The Efficacy of Selected Probiotic Strains and Their Combination to Inhibit the Interaction of Adherent-Invasive Escherichia coli (AIEC) with a Co-Culture of Caco-2:HT29-MTX Cells.

The gastrointestinal tract's microbiota plays a crucial role in human health, with dysbiosis linked to the development of diseases such as inflammatory bowel disease (IBD). Whilst the pathogenic mechanisms underlying IBD remain poorly characterised, adherent-invasive Escherichia coli (AIEC) has been implicated as a microbiological factor in disease pathogenesis. These strains show an enhanced ability to diffusely adhere to and invade intestinal epithelial cells, along with the ability to survive and replicate within macrophages. Probiotics, such as Lactobacillus strains, have been identified as potential treatment options due to their abilities to compete with pathogens for binding sites and regulate the host immune response. In this study, we used four well-characterised Lactobacillus strains and their combination to test their ability to inhibit the adhesion, invasion, and translocation of a well-characterized AIEC strain, F44A-1, in a co-culture of Caco-2 and HT29-MTX cell lines representing the gut epithelium. The results demonstrated that the pre-inoculation of the probiotic candidates 90 min prior to the introduction of the AIEC was more effective in inhibiting AIEC interaction than the co-inoculation of the strains. While the individual probiotic strains greatly reduced AIEC colonisation and invasion of the co-cultured cells, their combination was only more effective in reducing the translocation of the AIEC. These results suggest that probiotics are more effective when used prophylactically against pathogens and that the combination of strains may enhance their efficacy against AIEC translocation once used as a prophylactic measure.

Associations between Maternal Nutrition and the Concentrations of Human Milk Oligosaccharides in a Cohort of Healthy Australian Lactating Women.

Human milk oligosaccharides (HMOs) are complex glycans associated with positive infant health outcomes. The concentrations of HMOs in the milk of lactating women are associated with substantial intra- and inter-individual differences and may be influenced by maternal physiological and/or nutrition-related factors. The primary aim of this study was to explore potential influences of short-term maternal diet and current body composition on HMO profiles in mature human milk. Milk samples were collected at 3-4 months postpartum from 101 healthy Australian women using standardised procedures, and analysed for macronutrients (lactose, fat, and protein). In addition, HMO concentrations were analysed using liquid-chromatography mass-spectrometry (LC-MS). Maternal dietary data were collected using three validated 24-h dietary recalls, and the body composition of a subgroup of mothers was assessed by DEXA scans (n = 30). Most (79%) of the women were secretor-positive. Individual nutrients were not significantly correlated with HMO concentrations after correction for multiple comparisons (p > 0.05), except for dietary folate intake. DEXA scans revealed no associations between HMO profiles and maternal body composition during established lactation. The study findings suggest a lack of clear and consistent associations between maternal nutrition and HMO concentrations in mature human milk from healthy lactating women with adequate dietary intake. The prevailing influence of genetic variation in lactating mothers may overshadow any impact of maternal nutritional and/or physiological status on HMO composition in mature human milk.

Efficacy of Selected Live Biotherapeutic Candidates to Inhibit the Interaction of an Adhesive-Invasive Escherichia coli Strain with Caco-2, HT29-MTX Cells and Their Co-Culture.

Adherent-invasive Escherichia coli (AIEC) has been implicated as a microbiological factor in the pathogenesis of inflammatory bowel disease (IBD). We evaluated the ability of six live biotherapeutic products (LBPs) to inhibit the interaction of an AIEC strain to three cell lines representing human gut epithelium. Co-inoculation of LBPs with AIEC showed a reduction in adhesion (up to 73%) and invasion of AIEC (up to 89%). Pre-inoculation of LBPs in HT-29-MTX and Caco-2 cells before challenging with AIEC further reduced the adhesion and invasion of the AIEC, with three LBPs showing significantly (p < 0.0001) higher efficiency in reducing the adhesion of AIEC. In co-inoculation experiments, the highest reduction in adhesion (73%) of AIEC was observed in HT-29-MTX cells, whereas the highest reduction in invasion (89%) was seen in HT-29-MTX and the co-culture of cells. Pre-inoculation of LBPs further reduced the invasion of AIEC with highest reduction (97%) observed in co-culture of cells. Our results indicated that whilst there were differences in the efficacy of LBPs, they all reduced interaction of AIEC with cell lines representing gut epithelium. Their efficiency was higher when they were pre-inoculated onto the cells, suggesting their potential as candidates for alleviating pathogenesis of AIEC in patients with IBD.

Fingolimod does not prevent syndecan-4 shedding from the endothelial glycocalyx in a cultured human umbilical vein endothelial cell model of vascular injury.

BACKGROUND: Shedding of the endothelial glycocalyx (EG) is associated with poor outcomes in a range of conditions including sepsis. Fresh frozen plasma (FFP) restores the damaged EG to baseline thickness, however the mechanism for this effect is unknown, and some components of FFP have adverse effects unrelated to the EG. There is some limited evidence that sphingosine-1-phosphate (S1P) within FFP restores the EG by activating the endothelial cell S1P receptor 1 (S1PR1). However, there are disadvantages to using S1P clinically as an EG restorative therapy. A potential alternative is the S1PR agonist fingolimod (FTY720). The aim of this study was to assess whether FTY720 prevents EG shedding in injured cultured human umbilical vein endothelial cells. METHODS: Shedding of the EG was induced in cultured human umbilical vein endothelial cells (HUVECs) by exposure to adrenaline, TNF-α and H2O2. The cells were then assigned to one of six conditions for 4 h: uninjured and untreated, injured and untreated, injured and treated with FTY720 with and without the S1PR1 inhibitor W146, and injured and treated with 25% FFP with and without W146. Syndecan-4, a component of the EG, was measured in cell supernatants, and syndecan-4 and thrombomodulin mRNA expression was quantitated in cell lysates. RESULTS: The injury resulted in a 2.1-fold increase in syndecan-4 (p < 0.001), consistent with EG shedding. Syndecan-4 and thrombomodulin mRNA expression was increased (p < 0.001) and decreased (p < 0.05), respectively, by the injury. Syndecan-4 shedding was not affected by treatment with FTY720, whereas FFP attenuated syndecan-4 shedding back to baseline levels in the injured cells and this was unaffected by W146. Neither treatment affected syndecan-4 or thrombomodulin mRNA expression. CONCLUSIONS: FTY720 did not prevent syndecan-4 shedding from the EG in the HUVEC model of endothelial injury, suggesting that activation of S1PR does not prevent EG damage. FFP prevented syndecan-4 shedding from the EG via a mechanism that was independent of S1PR1 and upregulation of SDC-4 production. Further studies to examine whether FTY720 or another S1PR agonist might have EG-protective effects under different conditions are warranted, as are investigations seeking the mechanism of EG protection conferred by FFP in this experimental model.

Beyond the BMI: Validity and Practicality of Postpartum Body Composition Assessment Methods during Lactation: A Scoping Review.

The assessment of body composition during lactation is an important indicator of maternal nutritional status, which is central to the overall health of the mother and child. The lactating woman's nutritional status potentially impacts on breastmilk composition and the process of lactation itself. The purpose of this scoping review was to synthesize comparative studies that sought to validate various body composition assessment techniques for use in lactating women in the postpartum period. Using the PRISMA-ScR guidelines, a comprehensive, systematic literature search was conducted using Scopus, Web of Science, and PubMed. Eight comparative studies were included in the review, with data from 320 postpartum women. The design methodologies varied substantially across studies, and included a range of simple techniques to advanced multi-compartment models for assessing body composition. The validity and reliability of measurement tools must be considered alongside issues of safety, practicality, and appropriateness to guide the research design when applied to lactating women.

Microbial Diversity Profiling of Gut Microbiota of Macropus giganteus Using Three Hypervariable Regions of the Bacterial 16S rRNA.

Animal faecal contamination of surface waters poses a human health risk, as they may contain pathogenic bacteria or viruses. Of the numerous animal species residing along surface waterways in Australia, macropod species are a top contributor to wild animals' faecal pollution load. We characterised the gut microbiota of 30 native Australian Eastern Grey Kangaroos from six geographical regions (five kangaroos from each region) within South East Queensland in order to establish their bacterial diversity and identify potential novel species-specific bacteria for the rapid detection of faecal contamination of surface waters by these animals. Using three hypervariable regions (HVRs) of the 16S rRNA gene (i.e., V1-V3, V3-V4, and V5-V6), for their effectiveness in delineating the gut microbial diversity, faecal samples from each region were pooled and microbial genomic DNA was extracted, sequenced, and analysed. Results indicated that V1-V3 yielded a higher taxa richness due to its larger target region (~480 bp); however, higher levels of unassigned taxa were observed using the V1-V3 region. In contrast, the V3-V4 HVR (~569 bp) attained a higher likelihood of a taxonomic hit identity to the bacterial species level, with a 5-fold decrease in unassigned taxa. There were distinct dissimilarities in beta diversity between the regions, with the V1-V3 region displaying the highest number of unique taxa (n = 42), followed by V3-V4 (n = 11) and V5-V6 (n = 8). Variations in the gut microbial diversity profiles of kangaroos from different regions were also observed, which indicates that environmental factors may impact the microbial development and, thus, the composition of the gut microbiome of these animals.

Human Milk Oligosaccharide Profiles and Associations with Maternal Nutritional Factors: A Scoping Review.

Human milk oligosaccharides (HMOs) are complex unconjugated glycans associated with positive infant health outcomes. This study has examined current knowledge of the effect of maternal diet and nutritional status on the composition of HMOs in breast milk. Using the PRISMA-ScR guidelines, a comprehensive, systematic literature search was conducted using Scopus, Web of Science, Global Health (CABI), and MEDLINE. Titles and abstracts were screened independently by two reviewers against predefined inclusion and exclusion criteria. Fourteen studies met the inclusion criteria and reported on maternal dietary intake (n = 3), maternal body composition indices (n = 9), and dietary supplementation interventions (n = 2). In total, data from 1388 lactating mothers (4011 milk samples) were included. Design methodologies varied substantially across studies, particularly for milk sample collection, HMO analysis, dietary and body composition assessment. Overall, this review has identified potential associations between maternal dietary intake and nutritional status and the HMO composition of human milk, though an abundance and sufficiency of evidence is lacking. Standardised procedures for human milk sample collection and HMO analysis, along with robust and validated nutrition assessment techniques, should be employed to further investigate the impact of maternal nutritional factors on HMO composition.

Endotoxin Tolerance in Abdominal Aortic Aneurysm Macrophages, In Vitro: A Case-Control Study.

Macrophages are implicated in the pathogenesis of abdominal aortic aneurysm (AAA). This study examined the environmentally conditioned responses of AAA macrophages to inflammatory stimuli. Plasma- and blood-derived monocytes were separated from the whole blood of patients with AAA (30-45 mm diameter; n = 33) and sex-matched control participants (n = 44). Increased concentrations of pro-inflammatory and pro-oxidant biomarkers were detected in the plasma of AAA patients, consistent with systemic inflammation and oxidative stress. However, in monocyte-derived macrophages, a suppressed cytokine response was observed in AAA compared to the control following stimulation with lipopolysaccharide (LPS) (tumor necrosis factor alpha (TNF-α) 26.9 ± 3.3 vs. 15.5 ± 3.2 ng/mL, p < 0.05; IL-6 3.2 ± 0.6 vs. 1.4 ± 0.3 ng/mL, p < 0.01). LPS-stimulated production of 8-isoprostane, a biomarker of oxidative stress, was also markedly lower in AAA compared to control participants. These findings are consistent with developed tolerance in human AAA macrophages. As Toll-like receptor 4 (TLR4) has been implicated in tolerance, macrophages were examined for changes in TLR4 expression and distribution. Although TLR4 mRNA and protein expression were unaltered in AAA, cytosolic internalization of receptors and lipid rafts was found. These findings suggest the inflamed, pro-oxidant AAA microenvironment favors macrophages with an endotoxin-tolerant-like phenotype characterized by a diminished capacity to produce pro-inflammatory mediators that enhance the immune response.

A Comparative Study of the Adherent-Invasive Escherichia coli Population and Gut Microbiota of Healthy Vegans versus Omnivores.

Adherent-invasive Escherichia coli (AIEC) strains carry virulence genes (VGs) which are rarely found in strains other than E. coli. These strains are abundantly found in gut mucosa of patients with inflammatory bowel disease (IBD); however, it is not clear whether their prevalence in the gut is affected by the diet of the individual. Therefore, in this study, we compared the population structure of E. coli and the prevalence of AIEC as well as the composition of gut microbiota in fecal samples of healthy participants (n = 61) on either a vegan (n = 34) or omnivore (n = 27) diet to determine whether diet is associated with the presence of AIEC. From each participant, 28 colonies of E. coli were typed using Random Amplified Polymorphic DNA (RAPD)-PCR. A representative of each common type within an individual was tested for the presence of six AIEC-associated VGs. Whole genomic DNA of the gut microbiota was also analyzed for its diversity profiles, utilizing the V5-V6 region of the16S rRNA gene sequence. There were no significant differences in the abundance and diversity of E. coli between the two diet groups. The occurrence of AIEC-associated VGs was also similar among the two groups. However, the diversity of fecal microbiota in vegans was generally higher than omnivores, with Prevotella and Bacteroides dominant in both groups. Whilst 88 microbial taxa were present in both diet groups, 28 taxa were unique to vegans, compared to seven unique taxa in the omnivores. Our results indicate that a vegan diet may not affect the number and diversity of E. coli populations and AIEC prevalence compared to omnivores. The dominance of Prevotella and Bacteroides among omnivores might be accounted for the effect of diet in these groups.

The Gut Microbial Profile of Preclinical Crohn's Disease Is Similar to That of Healthy Controls.

BACKGROUND AND AIMS: It is unclear whether microbial dysbiosis plays an etiologic role in Crohn's disease (CD) or is the result of protracted inflammation. Here, we test the hypothesis that dysbiosis predates clinical CD in asymptomatic first-degree relatives (FDRs) of CD patients: normal (FDR1), with borderline inflammation (FDR2), and with frank, very early inflammation (FDR3). METHODS: The gut microbial diversity was tested in ileocecal biopsies through next generation sequencing of the 16S rRNA gene in 10 healthy controls (HCs), 22 patients with active, untreated CD, and 25 FDRs (9 FDR1; 12 FDR2; 4 FDR3). The metagenomic functions of 41 microbiome-related processes were inferred by Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis. RESULTS: Compared with HCs, alpha diversity in CD patients was decreased, with an observed decrease in Faecalibacterium prausnitzii and increase in Bacteroides fragilis. In FDRs, microbial diversity was unchanged compared with HCs. In Operational Taxonomic Units and PICRUSt Principal coordinates and component analyses, the ellipse centroid of FDRs was diagonally opposed to that of CD patients, but close to the HC centroid. In both analyses, statistically significant differences in terms of beta diversity were found between CD and HC but not between FDR and HC. CONCLUSIONS: In FDRs (including FDR3-who bear preclinical/biologic onset disease), we found that the microbial profile is remarkably similar to HC. If confirmed in larger studies, this finding suggests that clinical CD-associated dysbiosis could result from the changed microenvironment due to disease evolution over time.

Omega-3 fatty acids decrease oxidative stress and inflammation in macrophages from patients with small abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is associated with inflammation and oxidative stress, the latter of which contributes to activation of macrophages, a prominent cell type in AAA. Omega-3 polyunsaturated fatty acids (n-3 PUFAs) have been reported to limit oxidative stress in animal models of AAA. The aim of this study was to evaluate the effect of the n-3 PUFA docosahexaenoic acid (DHA) on antioxidant defence in macrophages from patients with AAA. Cells were obtained from men with small AAA (diameter 3.0-4.5 cm, 75 ± 6 yr, n = 19) and age- matched male controls (72 ± 5 yr, n = 41) and incubated with DHA for 1 h before exposure to 0.1 µg/mL lipopolysaccharide (LPS) for 24 h. DHA supplementation decreased the concentration of tumour necrosis factor-α (TNF-α; control, 42.1 ± 13.6 to 5.1 ± 2.1 pg/ml, p < 0.01; AAA, 25.2 ± 9.8 to 1.9 ± 0.9 pg/ml, p < 0.01) and interleukin-6 (IL-6; control, 44.9 ± 7.7 to 5.9 ± 2.0 pg/ml, p < 0.001; AAA, 24.3 ± 5.2 to 0.5 ± 0.3 pg/ml, p < 0.001) in macrophage supernatants. DHA increased glutathione peroxidase activity (control, 3.2 ± 0.3 to 4.1 ± 0.2 nmol/min/ml/µg protein, p = 0.004; AAA, 2.3 ± 0.5 to 3.4 ± 0.5 nmol/min/ml/µg protein, p = 0.008) and heme oxygenase-1 mRNA expression (control, 1.5-fold increase, p < 0.001). The improvements in macrophage oxidative stress status serve as a stimulus for further investigation of DHA in patients with AAA.

Compositional and temporal stability of fecal taxon libraries for use with SourceTracker in sub-tropical catchments.

Characterization of microbial communities using high-throughput amplicon sequencing is an emerging approach for microbial source tracking of fecal pollution. This study used SourceTracker software to examine temporal and geographical variability of fecal bacterial community profiles to identify pollutant sources in three freshwater catchments in sub-tropical Australia. Fecal bacterial communities from 10 animal species, humans, and composite wastewater samples from six sewage treatment plants were characterized and compared to freshwater samples using Illumina amplicon sequencing of the V5-V6 regions of the 16S rRNA gene. Source contributions were calculated in SourceTracker using new fecal taxon libraries as well as previously generated libraries to determine the effects of geographic and temporal variability on source assignments. SourceTracker determined 16S rRNA bacterial communites within freshwater samples, shared taxonomic similarities to that of wastewater at low levels (typically <3%). SourceTraker also predicted occasional fecal detection of deer and flying fox sources in the water samples. No significant differences in source contributions were observed within sequences from current and previously characterized fecal samples (P ≥ 0.107). However, significant differences were observed between previously characterized and newly characterized source communities (ANOSIM P ≤ 0.001), which shared <15% community composition. Results suggest temporal instability of fecal taxon libraries among tested sources and highlight continual evaluation of community-based MST using confirmatory qPCR analyses of marker genes.

A simple and effective method for the isolation and culture of human monocytes from small volumes of peripheral blood.

Innate immune cell defects contribute to severe autoimmunity and the pathogenesis of inflammatory disease. Monocyte-derived macrophages typically retain disease related signatures and represent an excellent in vitro model to uncover and validate mechanisms contributing to specific pathological states. Monocyte isolation procedures vary widely in terms of purity, yield, cost, degree of technical difficulty and volume of peripheral blood needed. This paper outlines a novel isolation method that yields monocytes through density gradient centrifugation (Ficoll® and hyperosmotic Percoll®). The protocol has been optimised for small volumes of blood (42 ml) and is simple, reproducible and inexpensive compared to other methods. Monocyte recovery is 70% (relative to monocyte numbers within the buffy coat) and the highly functional macrophages produced are characterised by excellent purity (98.6 ±â€¯0.6%) and intact activation and phagocytic capacities. The method is well suited to investigations involving patient populations where a particular subset of immune cells is known to contribute to the pathogenesis of a specific disease or is aberrant as a consequence of that disease.

Marker genes of fecal indicator bacteria and potential pathogens in animal feces in subtropical catchments.

We investigated the abundance of marker genes of two fecal indicator bacteria (FIB) and eight potential pathogens in fecal samples of humans (n = 14) and 10 domestic and native wild animals (n = 134). For each target animal, between 10 and 14 individual fecal samples were collected (n = 148 individual fecal samples in total). The abundance of FIB and potential pathogens within each sample was determined using quantitative PCR (qPCR) assays. All animals tested were positive for Escherichia coli (EC) and the concentrations ranged from 6.13 (flying fox) to 8.87 (chicken) log10 GC/g of feces. These values for Enterococcus spp. (ENT) were 5.25 log10 GC/g for flying fox and 8.12 log10 GC/g of feces for chicken. Moderate correlations were observed between EC with P. aeruginosa, EC O157 and Cryptosporidium parvum, whereas weak correlations were observed between EC and Salmonella spp. and Giardia lamblia, Mycobacterium avium complex (MAC) and Campylobacter spp. The prevalence of MAC and P. aeruginosa were low in dog (14.3% each) and moderate (57.2%, MAC; 42.9% P. aeruginosa) in Eastern grey kangaroo fecal samples. Cryptosporidium parvum was detected in one cattle and one human fecal sample, while G. lamblia was detected in one dog, one flying fox, and one pig fecal samples. Among the eight potential pathogens tested, five pathogens were detected in chicken and dog fecal samples. The remaining animal species contained up to three potential pathogens in their feces. The data generated in this study may aid in the calculation of pathogen loads in the environment, and hence to assess the risks from human and animal fecal contamination of source waters.

Yearly, pond, lineage and family variation of hepatopancreatic parvo-like virus (HPV) copy number in banana shrimp Fenneropenaeus merguiensis.

Hepatopancreatic parvo-like virus (HPV) has been reported from a variety of shrimp species around the world, including Australia, and thought to impact negatively on production, but until now there was scant information available on variation of HPV over time, ponds and shrimp lineages or families, information that could be used to manage or reduce virus levels. Here we report HPV copy number estimated using qPCR from 1500 individual shrimp sampled over three years and encompassing 91 ponds, 21 breeding groups or lineages and 40 families. HPV copy number variation between ponds was used by farm management as a criterion to choose prospective broodstock (candidates were taken from low HPV ponds). Despite such choice, HPV levels in farmed animals were not reduced from 2011 to 2013. Accordingly, the hypothesis that HPV levels can be reduced over time simply by considering average HPV levels in ponds alone is rejected. Different lines of shrimp within the same farm had different HPV levels, but as lines were raised separately, the line differences could be due to either genetic or environmental differences, the latter including possible different rearing effects and differences in vertical transmission. There were large (up to 2-3 LOG fold) differences of HPV levels between families bred and grown together contemporaneously, and the heritability for HPV copy number was estimated to be moderate to large (0.40 ± 0.13). Apart from genetic differences, differences of vertical transmission from dams may contribute to the between family differences, in any case we postulate that selection between families could be an effective method to reduce HPV levels. HPV levels were not genetically correlated with performance traits such as body weight or length, so selection for HPV level should not adversely affect production characteristics. This is the first evidence for an aquacultured species that viral levels, as opposed to survival/resistance to viruses, may have a substantial host genetic component. The heritability reported here for virus copy number was higher that most heritabilities reported for survival to specific pathogens such as white spot, raising the general postulate that selection for virus copy number may be more effective and repeatable than selection for survival to pathogen challenge.

Molecular insights into land snail neuropeptides through transcriptome and comparative gene analysis.

BACKGROUND: Snails belong to the molluscan class Gastropoda, which inhabit land, freshwater and marine environments. Several land snail species, including Theba pisana, are crop pests of major concern, causing extensive damage to agriculture and horticulture. A deeper understanding of their molecular biology is necessary in order to develop methods to manipulate land snail populations. RESULTS: The present study used in silico gene data mining of T. pisana tissue transcriptomes to predict 24,920 central nervous system (CNS) proteins, 37,661 foot muscle proteins and 40,766 hepatopancreas proteins, which together have 5,236 unique protein functional domains. Neuropeptides, metabolic enzymes and epiphragmin genes dominated expression within the CNS, hepatopancreas and muscle, respectively. Further investigation of the CNS transcriptome demonstrated that it might contain as many as 5,504 genes that encode for proteins destined for extracellular secretion. Neuropeptides form an important class of cell-cell messengers that control or influence various complex metabolic events. A total of 35 full-length neuropeptide genes were abundantly expressed within T. pisana CNS, encoding precursors that release molluscan-type bioactive neuropeptide products. These included achatin, allototropin, conopressin, elevenin, FMRFamide, LFRFamide, LRFNVamide, myomodulins, neurokinin Y, PKYMDT, PXFVamide, sCAPamides and several insulin-like peptides. Liquid chromatography-mass spectrometry of neural ganglia confirmed the presence of many of these neuropeptides. CONCLUSIONS: Our results provide the most comprehensive picture of the molecular genes and proteins associated with land snail functioning, including the repertoire of neuropeptides that likely play significant roles in neuroendocrine signalling. This information has the potential to expedite the study of molluscan metabolism and potentially stimulate advances in the biological control of land snail pest species.

Molecular characterisation of colour formation in the prawn Fenneropenaeus merguiensis.

INTRODUCTION: Body colouration in animals can have a range of functions, with predator protection an important aspect of colour in crustaceans. Colour determination is associated with the carotenoid astaxanthin, which is taken up through the diet and stabilised in the tissues by the protein crustacyanin. As a variety of genes are found to play a role in colour formation in other systems, a holistic approach was employed in this study to determine the factors involved in Fenneropenaeus merguiensis colouration. RESULTS: Full length F. merguiensis crustacyanin subunit A and C sequences were isolated. Crustacyanin subunit A and C were found in the F. merguiensis transcriptomes of the muscle/cuticle tissue, hepatopancreas, eye stalk and nervous system, using 454 next generation sequencing technology. Custom microarray analysis of albino, light and dark F. merguiensis cuticle tissue showed genes encoding actin, sarcoplasmic calcium-binding protein and arginine kinase to be 4-fold or greater differentially expressed (p<0.05) and down-regulated in albinos when compared to light and dark samples. QPCR expression analysis of crustacyanin and total astaxanthin pigment extraction revealed significantly (p<0.05) lower crustacyanin subunit A and C gene transcript copy numbers and total astaxanthin levels in albinos than in the light and dark samples. Additionally, crustacyanin subunit A and C expression levels correlated positively with each other. CONCLUSIONS: This study identified gene products putatively involved in crustacean colouration, such as crustacyanin, sarcoplasmic calcium-binding protein and forms of actin, and investigated differences in gene expression and astaxanthin levels between albino, light and dark coloured prawns. These genes open a path to enhance our understanding of the biology and regulation of colour formation.

Moult cycle specific differential gene expression profiling of the crab Portunus pelagicus.

BACKGROUND: Crustacean moulting is a complex process involving many regulatory pathways. A holistic approach to examine differential gene expression profiles of transcripts relevant to the moulting process, across all moult cycle stages, was used in this study. Custom cDNA microarrays were constructed for Portunus pelagicus. The printed arrays contained 5000 transcripts derived from both the whole organism, and from individual organs such as the brain, eyestalk, mandibular organ and Y-organ from all moult cycle stages. RESULTS: A total of 556 clones were sequenced from the cDNA libraries used to construct the arrays. These cDNAs represented 175 singletons and 62 contigs, resulting in 237 unique putative genes. The gene sequences were classified into the following biological functions: cuticular proteins associated with arthropod exoskeletons, farnesoic acid O-methyltransferase (FaMeT), proteins belonging to the hemocyanin gene family, lectins, proteins relevant to lipid metabolism, mitochondrial proteins, muscle related proteins, phenoloxidase activators and ribosomal proteins. Moult cycle-related differential expression patterns were observed for many transcripts. Of particular interest were those relating to the formation and hardening of the exoskeleton, and genes associated with cell respiration and energy metabolism. CONCLUSIONS: The expression data presented here provide a chronological depiction of the molecular events associated with the biological changes that occur during the crustacean moult cycle. Tracing the temporal expression patterns of a large variety of transcripts involved in the moult cycle of P. pelagicus can provide a greater understanding of gene function, interaction, and regulation of both known and new genes with respect to the moulting process.

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.