Differential expression profiling of components associated with exoskeletal hardening in crustaceans

Background: Exoskeletal hardening in crustaceans can be attributed to mineralization and sclerotization of the organic matrix. Glycoproteins have been implicated in the calcification process of many matrices. Sclerotization, on the other hand, is catalysed by phenoloxidases, which also play a role in melanization and the immunological response in arthropods. Custom cDNA microarrays from Portunus pelagicus were used to identify genes possibly associated with the activation pathways involved in these processes. Results: Two genes potentially involved in the recognition of glycosylation, the C-type lectin receptor and the mannose-binding protein, were found to display molt cycle-related differential expression profiles. C-type lectin receptor up-regulation was found to coincide with periods associated with new uncalcified cuticle formation, while the up-regulation of mannose-binding protein occurred only in the post-molt stage, during which calcification takes place, implicating both in the regulation of calcification. Genes presumed to be involved in the phenoloxidase activation pathway that facilitates sclerotization also displayed molt cycle-related differential expression profiles. Members of the serine protease superfamily, trypsin-like and chymotrypsin-like, were up-regulated in the intermolt stage when compared to post-molt, while trypsin-like was also up-regulated in pre-molt compared to ecdysis. Additionally, up-regulation in pre- and intermolt stages was observed by transcripts encoding other phenoloxidase activators including the putative antibacterial protein carcinin-like, and clotting protein precursor-like. Furthermore, hemocyanin, itself with phenoloxidase activity, displayed an identical expression pattern to that of the phenoloxidase activators, i.e. up-regulation in pre- and intermolt. Conclusion: Cuticle hardening in crustaceans is a complex process that is precisely timed to occur in the post-molt stage of the molt cycle. We have identified differential expression patterns of several genes that are believed to be involved in biomineralization and sclerotization and propose possible regulatory mechanisms for these processes based on their expression profiles, such as the potential involvement of C-type lectin receptors and mannose binding protein in the regulation of calcification.

Screening of new lines of cereal grains for inclusion in Near Infra Red Scan calibrations

The project will provide enough data for a reliable and robust NIRs. It will more fully develop the in vitro method to enable less costly assessment of grains in the future. It will also provide a reliable assessment for DE which is the most expensive component of pig feed.

A Differential typing of Campylobacter

Establish a DNA-based typing scheme that allows the allocation of strains of Campylobacter to types that are host specific and/or non-host specific.

Effect of monensin inclusion in supplements for cattle consuming low quality tropical forage

A pen feeding study was carried out over 70 days to determine the effects of monensin (M) inclusion in two commercial supplements designed to provide different planes of nutrition to recently weaned steers. Thirty Bos indicus crossbred steers (191.4 +/- s.d. 7.1 kg) were individually fed a low quality pangola grass hay (57 g crude protein/kg DM; 497 g/kg DM digestibility) ad libitum (Control) with either a urea/molasses-based supplement of Rumevite Maxi-graze 60 Block (B), fed at 100 g/day, or grain-based Rumevite Weaner Pellets (WP), fed at 7.5 g/kg liveweight (W).day, both with and without M, viz. B, B+M, WP and WP+M, respectively. There were no significant interactions between supplement type and M inclusion for any measurement. Growth rates (main effects) averaged 0.17, 0.35 and 0.58 kg/day for the Control, B and WP supplements, respectively, with all means different (P < 0.05), while the response (P < 0.05) to M across supplement type was 0.11 kg/day. Hay DM intake was similar for the Control and B treatments (18.6 and 19.6 g/kg W.day) but was reduced (P < 0.05) with the WP supplement (16.8 g/kg W.day) while corresponding total DM intakes increased from 18.6 to 20.0 to 23.5 g/kg W.day (all differences P < 0.05), respectively. Monensin inclusion in the supplements did not affect supplement, hay or total DM intake. Inclusion of of M in supplements for grazing weaners in northern Australia may increase survival rates although the effect of M with cattle at liveweight maintenance or below requires further investigation.

Differential recognition by tick-resistant cattle of the recombinantly expressed Rhipicephalus microplus serine protease inhibitor-3 (RMS-3)

Rhipicephalus micro plus is an important bovine ectoparasite, widely distributed in tropical and subtropical regions of the world causing large economic losses to the cattle industry. Its success as an ectoparasite is associated with its capacity to disarm the antihemostatic and anti-inflammatory reactions of the host. Serpins are protease inhibitors with an important role in the modulation of host-parasite interactions. The cDNA that encodes for a R. microplus serpin was isolated by RACE and subsequently cloned into the pPICZ alpha A vector. Sequence analysis of the cDNA and predicted amino acid showed that this cDNA has a conserved serpin domain. B- and T-cell epitopes were predicted using bioinformatics tools. The recombinant R. microplus serpin (rRMS-3) was secreted into the culture media of Pichia pastoris after methanol induction at 0.2 mg l(-1) qRT-PCR expression analysis of tissues and life cycle stages demonstrated that RMS-3 was mainly expressed in the salivary glands of female adult ticks. Immunological recognition of the rRMS-3 and predicted B-cell epitopes was tested using tick-resistant and susceptible cattle sera. Only sera from tick-resistant bovines recognized the B-cell epitope AHYNPPPPIEFT (Seq7). The recombinant RMS-3 was expressed in P. pastoris, and ELISA screening also showed higher recognition by tick-resistant bovine sera. The results obtained suggest that RMS-3 is highly and specifically secreted into the bite site of R. microplus feeding on tick-resistant bovines. Capillary feeding of semi-engorged ticks with anti-AHYNPPPPIEFT sheep sera led to an 81.16% reduction in the reproduction capacity of R. microplus. Therefore, it is possible to conclude that R. microplus serpin (RMS-3) has an important role in the host-parasite interaction to overcome the immune responses in resistant cattle. (C) 2012 Elsevier GmbH. All rights reserved.

Validation of a new spot form of net blotch differential set and evidence for hybridisation between the spot and net forms of net blotch in Australia

A recently developed spot form of blotch differential set of 16 barley lines was tested for reaction response to 60 Pyrenophora teres f. maculata isolates from geographically disperse barley crops of Australia. Twelve barley lines (Arimont, Barque, Chebec, CI5286, CI5791, CI9214, CII6150, Dairokkaku, Esperance Orge 289, Galleon, Keel, Skiff, Torrens and TR250) provided differential response between the isolates. The susceptible controls Gairdner and Kombar provided indication of isolate virulence or avirulence. Abundant pathogenic diversity was revealed with 33 designated pathotypes, some of which related to geographic region. AFLP analysis also revealed abundant diversity with each of the isolates representing a unique genotype and one isolate that contained both AFLP bands unique to P. teres f. maculata and P. teres f. teres, the cause of spot form and net form of net blotch respectively, suggesting that sexual recombination between the net form and spot form isolates may have occurred naturally in the field.

Programming rumen bacterial communities in newborn Merino lambs

Establishment of the rumen microbiome can be affected by both early-life dietary measures and rumen microbial inoculation. This study used a 2 × 3 factorial design to evaluate the effects of inclusion of dietary fat type and the effects of rumen inoculum from different sources on ruminal bacterial communities present in early stages of the lambs’ life. Two different diets were fed ad libitum to 36 pregnant ewes (and their lambs) from 1 month pre-lambing until weaning. Diets consisted of chaffed lucerne and cereal hay and 4% molasses, with either 4% distilled coconut oil (CO) provided as a source of rumen-active fat or 4% Megalac® provided as a source of rumen-protected fat (PF). One of three inoculums was introduced orally to all lambs, being either (1) rumen fluid from donor ewes fed the PF diet; (2) rumen fluid from donor ewes fed CO; or (3) a control treatment of MilliQ-water. After weaning at 3 months of age, each of the six lamb treatment groups were grazed in spatially separated paddocks. Rumen bacterial populations of ewes and lambs were characterised using 454 amplicon pyrosequencing of the V3/V4 regions of the 16S rRNA gene. Species richness and biodiversity of the bacterial communities were found to be affected by the diet in ewes and lambs and by inoculation treatment of the lambs. Principal coordinate analysis and analysis of similarity (ANOSIM) showed between diet differences in bacterial community groups existed in ewes and differential bacterial clusters occurred in lambs due to both diet and neonatal inoculation. Diet and rumen inoculation acted together to clearly differentiate the bacterial communities through to weaning, however the microbiome effects of these initial early life interventions diminished with time so that rumen bacterial communities showed greater similarity 2 months after weaning. These results demonstrate that ruminal bacterial communities of newborn lambs can be altered by modifying the diet of their mothers. Moreover, the rumen microbiome of lambs can be changed by diet while they are suckling or by inoculating their rumen, and resulting changes in the rumen bacterial microbiome can persist beyond weaning.

Sequence diversity and differential expression of major phenylpropanoid-flavonoid biosynthetic genes among three mango varieties

Background: Mango fruits contain a broad spectrum of phenolic compounds which impart potential health benefits; their biosynthesis is catalysed by enzymes in the phenylpropanoid-flavonoid (PF) pathway. The aim of this study was to reveal the variability in genes involved in the PF pathway in three different mango varieties Mangifera indica L., a member of the family Anacardiaceae: Kensington Pride (KP), Irwin (IW) and Nam Doc Mai (NDM) and to determine associations with gene expression and mango flavonoid profiles. Results: A close evolutionary relationship between mango genes and those from the woody species poplar of the Salicaceae family (Populus trichocarpa) and grape of the Vitaceae family (Vitis vinifera), was revealed through phylogenetic analysis of PF pathway genes. We discovered 145 SNPs in total within coding sequences with an average frequency of one SNP every 316bp. Variety IW had the highest SNP frequency (one SNP every 258bp) while KP and NDM had similar frequencies (one SNP every 369bp and 360bp, respectively). The position in the PF pathway appeared to influence the extent of genetic diversity of the encoded enzymes. The entry point enzymes phenylalanine lyase (PAL), cinnamate 4-mono-oxygenase (C4H) and chalcone synthase (CHS) had low levels of SNP diversity in their coding sequences, whereas anthocyanidin reductase (ANR) showed the highest SNP frequency followed by flavonoid 3'-hydroxylase (F3'H). Quantitative PCR revealed characteristic patterns of gene expression that differed between mango peel and flesh, and between varieties. Conclusions: The combination of mango expressed sequence tags and availability of well-established reference PF biosynthetic genes from other plant species allowed the identification of coding sequences of genes that may lead to the formation of important flavonoid compounds in mango fruits and facilitated characterisation of single nucleotide polymorphisms between varieties. We discovered an association between the extent of sequence variation and position in the pathway for up-stream genes. The high expression of PAL, C4H and CHS genes in mango peel compared to flesh is associated with high amounts of total phenolic contents in peels, which suggest that these genes have an influence on total flavonoid levels in mango fruit peel and flesh. In addition, the particularly high expression levels of ANR in KP and NDM peels compared to IW peel and the significant accumulation of its product epicatechin gallate (ECG) in those extracts reflects the rate-limiting role of ANR on ECG biosynthesis in mango. © 2015 Hoang et al.

Controlled release of ethylene gas from the ethylene-α-cyclodextrin inclusion complex powder with deliquescent salts

Deliquescent calcium chloride (CaCl2) and magnesium chloride (MgCl2) were investigated for their practical application to release ethylene gas from an ethylene-α-cyclodextrin inclusion complexes (CD IC) powder at relative humidities (RHs) between 11.2 and 93.6 % at 18 °C. The IC powder and deliquescent salts were mixed at a ratio of 1:5, respectively. CaCl2 and MgCl2 started to deliquesce at 32.7 % RH. The IC powder dissolved in the concentrated salt solutions to release ethylene gas. Increasing the RH accelerated the release rate. Maximum release of ethylene gas was achieved after 24 h at 75.5 and 93.6 % RH for both IC powder-deliquescent salts mixture. The deliquescent salts proved to be a simple option for releasing ethylene gas from the IC powder.

Genome-wide association analysis and differential expression analysis of resistance to Sclerotinia stem rot in Brassica napus

Brassica napus is one of the most important oil crops in the world, and stem rot caused by the fungus Sclerotinia sclerotiorum results in major losses in yield and quality. To elucidate resistance genes and pathogenesis-related genes, genome-wide association analysis of 347 accessions was performed using the Illumina 60K Brassica SNP (single nucleotide polymorphism) array. In addition, the detached stem inoculation assay was used to select five highly resistant (R) and susceptible (S) B. napus lines, 48 h postinoculation with S. sclerotiorum for transcriptome sequencing. We identified 17 significant associations for stem resistance on chromosomes A8 and C6, five of which were on A8 and 12 on C6. The SNPs identified on A8 were located in a 409-kb haplotype block, and those on C6 were consistent with previous QTL mapping efforts. Transcriptome analysis suggested that S. sclerotiorum infection activates the immune system, sulphur metabolism, especially glutathione (GSH) and glucosinolates in both R and S genotypes. Genes found to be specific to the R genotype related to the jasmonic acid pathway, lignin biosynthesis, defence response, signal transduction and encoding transcription factors. Twenty-four genes were identified in both the SNP-trait association and transcriptome sequencing analyses, including a tau class glutathione S-transferase (GSTU) gene cluster. This study provides useful insight into the molecular mechanisms underlying the plant's response to S. sclerotiorum.

Uses of an innovative ethylene-α-cyclodextrin inclusion complex powder for ripening of mango fruit

A novel ethylene-α-cyclodextrin (α-CD) inclusion complex (IC) powder was investigated to ripen Calypso mango fruit. Modulated release of ethylene gas from the IC powder was achieved by admixture with deliquescent salt CaCl2 at RHs of 75.5% and 93.6%. The IC powder was tested in the laboratory and for in-transit ripening of mango fruit over two seasons. In the laboratory experiment, ethylene gas started to release from the IC powder in 2 h and complete release was achieved in 24 h. Assessments of fruit colour and firmness showed that encapsulated ethylene and commercial grade ethylene from pressurised cylinder similarly shortened the ripening time to 9–10 days (after harvest) for treated fruit as compared with 15 days for untreated mango. Mango fruit treated in both ways with ethylene showed more uniform ripening than the control. For the in-transit ripening using the IC powder, ethylene was found to be between 4.9 and 10.5 μL L−1 in the headspace of the truck containers over 48 h. Mango fruit from the treated containers shortened the ripening time by 3–6 days as compared to the untreated control fruit. Thus, the safe and convenient IC powder has demonstrated promise for in-transit fruit ripening.