Morphological and molecular characterization of Isospora elliotae n. sp. (Apicomplexa: Eimeriidae) from the Australian magpie Gymnorhina tibicen (Latham, 1801) (Passeriformes: Artamidae) in Western Australia.

A new coccidian species, Isospora elliotae n. sp., from the Australian magpie Gymnorhina tibicen (Latham, 1801) in Western Australia, is described and characterized morphologically and molecularly. Microscopic analysis of a faecal sample identified subspheroidal oocysts (n = 20), 20-22 × 18-20 (20.7 × 18.7); length/width (L/W) ratio 1.05-1.14 (1.10). Wall bi-layered, 1.0-1.3 (1.2) thick, outer layer smooth, c. 2/3 of total thickness. Micropyle and oocyst residuum absent, but usually two polar granules are present. Sporocysts (n = 28) ovoidal, 12-13 × 9-11 (12.6 × 9.7); L/W ratio 1.22-1.35 (1.30). Stieda body present, flattened to half-moon-shaped, c. 0.5 deep × 2.0 wide; sub-Stieda indistinct or barely discernible, c. 1.0 deep × 2.5 wide; para-Stieda body absent; sporocyst residuum present, composed of granules dispersed among the sporozoites. Sporozoites vermiform, with anterior and posterior refractile bodies and nucleus. Segments of three gene loci (18S rRNA, 28S rRNA and COI) were sequenced and I. elliotae n. sp. exhibited 99.8% genetic similarity to Isospora sp. MAH-2013a (KF648870) followed by 99.7% genetic similarity to Isospora neochmiae (Yang, Brice & Ryan, 2016) (KT224380) at the 18S rRNA gene locus. It shared 97.0% genetic similarity with an unnamed Isospora sp. (AY283852) at the 28S rRNA gene locus and it also shared the highest genetic similarity of 99.8% with the unnamed Isospora sp. from an American crow (OL999120) at the COI gene locus. Based on morphological and molecular data, this isolate is a new species named as I. elliotae n. sp.

The putative vacuolar processing enzyme gene TaVPE3cB is a candidate gene for wheat stem pith-thickness.

KEY MESSAGE: The vacuolar processing enzyme gene TaVPE3cB is identified as a candidate gene for a QTL of wheat pith-thickness on chromosome 3B by BSR-seq and differential expression analyses. The high pith-thickness (PT) of the wheat stem could greatly enhance stem mechanical strength, especially the basal internodes which support the heavier upper part, such as upper stems, leaves and spikes. A QTL for PT in wheat was previously discovered on 3BL in a double haploid population of 'Westonia' × 'Kauz'. Here, a bulked segregant RNA-seq analysis was applied to identify candidate genes and develop associated SNP markers for PT. In this study, we aimed at screening differentially expressed genes (DEGs) and SNPs in the 3BL QTL interval. Sixteen DEGs were obtained based on BSR-seq and differential expression analyses. Twenty-four high-probability SNPs in eight genes were identified by comparing the allelic polymorphism in mRNA sequences between the high PT and low PT samples. Among them, six genes were confirmed to be associated with PT by qRT-PCR and sequencing. A putative vacuolar processing enzyme gene TaVPE3cB was screened out as a potential PT candidate gene in Australian wheat 'Westonia'. A robust SNP marker associated with TaVPE3cB was developed, which can assist in the introgression of TaVPE3cB.b in wheat breeding programs. In addition, we also discussed the function of other DEGs which may be related to pith development and programmed cell death (PCD). A five-level hierarchical regulation mechanism of stem pith PCD in wheat was proposed.

Morphological and molecular characterization of a novel eimerian species (Apicomplexa: Eimeriidae) from the Australian pelican Pelecanus conspicillatus Temminck, 1824 (Pelecaniformes: Pelecanidae) in Western Australia.

A new Eimeria Schneider, 1875 species is described from an Australian pelican Pelecanus conspicillatus Temminck, 1824 in Western Australia. Sporulated oocysts (n = 23) subspheroidal, 33-35 × 31-33 (34.1 × 32.0) µm; length/width (L/W) ratio 1.0-1.1 (1.07). Wall bi-layered, 1.2-1.5 (1.4) µm thick, outer layer smooth, c.2/3 of total thickness. Micropyle absent, but 2 or 3 polar granules surrounded by a thin membrane, apparently residual, are present. Sporocysts (n = 23) elongate ellipsoidal or capsule shaped, 19-20 × 5-6 (19.5 × 5.6) µm; L/W ratio 3.4-3.8 (3.51). Stieda body vestigial and barely discernible, 0.5 × 1.0 µm; sub-Stieda and para-Stieda bodies absent; sporocyst residuum present, composed of a few dense spherules dispersed among the sporozoites. Sporozoites with robust anterior and posterior refractile bodies and centrally located nucleus. Molecular analysis was conducted at three loci; the 18S and 28S ribosomal RNA genes and the cytochrome c oxidase subunit I (COI) gene. At the 18S locus, the new isolate shared 98.6% genetic similarity with Eimeria fulva Farr, 1953 (KP789172), which was identified from a goose in China. At the 28S locus, the new isolate shared the highest similarity of 96.2% with Eimeria hermani Farr, 1953 (MW775031) identified from a whooper-swan (Cygnus cygnus (Linnaeus, 1758)) in China. At the COI gene locus, this new isolate was most closely related to Isospora sp. isolate COI-178 and Eimeria tiliquae [25,26], presented 96.5% and 96.2% genetic similarity, respectively. Based on the morphological and molecular data, this isolate is a new species of coccidian parasite, which is named Eimeria briceae n. sp.

Next-generation sequencing amplicon analysis of the genetic diversity of Eimeria populations in livestock and wildlife samples from Australia.

Eimeria is an important coccidian enteric parasite that infects a wide range of hosts and can cause substantial economic losses in the poultry and livestock industries. It is common for multiple Eimeria species to infect individual hosts, and this can make species identification difficult due to morphological similarities between species and mixed chromatograms when using Sanger sequencing. Relatively few studies have applied next-generation amplicon sequencing (NGS) to determining the genetic diversity of Eimeria species in different hosts. The present study screened 408 faecal samples from a range of hosts including livestock and wildlife using a previously developed quantitative polymerase chain reaction (qPCR) at the 18S locus and conducted amplicon NGS on the positives using a ~ 455-bp fragment of the 18S locus. A total of 41 positives (10.1%) were identified by qPCR from various hosts and NGS was successful for 38 of these positives. Fifteen Eimeria species and three genotypes were detected by NGS: E. ferrisi, E. kanyana, E. potoroi, E. quokka, E. setonicis, E. trichosuri, E. reichenowi, E. angustus, E. ahsata, E. auburnensis, E. bovis, E. brasiliensis, E. christenseni, E. crandallis, E. ovinoidalis, Eimeria sp. (JF419345), Eimeria sp. (JF419349) and Eimeria sp. (JF419351). Mixed infections were detected in 55.3% (21/38) of positive samples. The most striking finding was the identification of the same species in different hosts. This could be due to contamination and/or mechanical transmission or may provide support for previous studies suggesting that Eimeria species can infect not just closely related hosts but different genera and further research is required. This is also the first study to audit Eimeria populations in livestock (sheep and cattle) by NGS and could be applied in the future to determine the extent of pathogenic species and outcomes of Eimeria control strategies.

Systemic long-term metabolic effects of acute non-severe paediatric burn injury.

A growing body of evidence supports the concept of a systemic response to non-severe thermal trauma. This provokes an immunosuppressed state that predisposes paediatric patients to poor recovery and increased risk of secondary morbidity. In this study, to understand the long-term systemic effects of non-severe burns in children, targeted mass spectrometry assays for biogenic amines and tryptophan metabolites were performed on plasma collected from child burn patients at least three years post injury and compared to age and sex matched non-burn (healthy) controls. A panel of 12 metabolites, including urea cycle intermediates, aromatic amino acids and quinolinic acid were present in significantly higher concentrations in children with previous burn injury. Correlation analysis of metabolite levels to previously measured cytokine levels indicated the presence of multiple cytokine-metabolite associations in the burn injury participants that were absent from the healthy controls. These data suggest that there is a sustained immunometabolic imprint of non-severe burn trauma, potentially linked to long-term immune changes that may contribute to the poor long-term health outcomes observed in children after burn injury.

Eimeria spp. and Tyzzeria perniciosa Allen, 1936 (Apicomplexa: Eimeriidae) from a Pacific black duck, Anas superciliosa Gmelin (Aves: Anseriformes), in western Australia.

Four species of the Eimeriidae, Eimeria anatis Scholtyseck, 1955, Eimeria aythyae Farr, 1965, Eimeria krylovi Svanbaev & Rakhmatullina, 1967 and Tyzzeria perniciosa Allen, 1936, were morphologically identified from oöcysts recovered from a Pacific black duck, Anas superciliosa Gmelin. Additionally, genotypic characterization of E. anatis is provided via sequencing of the mitochondrial cytochrome c oxidase subunit 1 (cox1) and the small subunit ribosomal RNA (18S) genes. The four species are redescribed, providing additional morphological details. The validity of genera and coccidian species parasitizing birds of the order Anseriformes such as Wenyonella Hoare, 1933 and some Tyzzeria spp. are discussed. Molecular phylogenetic analyses for the cox1 and 18S rRNA genes resulted in monophylies of Eimeria spp. from Anseriformes which included the sequences obtained from E. anatis oöcysts.

Yield-Related QTL Clusters and the Potential Candidate Genes in Two Wheat DH Populations.

In the present study, four large-scale field trials using two doubled haploid wheat populations were conducted in different environments for two years. Grain protein content (GPC) and 21 other yield-related traits were investigated. A total of 227 QTL were mapped on 18 chromosomes, which formed 35 QTL clusters. The potential candidate genes underlying the QTL clusters were suggested. Furthermore, adding to the significant correlations between yield and its related traits, correlation variations were clearly shown within the QTL clusters. The QTL clusters with consistently positive correlations were suggested to be directly utilized in wheat breeding, including 1B.2, 2A.2, 2B (4.9-16.5 Mb), 2B.3, 3B (68.9-214.5 Mb), 4A.2, 4B.2, 4D, 5A.1, 5A.2, 5B.1, and 5D. The QTL clusters with negative alignments between traits may also have potential value for yield or GPC improvement in specific environments, including 1A.1, 2B.1, 1B.3, 5A.3, 5B.2 (612.1-613.6 Mb), 7A.1, 7A.2, 7B.1, and 7B.2. One GPC QTL (5B.2: 671.3-672.9 Mb) contributed by cultivar Spitfire was positively associated with nitrogen use efficiency or grain protein yield and is highly recommended for breeding use. Another GPC QTL without negatively pleiotropic effects on 2A (50.0-56.3 Mb), 2D, 4D, and 6B is suggested for quality wheat breeding.

Morphological and molecular characterization of Isospora amphiboluri (Apicomplexa: Eimeriidae), a coccidian parasite, in a central netted dragon (Ctenophorus nuchalis) (De Vis, 1884) in Australia.

An Isospora species, Isospora amphiboluri, originally described by Canon in 1967 and later by McAllister et al. (1995), was isolated from a central netted dragon (Ctenophorus nuchalis) housed at a wildlife rehabilitation centre in Perth, Western Australia. Sporulated oocysts of Isospora amphiboluri (n = 30) are spherical, 24.2 (26.5-23.0) µm in length and 23.9 (22.4-25.9) µm in width, with a shape index of 1.01. The bilayered oocyst wall is smooth and light-yellow in color. Polar granule, oocyst residuum and micropyle are absent. The sporocysts are lemon-shaped, 15.7 (15.2-18.0) × 10.2 (8.9-11.2) µm, with a shape index (length/width) of 1.53. Stieda and substieda bodies are present, the Stieda body being small and hemidome-shaped and the substieda half-moon-shaped. Each sporocyst contains four vermiform sporozoites arranged head to tail. The sporozoites are 11.7 (9.9-16.2) × 3.0 (2.4-3.5) µm, with a shape index (length/width) of 3.87. A sporocyst residuum is present. Sporozoites contain a central nucleus with a finely distributed granular residuum. Comparison of oocyst measurements and their features with other valid Isospora species from hosts in the Agamid family confirmed that this Isospora species is Isospora amphiboluri. Molecular characterization of I. amphiboluri at the 18S rRNA and MTCOI loci showed the highest similarity with I. amphiboluri from the central bearded dragon, 99.8% and 99.7% respectively. This is the first report of I. amphiboluri from a central netted dragon in Australia.

Incomplete Systemic Recovery and Metabolic Phenoreversion in Post-Acute-Phase Nonhospitalized COVID-19 Patients: Implications for Assessment of Post-Acute COVID-19 Syndrome.

We present a multivariate metabotyping approach to assess the functional recovery of nonhospitalized COVID-19 patients and the possible biochemical sequelae of "Post-Acute COVID-19 Syndrome", colloquially known as long-COVID. Blood samples were taken from patients ca. 3 months after acute COVID-19 infection with further assessment of symptoms at 6 months. Some 57% of the patients had one or more persistent symptoms including respiratory-related symptoms like cough, dyspnea, and rhinorrhea or other nonrespiratory symptoms including chronic fatigue, anosmia, myalgia, or joint pain. Plasma samples were quantitatively analyzed for lipoproteins, glycoproteins, amino acids, biogenic amines, and tryptophan pathway intermediates using Nuclear Magnetic Resonance (NMR) spectroscopy and mass spectrometry. Metabolic data for the follow-up patients (n = 27) were compared with controls (n = 41) and hospitalized severe acute respiratory syndrome SARS-CoV-2 positive patients (n = 18, with multiple time-points). Univariate and multivariate statistics revealed variable patterns of functional recovery with many patients exhibiting residual COVID-19 biomarker signatures. Several parameters were persistently perturbed, e.g., elevated taurine (p = 3.6 × 10-3 versus controls) and reduced glutamine/glutamate ratio (p = 6.95 × 10-8 versus controls), indicative of possible liver and muscle damage and a high energy demand linked to more generalized tissue repair or immune function. Some parameters showed near-complete normalization, e.g., the plasma apolipoprotein B100/A1 ratio was similar to that of healthy controls but significantly lower (p = 4.2 × 10-3) than post-acute COVID-19 patients, reflecting partial reversion of the metabolic phenotype (phenoreversion) toward the healthy metabolic state. Plasma neopterin was normalized in all follow-up patients, indicative of a reduction in the adaptive immune activity that has been previously detected in active SARS-CoV-2 infection. Other systemic inflammatory biomarkers such as GlycA and the kynurenine/tryptophan ratio remained elevated in some, but not all, patients. Correlation analysis, principal component analysis (PCA), and orthogonal-partial least-squares discriminant analysis (O-PLS-DA) showed that the follow-up patients were, as a group, metabolically distinct from controls and partially comapped with the acute-phase patients. Significant systematic metabolic differences between asymptomatic and symptomatic follow-up patients were also observed for multiple metabolites. The overall metabolic variance of the symptomatic patients was significantly greater than that of nonsymptomatic patients for multiple parameters (χ2p = 0.014). Thus, asymptomatic follow-up patients including those with post-acute COVID-19 Syndrome displayed a spectrum of multiple persistent biochemical pathophysiology, suggesting that the metabolic phenotyping approach may be deployed for multisystem functional assessment of individual post-acute COVID-19 patients.

Morphological and molecular description of a new species of Isospora (Apicomplexa) from a New Holland honeyeater (Phylidonyris novaehollandiae).

A new Isospora species is described from New Holland honeyeaters (Phylidonyris novaehollandiae). Sporulated oocysts (n = 25) were characterised as subspheroidal, 29-32 × 28-31 (29.8 × 29.4); length/width (L/W) ratio 1.01-1.02 (1.01). Wall bi-layered, 1.3-1.6 (1.5) thick, outer layer smooth, c.2/3 of total thickness. Micropyle and oocyst residuum absent, but usually two polar granules are present. Sporocysts (n = 25) ovoidal, 18-19 × 12-14 (18.4 × 12.3); L/W ratio 1.42-1.53 (1.50). Stieda body present, flattened, c.0.5 deep × 2.5 wide; sub-Stieda present, rounded, c.2.5 deep × 3.5 wide; para-Stieda body absent; sporocyst residuum present, usually a distinctly irregular body consisting of numerous small granules that appear to be membrane-bound. Sporozoites vermiform, with robust anterior and posterior refractile bodies. Molecular characterization was conducted at the 18S and 28S ribosomal RNA and the mitochondrial (mt) cytochrome oxidase (COI) loci. Phylogenetic analysis of genomic 18S and mt COI sequences indicated that Isospora phylidonyrisae n. sp. was genetically similar to Isospora coronoideae, isolated from an Australian raven (Corvus coronoides) in Western Australia, with a 99.3% and 98.4% homology, respectively. The 28S rRNA sequence was most similar to Isospora anthochaerae (KF766053) and Isospora manorinae (KT224381), both with a 98.2% genetic similarity. Based on morphological and genetic data, this isolate is a new species of Isospora, which is named Isospora phylidonyrisae n. sp. after its host.

Systemic Perturbations in Amine and Kynurenine Metabolism Associated with Acute SARS-CoV-2 Infection and Inflammatory Cytokine Responses.

We performed quantitative metabolic phenotyping of blood plasma in parallel with cytokine/chemokine analysis from participants who were either SARS-CoV-2 (+) (n = 10) or SARS-CoV-2 (-) (n = 49). SARS-CoV-2 positivity was associated with a unique metabolic phenotype and demonstrated a complex systemic response to infection, including severe perturbations in amino acid and kynurenine metabolic pathways. Nine metabolites were elevated in plasma and strongly associated with infection (quinolinic acid, glutamic acid, nicotinic acid, aspartic acid, neopterin, kynurenine, phenylalanine, 3-hydroxykynurenine, and taurine; p < 0.05), while four metabolites were lower in infection (tryptophan, histidine, indole-3-acetic acid, and citrulline; p < 0.05). This signature supports a systemic metabolic phenoconversion following infection, indicating possible neurotoxicity and neurological disruption (elevations of 3-hydroxykynurenine and quinolinic acid) and liver dysfunction (reduction in Fischer's ratio and elevation of taurine). Finally, we report correlations between the key metabolite changes observed in the disease with concentrations of proinflammatory cytokines and chemokines showing strong immunometabolic disorder in response to SARS-CoV-2 infection.

Morphological and genetic characterization of the first Isospora species (I. lugensae n. sp.) from a Kerguelen petrel (Lugensa brevirostris).

A new coccidian species, Isospora lugensae n. sp., was described from a single Kerguelen petrel (Lugensa brevirostris). Sporulated oocysts (n = 25) were characterized as subspheroidal to ellipsoidal measuring 24-25 µm × 21-23 µm (24.8 × 22.2 µm) in length/width (L/W), respectively, with a ratio of 1.07-1.14 µm (1.12). They contained a bi-layered wall with a thickness of 0.8-1.2 µm (1.0) and the outer layer smooth, with c.2/3 of total thickness. The oocyst contained two polar granules with both micropyle and oocyst residuum absent. Ovoidal sporocysts (n = 25) measured 15-16 µm × 10-11 µm (15.7 × 10.8 µm) in L/W, with a ratio of 1.41-1.49 µm (1.46). A flattened to knob-like Stieda body was present (c.0.5 µm deep × 2.5 µm wide) as well as a rounded to trapezoidal sub-Stieda (c.1.5 µm deep × 3.0 µm wide); however, no para-Stieda body was detected. The sporocyst residuum was composed of scattered spherules of different sizes, while vermiform sporozoites contained a refractile body, nucleus and visible striations. Analysis of the full-length mitochrondrial (mtDNA) genome revealed 3 protein-coding genes, (CytB, COI and COIII), 18 LSU and 14 small subunit (SSU) rDNA fragments, without transfer RNA genes with a total length of 6257 bp. Phylogenetic analysis of genomic SSU ribosomal sequences indicated that Isospora lugensae n. sp. is genetically similar to Eimeria reichenowi, isolated from a red-crowned crane (Grus japonensis) from Japan, with a 96.6% homology. The mtDNA sequence is most similar to Isospora serinuse with a 95.8% genetic similarity. Based on morphological and molecular data, this isolate is a new species of coccidian parasite that to date has only been found in a Kerguelen petrel.

A simultaneous exploratory and quantitative amino acid and biogenic amine metabolic profiling platform for rapid disease phenotyping via UPLC-QToF-MS.

Metabolic phenotyping using mass spectrometry (MS) is being applied to ever increasing sample numbers in clinical and epidemiology studies. High-throughput and robust methods are being developed for the accurate measurement of metabolites associated with disease. Traditionally, quantitative assays have utilized triple quadrupole (QQQ) MS based methods; however, the use of such focused methods removes the ability to perform discovery-based metabolic phenotyping. An integrated workflow for the hybrid simultaneous quantification of 34 biogenic amines in combination with full scan high-resolution accurate mass (HRAM) exploratory metabolic phenotyping is presented. Primary and secondary amines are derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate prior to revered-phase liquid chromatographic separation and mass spectrometric detection. Using the HRAM-MS data, retrospective phenotypic data mining could be performed, demonstrating the versatility of HRAM-MS instrumentation in a clinical and molecular epidemiological environment. Quantitative performance was assessed using two MS detector platforms: Waters TQ-XS (QQQ; n = 3) and Bruker Impact II QToF (HRAMS-MS; n = 2) and three human biofluids (plasma, serum and urine). Finally, each platform was assessed using a certified external reference sample (NIST SRM 1950 plasma). Intra- and inter-day accuracy and precision were comparable between the QQQ and QToF instruments (<15%), with excellent linearity (R2 > 0.99) over the quantification range of 1-400 µmol L-1. Quantitative values were comparable across all instruments for human plasma, serum and urine samples, and calculated concentrations were verified against certified reference values for NIST SRM 1950 plasma as an external reference. As a real-life biological exemplar, the method was applied to plasma samples obtained from SARS-CoV-2 positive patients versus healthy controls. Both the QQQ and QToF approaches were equivalent in being able to correctly classify SARS-CoV-2 positivity. Critically, the use of HRAM full scan data was also assessed for retrospective exploratory mining of data to extract additional biogenic amines of biomarker interest beyond the 34 quantified targets.

Morphological and molecular characterization of a new species of Isospora Schneider, 1881 (Apicomplexa: Eimeriidae) from the western wattlebird Anthochaera lunulata Gould in Western Australia.

A new coccidian species, Isospora lunulatae n. sp., from the western wattlebird Anthochaera lunulata Gould in Western Australia is described and characterised molecularly. Microscopic analysis of a faecal sample identified subspheroidal oöcysts measuring 27-34 × 26-31 (30.6 × 29.4) µm (n = 20), with a length/width (L/W) ratio of 1.0-1.1 (1.0). Oöcysts have a bi-layered wall, 0.9-1.2 (1.0) µm thick; the outer layer is smooth, representing c.2/3 of total thickness. Micropyle and oöcyst residuum are both absent, but a polar granule is present. Sporocysts are ovoidal, 17-19 × 10-12 (18.3 × 10.7) µm, with a L/W ratio of 1.6-1.8 (1.7) and occupying about 21% of the area (each one) within the oöcyst. Stieda body is flattened to rounded, measuring on average 0.9 × 1.8 µm; sub-Stieda body is rounded to rectangular, measuring on average 1.5 × 2.6 µm; para-Stieda body is absent. Sporocyst residuum has an irregular shape consisting of numerous granules and appears membrane-bound. Sporozoites are vermiform 12.8 × 3.0 µm on average, with prominent striations at the more pointed end and two refractile bodies below striations. Segments of three gene loci (18S rRNA, 28S rRNA and cox1) were sequenced and I. lunulatae n. sp. exhibited 99.6% genetic similarity to Isospora phylidonyrisae Yang, Brice, Berto & Ryan, 2021 at the 18S rRNA gene locus, 99.8% genetic similarity to Isospora anthochaerae Yang, Brice & Ryan, 2014 and shared a 98.1% genetic similarity with Isospora manorinae Yang, Brice, Jian & Ryan, 2016 at the cox1 gene locus. Morphological and molecular data support the distinct species status of the new species.

Transcriptomic Study for Identification of Major Nitrogen Stress Responsive Genes in Australian Bread Wheat Cultivars.

High nitrogen use efficiency (NUE) in bread wheat is pivotal to sustain high productivity. Knowledge about the physiological and transcriptomic changes that regulate NUE, in particular how plants cope with nitrogen (N) stress during flowering and the grain filling period, is crucial in achieving high NUE. Nitrogen response is differentially manifested in different tissues and shows significant genetic variability. A comparative transcriptome study was carried out using RNA-seq analysis to investigate the effect of nitrogen levels on gene expression at 0 days post anthesis (0 DPA) and 10 DPA in second leaf and grain tissues of three Australian wheat (Triticum aestivum) varieties that were known to have varying NUEs. A total of 12,344 differentially expressed genes (DEGs) were identified under nitrogen stress where down-regulated DEGs were predominantly associated with carbohydrate metabolic process, photosynthesis, light-harvesting, and defense response, whereas the up-regulated DEGs were associated with nucleotide metabolism, proteolysis, and transmembrane transport under nitrogen stress. Protein-protein interaction and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis further revealed that highly interacted down-regulated DEGs were involved in light-harvesting and photosynthesis, and up-regulated DEGs were mostly involved in steroid biosynthesis under N stress. The common down-regulated genes across the cultivars included photosystem II 10 kDa polypeptide family proteins, plant protein 1589 of uncharacterized protein function, etc., whereas common up-regulated genes included glutamate carboxypeptidase 2, placenta-specific8 (PLAC8) family protein, and a sulfate transporter. On the other hand, high NUE cultivar Mace responded to nitrogen stress by down-regulation of a stress-related gene annotated as beta-1,3-endoglucanase and pathogenesis-related protein (PR-4, PR-1) and up-regulation of MYB/SANT domain-containing RADIALIS (RAD)-like transcription factors. The medium NUE cultivar Spitfire and low NUE cultivar Volcani demonstrated strong down-regulation of Photosystem II 10 kDa polypeptide family protein and predominant up-regulation of 11S globulin seed storage protein 2 and protein transport protein Sec61 subunit gamma. In grain tissue, most of the DEGs were related to nitrogen metabolism and proteolysis. The DEGs with high abundance in high NUE cultivar can be good candidates to develop nitrogen stress-tolerant variety with improved NUE.

Morphological and genetic characterization of Eimeria chalcoptereae n. sp. (Apicomplexa: Eimeriidae) in a common bronzewing pigeon (Phaps chalcoptera) (Latham, 1790) in Western Australia.

A new Eimeria species is described from a common bronzewing pigeon (Phaps chalcoptera) (Latham, 1790) in Western Australia. Sporulated oocysts of Eimeria chalcoptereae n. sp. (n = 30) are subspheroidal, 22-25 × 21-24 (23.5 × 22.6) µm; length/width (L/W) ratio 1.0-1.1 (1.04) µm. Wall bi-layered, 1.0-1.4 (1.2) µm thick, outer layer smooth, c.2/3 of total thickness. Micropyle barely discernible. Oocyst residuum is absent, but 2 to 3 small polar granules are present. Sporocysts (n = 30) ellipsoidal, 13-14 × 7-8 (13.5 × 7.2) µm; L/W ratio 1.8-2.0 (1.88). Stieda body present, flattened to half-moon-shaped, 0.5 × 2.0 µm; sub-Stieda present, rounded to trapezoidal, 1.5 × 2.5 µm; para-Stieda body absent; sporocyst residuum present, usually as an irregular body consisting of numerous small granules that appear to be membrane-bound. Sporozoites vermiform, with a robust refractile body and centrally located nucleus. Isolated Eimeria oocysts were analysed at the 18S and 28S ribosomal RNA and the mitochondrial cytochrome oxidase (COI) loci. Analyses revealed that Eimeria chalcoptereae n. sp. shared the highest number of molecular features with an Eimeria sp. previously identified from a domestic pigeon in Australia (KT305927-29), with similarities at these three loci of 98.53%, 97.32% and 94.93%, respectively. According to morphological and molecular analysis, the isolated coccidian parasite is a new species of Eimeria named Eimeria chalcoptereae n. sp. after its host, the common bronzewing pigeon (Phaps chalcoptera) (Columbiformes: Columbidae) (Latham, 1790).

COCCIDIOSIS IN GREEN TURTLES (CHELONIA MYDAS) IN AUSTRALIA: PATHOGENESIS, SPATIAL AND TEMPORAL DISTRIBUTION, AND CLIMATE-RELATED DETERMINANTS OF DISEASE OUTBREAKS.

An epizootic of coccidiosis in free-ranging green turtles (Chelonia mydas) occurred in Australia in 1991 and the parasites were thought to be Caryospora cheloniae. Recurring outbreaks over an increased geographic range followed. We used medical records and temporal and spatial data of turtles diagnosed with coccidiosis between 1991 and 2014 to characterize the disease and factors associated with outbreaks. Most affected animals were subadults or older. Neurologic signs with intralesional cerebral coccidia were observed. Coccidia associated with inflammation and necrosis were predominantly found in the intestine, brain, kidney, and thyroid. Cases occurred in the spring and summer. Three major outbreaks (1991, 2002, and 2014) were concentrated in Port Stephens, New South Wales (NSW) and Moreton Bay, Queensland, but cases occurred as far south as Sydney, NSW. Coccidiosis cases were more likely during, or 1 mo prior to, El Niño-like events. Molecular characterization of the 18S rRNA locus of coccidia from tissues of 10 green turtles collected in 2002 and 2004 in Port Stevens and Sydney imply that they were Schellackia-like organisms. Two genotypes were identified. The Genotype 3 sequence was most common (in eight of 10 turtles), with 98.8% similarity to the 18S sequence of Schellackia orientalis. The Genotype 4 sequence was less common (in two of 10 turtles) with 99.7% similarity to the 18S sequence of the most common genotype (Genotype 1) detected in turtles from the 2014 Moreton Bay outbreak. Our study will help with the identification and management of future outbreaks and provide tools for identification of additional disease patterns in green turtles.

Molecular and morphological analysis of a Caryospora-like isolate (Apicomplexa: Eimeriidae) from the magpie-lark (Grallina cyanoleuca) (Latham, 1801) in Western Australia.

A new Caryospora-like isolate is described from a magpie-lark (Grallina cyanoleuca) in Western Australia. Sporulated oocysts of the Caryospora-like isolate (n = 35) are subspherical with a shape index of 1.13 ((21.5 (19.7-23.6) × 19.0 (18.1-19.8) µm). The bilayered oocyst wall is smooth. Micropyle, polar granule and oocyst residuum are absent. The sporocyst is ellipsoidal, 18.9 (17.2-20.8) × 12.3 (11.9-12.8) µm, with a shape index (length/width) of 1.54. The sporocyst wall is bilayered. Stieda and substieda bodies are present, the Stieda body is small and flattened and the substieda is trapezoidal. Sporocyst with eight sporozoites arranged head to tail. The sporozoites are vermiform, 18.9 (17.2-20.8) × 12.3 (11.9-12.8) µm and have striations at the anterior end. Each sporozoite has both anterior and posterior refractile bodies. A sporocyst residuum is present. Molecular characterization of the isolated Caryospora-like oocysts was conducted at the 18S ribosomal RNA and the mitochondrial cytochrome oxidase (COI) loci. At the 18S rRNA locus, the Caryospora-like isolate exhibited 88.8% to 96.5% similarity with other Caryospora spp. from different hosts. At the COI locus, it showed 91.5% similarity to Caryospora cf. bigenetica JB-2013 (KF859856) from the rattlesnake, Sistrurus catenatus.

Identification of a novel species of Eimeria Schneider, 1875 from the woylie, Bettongia penicillata Gray (Diprotodontia: Potoroidae) and the genetic characterisation of three Eimeria spp. from other potoroid marsupials.

Faecal samples (n = 1,093) collected from the woylie Bettongia penicillata Gray, in south-western Australia were examined for the presence of coccidian parasites. Eimeria sp. oöcysts were detected in 15.2% of samples. Faecal samples obtained from the eastern bettong Bettongia gaimardi (Desmarest) (n = 4) and long-nosed potoroo Potorous tridactylus (Kerr) (n = 12) in Tasmania, were also screened for the presence of Eimeria spp. (prevalence 50% and 41.7%, respectively). Morphological and genetic comparison with other known species of Eimeria indicates that the material identified in woylies is novel. This study aimed to (i) morphologically describe and genetically characterise Eimeria woyliei n. sp. found in woylies; and (ii) genetically characterise Eimeria gaimardi Barker, O'Callaghan & Beveridge, 1988, Eimeria potoroi Barker, O'Callaghan & Beveridge, 1988, and Eimeria mundayi Barker, O'Callaghan & Beveridge, 1988, from other potoroid marsupials. Molecular phylogenetic analyses conducted at the 18S rDNA and mitochondrial cytochrome c oxidase subunit 1 (cox1) loci revealed that E. woyliei n. sp. was most closely related to Eimeria setonicis Barker, O'Callaghan & Beveridge, 1988, at the 18S rDNA locus, and Eimeria trichosuri O'Callaghan & O'Donoghue, 2001, at the cox1 locus. Eimeria woyliei n. sp. is the sixth species of Eimeria to be formally described from potoroid marsupials.

Isospora coronoideae n. sp. (Apicomplexa: Eimeriidae) from the Australian raven (Corvus coronoides) (Passeriformes: Corvidae) (Linnaeus, 1758) in Western Australia.

A new Isospora (Apicomplexa: Eimeriidae) species is described from an Australian raven (Corvus coronoides) in Western Australia. Sporulated oocysts (n = 21) are ovoid, 21.2 (18.4-23.9) µm in length and 18.8 (16.9-20.6) µm in width, with a shape index of 1.13. The bi-layered oocyst wall is smooth and colourless, 1.2 µm thick. A polar granule and oocyst residuum is present, but the micropyle is absent. The sporocysts are ovoid-shaped, 16.3 (13.7-18.9) × 10.7 (8.4-12.9) µm, with a shape index (length/width) of 1.52. Stieda and substieda bodies are present, the Stieda body being small and hemidome-shaped and the substieda being indistinct. Each sporocyst with four vermiform sporozoites arranged head to tail. The sporozoites are crescent-shaped, 9.0 (8.9-9.2) × 2.7 (2.3-3.0) µm, with a shape index (length/width) of 3.33. The sporocyst residuum is present. The isolated oocysts had different morphological characteristics when compared with all known Isospora spp. The coccidian parasite was analysed at the 18S and 28S ribosomal RNA and the mitochondrial cytochrome oxidase (COI) loci. At the 18S locus, I. coronoideae n. sp. exhibited 98.9% similarity to I. neochmiae from a captive-bred red-browed finch (KT224380) and Isospora sp. from domestic pigeons (Columba livia domestica) (AB757860), 98.5% similarity to I. gryphoni (AF080613) from an American goldfinch and 98.3% similarity to I. manorinae (KT224379) from a yellow-throated miner. At the 28S locus, it exhibited 95.4% and 94.8% similarity to I. manorinae (KT224381) and I. anthochaerae (KF766053), respectively. At the COI locus, it exhibited 99.8% and 99.7% similarity to I. butcherae (KY801687) and I. neochmiae (KT224378), respectively. Based on morphological and molecular data, this isolate is a new species of Isospora, which is named Isospora coronoideae n. sp. after its host, the Australian raven (Corvus coronoides) (Passeriformes: Corvidae) (Linnaeus, 1758).