Transcriptomic responses in the nervous system and correlated behavioural changes of a cephalopod exposed to ocean acidification.

BACKGROUND: The nervous system is central to coordinating behavioural responses to environmental change, likely including ocean acidification (OA). However, a clear understanding of neurobiological responses to OA is lacking, especially for marine invertebrates. RESULTS: We evaluated the transcriptomic response of the central nervous system (CNS) and eyes of the two-toned pygmy squid (Idiosepius pygmaeus) to OA conditions, using a de novo transcriptome assembly created with long read PacBio ISO-sequencing data. We then correlated patterns of gene expression with CO2 treatment levels and OA-affected behaviours in the same individuals. OA induced transcriptomic responses within the nervous system related to various different types of neurotransmission, neuroplasticity, immune function and oxidative stress. These molecular changes may contribute to OA-induced behavioural changes, as suggested by correlations among gene expression profiles, CO2 treatment and OA-affected behaviours. CONCLUSIONS: This study provides the first molecular insights into the neurobiological effects of OA on a cephalopod and correlates molecular changes with whole animal behavioural responses, helping to bridge the gaps in our knowledge between environmental change and animal responses.

The bacterial gut microbiome of probiotic-treated very-preterm infants: changes from admission to discharge.

BACKGROUND: Preterm birth is associated with the development of acute and chronic disease, potentially, through the disruption of normal gut microbiome development. Probiotics may correct for microbial imbalances and mitigate disease risk. Here, we used amplicon sequencing to characterise the gut microbiome of probiotic-treated premature infants. We aimed to identify and understand variation in bacterial gut flora from admission to discharge and in association with clinical variables. METHODS: Infants born <32 weeks gestation and <1500 g, and who received probiotic treatment, were recruited in North Queensland Australia. Meconium and faecal samples were collected at admission and discharge. All samples underwent 16S rRNA short amplicon sequencing, and subsequently, a combination of univariate and multivariate analyses. RESULTS: 71 admission and 63 discharge samples were collected. Univariate analyses showed significant changes in the gut flora from admission to discharge. Mixed-effects modelling showed significantly lower alpha diversity in infants diagnosed with either sepsis or retinopathy of prematurity (ROP) and those fed formula. In addition, chorioamnionitis, preeclampsia, sepsis, necrotising enterocolitis and ROP were also all associated with the differential abundance of several taxa. CONCLUSIONS: The lower microbial diversity seen in infants with diagnosed disorders or formula-fed, as well as differing abundances of several taxa across multiple variables, highlights the role of the microbiome in the development of health and disease. This study supports the need for promoting healthy microbiome development in preterm neonates. IMPACT: Low diversity and differing taxonomic abundances in preterm gut microbiota demonstrated in formula-fed infants and those identified with postnatal conditions, as well as differences in taxonomy associated with preeclampsia and chorioamnionitis, reinforcing the association of the microbiome composition changes due to maternal and infant disease. The largest study exploring an association between the preterm infant microbiome and ROP. A novel association between the preterm infant gut microbiome and preeclampsia in a unique cohort of very-premature probiotic-supplemented infants.

Exploring the long-term colonisation and persistence of probiotic-prophylaxis species on the gut microbiome of preterm infants: a pilot study.

Preterm infants suffer from a higher incidence of acute diseases such as necrotising enterocolitis and sepsis. This risk can be mitigated through probiotic prophylaxis during admission. This reduction in risk is likely the result of acute modulation of the gut microbiome induced by probiotic species, which has been observed to occur up until discharge. We aimed to determine if this modulation, and the associated probiotic species, persisted beyond discharge. We conducted both a cross-sectional analysis (n = 18), at ~ 18 months of age, and a longitudinal analysis (n = 6), from admission to 18 months of the gut microbiome of preterm infants using both shotgun metagenomics and 16S rRNA profiling respectively. The 16S amplicon sequencing revealed that the microbial composition of the probiotic-supplemented infants changed dramatically over time, stabilising at discharge. However, species from the probiotic Infloran®, as well as positive modulatory effects previously associated with supplementation, do not appear to persist beyond discharge and once prophylaxis has stopped.    Conclusions: Although differences exist between supplemented and non-supplemented groups, the implications of these differences remain unclear. Additionally, despite a lack of long-term colonisation, the presence of probiotics during early neonatal life may still have modulatory effects on the microbiome assembly and immune system training. What is Known: • Evidence suggests modulation of the microbiome occurs during probiotic prophylaxis, which may support key taxa that exert positive immunological benefits. • Some evidence suggests that this modulation can persist post-prophylaxis. What is New: • We present support for long-term modulation in association with probiotic prophylaxis in a cohort of infants from North Queensland Australia. • We also observed limited persistence of the probiotic species post-discharge.

Microbiome diversity and composition varies across body areas in a freshwater turtle.

There is increasing recognition that microbiomes are important for host health and ecology, and understanding host microbiomes is important for planning appropriate conservation strategies. However, microbiome data are lacking for many taxa, including turtles. To further our understanding of the interactions between aquatic microbiomes and their hosts, we used next generation sequencing technology to examine the microbiomes of the Krefft's river turtle (Emydura macquarii krefftii). We examined the microbiomes of the buccal (oral) cavity, skin on the head, parts of the shell with macroalgae and parts of the shell without macroalgae. Bacteria in the phyla Proteobacteria and Bacteroidetes were the most common in most samples (particularly buccal samples), but Cyanobacteria, Deinococcus-thermus and Chloroflexi were also common (particularly in external microbiomes). We found significant differences in community composition among each body area, as well as significant differences among individuals. The buccal cavity had lower bacterial richness and evenness than any of the external microbiomes, and it had many amplicon sequence variants (ASVs) with a low relative abundance compared to other body areas. Nevertheless, the buccal cavity also had the most unique ASVs. Parts of the shell with and without algae also had different microbiomes, with particularly obvious differences in the relative abundances of the families Methylomonaceae, Saprospiraceae and Nostocaceae. This study provides novel, baseline information about the external microbiomes of turtles and is a first step in understanding their ecological roles.

Novel Allergen Discovery through Comprehensive De Novo Transcriptomic Analyses of Five Shrimp Species.

Shellfish allergy affects 2% of the world's population and persists for life in most patients. The diagnosis of shellfish allergy, in particular shrimp, is challenging due to the similarity of allergenic proteins from other invertebrates. Despite the clinical importance of immunological cross-reactivity among shellfish species and between allergenic invertebrates such as dust mites, the underlying molecular basis is not well understood. Here we mine the complete transcriptome of five frequently consumed shrimp species to identify and compare allergens with all known allergen sources. The transcriptomes were assembled de novo, using Trinity, from raw RNA-Seq data of the whiteleg shrimp (Litopenaeus vannamei), black tiger shrimp (Penaeus monodon), banana shrimp (Fenneropenaeus merguiensis), king shrimp (Melicertus latisulcatus), and endeavour shrimp (Metapenaeus endeavouri). BLAST searching using the two major allergen databases, WHO/IUIS Allergen Nomenclature and AllergenOnline, successfully identified all seven known crustacean allergens. The analyses revealed up to 39 unreported allergens in the different shrimp species, including heat shock protein (HSP), alpha-tubulin, chymotrypsin, cyclophilin, beta-enolase, aldolase A, and glyceraldehyde-3-phosphate dehydrogenase (G3PD). Multiple sequence alignment (Clustal Omega) demonstrated high homology with allergens from other invertebrates including mites and cockroaches. This first transcriptomic analyses of allergens in a major food source provides a valuable resource for investigating shellfish allergens, comparing invertebrate allergens and future development of improved diagnostics for food allergy.

Bacteriophage versus antibiotic therapy on gut bacterial communities of juvenile green turtle, Chelonia mydas.

Green turtles are endangered marine herbivorous hindgut fermenters that contribute to a variety of marine ecosystems. Debilitated turtles are often rehabilitated in turtle hospitals. Since accurate diagnosis of disease is difficult, broad-spectrum antibiotics are routinely used as a general treatment, potentially causing collateral damage to the gut microbiome of the patient. Here, we evaluated the concept of the application of bacteriophage (phages) to eliminate targeted intestinal bacteria as an alternative to a broad-spectrum antibiotic (enrofloxacin) in clinically healthy, captive green turtles. Additionally, the impact of a broad-spectrum antibiotic (enrofloxacin) and phage therapy on the gut bacterial communities of green turtles was evaluated. Gut bacterial communities in faecal samples were analysed by sequencing the V1-V3 regions of the bacterial 16S rRNA. Bacteria-specific phage cocktails significantly (P < 0.05) reduced targeted Acinetobacter in phage-treated turtles during the therapy. Compared to control, no significant difference was observed in the bacterial diversity and compositions in phage-treated turtles. In contrast, bacterial diversity was significantly (P < 0.05) reduced in antibiotic-treated turtles at day 15 and throughout the trial. The alteration in the bacterial microbiota of antibiotic-treated turtles was largely due to an increase in abundance of Gram-positive Firmicutes and a concurrent decrease in Gram-negative Bacteroidetes, Proteobacteria and Verrucomicrobia. Additionally, we observed the relative abundance of several bacteria at lower taxonomic level was much less affected by phages than by antibiotics. These data offer the proof of concept of phage therapy to manipulate transient as well as indigenous bacterial flora in gut-related dysbiosis of turtles.

Toxic effects of polyethylene terephthalate microparticles and Di(2-ethylhexyl)phthalate on the calanoid copepod, Parvocalanus crassirostris.

Large amounts of plastic end up in the oceans every year where they fragment into microplastics over time. During this process, microplastics and their associated plasticizers become available for ingestion by different organisms. This study assessed the effects of microplastics (Polyethylene terephthalate; PET) and one plasticizer (Di(2-ethylhexyl)phthalate; DEHP) on mortality, productivity, population sizes and gene expression of the calanoid copepod Parvocalanus crassirostris. Copepods were exposed to DEHP for 48h to assess toxicity. Adults were very healthy following chemical exposure (up to 5120µg L-1), whereas nauplii were severely affected at very low concentrations (48h LC50value of 1.04 ng L-1). Adults exposed to sub-lethal concentrations of DEHP (0.1-0.3µg L-1) or microplastics (10,000-80,000 particles mL-1) exhibited substantial reductions in egg production. Populations were exposed to either microplastics or DEHP for 6 days with 18 days of recovery or for 24 days. Populations exposed to microplastics for 24 days significantly depleted in population size (60±4.1%, p<0.001) relative to controls, whilst populations exposed for only 6 days (with 18 days of recovery) experienced less severe depletions (75±6.0% of control, p<0.05). Populations exposed to DEHP, however, exhibited no recovery and both treatments (6 and 24 days) yielded the same average population size at the termination of the experiment (59±4.9% and 59±3.4% compared to control; p<0.001). These results suggest that DEHP may induce reproductive disorders that can be inherited by subsequent generations. Histone 3 (H3) was significantly (p<0.05) upregulated in both plastic and DEHP treatments after 6 days of exposure, but not after 18 days of recovery. Hsp70-like expression showed to be unresponsive to either DEHP or microplastic exposure. Clearly, microplastics and plasticizers pose a serious threat to zooplankton and potentially to higher trophic levels.

Responses of mixed methanotrophic consortia to variable Cu2+/Fe2+ ratios.

Methane mitigation in landfill top cover soils is mediated by methanotrophs whose optimal methane (CH4) oxidation capacity is governed by environmental and complex microbial community interactions. Optimization of CH4 remediating bio-filters need to take microbial responses into account. Divalent copper (Cu2+) and iron (Fe2+) are present in landfills at variable ratios and play a vital role in methane oxidation capacity and growth of methanotrophs. This study, as a first of its kind, therefore quantified effects of variable Cu2+ and Fe2+ (5:5, 5:25 and 5:50 µM) ratios on mixed methanotrophic communities enriched from landfill top cover (LB) and compost soils (CB). CH4 oxidation capacity, CH4 removal efficiencies, fatty acids content/profiles and polyhydroxybutyrate (PHB; a biopolymer) contents were also analysed to quantify performance and potential co-product development. Mixed methanotroph cultures were raised in 10 L continuous stirred tank reactors (CSTRs, Bioflo® & Celligen® 310 Fermentor/Bioreactor; John Morris Scientific, Chatswood, NSW, Australia). Community structure was determined by amplifying the V3-V4 region of 16s rRNA gene. Community structure and, consequently, fatty acid-profiles changed significantly with increasing Cu2+/Fe2+ ratios, and responses were different for LB and CB. Effects on methane oxidation capacities and PHB content were similar in the LB- and CB-CSTR, decreasing with increasing Cu2+/Fe2+ ratios, while biomass growth was unaffected. In general, high Fe2+ concentration favored growth of the type -II methanotroph Methylosinus in the CB-CSTR, but methanotroph abundances decreased in the LB-CSTR. Increase in Cu2+/Fe2+ ratio increased the growth of Sphingopyxis in both systems, while Azospirllum was co-dominant in the LB- but absent in the CB-CSTR. After 13 days, methane oxidation capacities and PHB content decreased by ∼50% and more in response to increasing Fe2+ concentrations. Although methanotroph abundance was ∼2% in the LB- (compared to >50% in CB-CSTR), methane oxidation capacities were comparable in the two systems, suggesting that methane oxidation capacity was maintained by the dominant Azospirllum and Sphingopyxis in the LB-CSTR. Despite similar methanotroph inoculum community composition and controlled environmental variables, increasing Cu2+/Fe2+ ratios resulted in significantly different microbial community structures in the LB- and CB-CSTR, indicative of complex microbial interactions. In summary, our results suggest that a detailed understanding of allelopathic interactions in mixed methanotrophic consortia is vital for constructing robust bio-filters for CH4 emission abatement.

Microbiome manipulation by corals and other Cnidaria via quorum quenching.

A dynamic mucous layer containing numerous micro-organisms covers the surface of corals and has multiple functions including both removal of sediment and "food gathering."1 It is likely to also act as the primary barrier to infection; various proteins and compounds with antimicrobial activity have been identified in coral mucus, though these are thought to be largely or exclusively of microbial origin. As in Hydra,2 anti-microbial peptides (AMPs) are likely to play major roles in regulating the microbiomes of corals.3,4 Some eukaryotes employ a complementary but less obvious approach to manipulate their associated microbiome by interfering with quorum signaling, effectively preventing bacteria from coordinating gene expression across a population. Our investigation of immunity in the reef-building coral Acropora millepora,5 however, led to the discovery of a coral gene referred to here as AmNtNH1 that can inactivate a range of acyl homoserine lactones (AHLs), common bacterial quorum signaling molecules, and is induced on immune challenge of adult corals and expressed during the larval settlement process. Closely related proteins are widely distributed within the Scleractinia (hard corals) and some other cnidarians, with multiple paralogs in Acropora, but their closest relatives are bacterial, implying that these are products of one or more lateral gene transfer events post-dating the cnidarian-bilaterian divergence. The deployment by corals of genes used by bacteria to compete with other bacteria reflects a mechanism of microbiome manipulation previously unknown in Metazoa but that may apply more generally.

Comprehensive guide to acetyl-carboxylases in algae.

Lipids from microalgae have become an important commodity in the last 20 years, biodiesel and supplementing human diets with ω-3 fatty acids are just two of the many applications. Acetyl-CoA carboxylase (ACCase) is a key enzyme in the lipid synthesis pathway. In general, ACCases consist of four functional domains: the biotin carboxylase (BC), the biotin carboxyl binding protein (BCCP), and α-and ß-carboxyltransferases (α-and ß-CT). In algae, like in plants, lipid synthesis is another function of the chloroplast. Despite being well researched in plants and animals, there is a distinct lack of information about this enzyme in the taxonomically diverse algae. In plastid-containing organisms, ACCases are present in the cytosol and the plastid (chloroplasts) and two different forms exist, the heteromeric (prokaryotic) and homomeric (eukaryotic) form. Despite recognition of the existence of the two ACCase forms, generalized published statements still list the heteromeric form as the one present in algal plastids. In this study, the authors show this is not the case for all algae. The presence of heteromeric or homomeric ACCase is dependent on the origin of plastid. The authors used ACCase amino acid sequence comparisons to show that green (Chlorophyta) and red (Rhodophyta) algae, with the exception of the green algal class Prasinophyceae, contain heteromeric ACCase in their plastids, which are of primary symbiotic origin and surrounded by two envelope membranes. In contrast, algal plastids surrounded by three to four membranes were derived through secondary endosymbiosis (Heterokontophyta and Haptophyta), as well as apicoplast containing Apicomplexa, contain homomeric ACCase in their plastids. Distinctive differences in the substrate binding regions of heteromeric and homomeric α-CT and ß-CT were discovered, which can be used to distinguish between the two ACCase types. Furthermore, the acetyl-CoA binding region of homomeric α-CT can be used to distinguish between cytosolic and plastidial ACCase. The information provided here will be of fundamental importance in ACCase expression and activity research to unravel impacts of environmental and physicochemical parameters on lipid content and productivity.

Development of a real-time PCR (qPCR) method for the identification of the invasive paddle crab Charybdis japonica (Crustacea, Portunidae).

Crabs can be transported beyond their native range via anthropogenic-mediated means such as aquarium trade, live seafood trade and shipping. Once introduced into new locations, they can establish persisting populations and become invasive, often leading to negative impacts on the recipient environment and native species. Molecular techniques are increasingly being used as complementary tools in biosecurity surveillance and monitoring plans for invasive species. Molecular tools can be particularly useful for early detection, rapid identification and discrimination of closely related species, including when diagnostic morphological characters are absent or challenging, such as early life stages, or when only part of the animal is available. In this study, we developed a species-specific qPCR assay, which targets the cytochrome c oxidase subunit 1 (CO1) region of the Asian paddle crab Charybdis japonica. In Australia, as well as many parts of the world, this species is considered invasive and routine biosecurity surveillance is conducted to reduce the risk of establishment. Through rigorous testing of tissue from target and non-target species we demonstrate that this assay is sensitive enough to detect as little as two copies per reaction and does not cross amplify with other closely related species. Field samples and environmental samples spiked with C. japonica DNA in high and low concentrations indicate that this assay is also a promising tool for detecting trace amounts of C. japonica eDNA in complex substrates, making it a useful complementary tool in marine biosecurity assessments.

To Probiotic or Not to Probiotic: A Metagenomic Comparison of the Discharge Gut Microbiome of Infants Supplemented With Probiotics in NICU and Those Who Are Not.

Background: Preterm birth is associated with the development of both acute and chronic disease, and the disruption of normal gut microbiome development. Recent studies have sought to both characterize and understand the links between disease and the microbiome. Probiotic treatment may correct for these microbial imbalances and, in turn, mitigate disease. However, the criteria for probiotic supplementation in NICU's in North Queensland, Australia limits its usage to the most premature (<32 weeks gestation) and small for gestational age infants (<1,500 g). Here we use a combination of amplicon and shotgun metagenomic sequencing to compare the gut microbiome of infants who fulfill the criteria for probiotic-treatment and those who do not. The aims of this study were to determine if probiotic-supplemented preterm infants have significantly different taxonomic and functional profiles when compared to non-supplemented preterm infants at discharge. Methods: Preterm infants were recruited in North Queensland, Australia, with fecal samples collected just prior to discharge (36 ± 0.5 weeks gestation), to capture potential changes that could be probiotic induced. All samples underwent 16S rRNA gene amplicon sequencing, with a subset also used for shotgun metagenomics. Mixed effects models were used to assess the effect of probiotics on alpha diversity, beta diversity and taxonomic abundance, whilst accounting for other known covariates. Results: Mixed effects modeling demonstrated that probiotic treatment had a significant effect on overall community composition (beta diversity), characterized by greater alpha diversity and differing abundances of several taxa, including Bifidobacterium and Lactobacillus, in supplemented infants. Conclusion: Late preterm-infants who go without probiotic-supplementation may be missing out on stabilizing-effects provided through increased alpha diversity and the presence of commensal microbes, via the use of probiotic-treatment. These findings suggest that late-preterm infants may benefit from probiotic supplementation. More research is needed to both understand the consequences of the differences observed and the long-term effects of this probiotic-treatment.

Genome assembly of the Australian black tiger shrimp (Penaeus monodon) reveals a novel fragmented IHHNV EVE sequence.

Shrimp are a valuable aquaculture species globally; however, disease remains a major hindrance to shrimp aquaculture sustainability and growth. Mechanisms mediated by endogenous viral elements have been proposed as a means by which shrimp that encounter a new virus start to accommodate rather than succumb to infection over time. However, evidence on the nature of such endogenous viral elements and how they mediate viral accommodation is limited. More extensive genomic data on Penaeid shrimp from different geographical locations should assist in exposing the diversity of endogenous viral elements. In this context, reported here is a PacBio Sequel-based draft genome assembly of an Australian black tiger shrimp (Penaeus monodon) inbred for 1 generation. The 1.89 Gbp draft genome is comprised of 31,922 scaffolds (N50: 496,398 bp) covering 85.9% of the projected genome size. The genome repeat content (61.8% with 30% representing simple sequence repeats) is almost the highest identified for any species. The functional annotation identified 35,517 gene models, of which 25,809 were protein-coding and 17,158 were annotated using interproscan. Scaffold scanning for specific endogenous viral elements identified an element comprised of a 9,045-bp stretch of repeated, inverted, and jumbled genome fragments of infectious hypodermal and hematopoietic necrosis virus bounded by a repeated 591/590 bp host sequence. As only near complete linear ∼4 kb infectious hypodermal and hematopoietic necrosis virus genomes have been found integrated in the genome of P. monodon previously, its discovery has implications regarding the validity of PCR tests designed to specifically detect such linear endogenous viral element types. The existence of joined inverted infectious hypodermal and hematopoietic necrosis virus genome fragments also provides a means by which hairpin double-stranded RNA could be expressed and processed by the shrimp RNA interference machinery.

Testudines as Sentinels for Monitoring the Dissemination of Antibiotic Resistance in Marine Environments: An Integrative Review.

Dissemination of antibiotic resistance (AR) in marine environments is a global concern with a propensity to affect public health and many ecosystems worldwide. We evaluated the use of sea turtles as sentinel species for monitoring AR in marine environments. In this field, antibiotic-resistant bacteria have been commonly identified by using standard culture and sensitivity tests, leading to an overrepresentation of specific, culturable bacterial classes in the available literature. AR was detected against all major antibiotic classes, but the highest cumulative global frequency of resistance in all represented geographical sites was against the beta-lactam class by a two-fold difference compared to all other antibiotics. Wastewater facilities and turtle rehabilitation centres were associated with higher incidences of multidrug-resistant bacteria (MDRB) accounting for an average of 58% and 49% of resistant isolates, respectively. Furthermore, a relatively similar prevalence of MDRB was seen in all studied locations. These data suggest that anthropogenically driven selection pressures for the development of AR in sea turtles and marine environments are relatively similar worldwide. There is a need, however, to establish direct demonstrable associations between AR in sea turtles in their respective marine environments with wastewater facilities and other anthropogenic activities worldwide.

Methods for exploring the faecal microbiome of premature infants: a review.

The premature infant gut microbiome plays an important part in infant health and development, and recognition of the implications of microbial dysbiosis in premature infants has prompted significant research into these issues. The approaches to designing investigations into microbial populations are many and varied, each with its own benefits and limitations. The technique used can influence results, contributing to heterogeneity across studies. This review aimed to describe the most common techniques used in researching the preterm infant microbiome, detailing their various limitations. The objective was to provide those entering the field with a broad understanding of available methodologies, so that the likely effects of their use can be factored into literature interpretation and future study design. We found that although many techniques are used for characterising the premature infant microbiome, 16S rRNA short amplicon sequencing is the most common. 16S rRNA short amplicon sequencing has several benefits, including high accuracy, discoverability and high throughput capacity. However, this technique has limitations. Each stage of the protocol offers opportunities for the injection of bias. Bias can contribute to variability between studies using 16S rRNA high throughout sequencing. Thus, we recommend that the interpretation of previous results and future study design be given careful consideration.

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.

Next Generation Sequencing of Single Nucleotide Polymorphic DNA-Markers in Selecting for Intramuscular Fat, Fat Melting Point, Omega-3 Long-Chain Polyunsaturated Fatty Acids and Meat Eating Quality in Tattykeel Australian White MARGRA Lamb.

Meat quality data can only be obtained after slaughter when selection decisions about the live animal are already too late. Carcass estimated breeding values present major precision problems due to low accuracy, and by the time an informed decision on the genetic merit for meat quality is made, the animal is already dead. We report for the first time, a targeted next-generation sequencing (NGS) of single nucleotide polymorphisms (SNP) of lipid metabolism genes in Tattykeel Australian White (TAW) sheep of the MARGRA lamb brand, utilizing an innovative and minimally invasive muscle biopsy sampling technique for directly quantifying the genetic worth of live lambs for health-beneficial omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA), intramuscular fat (IMF), and fat melting point (FMP). NGS of stearoyl-CoA desaturase (SCD), fatty acid binding protein-4 (FABP4), and fatty acid synthase (FASN) genes identified functional SNP with unique DNA marker signatures for TAW genetics. The SCD g.23881050T>C locus was significantly associated with IMF, C22:6n-3, and C22:5n-3; FASN g.12323864A>G locus with FMP, C18:3n-3, C18:1n-9, C18:0, C16:0, MUFA, and FABP4 g.62829478A>T locus with IMF. These add new knowledge, precision, and reliability in directly making early and informed decisions on live sheep selection and breeding for health-beneficial n-3 LC-PUFA, FMP, IMF and superior meat-eating quality at the farmgate level. The findings provide evidence that significant associations exist between SNP of lipid metabolism genes and n-3 LC-PUFA, IMF, and FMP, thus underpinning potential marker-assisted selection for meat-eating quality traits in TAW lambs.

Growth, lipid content, productivity, and fatty acid composition of tropical microalgae for scale-up production.

Biomass and lipid productivity, lipid content, and quantitative and qualitative lipid composition are critical parameters in selecting microalgal species for commercial scale-up production. This study compares lipid content and composition, and lipid and biomass productivity during logarithmic, late logarithmic, and stationary phase of Nannochloropsis sp., Isochrysis sp., Tetraselmis sp., and Rhodomonas sp. grown in L1-, f/2-, and K-medium. Of the tested species, Tetraselmis sp. exhibited a lipid productivity of 3.9-4.8 g m(-2) day(-1) in any media type, with comparable lipid productivity by Nannochloropsis sp. and Isochrysis sp. when grown in L1-medium. The dry biomass productivity of Tetraselmis sp. (33.1-45.0 g m(-2) day(-1)) exceeded that of the other species by a factor 2-10. Of the organisms studied, Tetraselmis sp. had the best dry biomass and/or lipid production profile in large-scale cultures. The present study provides a practical benchmark, which allows comparison of microalgal production systems with different footprints, as well as terrestrial systems.

Resolving hemocyanin isoform complexity in haemolymph of black tiger shrimp Penaeus monodon - implications in aquaculture, medicine and food safety.

Hemocyanin (Hc) is a multifunctional macromolecule involved in oxygen transport and non-specific immunity in shrimp. Hc is crucial in physiology and nutrition linked with optimal performance in aquaculture production systems. In medicine, Hc has been approved for clinical use in humans as adjuvant and anticancer therapeutic. In contrast, Hc has also been identified as one of the proteins causing anaphylaxis following shrimp consumption. The role of individual Hc isoforms remains unknown due to a lack of resolved Hc isoforms. We successfully identified eleven different Penaeus monodon hemocyanin (PmoHc) γ isoforms including two truncated isoforms (50 and 20 kDa) and one PmoHc ß isoform in haemolymph using proteomics informed by transcriptomics. Amino acid sequence homology ranged from 24 to 97% between putative PmoHc gene isoforms. Hc isoforms showed specific patterns of transcript expression in shrimp larval stages and adult hepatopancreas. These findings enable isoform level investigations aiming to define molecular mechanisms underpinning Hc functionality in shrimp physiology and immunity, as well as their individual immunogenic role in human allergy. Our research demonstrates the power of proteomics informed by transcriptomics to resolve isoform complexity in non-model organisms and lay the foundations for improved performance within the aquaculture industry and advance allergenic applications in medicine. SIGNIFICANCE: The roles of hemocyanin (Hc) in shrimp homeostasis and immunity as well as in human allergy are not well understood because the complexity of Hc isoforms has remained unresolved. Our results have confirmed the existence of at least 12 individual Hc isoforms in shrimp haemolymph and validated putative Hc gene assemblies from transcriptomics. Our findings will enable monitoring the expression of specific Hc isoforms in shrimp haemolymph during different environmental, nutritional and pathogenic conditions, thus providing insights into isoform specific functional roles. In medicine, the potential allergenicity of each Hc isoform could be determined and advance allergenic applications. Lastly, since Hc comprises up to 95% of the total protein in haemolymph, these isoforms become ideal targets for prawn provenance, traceability and food contamination studies.