvious degree condition by adding a bounded number of edges. The condition for having no edge-cut of order 3 is necessary. A simple application of this theorem shows that for every graph H of maximum degree d ≥ 4 , there exists an integer c such that for every positive integer m , there are at most c m unlabeled d -edge-connected H -immersion free m -edge graphs with no isolated vertex, while there are superexponentially many unlabeled (d − 1) -edge-connected H -immersion free m -edge graphs with no isolated vertex. Our structure theorem will be applied in a forthcoming paper about determining the clustered chromatic number of the class of H -immersion free graphs. [ABSTRACT FROM AUTHOR]

The clustering of a graph coloring is the maximum size of monochromatic components. This paper studies colorings with bounded clustering in graph classes with bounded layeredtreewidth, which include planar graphs, graphs of bounded Euler genus, graphs embeddable on a fixed surface with a bounded number of crossings per edge, map graphs, amongst other examples. Our main theorem says that every graph with layered treewidth at most k and with maximum degree at most Δ is 3-colorable with clustering O (k 19 Δ 37). This is the first known polynomial bound on the clustering. This greatly improves upon a corresponding result of Esperet and Joret for graphs of bounded genus. [ABSTRACT FROM AUTHOR]

Hajós conjectured that every graph containing no subdivision of the complete graph K s + 1 is properly s -colorable. This conjecture was disproved by Catlin. Indeed, the maximum chromatic number of such graphs is Ω (s 2 / log ⁡ s). We prove that O (s) colors are enough for a weakening of this conjecture that only requires every monochromatic component to have bounded size (so-called clustered coloring). Our approach leads to more results, many of which only require a much weaker assumption that forbids an 'almost (⩽ 1) -subdivision' (where at most one edge is subdivided more than once). This assumption is best possible, since no bound on the number of colors exists unless we allow at least one edge to be subdivided arbitrarily many times. We prove the following (where s ⩾ 2): 1. Graphs of bounded treewidth and with no almost (⩽ 1) -subdivision of K s + 1 are s -choosable with bounded clustering. 2. For every graph H , graphs with no H -minor and no almost (⩽ 1) -subdivision of K s + 1 are (s + 1) -colorable with bounded clustering. 3. For every graph H of maximum degree at most d , graphs with no H -subdivision and no almost (⩽ 1) -subdivision of K s + 1 are max ⁡ { s + 3 d − 5 , 2 } -colorable with bounded clustering. 4. For every graph H of maximum degree d , graphs with no K s , t subgraph and no H -subdivision are max ⁡ { s + 3 d − 4 , 2 } -colorable with bounded clustering. 5. Graphs with no K s + 1 -subdivision are (4 s − 5) -colorable with bounded clustering. The first result is tight and shows that the clustered analogue of Hajós' conjecture is true for graphs of bounded treewidth. The second result implies an upper bound for the clustered version of Hadwiger's conjecture that is only one color away from the known lower bound, and shows that the number of colors is independent of the forbidden minor. The final result is the first O (s) bound on the clustered chromatic number of graphs with no K s + 1 -subdivision. [ABSTRACT FROM AUTHOR]

A graph G contains another graph H as an immersion if H can be obtained from a subgraph of G by splitting off edges and removing isolated vertices. In this paper, we prove an edge-variant of the Erdős-Pósa property with respect to the immersion containment in 4-edge-connected graphs. More precisely, we prove that for every graph H , there exists a function f such that for every 4-edge-connected graph G , either G contains k pairwise edge-disjoint subgraphs each containing H as an immersion, or there exists a set of at most f (k) edges of G intersecting all such subgraphs. This theorem is best possible in the sense that the 4-edge-connectivity cannot be replaced by the 3-edge-connectivity. [ABSTRACT FROM AUTHOR]

Since the mechanisms by which cellular factors modulate replication of the shrimp viral pathogen white spot syndrome virus (WSSV) are still largely unknown, here we consider the sirtuins, a family of NAD+-dependent protein deacetylases that are known to function as regulatory factors that activate or suppress viral transcription and replication in mammals. In particular, we focus on SIRT1 by isolating and characterizing LvSIRT1 from white shrimp (Litopenaeus vannamei) and investigating its involvement in WSSV infection. DsRNA-mediated gene silencing led to the expression of WSSV genes and the replication of genomic DNAs being significantly decreased in LvSIRT1 -silenced shrimp. The deacetylase activity of LvSIRT1 was significantly induced at the early stage (2 hpi) and the genome replication stage (12 hpi) of WSSV replication, but decreased at the late stage of WSSV replication (24 hpi). Treatment with the SIRT1 activator resveratrol further suggested that LvSIRT1 activation increased the expression of several WSSV genes (IE1 , VP28 and ICP11). Lastly, we used transfection and dual luciferase assays in Sf9 insect cells to show that while the overexpression of LvSIRT1 facilitates the promoter activity of WSSV IE1, this enhancement of WSSV IE1 expression is achieved by a transactivation pathway that is NF-κB-independent. • CDNA encoding SIRT1 was identified and characterized from white shrimp (Litopenaeus vannamei). • LvSIRT1 deacetylase activity was induced at the early stage and genome replication stage of WSSV replication. • LvSIRT1 silencing led to the significant decrease in WSSV genes and WSSV genomic DNAs. • LvSIRT1 activation by SIRT1 activator resveratrol increased the expression of several WSSV genes (IE1 , VP28 and ICP11). • Shrimp SIRT1 activates of the WSSV IE1 promoter independently of the NF-κB binding site. [ABSTRACT FROM AUTHOR]

The use of probiotics as alternatives to antibiotics for disease control is a relatively eco-friendly approach in aquaculture; hence, studies isolating and assessing the benefit of potential probiotics to fish farming are common. The zebrafish is an excellent model system for validating beneficial functions of potential probiotics before their practical application in aquaculture. Here, a potentially probiotic Chromobacterium aquaticum was isolated from lake water samples and characterized by biochemical analysis and 16S rDNA sequencing. The probiotic produced extracellular enzymes (protease and xylanase) and a bacteriocin-like substance, which exhibited tolerance to extreme pH and high-temperature conditions and broad-spectrum bactericidal activity against diverse pathogens, including aquatic, foodborne, clinical and plant pathogens. The effects of C. aquaticum on zebrafish nutrient metabolism, growth performance and innate immunity were evaluated by measuring the expression of indicator genes after C. aquaticum feeding for 8 weeks. Fish administered the probiotic exhibited significantly increased hepatic mRNA expression of carbohydrate metabolism-related genes, including glucokinase (GK), hexokinase (HK), glucose-6-phosphatase (G6Pase), and pyruvate kinase (PK-L), and growth-related genes, including the growth hormone receptor (GHR) and insulin-like growth factor-1 (IGF-1). Innate immune-related genes (IL-1β, IL-6, TNF-α, IL-10, IL-21, NF-κb, lysozyme and complement C3b) were induced in fish with probiotic supplementation. Probiotic-treated fish exhibited a higher survival rate than control fish after challenge with Aeromonas hydrophila and Streptococcus iniae. Together, these data suggest that C. aquaticum , as a probiotic feed supplement, could enhance nutrient metabolism and growth performance and could modulate innate immunity against A. hydrophila and S. iniae in zebrafish. • Chromobacterium aquaticum with bacteriocin-like activity was used as a probiotic. • C. aquaticum enhanced the hepatic expression levels of metabolism- and growth-related genes in fish. • Fish fed a C. aquaticum -supplemented diet exhibited disease resistance against A. hydrophila and S. iniae infections. • C. aquaticum modulated innate immunity and enhanced the defense against bacterial infection. [ABSTRACT FROM AUTHOR]

Abstract Bacteria-induced diseases are a major cause of mortality in aquaculture. Probiotics have commonly been used to replace antibiotics for prophylactic biocontrol in aquaculture. In the present study, Paenibacillus ehimensis NPUST1 was isolated from a tilapia culture pond. This probiotic has bacteriocin-like activities against Aeromonas hydrophila and was characterized by biochemical analysis and 16S rDNA sequencing. The physiochemical properties of a crude extract of the bacteriocin-like substance revealed low pH and high thermal tolerance. The substance exhibited broad-spectrum antimicrobial activity against diverse aquatic pathogens, food spoilage, clinical pathogens, and plant pathogens. The effect of dietary supplementation with P. ehimensis NPUST1 was evaluated in regard to the growth of Nile tilapia (Oreochromis niloticus) and immunity against pathogenic infection. The results showed significantly increased weight gain (WG), feed conversion ratio (FCR), and feed efficiency (FE) in Nile tilapia fed P. ehimensis NPUST1 for 2 months compared with fish fed a control diet. When challenged with A. hydrophila and S. iniae, the fish fed P. ehimensis NPUST1 also exhibited a higher survival rate than fish fed the control diet. The immune parameters revealed that the P. ehimensis NPUST1-fed fish had significantly higher phagocytic activity, respiratory burst, and superoxide dismutase (SOD) of the head kidney leukocytes, as well as higher serum lysozyme activity and expression of cytokines TNF-α and IL-1β than the fish fed the control diet. These results indicate that dietary supplementation with P. ehimensis NPUST1 improved the growth performance, immunity, and disease resistance in Nile tilapia. Highlights • Paenibacillus ehimensis NPUST1 produces a bacteriocin-like substance and was investigated as a probiotic. • Dietary supplementation of P. ehimensis NPUST1 enhanced fish growth. • When used as a probiotic, P. ehimensis NPUST1 increased the survival of fish challenged with A. hydrophila and S. iniae. • P. ehimensis NPUST1 also improved innate immunity against bacterial infection when used as a probiotic. [ABSTRACT FROM AUTHOR]

Abstract Let H be a fixed graph. What can be said about graphs G that have no subgraph isomorphic to a subdivision of H ? Grohe and Marx proved that such graphs G satisfy a certain structure theorem that is not satisfied by graphs that contain a subdivision of a (larger) graph H 1. Dvořák found a clever strengthening—his structure is not satisfied by graphs that contain a subdivision of a graph H 2 , where H 2 has "similar embedding properties" as H. Building upon Dvořák's theorem, we prove that said graphs G satisfy a similar structure theorem. Our structure is not satisfied by graphs that contain a subdivision of a graph H 3 that has similar embedding properties as H and has the same maximum degree as H. This will be important in a forthcoming application to well-quasi-ordering. [ABSTRACT FROM AUTHOR]

There has been extensive research on cycle lengths in graphs with large minimum degree. In this paper, we obtain several results that are tight in this area. Let G be a graph with minimum degree at least k + 1 . We prove that if G is bipartite, then there are k cycles in G whose lengths form an arithmetic progression with common difference two. For a general graph G , we show that G contains ⌊ k / 2 ⌋ cycles with consecutive even lengths and k − 3 cycles whose lengths form an arithmetic progression with common difference one or two. In addition, if G is 2-connected and non-bipartite, then G contains ⌊ k / 2 ⌋ cycles with consecutive odd lengths. Thomassen (1983) made two conjectures on cycle lengths modulo a fixed integer k : (1) every graph with minimum degree at least k + 1 contains cycles of all even lengths modulo k ; (2) every 2-connected non-bipartite graph with minimum degree at least k + 1 contains cycles of all lengths modulo k . These two conjectures, if true, are best possible. Our results confirm both conjectures when k is even. When k is odd, we show that minimum degree at least k + 4 suffices. This improves all previous results in this direction. Moreover, our results derive new upper bounds of the chromatic number in terms of the longest sequence of cycles with consecutive (even or odd) lengths. [ABSTRACT FROM AUTHOR]

We prove that for every graph H , if a graph G has no (odd) H minor, then its vertex set V ( G ) can be partitioned into three sets X 1 , X 2 , X 3 such that for each i , the subgraph induced on X i has no component of size larger than a function of H and the maximum degree of G . This improves a previous result of Alon, Ding, Oporowski and Vertigan (2003) [1] stating that V ( G ) can be partitioned into four such sets if G has no H minor. Our theorem generalizes a result of Esperet and Joret (2014) [9] , who proved it for graphs embeddable on a fixed surface and asked whether it is true for graphs with no H minor. As a corollary, we prove that for every positive integer t , if a graph G has no K t + 1 minor, then its vertex set V ( G ) can be partitioned into 3 t sets X 1 , … , X 3 t such that for each i , the subgraph induced on X i has no component of size larger than a function of t . This corollary improves a result of Wood (2010) [21] , which states that V ( G ) can be partitioned into ⌈ 3.5 t + 2 ⌉ such sets. [ABSTRACT FROM AUTHOR]

Given an infinite family G of graphs and a monotone property P , an (upper) threshold for G and P is a "fastest growing" function p : N → [ 0 , 1 ] such that lim n → ∞ ⁡ Pr ⁡ (G n (p (n)) ∈ P) = 1 for any sequence (G n) n ∈ N over G with lim n → ∞ ⁡ | V (G n) | = ∞ , where G n (p (n)) is the random subgraph of G n such that each edge remains independently with probability p (n). In this paper we study the upper threshold for the family of H -minor free graphs and the property of being (r − 1) -degenerate and apply it to study the thresholds for general minor-closed families and the properties for being r -choosable and r -colorable. Even a constant factor approximation for the upper threshold for all pairs (r , H) is expected to be challenging by its close connection to a major open question in extremal graph theory. We determine asymptotically the thresholds (up to a constant factor) for being (r − 1) -degenerate (and r -choosable, respectively) for a large class of pairs (r , H) , including all graphs H of minimum degree at least r and all graphs H with no vertex-cover of size at most r , and provide lower bounds for the rest of the pairs of (r , H). [ABSTRACT FROM AUTHOR]

A feedback vertex set of a graph is a subset of vertices intersecting all cycles. We provide tight upper bounds on the size of a minimum feedback vertex set in planar graphs of girth at least five. We prove that if G is a connected planar graph of girth at least five on n vertices and m edges, then G has a feedback vertex set of size at most 2 m − n + 2 7 . By Euler’s formula, this implies that G has a feedback vertex set of size at most m 5 and n − 2 3 . These results not only improve a result of Dross, Montassier and Pinlou and confirm the girth-5 case of one of their conjectures, but also make the best known progress towards a conjecture of Kowalik, Lužar and Škrekovski and solve the subcubic case of their conjecture. An important step of our proof is providing an upper bound on the size of minimum feedback vertex sets of subcubic graphs with girth at least five with no induced subdivision of members of a finite family of non-planar graphs. [ABSTRACT FROM AUTHOR]

Abstract: A linear coloring of a graph is a proper coloring of the vertices of the graph so that each pair of color classes induces a union of disjoint paths. In this paper, we prove that for every connected graph with maximum degree at most three and every assignment of lists of size four to the vertices of the graph, there exists a linear coloring such that the color of each vertex belongs to the list assigned to that vertex and the neighbors of every degree-two vertex receive different colors, unless the graph is or . This confirms a conjecture raised by Esperet, Montassier and Raspaud [L. Esperet, M. Montassier, and A. Raspaud, Linear choosability of graphs, Discrete Math. 308 (2008) 3938–3950]. Our proof is constructive and yields a linear-time algorithm to find such a coloring. [Copyright &y& Elsevier]

Abstract: The aim of this study was to improve the growth performance, immune response and disease resistance of grouper, Epinephelus coioides by using probiotic, Bacillus subtilis E20. The percent weight gain (PWG) and feeding efficiency (FE) of grouper administered the probiotic B. subtilis E20 were calculated. Survival of B. subtilis E20 in the posterior intestines was determined using a specific primer pair of BPHYF/BPHYR, as were the non-specific immune parameters of grouper, and its susceptibility to Streptococcus sp. and an iridovirus when fish were fed diets containing B. subtilis at 0 (control), 104, 106, and 108 colony-forming units (cfu) g−1 up to 28 days. Results showed that grouper fed a diet containing B. subtilis at the levels of 104, 106, and 108 cfu g−1 had significantly increased PGW (203.0%, 229.6%, and 238.0%) and FE (1.15, 1.20, and 1.22) compared to control (191.8% and 1.0), and these directly increased in a dose-dependent manner with B. subtilis concentrations. B. subtilis was able to survive in the fish''s posterior intestines during the feeding period. The survival rate increased in grouper challenged with Streptococcus sp. or an iridovirus when the fish were fed B. subtilis at 104, 106, and 108 cfu g−1 for 14 and 28 days, and it was higher at 28 days than at 14 days. After 28 days of feeding, the relative survival percentages of fish challenged with Streptococcus sp. and an iridovirus were 22.8, 40.9 and 45.5, and 21.7, 30.4, and 52.2, respectively. The phagocytic activity, respiratory bursts, and superoxide dismutase (SOD) level of head kidney leucocytes as well as serum lysozyme activity and serum alternative complement activity (ACH50) of fish fed diets containing B. subtilis at 104, 106 and 108 cfu g−1 were significantly and dose-dependently higher than those of fish fed the control diet for 28 days. We therefore recommend dietary B. subtilis E20 administration of 104 – 108 cfu g−1 to E. coioides to promote growth, and enhance immunity and resistance against Streptococcus sp. and an iridovirus. The best results were seen in the 108 cfu g−1 group fed for 28 days. [Copyright &y& Elsevier]

Abstract: Zingerone, one of the active components of ginger, is a phenolic alkanone with antioxidant and anti-inflammatory properties. The effects of zingerone supplementation on the growth, immunity, and disease resistance of Pacific white shrimp (Litopenaeus vannamei) juveniles were studied. Four experimental diets, including a control diet (without zingerone enrichment) and 1, 2.5, and 5 mg zingerone (kg diet)−1 were used. After 56 days of culture, shrimp fed diets supplemented with 1, 2.5, and 5 mg zingerone (kg diet)−1 had significantly greater weight gain and feed efficiency than the controls. Furthermore, after 56 days of culture, shrimp fed all doses of the zingerone diet had higher survival rates compared to the controls after 24–72 h of challenge by the pathogen, Vibrio alginolyticus. Significantly increased phenoloxidase levels were found in shrimp fed the zingerone diets at all doses, and respiratory bursts, lysozyme and phagocytic activities of shrimp fed 2.5 and 5 mg zingerone (kg diet)−1 also significantly increased. Neither the total hemocyte count nor superoxide dismutase activity of the experimental and control groups revealed significant differences at any dose. The results indicate that zingerone can be recommended as a supplement to shrimp feed to increase growth, immunity, and disease resistance against the pathogen, V. alginolyticus. Use of zingerone as appetizer and immunostimulant in shrimp is promising. [Copyright &y& Elsevier]

Abstract: Complementary (c)DNA encoding transglutaminase (TG) messenger (m)RNA of the giant freshwater prawn, Macrobrachium rosenbergii, was cloned from haemocytes by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) using oligonucleotide primers based on the TG sequence of the horseshoe crab, Tachypleus tridentatus; tiger shrimp, Penaeus monodon; kuruma shrimp, Marsupenaeus japonicus; and crayfish, Pacifastacus leniusculus. The 2722-bp cDNA contained an open reading frame (ORF) of 2334 bp, a 72-bp 5′-untranslated region (UTR), and a 316-bp 3′-UTR containing a stop codon and a poly A tail. The molecular mass of the deduced amino acid (aa) sequence (778 aa) was 86.67 kDa with an estimated pI of 5.4. The M. rosenbergii TG (abbreviated MrTG, accession no.: JF309296) contains a typical transglutaminase-like homologue, two putative integrin-binding motifs (RGD), ten glycosylation sites, and four calcium-binding sites; a catalytic triad is present as in arthropod TGs. Sequence comparison and a phylogenetic analysis revealed that shrimp TG can be separated into three subgroups, penaeid TG1, freshwater crustacean TG2 and marine crustacean TG2, and MrTG was more closely related to TG2 than to TG1. MrTG mRNA and TG activities were detected in all tested tissues of M. rosenbergii, with MrTG mainly being synthesised by haemocytes. There was a negative correlation between clotting time of haemolymph, and MrTG expression and TG activity of haemocytes in prawn injected with Lactococcus garvieae. The pattern of MrTG mRNA expression and TG activity in haemocytes exhibited a contrary tendency with clotting time of haemolymph during the moult stages. Those results indicate that cloned MrTG is involved in the defence response, and is probably the major functional TG for haemolymph coagulation in M. rosenbergii. [Copyright &y& Elsevier]

Abstract: Respiratory burst, lysozyme and phagocytic activities, and immunoglobulin levels in response to the pathogen Aeromonas hydrophila were examined in tilapia (Oreochromis mossanbicus, 10.65 ± 2.5 g) injected individually with hot-water extract of Toona sinensis at 4 or 8 μg g−1. Tilapia receiving the hot-water extract of T. sinensis at either dose had significantly increased respiratory burst, phagocytic activity and lysozyme activity towards A. hydrophila by 1 and 2 days post injection. No significant differences in total immunoglobulin levels were observed among the tilapia that received the two different doses of hot-water extract of T. sinensis at 4 and 8 μg g−1. In another experiment, a Tilapia that had been injected with hot-water extract of T. sinensis was challenged with A. hydrophila at 5 × 107 colony-forming units (cfu) fish−1. The survival of tilapia that received the hot-water extract of T. sinensis at 8 μg g−1 was significantly higher than fish that received phosphate buffered saline and the control fish after 2 days, and at the termination of the experiment (7 days after the challenge). It was concluded that the hot-water extract of T. sinensis at 8 μg g−1 or less had increased the immune response and resistance to A. hydrophila infection in tilapia. [Copyright &y& Elsevier]

Abstract: Complementary (c)DNA encoding transglutaminase (TG) messenger (m)RNA of white shrimp, Litopenaeus vannamei, was cloned from haemocytes by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) using oligonucleotide primers based on the TG sequence of the horseshoe crab, Tachypleus tridentatus (accession no.: BAA02134); tiger shrimp, Penaeus monodon (AAL78166); and Pacifastacus leniusculus (AF336805). The 2638-bp cDNA contained an open reading frame (ORF) of 2172 bp, a 55-bp 5′-untranslated region (UTR), and a 411-bp 3′-UTR containing a poly A tail. The molecular mass of the deduced amino acid (aa) sequence (757 aa) was 84.9 kDa with an estimated pI of 5.2. The L. vannamei TG (abbreviated LvTG) contains a typical transglutaminase-like homologue, a putative integrin-binding motif (RGD), and four calcium-binding sites; a catalytic triad is present as in arthropod TG. Sequence comparison and phylogenetic analysis revealed that shrimp TG can be separated into two subgroups, STGS1 and STGS2, and LvTG is more closely related to STGS1 than to STGS2. LvTG mRNA and TG activities were detected in all tested tissues of L. vannamei, with LvTG mainly being synthesised in haemocytes. However, the pattern of LvTG mRNA expression was not directly correlated with TG activity. The haemocytes of L. vannamei injected with Vibrio alginolyticus showed a significant decrease of TG activity at 3 h and a significant increase of LvTG mRNA expression at 6 h followed by a notable decrease from 12 to 24 h, which indicated that cloned LvTG was involved in the immune response of shrimp. The results also imply that more than one type of TG may be involved in the defense response in L. vannamei. [Copyright &y& Elsevier]

Abstract: cDNA encoding glutathione peroxidase (GPx) mRNA of the white shrimp Litopenaeus vannamei was obtained from haemocytes by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA (RACE) using oligonucleotide primers based on the GPx sequence of Homo sapiens (NM002083), Mus musculus (NM008160), Arabidopsis thaliana (U94495), Bos taurus (NM174770), and Capsicum chinense (AJ973135). The 727-bp cDNA contained an open reading frame (ORF) of 567bp, a 101-bp 5′-untranslated region, and a 59-bp 3′-untranslated region containing the poly A tail. The molecular mass of the deduced amino acid (aa) sequence (189aa) was 19.25kDa long with an estimated pI of 8.39. It contains a putative selenocysteine residue which is encoded by the unusual stop codon, TGA, and forms the active site with residues Glu75 and Trp153. Comparison of amino acid sequences showed that white shrimp GPx is more closely related to GPx1 and GPx2 than to GPx3 and GPx4 of various animals. The GPx cDNA was synthesized in haemocytes, gills, the hepatopancreas, intestines, and muscles. The respiratory bursts of shrimp increased significantly after a Vibrio alginolyticus injection in order to kill the pathogen, and then induced increases in the activities of SOD and GPx to protect cells against damage from oxidation. However, GPx activity increased as a result of upregulated expression of GPx mRNA which was induced by the increase in H2O2. [Copyright &y& Elsevier]

Abstract: Peroxinectin (PE) gene expressions were determined using real-time PCR in the giant freshwater prawn Macrobrachium rosenbergii based on moulting; prawns were fed diets containing different concentrations of sodium alginate, and were exposed to different concentrations of copper sulphate, benzalkonium chloride (BKC), and trichlorfon. Results showed that PE mRNA expression of prawns was the highest in stage A, significantly decreased in stage B, and reached the lowest level in stages D0/D1. The PE transcript was significantly higher in prawns fed the 1.0gkg−1 sodium alginate-containing diet than those fed the 2.0gkg−1 sodium alginate-containing diet and those fed the control diet. PE transcripts significantly decreased in prawns exposed to 0.1–0.4mgL−1 copper sulphate after 96h, 0.3–1.0mgL−1 BKC after 96h, and 0.2–0.4mgL−1 trichlorfon after 48h. It was concluded that the status of PE gene expression was seriously affected by the moult cycle, immunostimulant, and chemotherapeutants. [Copyright &y& Elsevier]

Abstract: The total haemocyte count (THC), phenoloxidase (PO) activity, respiratory bursts (release of superoxide anions), and superoxide dismutase (SOD) activity, as well as expressions of β-1,3-glucan-binding protein (βGBP), prophenoloxidase (proPO), peroxinectin (PE), cytosolic SOD (cyt-SOD), penaeidin-5 (PA-5), and a single whey acidic protein (WAP) domain protein (SWDP) gene were determined in the tiger shrimp Penaeus monodon (15.6–19.5g) which had individually been fed diets containing sodium alginate at 0, 1.0, or 2.0gkg−1 for 5months. Results showed that shrimp fed a diet containing 1.0 and 2.0gkg−1 sodium alginate had significantly increased SOD activity but decreased respiratory bursts. The expressions of βGBP, PE, cyt-SOD, PA-5, and SWDP were significantly elevated in shrimp fed the ≤2.0gkg−1 sodium alginate-containing diet for 5months. However, no significant differences in THC, PO activity, or proPO mRNA transcription in shrimp were observed among the three treatments. It was concluded that sodium alginate can be used as an immunomodulator for shrimp through dietary administration to modify immune genes expression of shrimp. [Copyright &y& Elsevier]

Abstract: 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 2547-bp cDNA contained an open reading frame (ORF) of 2013bp, 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 76.7kDa 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 D2/D3 stage. [Copyright &y& Elsevier]

Abstract: 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 2403bp 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 88.7kDa 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. [Copyright &y& Elsevier]

Abstract: Peroxinectin mRNA expression in the different types of haemocytes of white shrimp Litopenaeus vannamei was studied by in situ hybridisation using digoxigenin-UTP-labelled riboprobes and reverse transcription-polymerase chain reaction (RT-PCR). Granular cells (GC) and a mixture of semi-granular cells (SGC) and hyaline cells (HC) were separated by 70% Percoll gradient centrifugation. Peroxinectin was synthesised in both GC and the mixture of SGC-HC. An in situ hybridisation assay indicated that peroxinectin mRNA expression occurred in GC and SGC, but not in HC. Peroxinectin transcript up-regulated significantly, whereas haemocyte count decreased significantly at 6, 12 and 24 h post Vibrio alginolyticus-injection with slower restoration as compared to that of saline-injected shrimp. The RT-PCR assay indicated that peroxinectin exists extensively in several species of decapod crustaceans including L. vannamei, freshwater prawn Macrobrachium rosenbergii, common caridina Caridina pseudodenticulata, stone crab Thalamita crenata and mud crab Scylla serrata suggesting that this protein plays an important role in defence against pathogens. [Copyright &y& Elsevier]

Abstract: A lipopolysaccharide- and β-1,3-glucan binding protein (LGBP) cDNA was cloned from the haemocyte and hepatopancreas of white shrimp Litopenaeus vannamei using oligonucleotide primers and RT-PCR. Both 3′- and 5′-regions were isolated by rapid amplification of cDNA end RACE method. Analysis of nucleotide sequence revealed that the cDNA clone has an open reading frame of 1101bp encoding a protein of 367 amino acids including a 17 amino acid signal peptide. The calculated molecular mass of the mature proteins (350 amino acids) is 39.92kDa with an estimated pI of 4.37. Two putative integrin binding motifs (cell adhesion site), RGD (Arg-Gly-Asp) and a potential recognition motif for β- (1→3) linkage of polysaccharides were observed in the LGBP. Sequence comparison showed that LGBP deduced amino acid of L. vannamei has an overall similarity of 95%, 92% and 61% to that of blue shrimp Litopenaeus stylirostris LGBP, tiger shrimp Penaeus monodon BGBP and crayfish Pacifastacus leniusculus LGBP, respectively. Quantitative real-time RT-PCR analysis showed that LGBP transcript in haemocyte of L. vannamei increased in 3- and 6-h post Vibrio alginolyticus injection. [Copyright &y& Elsevier]

Haemocyte count, phenoloxidase activity, respiratory burst (release of superoxide anion), superoxide dismutase (SOD) activity, glutathione peroxidase (GPX) activity, phagocytic activity and clearance efficiency to the pathogen Vibrio alginolyticus were measured in white shrimp Litopenaeus vannamei juveniles (12.3 ± 1.2 g) which had been fed diets containing sodium alginate at 0.5, 1.0, 2.0 g kg-1 after five months. L. vannamei fed a diet containing 2.0 g kg-1 sodium alginate had increased phenoloxidase activity, respiratory burst and SOD activity, but decreased GPX activity significantly. L. vannamei fed a diet containing 2.0 g kg-1 sodium alginate had increased phagocytic activity and the shrimp fed a diet containing sodium alginate at 0.5, 1.0 or 2.0 g kg-1 had increased clearance efficiency to V. alginolyticus. In another experiment, L. vannamei, which had been fed control diet, or sodium alginate-containing diets after 5 months, were challenged with V. alginolyticus at 2 × 106 colony-forming units (CFU) shrimp-1 and then placed in seawater of 15‰. The survival of shrimp fed a diet containing 2.0 g kg-1 after one day, and the survival of shrimp fed diets containing sodium alginate at 0.5 and 1.0 g kg-1 after 2–4 days increased significantly, as compared to that of shrimp fed control diet. It is therefore concluded that administration of sodium alginate in the diet at 2.0 g kg-1 or less could enhance the immune ability of L. vannamei and increase its resistance to V. alginolyticus infection. [Copyright &y& Elsevier]

White shrimp Litopenaeus vannamei (Boone) held in 35‰ seawater were challenged with Vibrio alginolyticus at a dose of 3 × 105 colony-forming units (cfu) shrimp-1, and then placed in water containing concentrations of Cu2+ at 0 (control), 1, 5, 10 and 20 mg l-1. Mortality of shrimp in 5, 10 and 20 mg l-1 Cu2+ was significantly higher than those in 1 mg l-1 Cu2+ and the control solution after 24–96 h. In another experiment, L. vannamei which had been exposed to control, 1, 5, 10 and 20 mg l-1 Cu2+ for 24, 48 and 96 h were examined for THC (total haemocyte count), phenoloxidase activity, respiratory burst (release of superoxide anion), phagocytic activity and clearance efficiency to V. alginolyticus. Copper concentrations at 1 mg l-1 or greater for 24 h resulted in decreased THC, phenoloxidase activity, phagocytic activity and clearance efficiency, whereas copper concentration at 20 mg l-1 caused significant increase in respiratory burst of L. vannamei. In conclusion, concentration of Cu2+ at 1 mg l-1 or greater increased the susceptibility of L. vannamei to V. alginolyticus infection by a depression in immune ability. The release of superoxide anion by L. vannamei exposed to 20 mg l-1 Cu2+ was considered to be cytotoxic to the host. [Copyright &y& Elsevier]

Expression of peroxinectin cDNA was obtained from haemocytes of white shrimp Litopenaeus vannamei using oligonucleotide primers based on the peroxinectin sequence of tiger shrimp Penaeus monodon and freshwater crayfish Pacifastacus leniusculus. The peroxinectin of L. vannamei was constitutively expressed. The cloning of peroxinectin cDNA fragment was subjected to RT–PCR using degenerated primers. Both 3′- and 5′-regions were isolated by rapid amplification of cDNA end RACE method. Analysis of nucleotide sequence revealed that the cDNA clone has an open reading frame of 2415 bp encoding a protein of 805 amino acids including a 20 amino acid signal peptide. The calculated molecular mass of the mature protein (758 amino acids) is 89.1 kDa with an estimated pI of 7.5. Two putative integrin binding motifs, RGD (Arg–Gly–Asp) and KGD (Lys–Gly–Asp) were observed in L. vannamei peroxinectin. Sequence comparison showed that nucleotide homology has an overall similarity of 91% and 60% to that of P. monodon and P. leniusculus, respectively. Sequence comparison showed that peroxinectin deduced amino acid of L. vannamei has an overall similarity of 92% and 60% to that of P. monodon and P. leniusculus, respectively. The 432 bp fragment (partial cDNA) and 1016 bp fragment (genomic DNA) were obtained using specific primers PE2F and PE2R. There were three introns in the 1016 bp (genomic DNA) fragment. Peroxinectin transcript was significantly reduced when the shrimp was transferred to high temperature (34 °C) as compared to the shrimp transferred to 18 °C and the control shrimp (26 °C). However, peroxinectin transcript did not change in 24 h-post zymosan injection. [Copyright &y& Elsevier]

The total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase activity, respiratory burst (release of superoxide anion), superoxide dismutase activity, phagocytic activity and clearance efficiency to the pathogen Vibrio alginolyticus were measured when the white shrimp Litopenaeus vannamei (9.4–11.3 g) were injected individually with sodium alginate at 10, 20 or 50 μg g-1. No significant differences in THC, DHC and superoxide dismutase activity were observed among the shrimp injected with saline and those injected with sodium alginate at 10, 20 or 50 μg g-1. However, L. vannamei injected with sodium alginate at 20 μg g-1increased its phenoloxidase activity and respiratory burst after 2 days and one day, respectively. L. vannamei injected with sodium alginate at 50 μg g-1maintained a higher phagocytic activity and clearance efficiency to V. alginolyticus after 4 days. In another experiment, L. vannamei which had been injected with sodium alginate, were challenged with V. alginolyticus at 2×105colony-forming units (CFU) shrimp-1and then placed in seawater of 34‰. The survival of shrimp that received sodium alginate at either dose was significantly higher than that of control shrimp at the termination of the experiment (6 days after the challenge). It is therefore concluded that L. vannamei received sodium alginate at 10 μg g-1or more and increased its immune ability and resistance from V. alginolyticus infection. [Copyright &y& Elsevier]

Growth of Vibrio alginolyticus was not affected by TSB medium containing ammonia-N concentration in the range of 0–20 mg l−1. White shrimp Litopenaeus vannamei (7–12 g in the intermolt stage) were challenged with V. alginolyticus, which had been incubated for 24 h in the TSB medium containing different concentrations of ammonia-N (0, 1, 5. 10 and 20 mg l−1). There was no significant difference in cumulative mortality for shrimp incubated in the TSB medium containing 0, 1, 5, 10 and 20 mg l−1ammonia-N after 120 h of challenge. The shrimps were challenged with V. alginolyticus previously incubated in the TSB medium for 24 h, then placed in water containing concentrations of ammonia-N at 0.01 mg l−1(control), 1.10, 5.24, 11.10 and 21.60 mg l−1. Mortality of shrimp in 5.24, 11.10 and 21.60 mg l−1was significantly higher than those in the control solution (0.01 mgl−1) after 48–168 h. Shrimps which had been exposed to control, 1.10, 5.24, 11.10 and 21.60 mg l−1ammonia-N for 7 days were examined for THC (total haemocyte count), granular cells, hyaline cells, phenoloxidase activity, release of superoxide anion, superoxide dismutase (SOD) activity, phagocytic activity and clearance efficiency to V. alginolyticus. No significant difference in THC, hyaline cells and granular cells were observed among shrimps at different ammonia-N concentrations. Phenoloxidase activity however, decreased when the shrimps were exposed to 5.24 mg l−1ammonia-N and greater after 7 days. The release of superoxide anion increased significantly, whereas SOD activity decreased significantly at 21.60 mg l−1ammonia-N. With shrimps exposed to 11.21 and 21.22 mg l−1ammonia-N for 7 days, phagocytic activity and clearance efficiency to V. alginolyticus significantly decreased. It is therefore suggested that ammonia in water caused a depression in the immune response and an increase in mortality of L. vannamei from the V. alginolyticus infection. [Copyright &y& Elsevier]

The white shrimp Litopenaeus vannamei (8.0–14.4 g) was examined for haemocyte count, phenoloxidase activity, respiratory burst (release of superoxide anion), phagocytic activity, and clearance efficiency to the pathogen Vibrio alginolyticus in relation with moult cycle (postmoult, A, B; intermoult, C; premoult, D0/D1D2/D3). Granular cells were the highest at C and D0/D1stage, and the lowest at A stage. Hyaline cells and THC (total haemocyte count) were higher at C stage, but lower at postmoult stages. Phenoloxidase activity was the highest at C stage, and the lowest at A stage. Respiratory burst was lower at A stage. Phagocytic activity of shrimps against V. alginolyticus decreased significantly at postmoult and premoult stages. Additionally, the clearance efficiency of shrimps to V. alginolyticus was significantly lower for shrimps at A stage than those at C stage. In another experiment, L. vannamei at different moult stages were injected with tryptic soy broth (TSB)-grown V. alginolyticus (1×105cfu shrimp−1) and then held in 34% seawater. After 10 h, the mortality of V. alginolyticus-injected shrimps was significantly higher for shrimps at postmoult stage than those at intermoult stage. Over 48–120 h, the mortality of V. alginolyticus-injected shrimps was 50.0%, 33.3% and 40.0% at postmoult, intermoult and premoult stage, respectively. It is concluded that L. vannamei showed a decrease in resistance at A stage through a reduction of its haemocyte count, phenoloxidase activity, respiratory burst, phagocytic activity and clearance efficiency against V. alginolyticus. [Copyright &y& Elsevier]

Giant freshwater prawns Macrobrachium rosenbergii (14–19 g) were challenged withEnterococcus (3×105 cfu prawn−1) previously incubated in TSB medium for 24 h, then placed in water having concentrations of dissolved oxygen (DO) at 7·75, 4·75, 2·75 and 1·75 mg l−1. Onset of mortality occurred after 6 h exposure to 1·75 mg l−1 DO, and after 12 h exposure to 2·75 mg l−1 DO. Cumulative mortality of prawns at 1·75 mg l−1 DO was significantly higher than that at 4·75 and 2·75 mg l−1 DO, and cumulative mortality of prawns at 4·75 and 2·75 mg l−1 DO was significantly higher than that at 7·75 mg l−1 DO after 96 h. The prawns (20–30 g) which had been placed in water for 0 to 120 h at 7·75, 4·75, 2·75 and 1·75 mg l−1 DO were examined for the THC (total haemocyte counts), DHC (differential haemocyte counts), phenoloxidase activity, respiratory burst, percentage phagocytosis and clearance efficiency. No significant difference in semi-granular cells and granular cells of prawns was observed among four treatments. The prawns following 120 h exposure to 2·75 mg l−1 DO decreased significantly the hyaline cells and THC by 39% and 36%, respectively. Phenoloxidase activity and respiratory burst decreased significantly by 33% and 11% when the prawns were exposed to 2·75 mg l−1 DO after 24 h, respectively. Percentage phagocytosis and clearance efficiency to Enterococcus decreased significantly by 44% and 54% for the prawns following 12 h exposure to 2·75 mg l−1 DO, respectively. It is concluded that DO as low as 2·75 mg l−1 and 4·75 mg l−1 causes depression in immune system ofM. rosenbergii , and increases its susceptibility to Enterococcus infection. [Copyright &y& Elsevier]

Peritrophins are peritrophic membrane (PM) proteins that can interact with chitin fibers via chitin-binding domains. Peritrophins have essential roles in providing porosity and strength to the PM that lines the shrimp midgut. Acute hepatopancreatic necrosis disease (AHPND), caused by strains of V. parahaemolyticus, is known to initially colonize the shrimp stomach and simultaneously disrupt its structural barriers (e.g., cuticle or epithelial tissues) to reach the hepatopancreas. Although stomach and hepatopancreas were identified as target tissues involved in AHPND pathogenesis, our results indicated that peritrophin in peritrophic membrane has a crucial role in determining not only colonization of AHPND-causing bacteria but also their tissue distribution. As the interaction between Lv Peritrophin (LvPT) and WSSV (white spot syndrome virus) is not well understood, we noted that LvPT expression was upregulated in shrimp stomach challenged with either WSSV or AHPND. In an i n vitro pathogen binding assay, there was strong binding of recombinant LvPT WSSV and AHPND-causing V. parahaemolyticus , and various bacteria. Furthermore, dsRNA-mediated LvPT silencing inhibited WSSV gene expression and viral genome replication. However, downregulation of LvPT gene expression increased copies of AHPND-causing bacteria in shrimp digestive tract, and facilitated bacterial colonization in stomach. In conclusion, we speculated that LvPT might regulate bacterial colonization during AHPND, whereas in WSSV infection, LvPT silencing favored the host. Although recombinant LvPT had strong binding with WSSV, the precise role of LvPT in WSSV infection needs further investigation. These findings increased our understanding of host-pathogen interactions in AHPND and WSSV infection that can be applied in shrimp aquaculture for developing effective antibacterial and antiviral strategies. • A Peritrophin with 3 chitin-binding domains was identified from Litopenaeus vannamei. • Recombinant LvPT had strong binding ability to WSSV, and AHPND-causing V. parahaemolyticus. • Silencing LvPT in L. vannamei reduced WSSV replication. • LvPT silencing increased colonization of AHPND-causing bacteria in shrimp digestive tract. [ABSTRACT FROM AUTHOR]

Cobia (Rachycentron canadum), a commercially important marine fish, has been used to develop a novel gill cell line, designated CG, for the first time. The CG cell line was cultured in Leibovitz's-15 medium with 5% fetal bovine serum (FBS) and successfully sub-cultured more than 110 passages. It underwent verification through sequencing of the mitochondrial cytochrome C oxidase subunit I (COI) gene. Optimal growth rate was achieved when the CG cell line was cultured in a medium supplemented with 5% FBS, 1% Penicillin-Streptomycin (P/S), and 5 parts per thousand (ppt) of coral sea salt water, maintained at a temperature of 27 °C. The addition of 5 ppt of salt in the growth medium suggests that this cell line could be a viable in vitro tool for marine ecosystem toxicological studies or for culturing marine parasitic microorganisms. The CG cell line was also successfully transfected using the pTurbo-GFP plasmids, showing an 18% efficiency, with observable GFP expression. Furthermore, the cell line has been effectively cryopreserved. Gene expression analysis indicated that the CG cell line exhibits responsive regulation of immune gene expression when exposured to various stimulants, highlighting its potential as an in vitro platform for immune response studies. This makes it suitable for exploring dynamic immune signaling pathways and host-pathogen interactions, thereby offering valuable insights for therapeutic development. • A novel cell line derived from cobia gill (CG) has been successfully established. • The origin of the CG cell line was authenticated, and it has been sub-cultured for more than 110 passages. • Optimal culturing conditions for the CG cell line have been verified. • Transfection efficiency of the CG cell line was examined. • The CG cell line performs phagocytosis and responds to various immunostimulants. [ABSTRACT FROM AUTHOR]

We investigate the following conjecture of Hehui Wu: for every tournament S , the class of S -free tournaments has bounded domination number. We show that the conjecture is false in general, but true when S is 2-colourable (that is, its vertex set can be partitioned into two transitive sets); the latter follows by a direct application of VC-dimension. Our goal is to go beyond this; we give a non-2-colourable tournament S that satisfies the conjecture. The key ingredient here (perhaps more interesting than the result itself) is that we overcome the unboundedness of the VC-dimension by showing that the set of shattered sets is sparse. [ABSTRACT FROM AUTHOR]

Red seaweeds have several biofunctional properties, including immunomodulatory, antitumor, antioxidant, and antibacterial activities. In this study, we examined the effects of diets containing Sarcodia suae on the immune response, immune-related gene expressions, and disease resistance against Vibrio alginolyticus in white shrimp Litopenaeus vannamei. In addition, 1H NMR metabolomics was applied to analyze the metabolites extracted from shrimp fed with S. suae and their functions in regulating immunity. A diet containing only fish meal was used as the control diet (S 0), and three diets containing different concentrations of S. suae powder, 2.5% (S2.5), 5% (S5), and 7.5% (S7.5) were used as experimental diets. Shrimp were fed diets for 20 days. Compared to the control group (S0), results showed that (1) shrimp fed diets supplemented with 5–7.5% of S. suae powder significantly increased anti -V. alginolyticus activity; (2) phagocytic activity (PA) increased in all shrimp fed with S. suae , but total haemocyte count (THC) only increased in S7.5 group; and (3) the expression of glutathione peroxidase (GPx) in haemocyte were significantly higher in S7.5 groups. Results from the 1H NMR analysis revealed that 19 heapatopancreatic metabolites were matched and identified among groups. Based on the KEGG enrichment analysis, the up-regulated metabolites in the shrimp fed S5 and S7.5 diets were primarily due to the metabolism of purine and phenylalanine and their respective pathways. Results from these trials reveal that diets containing S. suae can increase immune response, thereby increasing shrimp resistance to V. alginolyticus. The purine and phenylalanine metabolic pathways may be considered as the relevant pathways for optimizing immunomodulatory responses. • Shrimp's immunity was improved by dietary Sarcodia suae. • Dietary S. suae increased shrimp's disease resistance to Vibrio. • Shrimp's health was improved via purine and phenylalanine metabolic pathways. [ABSTRACT FROM AUTHOR]

The growth performance, immunological status, and intestinal microbiology of white shrimp, Litopenaeus vannamei, were evaluated after dietary administration of the commercial probiotic SYNSEA. Shrimp were fed a control diet (without probiotic supplement) and two levels of SYNSEA probiotic, a low concentration of SYNSEA (LSL) containing 105 CFU (g diet)−1 Bacillus subtilis and 105 CFU (g diet)−1 lactic acid bacteria (LAB), and a high concentration of SYNSEA (LSH) containing 106 CFU (g diet)−1 B. subtilis and 106 CFU (g diet)−1 LAB, for 12 weeks. Shrimp fed with the LSL diet significantly increased growth performance as well as final weight and feed efficiency compared to the control, but not the LSH diet. After being orally challenged with Vibrio parahaemolyticus , shrimp fed with LSL diet prior to the challenge or fed with LSL and pathogen simultaneously showed significantly lower mortality compared to the control. SYNSEA probiotic significantly improved shrimp immune response, including lysozyme activity in LSL and LSH groups, and phagocytic activity in the LSL group in comparison to the control. In addition, the gene expressions of anti-lipopolysaccharide factor 2 in LSL and LSH groups, and penaeidin 4 in LSL were also up-regulated. Although there was no significant difference among groups for hepatopancreas and intestinal morphology, the muscular layer thickness and villi height were slightly improved in the intestines of shrimp fed SYNSEA. The 16S rDNA gene amplicon sequence analysis using next-generation sequencing revealed a significant decrease in α-diversity (Margalef's species richness) after oral administration of SYNSEA due to an increase in the relative abundance of beneficial bacteria in the gut flora of shrimp, such as Lactobacillus, Shewanella, and Bradymonadales and a decrease in harmful bacteria, such as Vibrio , Candidatus_Berkiella , and Acinetobacter baumannii. Together the data suggest that the provision of SYNSEA probiotic at 105 CFU (g diet)−1 B. subtilis and 105 CFU (g diet)−1 LAB can improve shrimp growth, enhance immunity, and disease resistance status of the host. In addition, these findings conclude that SYNSEA probiotic has great preventive and therapeutic potential for Vibrio infection in shrimp aquaculture. • SYNSEA probiotic was able to improve white shrimp's growth. • SYNSEA probiotic improved disease resistance of shrimp via modulating immunity and altering gut microbiota. • SYNSEA probiotic contains 105 CFU (g diet)−1 B. subtilis and 105 CFU (g diet)−1 LAB was recommended. [ABSTRACT FROM AUTHOR]

The aim of this study was to increase the efficacy of probiotic Bacillus subtilis E20 by encapsulating the probiotic in alginate and coating it with chitosan. The protective effect was evaluated by firstly ensuring the viability of encapsulated probiotics in simulated gastrointestinal fluid (SGF) and simulated intestinal fluid (SIF) conditions and then at different storage temperatures. In addition, the encapsulated probiotic was incorporated into the diet to improve the growth performance and health status of white shrimp, Litopenaeus vannamei. B. subtilis E20 has the ability to survive in SGF when encapsulated in 1.5–2% alginate and coated with 0.4% chitosan. Furthermore, viability increased significantly in SIF compared to the probiotic encapsulated in 1% alginate and coated with 0.4% chitosan and the non-encapsulated probiotic. Longer storage time and adverse conditions affected probiotics' survival, which was improved by the encapsulation with significantly higher viability than the non-encapsulated probiotic at different temperatures and storage duration. Encapsulation of B. subtilis E20 and dietary administration at 107 CFU kg−1 decreased shrimp mortality after a Vibrio infection, thereby improving shrimp's disease resistance, while the non-encapsulated probiotic required 109 CFU kg−1 to achieve better resistance. Although the best results of growth performance, immune response, and disease resistance against Vibrio alginolyticus were found in the shrimp fed with the diets supplemented with encapsulated probiotic at >108 CFU kg−1, shrimp's growth performance and health status improved after being fed 107 CFU kg−1 encapsulated probiotic for 56 days. Together, the results of this study prove that encapsulation could improve the viability of probiotic in different gastrointestinal conditions and adverse storage temperatures. Overall, lower concentrations of encapsulated probiotic B. subtilis E20 (107 CFU kg−1) was able to increase the growth performance and health status of shrimp. • Probiotic efficacy of Bacillus subtilis E20 was improved by encapsulation. • Encapsulated probiotic had better resistance against adverts conditions of gastrointestinal tract and storage. • Encapsulated probiotic in diet at a level of 107 CFU kg−1 were able to improve the growth and health status of shrimp. [ABSTRACT FROM AUTHOR]

This study investigated the effects of fish protein hydrolysate derived from barramundi on growth performance, muscle composition, immune response, disease resistance, histology and gene expression in white shrimp (Penaeus vannamei). In vitro studies demonstrated FPH enhanced mRNA expressions of key immune-related genes and stimulated reactive oxygen species (ROS) production and phagocytic activity in shrimp hemocytes. To evaluate the effects of substituting fish meal with FPH in vivo , four isoproteic (43 %), isolipidic (6 %), and isoenergetic diets (489 kcal/100 g) were formulated with fish meal substitution levels of 0 % (control), 30 % (FPH30), 65 % (FPH65), and 100 % (FPH100). After 8-week feeding, the growth performance of FPH65 and FPH100 were significantly lower than that of control and FPH30 (p < 0.05). Similarly, the midgut histological examination revealed the wall thickness and villi height of FPH100 were significantly lower than those of control (p < 0.05). The shrimps were received the challenge of AHPND + Vibrio parahaemolyticus at week 4 and 8. All FPH-fed groups significantly enhanced resistance against Vibrio parahaemolyticus at week 4 (p < 0.05). However, this protective effect diminished after long-period feeding. No significant difference of survival rate was observed among all groups at week 8 (p > 0.05). The expressions of immune-related genes were analyzed at week 4 before and after challenge. In control group, V. parahaemolyticus significantly elevated SOD in hepatopancreas and Muc 19, trypsin , Midline-fas , and GPx in foregut (p < 0.05). Moreover, hepatopancreatic SOD of FPH65 and FPH100 were significantly higher than that of control before challenge (p < 0.05). Immune parameters were measured at week 8. Compared with control, the phagocytic index of FPH 30 was significantly higher (p < 0.05). However, dietary FPH did not alter ROS production, phenoloxidase activity, phagocytic rate, and total hemocyte count (p > 0.05). These findings suggest that FPH30 holds promise as a feed without adverse impacts on growth performance while enhancing the immunological response of white shrimp. • FPH derived from barramundi byproducts enhances the immune in of white shrimp. • Diets containing FPH were found to enhance shrimp protection against Vibrio parahaemolyticus by week 4, with no adverse effects observed at a 30 % substitution level. • The protective effect of FPH diminishes and returns to the basal level over an 8-week feeding period. [ABSTRACT FROM AUTHOR]

Abstract: Gynura bicolor (Roxb. & Willd.) DC is widely distributed in certain areas of Asia and is very popular in vegetarian cuisine in Taiwan. To investigate the regulatory roles of G. bicolor in various functions in crustaceans, we examined innate non-specific immune responses (including total hemocyte count (THC), phenoloxidase activity (PO), respiratory bursts (RBs), and superoxide dismutase (SOD) activity), physiological responses (including haemolymph glucose, lactate, and lipids), and gene expressions (including prophenoloxidase (proPO), lipopolysaccharide- and b-1,3-glucan-binding protein (LGBP), and peroxinectin (PE) mRNA transcripts) to the pathogen Vibrio alginolyticus in white shrimp (Litopenaeus vannamei) that were individually injected with the water extract from G. bicolor at 2, 4, and 8 μg g−1. Results indicated that PO, RBs, SOD activity, proPO, LGBP, and PE mRNA transcripts of shrimps receiving the water extract of G. bicolor at 2, 4, and 8 μg g−1 significantly increased after challenge with V. alginolyticus for 96 h. However, no significant difference in the THC was seen at any dose. L. vannamei injected with the water extract of G. bicolor at all doses respectively maintained lower glucose, lactate, and lipid levels in response to V. alginolyticus challenge at 12–36, 24–36, and 24–48 h. Survival rates at 24–72 h of L. vannamei that received G. bicolor at any dose was significantly higher than those of shrimp that received saline. It was concluded that the water extract of G. bicolor can maintain physiological homeostasis and enhance immunity against V. alginolyticus infection in L. vannamei. [Copyright &y& Elsevier]

Abstract: Prophenoloxidase (proPO) is a melanin-synthesising enzyme that plays important roles in immune responses by crustaceans. Previously, we cloned and characterized proPO-I from white shrimp, Litopenaeus vannamei. In the present study, a novel prophenoloxidase-II (proPO-II) cDNA was also cloned from haemocytes of L. vannamei using oligonucleotide primers and reverse-transcriptase polymerase chain reaction (RT-PCR). Both 3′- and 5′-regions were isolated by the rapid amplification of complementary (c)DNA end (RACE) method. The 2504-bp cDNA contained an open reading frame (ORF) of 2073 bp, an 84-bp 5′-untranslated region, and a 347-bp 3′-untranslated region containing the poly A tail. The molecular mass of the deduced amino acid sequence (691 amino acids) was 78.8 kDa with an estimated pI of 6.07. It contains two putative tyrosinase copper-binding motifs and a conserved C-terminal region common to all known proPOs. Comparisons of the amino acid sequences showed that white shrimp proPO-II is more closely related to the proPO of other penaeids than to that of crayfish, lobsters, crab, or a freshwater prawn, and is the ancestor type of known penaeid proPOs. proPO-I and proPO-II messenger (m)RNAs of shrimp were located on different loci, and were constitutively expressed mainly in haemocytes. The transcriptional regulation of these two proPOs in shrimp at different molt stages, those administered dietary sodium alginate, and those challenged with Vibrio alginolyticus were surveyed. The results showed that the proPOs may be directly involved in the acute-phase immune defence, and proPO-II may contribute earlier to immune defence in shrimp injected with V. alginolyticus, and it may be regulated by ecdysone. However, a similar effect was found by stimulating proPO-I and proPO-II mRNA expression in shrimp fed a sodium alginate-containing diet. Results of this study provide a basis for developing a comprehensive understanding of expression/function relationships of individual proPOs in shrimp. [Copyright &y& Elsevier]

Abstract: The total haemocyte counts, phenoloxidase (PO) activity, respiratory bursts, superoxide dismutase (SOD) activity, and phagocytic activity and clearance efficiency to Vibrio alginolyticus, as well as prophenoloxidase (proPO), lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP), serine protein (SP), and peroxinectin (PE) mRNA transcription of L. vannamei, and its susceptibility to V. alginolyticus when the shrimp were fed diets containing Lactobacillus plantarum at 0 (control), 107, and 1010 cfu(kg diet) −1 for 48 and 168h were evaluated. The results indicated that PO activity, SOD activity, clearance efficiency to V. alginolyticus, proPO and PE mRNA transcription, and the survival rate after challenge with V. alginolyticus all significantly increased, but the total haemocyte counts significantly decreased in shrimp fed a diet containing Lac. plantarum at 1010 cfu(kg diet)−1 for 168h. However, no significant differences in phagocytosis, LGBP, or SP mRNA expression of shrimp were observed among the different treatments. It was concluded that administration of Lac. plantarum in the diet at 1010 cfu(kg diet)−1 induced immune modulation and enhanced the immune ability of L. vannamei, and increased its resistance to V. alginolyticus infection. [Copyright &y& Elsevier]

Abstract: A copper/zinc superoxide dismutase (Cu,Zn-SOD) cDNA was cloned from the hepatopancreas of giant freshwater prawn Macrobrachium rosenbergii using reverse transcription–polymerase chain reaction (RT–PCR) by degenerate primers. Both 3′- and 5′-regions were isolated by the rapid amplification of cDNA ends method. Analysis of nucleotide sequence revealed that the Cu,Zn-SOD cDNA clone consists of 845bp with an open reading frame of 603bp encoding a protein of 201 amino acids with a 22 amino acid signal peptide. The calculated molecular mass of the mature proteins (179 amino acids) is 21kDa with an estimated pI of 4.75. Two putative N-glycosylation sites, NXT and NXS, were observed in the Cu,Zn-SOD. Four conserved amino acids responsible for binding copper (H86, H89, H106 and H163) and four conserved amino acids responsible for binding zinc (H106, H114, H123 and D126) were observed. Sequence comparison showed that the Cu,Zn-SOD deduced amino acid sequence of M. rosenbergii has similarity of 60% and 64% to that of freshwater crayfish Pacifastacus leniusculus ecCu,Zn-SOD and blue crab Callinectes sapidus ecCu,Zn-SOD, respectively. Quantitative real-time RT–PCR analysis showed that Cu,Zn-SOD transcripts in haemocytes of M. rosenbergii increased 3h and 6h after injection of Lactococcus garvieae, whereas Cu,Zn-SOD transcripts decreased in the hepatopancreas 3h after L. garvieae injection. [Copyright &y& Elsevier]

Abstract: The total haemocyte count (THC), phenoloxidase activity, respiratory bursts (release of superoxide anions), superoxide dismutase (SOD) activity, and phagocytic activity and clearance efficiency to the pathogen Lactococcus garvieae were measured when freshwater giant prawns Macrobrachium rosenbergii (18.2±2.1g) were individually reared in water containing concentrations of trichlorfon of 0, 0.2, or 0.4mgL−1 for a 144-h period. No significant differences in the THC were observed among prawns at the beginning and following 144h of exposure to 0, 0.2, and 0.4mgL−1 trichlorfon. However, phenoloxidase activity significantly decreased when the prawns were exposed to trichlorfon at 0.2 and 0.4mgL−1, and no significant differences were observed between the two concentrations at any sampling time. The total production of superoxide anion by M. rosenbergii significantly increased with exposure to 0.2 and 0.4mgL−1 trichlorfon, and no significant differences were observed between the two concentrations during the 144-h exposure period. However, M. rosenbergii exposed to 0.2 and 0.4mgL−1 trichlorfon showed decreased SOD activity from 48 to 144h. Phagocytic activity and clearance efficiency to L. garvieae significantly decreased when prawns were exposed to 0.2 and 0.4mgL−1 trichlorfon for 48h. In another experiment, prawns were challenged with 5×105 colony-forming units (cfu)prawn−1 of L. garvieae, then reared in water containing different concentrations of trichlorfon. The onset of mortality was earlier in prawns exposed to trichlorfon compared to those exposed to the zero control. The cumulative mortality of prawns directly increased with ambient trichlorfon concentrations in the range of 0–0.4mgL−1 after 168h. It is concluded that exposure of M. rosenbergii to trichlorfon at 0.2mgL−1 or more caused cytotoxicity resulting in depression of the immune response, and increased its susceptibility to L. garvieae infection. [Copyright &y& Elsevier]

Abstract: A cytosolic manganese superoxide dismutase (cytMn-SOD) cDNA was cloned from the hepatopancreas of giant freshwater prawn Macrobrachium rosenbergii using reverse transcription polymerase chain reaction (RT-PCR) by degenerate primers. Both 3′- and 5′-regions were isolated by rapid amplification of cDNA end RACE method. Analysis of nucleotide sequence revealed that the cytMn-SOD cDNA clone consists of 1339bp with an open reading frame of 858bp encoding a protein of 286 amino acids. The calculated molecular mass of the mature proteins (286 amino acids) is 31kDa with an estimated pI of 5.52. Two putative N-glycosylation sites, NXT and NXS were observed in the cytMn-SOD. Four conserved amino acids responsible for binding manganese were observed (H110, H158, D243 and H247). Sequence comparison showed that the cytMn-SOD deduced amino acid sequence of M. rosenbergii has an overall similarity of 77% and 54% to that of blue crab Callinectes sapidus and tiger shrimp Penaeus monodon, respectively. Quantitative real-time RT-PCR analysis showed that cytMn-SOD transcript in hepatopancreas of M. rosenbergii decreased 3h after Lactococcus garvieae injection, but no significant change in cytMn-SOD transcript was observed in the haemocytes 3–24h after L. garvieae injection. [Copyright &y& Elsevier]

This study aimed to evaluate the effects of a synbiotic composite an extract from a by-product of king oyster mushroom, Pleurotus eryngii (KOME), and probiotic Lactobacillus plantarum 7–40 on the growth performance and health status of white shrimp, Litopenaeus vannamei. The KOME was able to stimulate the growth of probiotic, but not the growth of Vibrio pathogens, including V. alginolyticus , V. parahaemolyticus , and V. harveyi. Four diets were formulated, including a control diet supplemented without prebiotic and probiotic, a basal diet supplemented with KOME (5 g kg−1) (ME), a basal diet supplemented with probiotic (1 × 108 CFU kg−1) (LP), and a basal diet supplemented with KOME (5 g kg−1) and probiotic (1 × 108 CFU kg−1) (SYN). Shrimp fed the ME, LP, and SYN diets had significantly higher survival than that of shrimp fed with the control diet for 8 weeks. Shrimp in the SYN group also had a significantly higher weight gain and total final weight in comparison with the control and other treatments. In the intestinal tract, lactic acid bacteria count was significantly higher in the SYN group, whereas the Vibrio -like bacteria count was significantly higher in the ME group than in the control group. For the health status assessment, the disease resistance of shrimp against V. alginolyticus was improved in all treatments compared to the shrimp in control. Shrimps in the SYN group had significantly lower cumulative mortality due to the significant increase in immune responses, including phenoloxidase, respiratory burst, and lysozyme activity, and the gene expression of pexn and pen4 in the haemocytes, and lgbp , sp , propoii , pexn , pen3a , pen4 , and gpx in the haepatopancreas of shrimp as compared to the control. Therefore, it is suggested that a combination of KOME and probiotics can be used as a synbiotic to improve the growth performance and reduce the risk of infectious diseases caused by Vibrio and at the same time significantly contribute to the circular economy. • King oyster mushroom extract (KOME) was able to support the growth of probiotic, Lactobacillus plantarum rather than pathogenic Vbrio. • Synbiotic was able to increase the growth performance of shrimp. • Synbiotic was able to improve the immunity and disease resistance of shrimp. [ABSTRACT FROM AUTHOR]

Daphnia similis chitin and its derivative chitosan were prepared as immunostimulants to boost the immune response and determine the ability to control infectious disease caused by Vibrio alginolyticus in white shrimp, Litopenaeus vannamei. Three experimental diets supplemented with 0% chitin or chitosan (control) and 0.4% chitin or 0.4% chitosan were fed to shrimp for 56 days. Dietary inclusion of 0.4% chitosan accelerated shrimp growth compared to chitin and control. The survival and disease resistance of shrimp increased significantly when fed chitin and chitosan diets, after pathogenic injection, as indicated by the up-regulated immune responses in respiratory burst (RB), superoxide dismutase (SOD), and phagocytic activity (PA). There were no significant differences in the total haemocyte count (THC), phenoloxidase (PO)activity, and lysozyme (LYZ) activity among the groups. No significant differences were observed for prophenoloxidase system-related gene expressions among groups. However, shrimp fed chitin, and chitosan expressed significantly higher levels of antimicrobial proteins (penaeidin 3a, crustin, and anti-lipopolysaccharide factor 2) in the haemocytes than in control. The gene expressions of catalase and heat shock protein 70 increased in the hepatopancreas of shrimp fed chitosan diet compared to the chitin and control diet. The O -linked N -acetylglucosamine transferase (ogt) was significantly higher in the haemocytes of shrimp fed chitosan and chitin than the control, but ogt was only significantly higher in the hepatopancreas of shrimp fed chitosan. Dietary chitin and chitosan also showed positive effects on the transcription of peritrophin-like protein. These findings suggest that both chitin and chitosan from D. similis are efficacious at boosting the immunity of shrimp by preventing and controlling infectious diseases caused by Vibrio and have great potential to be used as a feasible immunostimulant that significantly contributes to the circular economy. • Chitin and chitosan derived from D. similis were able to improve the growth and immunity of white shrimp. • Chitin and chitosan derived from D. similis were able to increase the disease resistance of white shrimp. • 0.4% chitin and 0.4% chitosan in diets are recommended for the disease prevention of white shrimp. [ABSTRACT FROM AUTHOR]