Ivan Jakovlić

Molecular cloning and expression of toll-like receptor 4 (tlr4) in the blunt snout bream (Megalobrama amblycephala)

Toll-like receptors (TLRs) play a pivotal role in teleost innate immune system. In this study, Megalobrama amblycephala (ma) tlr4 gene was cloned, its putative polypeptide product characterized, and expression analysed. Matlr4 cDNA is 2862 bp long, with an open reading frame of 2364 bp encoding 787 amino acids. MaTlr4 is a typical TLR protein, including the extracellular part with nine leucine-rich repeat motifs, a transmembrane region and a cytoplasmic Toll/interleukin-1 receptor domain. MaTlr4 has the highest level of identity (94%) and similarity (97%) with the grass carp Tlr4.2 homolog. This was also corroborated by the phylogenetic analysis, which placed MaTlr4 in a cluster with other cyprinid homologs. Matlr4 mRNA was ubiquitously expressed in all examined tissues and during all sampled developmental stages. The observed peak in matlr4 mRNA expression during gastrula and somite stages is in good agreement with its proposed role in the development of the neural system. Temporal expression patterns of matlr4 and maMyD88 mRNAs and proteins were analyzed in liver, spleen, head kidney, trunk kidney and intestine after Aeromonas hydrophila infection. And mRNA expression varied between different time-points. Both MaTlr4 and MaMyD88 protein expressions at 12 hpi were significantly enhanced in head kidney and intestine. These results indicate that matlr4 is involved in the immune response in M. amblycephala, and that it is indeed a functional homologue of tlr4s described in other animal species.

Blunt Snout Bream (Megalobrama amblycephala) MyD88 and TRAF6: Characterisation, Comparative Homology Modelling and Expression

MyD88 and TRAF6 play an essential role in the innate immune response in most animals. This study reports the full-length MaMyD88 and MaTRAF6 genes identified from the blunt snout bream (Megalobrama amblycephala) transcriptome profile. MaMyD88 is 2501 base pairs (bp) long, encoding a putative protein of 284 amino acids (aa), including the N-terminal DEATH domain of 78 aa and the C-terminal TIR domain of 138 aa. MaTRAF6 is 2252 bp long, encoding a putative protein of 542 aa, including the N-terminal low-complexity region, RING domain (40 aa), a coiled-coil region (64 aa) and C-terminal MATH domain (147 aa). Coding regions of MaMyD88 and MaTRAF6 genomic sequences consisted of five and six exons, respectively. Physicochemical and functional characteristics of the proteins were analysed. Alpha helices were dominant in the secondary structure of the proteins. Homology models of the MaMyD88 and MaTRAF6 domains were constructed applying the comparative modelling method. RT-qPCR was used to analyse the expression of MaMyD88 and MaTRAF6 mRNA transcripts in response to Aeromonas hydrophila challenge. Both genes were highly upregulated in the liver, spleen and kidney during the first 24 h after the challenge. While MyD88 and TRAF6 have been reported in various aquatic species, this is the first report and characterisation of these genes in blunt snout bream. This research also provides evidence of the important roles of these two genes in the blunt snout bream innate immune system.

Succession and Fermentation Products of Grass Carp (Ctenopharyngodon idellus) Hindgut Microbiota in Response to an Extreme Dietary Shift

Dietary intake affects the structure and function of microbes in host intestine. However, the succession of gut microbiota in response to changes in macronutrient levels during a long period of time remains insufficiently studied. Here, we determined the succession and metabolic products of intestinal microbiota in grass carp (Ctenopharyngodon idellus) undergoing an abrupt and extreme diet change, from fish meal to Sudan grass (Sorghum sudanense). Grass carp hindgut microbiota responded rapidly to the diet shift, reaching a new equilibrium approximately within 11 days. In comparison to animal-diet samples, Bacteroides, Lachnospiraceae and Erysipelotrichaceae increased significantly while Cetobacterium decreased significantly in plant-diet samples. Cetobacterium was negatively correlated with Bacteroides, Lachnospiraceae and Erysipelotrichaceae, while Bacteroides was positively correlated with Lachnospiraceae. Predicted glycoside hydrolase and polysaccharide lyase genes in Bacteroides and Lachnospiraceae from the Carbohydrate-Active enZymes (CAZy) database might be involved in degradation of the plant cell wall polysaccharides. However, none of these enzymes was detected in the grass carp genome searched against dbCAN database. Additionally, a significant decrease of short chain fatty acids levels in plant-based samples was observed. Generally, our results suggest a rapid adaption of grass carp intestinal microbiota to dietary shift, and that microbiota are likely to play an indispensable role in nutrient turnover and fermentation.

Expression and functional characterization of interferon regulatory factors (irf2, irf7 and irf9) in the blunt snout bream (Megalobrama amblycephala)

ABSTRACT Interferon regulatory factors (irfs) are a family of genes that encode transcription factors with important roles in regulating the expression of Type I interferons (IFNs) and other genes associated with related pathways. irfs have multitudinous functions in growth, development and regulation of oncogenesis. In this study, three irf family members (irf2, irf7, irf9) were identified and characterized in Megalobrama amblycephala at the mRNA and amino acid levels. M. amblycephala irfs share a high sequence homology with other vertebrate irfs. Constitutive expression levels of the three genes were detected (using qPCR) in all studied tissues: low to medium in kidney, gills, heart and muscle, and high in liver, spleen, intestine and blood. qPCR was also used to analyze the dynamic expression patterns of irfs in different embryonic development stages: irf2 is not activated during the embryonic development, whereas irf9 appears to play important roles around hatching and during the larval development. Transcripts of all three studied irfs were upregulated after stimulation by Aeromonas hydrophila bacterium in liver, spleen, head kidney and trunk kidney, whereas downregulation was observed in intestine and gills. The results show that these three irfs are likely to be important factors in the blunt snout bream immune system. They also provide a foundation for studying the origin and evolution of the innate immune system in the blunt snout bream. HighlightsIrf2, irf7 and irf9 cDNA were cloned from Megalobrama amblycephala.Mairfs share significant structural homology with the known irfs homologs.Mairfs mRNA are constitutively expressed in a wide range of tissues in vivo.The expression patterns of irfs in different embryonic development stage were different.Aeromonas hydrophila infection alters mairfs mRNA expression.

Sequencing of the complete mitochondrial genomes of eight freshwater snail species exposes pervasive paraphyly within the Viviparidae family (Caenogastropoda)

Phylogenetic relationships among snails (Caenogastropoda) are still unresolved, and many taxonomic categories remain non-monophyletic. Paraphyly has been reported within a large family of freshwater snails, Viviparidae, where the taxonomic status of several species remains questionable. As many endemic Chinese viviparid species have become endangered during the last few decades, this presents a major obstacle for conservation efforts. Mitochondrial genomes (mitogenomes) carry a large amount of data, so they can often provide a much higher resolution for phylogenetic analyses in comparison to the traditionally used molecular markers. To help resolve their phylogenetic relationships, the complete mitogenomes of eight Chinese viviparid snails, Viviparus chui, Cipangopaludina chinensis, C. ussuriensis, C. dianchiensis (endangered), Margarya melanioides (endangered), M. monodi (critically endangered), Bellamya quadrata and B. aeruginosa, were sequenced and compared to almost all of the available caenogastropod mitogenomes. Viviparidae possess the largest mitogenomes (16 392 to 18 544 bp), exhibit the highest A+T bias (72.5% on average), and some exhibit unique gene orders (a rearrangement of the standard MYCWQGE box), among the Caenogastropoda. Apart from the Vermetidae family and Cerithioidea superfamily, which possessed unique gene orders, the remaining studied caenogastropod mitogenomes exhibited highly conserved gene order, with minimal variations. Maximum likelihood and Bayesian inference analyses, used to reconstruct the phylogenetic relationships among 49 almost complete (all 37 genes) caenogastropod mitogenomes, produced almost identical tree topologies. Viviparidae were divided into three clades: a) Margarya and Cipangopaludina (except C. ussuriensis), b) Bellamya and C. ussuriensis, c) Viviparus chui. Our results present evidence that some Cipangopaludina species (dianchiensis and cathayensis) should be renamed into the senior genus Margarya. The phylogenetic resolution obtained in this study is insufficient to fully resolve the relationships within the ‘b’ clade, but if C. chinensis proves to be a valid representative of the genus, C. ussuriensis may have to be reassigned a different genus (possibly Bellamya, or even a new genus). Non-monophyly also remains pervasive among the higher (above the family-level) Caenogastropod taxonomic classes. Gene order distance matrix produced a different phylogenetic signal from the nucleotide sequences, which indicates a limited usability of this approach for inferring caenogastropod phylogenies. As phenotypic homoplasy appears to be widespread among some viviparid genera, in order to effectively protect the rapidly diminishing endemic Viviparid populations in China, further detailed molecular phylogenetic studies are urgently needed to resolve the taxonomic status of several species.

Molecular characterization and immunological response analysis of toll-like receptors from the blunt snout bream (Megalobrama amblycephala)

ABSTRACT The innate immunity serves as the primary line of defense against pathogen invasion. Toll‐like receptors (TLRs) play a pivotal role in the innate immunity by sensing specific pathogen‐associated molecular patterns and activating immune responses. In this study, 14 TLRs (TLR1, 2, 3, 4, 5, 7, 8a, 8b, 9, 18, 19, 20, 21 and 22) were identified and characterized in a cyprinid fish Megalobrama amblycephala. A majority of MaTLRs possessed the typical structural features of the TLR protein family: LRR domain, TM region and TIR domain. Phylogenetic analyses confirmed the existence of six TLR families and revealed close relationships with other cyprinid orthologs. All TLRs were constitutively expressed in all eight examined tissues. After Aeromonas hydrophila challenge, the expression profiles of MaTLR mRNAs were analyzed in liver, spleen and head kidney: MaTLR1, 2, 5, 9, 21 and 22 were up‐regulated, MaTLR3, 7, 8a, 8b and 19 were down‐regulated, and MaTLR4, 18 and 20 expression patterns varied among tissues. These results indicate that most of MaTLRs are likely to be involved in the immune responses against bacterial infection. Highlights14 TLR genes were characterized and analyzed in Megalobrama amblycephala.Phylogenetic tree of MaTLRs obviously showed the existence of six TLR families.All MaTLRs were constitutively expressed with variations in eight tested tissues.The expression profiles of MaTLRs were analyzed after A. hydrophila infection.Results indicated that most of MaTLRs might involve in the immune responses.

Expression of Hox paralog group 13 genes in adult and developing Megalobrama amblycephala

Hox genes encode transcription factors that play a key role in specifying the body plan in most metazoans. HoxPG13 genes most probably played an important role in body length variation during the evolution of animals. This is the first report of the mRNA expression patterns of the entire Hox paralog group 13 (HoxA13a, HoxA13b, HoxB13a, HoxC13a, HoxC13b, HoxD13a) in fish. Expression was studied by qPCR in five tissues of adult Megalobrama amblycephala specimens (spleen, liver, kidney, intestine and gills) and during development (17 stages: fertilised egg to 90 days-old juveniles). Expression in tissues (for all six genes) was generally very low in gills (0.0006-0.05), spleen (0.006-0.09) and kidney (0.02-0.39); the highest in intestine (from 2.28 for HoxC13b to 244.29 for HoxC13a). During the development, a peak in expression around the hatching was observed for all six genes. Results suggest a high maternal expression of HoxA13a, and low for HoxA13ab. HoxD13a paralog exhibited the lowest expression: 0.0006-2.63 in tissues and 0.0005-1.7 during development, suggesting the possibility of a gradual loss of functionality. Expression of HoxC13 paralogs corroborates the findings in zebrafish: HoxC13b is maternally expressed and more important during the development. In adults, it was the opposite: expression was low for HoxC13b and very variable for HoxC13a (0.06-244.29). Differences in expression levels between both pairs of paralogs (Aa/Ab and Ca/Cb) indicate the possibility of the existence of some redundancy afforded by maintaining both paralogs.

In silico characterisation, homology modelling and structure-based functional annotation of blunt snout bream (Megalobrama amblycephala) Hsp70 and Hsc70 proteins

BackgroundHeat shock proteins play an important role in protection from stress stimuli and metabolic insults in almost all organisms.MethodsIn this study, computational tools were used to deeply analyse the physicochemical characteristics and, using homology modelling, reliably predict the tertiary structure of the blunt snout bream (Ma-) Hsp70 and Hsc70 proteins. Derived three-dimensional models were then used to predict the function of the proteins.ResultsPreviously published predictions regarding the protein length, molecular weight, theoretical isoelectric point and total number of positive and negative residues were corroborated. Among the new findings are: the extinction coefficient (33725/33350 and 35090/34840 - Ma-Hsp70/ Ma-Hsc70, respectively), instability index (33.68/35.56 – both stable), aliphatic index (83.44/80.23 – both very stable), half-life estimates (both relatively stable), grand average of hydropathicity (−0.431/-0.473 – both hydrophilic) and amino acid composition (alanine-lysine-glycine/glycine-lysine-aspartic acid were the most abundant, no disulphide bonds, the N-terminal of both proteins was methionine). Homology modelling was performed by SWISS-MODEL program and the proposed model was evaluated as highly reliable based on PROCHECK’s Ramachandran plot, ERRAT, PROVE, Verify 3D, ProQ and ProSA analyses.ConclusionsThe research revealed a high structural similarity to Hsp70 and Hsc70 proteins from several taxonomically distant animal species, corroborating a remarkably high level of evolutionary conservation among the members of this protein family. Functional annotation based on structural similarity provides a reliable additional indirect evidence for a high level of functional conservation of these two genes/proteins in blunt snout bream, but it is not sensitive enough to functionally distinguish the two isoforms.

Characterization and expression of Megalobrama amblycephala toll‐like receptor 22 involved in the response to Aeromonas hydrophila

The toll-like receptors (TLR) tlr22 was identified and characterized for the first time in one of the economically most important freshwater fish species in China, Megalobrama amblycephala. The full-length cDNA (4039 bp) of M. amblycephala tlr22 contains an open reading frame of 2706 bp, encoding a 901 amino-acid long polypeptide. The putative polypeptide contains 16 leucine-rich repeat (LRR) motifs, an LRR C-terminal, a transmembrane region and a cytoplasmic toll-interleukin-1 receptor (TIR) domain. Phylogenetic analyses revealed that M. amblycephala Tlr22 shared the closest relationship with a grass carp ortholog. tlr22 was constitutively expressed in nine tissues and during 10 developmental stages studied, albeit with varying expression levels. Along with many pathological changes observed after Aeromonas hydrophila bacterium infection, tlr22 and myd88 mRNA were significantly upregulated in blood, head kidney, spleen and intestine, indicating that tlr22 is involved in the immune response. These results provide an insight into tlr22 regulation mechanisms in the innate immune response to bacterial infection.

Dietary Bile Salt Types Influence the Composition of Biliary Bile Acids and Gut Microbiota in Grass Carp

Lipid metabolism can influence host’s health. There is increasing evidence for interplay between two key regulating factors in lipid metabolism: bile acids (BAs) and gut microbiota. However, very little is known about how types of different diet-supplemented bile salts (BS) influence this interaction in vivo. We sought to explore these relationships using grass carp (Ctenopharyngodon idellus), which often suffers functional disorder of liver and gallbladder. We studied fluctuations of BAs in the gall and changes of microbial communities in the gut in response to seven different diets: five different BS, chelating BS agent, and control. The BS comprised two primary BS [sodium taurochololate (TCAS) and sodium taurochenodeoxycholate (TCDCAS)], sodium tauroursodeoxycholate (TUDCAS), and two secondary BS [sodium taurodeoxycholate (TDCAS) and sodium taurolithocholate (TLCAS)]. Supplementation of primary BS caused a more significant fluctuation of biliary BAs than secondary BS, and TCAS caused a more prominent increase than TCDCAS and TUDCAS. For the gut microbiota, primary BS tended to increase their diversity and induce community succession, secondary BS resulted in a higher firmicutes/bacteroidetes ratio, while TUDCAS had no significant effects. Changes of the gut microbiota triggered by different types of BS caused alteration in BAs biotransformation. Two-obesity-associated families, Lachnospiraceae and Ruminococcaceae were positively correlated with biliary cholic acid (CA), taurochenodeoxycholic acid (TCDCA), and deoxycholic acid (DCA). As both primary and secondary BS resulted in increased synthesis of toxic secondary Bas by the gut microbiota, future studies should pay closer attention to gut microbiota when considering BA treatment.