Characterisation of Nematoda and Digenea in selected Australian freshwater snails.

Freshwater snails are integral to local ecosystems as a primary food source for various vertebrate species, thereby contributing significantly to ecological food webs. However, their role as intermediate hosts also makes them pivotal in the transmission of parasites. In Australia, research on freshwater snails has predominantly focused on their role as intermediate hosts for livestock parasites, while there has been limited exploration of the impact of these parasites on snail health and population dynamics. The aim of this study was to determine parasitic infection in freshwater snails. This study was conducted in the south-eastern region of Australia, in 2022. A total of 163 freshwater snails from four different species were collected and examined in the Murrumbidgee catchment area in the southeastern part of Australia during the Southern Hemisphere summer and autumn months (February to May). The species included Isidorella hainesii, Glyptophysa novaehollandica, Bullastra lessoni (endemic species), and Physella acuta (an introduced species). Through the analysis of sequence data from the various regions of the nuclear ribosomal DNA, we determined that the Digenea species in this study belonged to three distinct species, including Choanocotyle hobbsi, Petasiger sp. and an unidentified species belonging to Plagiorchioidea. Additionally, analysis of the sequences from Nematoda found in this study, revealed they could be categorized into two separate taxa, including Krefftascaris sp. and an unidentified nematode closely associated with plant and soil nematodes. This research holds significant implications for the future understanding and conservation of Australian freshwater ecosystems. Most parasites found in the present study complete their life cycle in snails and turtles. As many of freshwater snail and turtle species in Australia are endemic and face population threats, exploring the potential adverse impacts of parasitic infections on snail and turtle health, is crucial for advancing our understanding of these ecosystems and also paving the way for future research and conservation efforts. While none of the native snail species in the present study have been listed as endangered or threatened, this may simply be attributed to the absence of regular population surveys.

Multidisciplinary approach to the diagnosis of Contracaecum magnipapillatum infections in Australian black noddies, Anous minutus (Charadriiformes: Laridae).

We provide the incidental necropsy findings associated with anisakid nematode infections of black noddy terns, Anous minutus Boie, 1844 (Charadriiformes: Laridae), from offshore islands in the southern Great Barrier Reef, Queensland, Australia. Specimens collected from the proventriculi were identified morphologically as Contracaecum magnipapillatum Chapin, 1925 (Rhabditida: Anisakidae), using light and scanning electron microscopy (SEM). The entire nuclear ribosomal DNA internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) was amplified by polymerase chain reaction (PCR) and sequenced to provide reference sequences for morphologically well-identified voucher specimens. Interestingly, after an alignment with closely related taxa using BLAST, sequences of the ITS1 and ITS2 were 100% identical to the sequences assigned to Contracaecum septentrionale Kreis, 1955, from a razorbill, Alca torda Linnaeus, 1758 (Charadriiformes: Alcidae), from Spain. These results either raise questions about the ITS as a genetic marker for some members of Contracaecum, or the identity of the specimens assigned to C. septentrionale, given that no supporting morphological data was associated with them. We highlight the need for a combined morphological and molecular approach to parasite diagnostics and the use of multiple genetic loci to resolve the molecular taxonomy of cryptic species. Morphological identifications should be taxonomically robust, transparent and precede the deposition of molecular barcodes in public repositories. The gross and histopathological findings of our investigation concur with previous reports of widespread Contracaecum infections in black noddies and support the contention that Contracaecum spp. are an unlikely primary cause of mortality.

Effect of a protease-activated receptor-2 antagonist (GB88) on inflammation-related loss of alveolar bone in periodontal disease.

BACKGROUND AND OBJECTIVE: Protease-activated receptor-2 (PAR2 ), a pro-inflammatory G-protein coupled receptor, has been associated with pathogenesis of periodontitis and the resulting bone loss caused by oral pathogens, including the keystone pathogen Porphyromonas gingivalis (P. gingivalis). We hypothesised that administration of a PAR2 antagonist, GB88, might prevent inflammation and subsequent alveolar bone resorption in a mouse model of periodontal disease. METHODS: Periodontitis was induced in mice by oral inoculations with P. gingivalis for a total of eight times over 24 days. The infected mice were treated with either GB88 or vehicle for the duration of the trial. Following euthanasia on day 56, serum was collected and used for the detection of mast cell tryptase. The right maxillae were defleshed and stained with methylene blue to measure the exposed cementum in molar teeth. The left maxillae were prepared for cryosections followed by staining for tartrate-resistant acid phosphatase to identify osteoclasts or with toluidine blue to identify mast cells. Reverse transcription quantitative PCR (RT-qPCR) was used to quantify the expression of inflammatory cytokines in the gingival tissue. Supernatants of T-lymphocyte cultures isolated from the regional lymph nodes were assayed using a cytometric bead array to measure the Th1/Th2/Th17 cytokine levels. RESULTS: Measurement of the exposed cementum showed that GB88 reduced P. gingivalis-induced alveolar bone loss by up to 69%. GB88 also prevented the increase in osteoclast numbers observed in the infected mice. Serum tryptase levels were significantly elevated in both the infected groups, and not altered by treatment. RT-qPCR showed that GB88 prevented the upregulation of Il1b, Il6, Ifng and Cd11b. In T-lymphocyte supernatants, only IFNγ and IL-17A levels were increased in response to infection, but this was prevented by GB88 treatment. CONCLUSIONS: GB88 significantly reduced osteoclastic alveolar bone loss in mice infected with P. gingivalis, seemingly by preventing the upregulation of several inflammatory cytokines. PAR2 antagonism may be an effective treatment strategy for periodontal disease.

Investigating potential anti-proliferative activity of different statins against five cancer cell lines.

Statins have been reported to have potential anti-proliferative effects through an unknown mechanism. This study aims to investigate the anti-proliferative activities of five statins, including simvastatin, rosuvastatin, fluvastatin, atorvastatin, and pravastatin, against five different cancer cell lines; cervical epithelial carcinoma DoTc2 4510, malignant melanoma A-375, muscle Ewing's sarcoma A-673, hepatocellular carcinoma HUH-7, as well as breast cancer cells MCF-7. At 100 µM, simvastatin and atorvastatin significantly inhibited 70% of cellular proliferation. At the same concentration, rosuvastatin and fluvastatin showed about 50% of inhibition only in A-375 and A-673 cancer cells in a time- and dose-dependent manner. Of all the statin drugs used, pravastatin had the least inhibitory effect on all the cancer cell lines. Western Blot analysis showed a decrease in mTOR level, and the expression of p53 tumour suppression and BCL-2 proteins was relatively elevated compared to the untreated cells. Simvastatin and atorvastatin may inhibit cellular proliferation via BCL-2/p53, Bax/Bak, and PI3K/Akt/mTOR signalling pathways. This is the first research to evaluate the anti-cancer effects of simvastatin, rosuvastatin, fluvastatin, atorvastatin, and pravastatin against five different cell lines from distinct origins and provided a relevant comparison of their efficacies for their anti-proliferative activity.

Dolichoperoides macalpini (Nicoll, 1914) (Digenea: Dolichoperoididae) infecting venomous snakes (Elapidae) across Australia: molecular characterisation and infection parameters.

Specimens of Dolichoperoides macalpini (Nicoll, 1914) (Digenea: Dolichoperoididae) were collected from Australian venomous snakes (Elapidae): Notechis scutatus Peters, 1861 and Austrelaps superbus (Günther, 1858) from Tasmania and surrounding islands and N. s. occidentalis Glauert, 1948 from wetlands near Perth, Western Australia. Despite variation in morphological measurements, genetic analysis showed that the one species of digeneans infected the snakes from all locations. This study presents the first DNA sequences for D. macalpini (internal transcribed spacer, 18S, 28S), confirming its placement in a family separate from the Reniferidae and Telorchiidae. Analysis of the infection dynamics of infection in Western Australian snakes showed significant differences in levels of infection between wetland locations, season and year of collection. Infection of D. macalpini was reported in the gastrointestinal tract, including the mouth, in freshly euthanised snakes in Western Australia, and in the lung in Tasmanian snakes, consistent with earlier reports. Differences in morphology and site of infection are suggested to be due to a combination of season and maturity of the digenean, with infection potentially occurring early in the season, as the snakes emerge from torpor. The need for research on the seasonal dynamics of infection with this parasite is discussed.

Coloured Rice Phenolic Extracts Increase Expression of Genes Associated with Insulin Secretion in Rat Pancreatic Insulinoma ß-cells.

Glucose-induced oxidative stress is associated with the overproduction of reactive oxygen species (ROS), which may dysregulate the expression of genes controlling insulin secretion leading to ß-cell dysfunction, a hallmark of type 2 diabetes mellitus (T2DM). This study investigated the impact of coloured rice phenolic extracts (CRPEs) on the expression of key genes associated with ß-cell function in pancreatic ß-cells (INS-1E). These genes included glucose transporter 2 (Glut2), silent mating type information regulation 2 homolog 1 (Sirt1), mitochondrial transcription factor A (Tfam), pancreatic/duodenal homeobox protein 1 (Pdx-1) and insulin 1 (Ins1). INS-1E cells were cultured in high glucose (25 mM) to induce glucotoxic stress conditions (HGSC) and in normal glucose conditions (NGC-11.1 mM) to represent normal ß-cell function. Cells were treated with CRPEs derived from two coloured rice cultivars, Purple and Yunlu29-red varieties at concentrations ranged from 50 to 250 µg/mL. CRPEs upregulated the expression of Glut2, Sirt1 and Pdx-1 significantly at 250 µg/mL under HGSC. CRPEs from both cultivars also upregulated Glut2, Sirt1, Tfam, Pdx-1 and Ins1 markedly at 250 µg/mL under NGC with Yunlu29 having the greatest effect. These data suggest that CRPEs may reduce ß-cell dysfunction in T2DM by upregulating the expression of genes involved in insulin secretion pathways.

Keratinocyte-specific ablation of protease-activated receptor 2 prevents gingival inflammation and bone loss in a mouse model of periodontal disease.

Chronic periodontitis is characterised by gingival inflammation and alveolar bone loss. A major aetiological agent is Porphyromonas gingivalis, which secretes proteases that activate protease-activated receptor 2 (PAR2 ). PAR2 expressed on oral keratinocytes is activated by proteases released by P. gingivalis, inducing secretion of interleukin 6 (IL-6), and global knockout of PAR2 prevents bone loss and inflammation in a periodontal disease model in mice. To test the hypothesis that PAR2 expressed on gingival keratinocytes is required for periodontal disease pathology, keratinocyte-specific PAR2 -null mice were generated using K14-Cre targeted deletion of the PAR2 gene (F2rl1). These mice were subjected to a model of periodontitis involving placement of a ligature around a tooth, combined with P. gingivalis infection ("Lig + Inf"). The intervention caused a significant 44% decrease in alveolar bone volume (assessed by microcomputed tomography) in wildtype (K14-Cre:F2rl1wt/wt ), but not littermate keratinocyte-specific PAR2 -null (K14-Cre:F2rl1fl/fl ) mice. Keratinocyte-specific ablation of PAR2 prevented the significant Lig + Inf-induced increase (2.8-fold) in the number of osteoclasts in alveolar bone and the significant up-regulation (2.4-4-fold) of the inflammatory markers IL-6, IL-1ß, interferon-γ, myeloperoxidase, and CD11b in gingival tissue. These data suggest that PAR2 expressed on oral epithelial cells is a critical regulator of periodontitis-induced bone loss and will help in designing novel therapies with which to treat the disease.

Dietary Polyphenols and Gene Expression in Molecular Pathways Associated with Type 2 Diabetes Mellitus: A Review.

Type 2 diabetes mellitus (T2DM) is a complex metabolic disorder with various contributing factors including genetics, epigenetics, environment and lifestyle such as diet. The hallmarks of T2DM are insulin deficiency (also referred to as ß-cell dysfunction) and insulin resistance. Robust evidence suggests that the major mechanism driving impaired ß-cell function and insulin signalling is through the action of intracellular reactive oxygen species (ROS)-induced stress. Chronic high blood glucose (hyperglycaemia) and hyperlipidaemia appear to be the primary activators of these pathways. Reactive oxygen species can disrupt intracellular signalling pathways, thereby dysregulating the expression of genes associated with insulin secretion and signalling. Plant-based diets, containing phenolic compounds, have been shown to exhibit remedial benefits by ameliorating insulin secretion and insulin resistance. The literature also provides evidence that polyphenol-rich diets can modulate the expression of genes involved in insulin secretion, insulin signalling, and liver gluconeogenesis pathways. However, whether various polyphenols and phenolic compounds can target specific cellular signalling pathways involved in the pathogenesis of T2DM has not been elucidated. This review aims to evaluate the modulating effects of various polyphenols and phenolic compounds on genes involved in cellular signalling pathways (both in vitro and in vivo from human, animal and cell models) leading to the pathogenesis of T2DM.

Rice Bran Phenolic Compounds Regulate Genes Associated with Antioxidant and Anti-Inflammatory Activity in Human Umbilical Vein Endothelial Cells with Induced Oxidative Stress.

Oxidative stress, inflammation and endothelial dysfunction are associated with the development of cardiovascular and metabolic diseases. Phenolic extracts derived from rice bran (RB) are recognised to have antioxidant and anti-inflammatory potential. However, the underlying mechanisms remain unknown. Therefore, this study aimed to evaluate the ability of RB-derived phenolic extracts to modulate genes associated with antioxidant and anti-inflammatory pathways in human umbilical vein endothelial cells (HUVECs) under induced oxidative stress conditions. HUVECs under oxidative stress were treated with varying concentrations of RB phenolic extracts (25-250 µg/mL). Using quantitative real-time polymerase chain reaction, the expression of candidate genes that regulate antioxidant and anti-inflammatory pathways were determined. This included nuclear factor erythroid 2-related factor 2 (Nrf2), nicotinamide adenine dinucleotide phosphate: quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO1), nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4), intercellular adhesion molecule 1 (ICAM1), endothelial nitric oxide synthase (eNOS), ectonucleoside triphosphate diphosphohydrolase 1 (CD39) and ecto-5'-nucleotidase (CD73). Phenolic extracts derived from RB down-regulated the expression of four genes, ICAM1, CD39, CD73 and NOX4 and up-regulated the expression of another four genes, Nrf2, NQO1, HO1 and eNOS, indicating an antioxidant/ anti-inflammatory effect for RB against endothelial dysfunction.

Black Sorghum Phenolic Extract Regulates Expression of Genes Associated with Oxidative Stress and Inflammation in Human Endothelial Cells.

Oxidative stress is one of the primary factors leading to endothelial dysfunction, a major underlying cause of vascular disorders. This study aims to understand the key signalling pathways regulated by sorghum (Shawaya short black 1 variety; characterised to be very high in its antioxidant activity) under oxidative stress in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were pre-treated with non-cytotoxic concentrations of phenolic-rich black sorghum extract (BSE) prior to induction of oxidative stress using hydrogen peroxide (H2O2). Treatment with BSE upregulated the expression of heme oxygenase 1 (HO1) and endothelial nitric oxide synthase (eNOS) and downregulated the levels of NADPH oxidase 4 (NOX4). BSE treatment significantly reduced the expression of pro-inflammatory mediators such as monocyte chemoattractant protein 1 (MCP1) and intracellular adhesion molecule 1 (ICAM1). Results from this study suggest that phenolic-rich BSE may reduce oxidative stress by regulating pro- and antioxidant signalling pathways and the expression of inflammatory mediators linked to endothelial dysfunction under oxidative stress.

Characterization of Echinostoma spp. (Trematoda: Echinostomatidae Looss, 1899) infecting ducks in south-eastern Australia.

Waterbirds, are one of the popular game animals and are of significant relevance to parasite spread due to their ability to fly and migrate great distances in relatively short periods of time. In Australia, however, the knowledge of parasites infecting native waterbirds is lacking with some of the last reports occurring over 50 years ago. The study aimed to characterise Echinostoma spp. infecting wild native Australian ducks found in the southern regions of the Murray Darling Basin (MDB). Ducks (n = 98) were collected from southern New South Wales within the MDB catchment. Three different species of native ducks were found including Anas superciliosa (n = 37), Anas gracilis (n = 47) and Chenonetta jubata (n = 14), of which 4.3 %, 2.7 % and 7.1 %, respectively, were found to be infected with adult stages Echinostoma spp. Examination of the parasites revealed the presence of two morphotypes. The 18S, 28S and ITS rRNA as well as the mitochondrial nad1 genes were sequenced for representative isolates of the two morphotypes. These sequences were then compared with existing sequences of Echinostoma spp. available in the GenBank. Phylogenetic analysis based on the ITS region indicated that the two morphotypes were genetically distinct. Although there are comparable sequences of Echinostoma spp. in Australia these morphotypes appear to be genetically distinct. Based on their distinct morphology and genetics we suggest that these two morphotypes are previously undescribed in Australia. This study sheds light on the presence of Echinostoma parasites in native Australian waterbirds and highlights the need for further research to better understand the diversity and prevalence of these parasites in the region.

Effect of gastrointestinal digestion on the stability, antioxidant activity, and Caco-2 cellular transport of pigmented grain polyphenols.

Grain polyphenols are known to possess several health properties. However, their digestive stability and intestinal absorption have not been fully elucidated. This study investigated the fate of pigmented grain polyphenols in the digestive system. Purple rice, purple barley, purple wheat, and blue wheat extracts were subjected to simulated gastric and intestinal phase digestion, followed by Caco-2 cellular transport. Phenolic profiling and antioxidant activity were determined using benchtop assays and an ultra-high-performance liquid chromatography-2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid free radical) (UHPLC-ABTS•+) system. The results demonstrated a decrease in the total phenolic content of extracts after digestion, with purple rice extract retaining the highest phenolic content (79%) and ABTS•+antioxidant activity (31%). Antioxidant activity was retained the most during the gastric phase; however, dominant antioxidant compounds were not detected after intestinal digestion. Significant variations in phenolic composition and radical scavenging activity were detected after digestion. Protocatechuic acid, vanillic acid, apigenin, and chrysoeriol were all transported across the intestinal barrier. The findings of this study provide novel insights into the in vitro stability and antioxidant activity of cereal grain polyphenols after simulated digestion.

First findings of Sarcocystis species in game deer and feral pigs in Australia.

Several wild game meat species, including deer and feral pigs are hunted and consumed in Australia. Feral pigs and deer are not indigenous to Australia, but they have proliferated extensively and established their presence in every state and territory. Following the report of a sambar deer displaying Sarcocystis like white cysts in its rump muscles, the present study was conducted to explore the prevalence of Sarcocystis infections in wild deer and feral pigs in the southeastern regions of Australia. Oesophagus, diaphragm, and heart tissue from 90 deer and eight feral pigs were examined visually for sarcocysts. All results were negative. PCR testing of randomly selected deer and feral pigs yielded positive results, which were subsequently supported by histopathology. This is the first study to report the presence of Sarcocystis spp. in deer and feral pigs in Australia. As no visual cysts were found on the heart or oesophagus that came back positive with PCR, infected animals, particularly those reared free-range, could be passing through meat quality checks unidentified. If people consume this meat without cooking it properly, it may lead to a human infection of Sarcocystis. However, a more targeted study focused on determining the parasite's prevalence and assessing its risks is necessary to determine if it constitutes a food safety issue. As this species has been found not only in feral pigs but also in domestic pigs, the potential for infection spreading between feral pigs and pigs in free-range livestock systems is high, potentially posing a large problem for the Australian pork industry, particularly with the increased emphasis on free-range pig husbandry. Future studies should concentrate on determining the species of Sarcocystis in feral animals commonly consumed as game meat to determine potential zoonotic risks. This could also include a more in-depth look at the prevalence of Sarcocystis infections in other game animals. Identifying where these parasites are present and to what extent, are important areas for future studies.

The first finding of Dictyocaulus cervi and Dictyocaulus skrjabini (Nematoda) in feral fallow deer (Dama dama) in Australia.

Feral deer are widespread throughout Australia with the capacity to impact livestock production via transmission of parasites. Samples of Dama dama (fallow deer), Rusa unicolor (sambar deer), Cervus elaphus (red deer) and an unidentified deer were sourced from various locations in south-eastern Australia for examination for parasites. Adult nematodes were collected from the lungs of all deer species across four separate geographical locations. The nematodes were identified as species of Dictyocaulus through both morphological and molecular means. Species identification based on morphological features was difficult, with many measurements from described species overlapping. Molecular analyses targeting three markers, namely 18S rRNA, ITS2, and cox1 revealed the presence of two distinct species: Dictyocaulus cervi and Dictyocaulus skrjabini. These are the first genetically confirmed reports of species of Dictyocaulus in feral deer in Australia, and although cross-transmission of species of Dictyocaulus with livestock has not yet been reported, it cannot be completely discounted without further research.

Description of two new species of Diplectanum Diesing, 1858 (Monogenea: Diplectanidae) collected from Protonibea diacanthus (Lacepède, 1802) (Teleostei: Sciaenidae) from waters off northern Australia.

Two new species of the family Diplectanidae Monticelli, 1903 from the gills of Protonibea diacanthus (Lacepède, 1802) (Teleostei: Sciaenidae) off the northern Australian coast are described. Previous studies have either morphological or genetic results, whereas this study combines morphological and advanced molecular methods to provide the first detailed descriptions for species of Diplectanum Diesing, 1858 from Australia utilising both methodologies. Two new species, Diplectanum timorcanthus n. sp. and Diplectanum diacanthi n. sp., are morphologically described and genetically characterised using the partial nuclear 28S ribosomal RNA gene (28S rRNA) and the internal transcribed spacer 1 (ITS1) partial sequence.

Occurrence of digenean parasites in freshwater snails in the Murrumbidgee catchment area, Australia.

Freshwater snails are important hosts in the life cycles of many medically important parasites, particularly for digenetic trematodes such as liver flukes and schistosomes. The current study was conducted to determine the infection of freshwater snails with parasites that can potentially be transmitted to humans within the Murrumbidgee catchment area which is an area of widespread intensive aquaculture in Australia. A total of 116 freshwater snails, belonging to three species (Isidorella hainesii, Glyptophysa novaehollandica and Bullastra lessoni), were examined for the presence of parasites in both man-made and natural environments. The analysis of sequence data, including the internal transcribed spacers (ITS) of nuclear ribosomal DNA, small subunit (18S) ribosomal DNA, and large subunit (28S) ribosomal DNA, indicated that the collected parasites belonged to two distinct genera, namely Clinostomum and Echinostoma. It is noteworthy that species of both of these digenean parasites have the potential to be zoonotic. Cercariae of both Clinostomum and Echinostoma were observed in snails collected from aquaculture settings. It is important to highlight that infectious stages of Clinostomum  has been previously detected in edible fish within Australia. This information raises concerns regarding the potential transmission of these parasites to humans through the consumption of contaminated fish. These findings emphasize the importance of monitoring and controlling the presence of Clinostomum and Echinostoma in aquaculture environments to minimise the risk of zoonotic infections and ensure food safety. Further research and surveillance are needed to better understand the prevalence, transmission dynamics, and potential public health implications associated with these parasites in the context of aquaculture in Australia.

Occurrence and characterisation of Eustrongylides species in Australian native birds and fish.

In Australia, nematodes belonging to the genus Eustrongylides were believed to be endemic species until the late 20th century when they were all considered to be E. excisus, invalid or inquirendae. Although these nematodes have frequently been reported in Australian fish, reptiles, and birds and cause disease or mortality among them, there has been no attempt to date to characterise them genetically. Globally, also, no one has validated or defined suitable genetic markers to distinguish between species of Eustrongylides. In this study, adult Eustrongylides from little black cormorant (Phalacrocorax sulcirostris; n = 3) and larvae from mountain galaxias (Galaxias olidus, n = 2) and a Murray cod (Maccullochella peelii, n = 1), and a Murray cod-trout cod hybrids (Maccullochella peelii x Maccullochella macquariensis, n = 1) were available for morphological examination and molecular characterisation. The adult nematodes from cormorants were identified as E. excisus. Sequences of the 18S and ITS regions were then obtained for all nematodes, which were identical among all specimens (larvae and adults) and also identical to those of E. excisus available in the GenBank. However, only one base pair difference exists between the 18S sequences of E. excisus and E. ignotus, with limited sequences available in GenBank accompanied with proper morphological data for the nematodes. With that limitation in mind, identifying our specimens as E. excisus suggests spill-over - that it is an introduced parasite species that has successfully established its life cycle among Australian native species - may have occurred. Our study is the first report of E. excisus in the little black cormorant, P. sulcirostris. Our results do not exclude the possibility of the occurrence of other species of Eustrongylides, either native or exotic, in Australia. This parasite is zoonotic and with increasing demand for fish and changing dietary preferences, such as the consumption of raw or undercooked fish, its occurrence in the flesh of the fish is concerning. This parasite is also associated with anthropogenic habitat alteration affecting the reproductive success of the infected hosts. Therefore, awareness among the relevant authorities of the presence of the parasite in Australia and its adverse impact on native animals is crucial for the success of conservation plans such as fish recovery and relocation efforts.

The effect of stress and exercise on the learning performance of horses.

Domestic horses are widely used for physically demanding activities but the effect of exercise on their learning abilities has not been explored. Horses are also frequently exposed to stressors that may affect their learning. Stress and exercise result in the release of glucocorticoids, noradrenaline and other neurotransmitters that can influence learning. It is not currently possible to directly measure concentrations of neurotransmitters in the brains of behaving horses, however the inference of neurobiological processes from peripheral markers have been widely used in studies of human cognition. We assigned 41 horses to either ridden exercise, uncontrollable stress or inactivity and evaluated their acquisition of an industry-style aversive instrumental learning task. Exercised horses achieved the learning criterion in the fewest number of trials compared to the stressed and inactive horses whose performance did not differ. The exercised horses' salivary cortisol concentrations decreased during learning whereas the concentrations of the other groups increased. Spearman's correlations revealed that horses with the highest cortisol concentrations required the most trials to reach the criterion. We present novel data that exercise prior to learning may enhance the acquisition of learning in horses. Conversely, activities that expose horses to uncontrollable stressors causing strong cortisol release may impair learning. It is proposed that these effects may be due to the influence of neurotransmitters such as cortisol and noradrenaline on brain regions responsible for learning.

Habit Formation and the Effect of Repeated Stress Exposures on Cognitive Flexibility Learning in Horses.

Horse training exposes horses to an array of cognitive and ethological challenges. Horses are routinely required to perform behaviours that are not aligned to aspects of their ethology, which may delay learning. While horses readily form habits during training, not all of these responses are considered desirable, resulting in the horse being subject to retraining. This is a form of cognitive flexibility and is critical to the extinction of habits and the learning of new responses. It is underpinned by complex neural processes which can be impaired by chronic or repeated stress. Domestic horses may be repeatedly exposed to multiples stressors. The potential contribution of stress impairments of cognitive flexibility to apparent training failures is not well understood, however research from neuroscience can be used to understand horses' responses to training. We trained horses to acquire habit-like responses in one of two industry-style aversive instrumental learning scenarios (moving away from the stimulus-instinctual or moving towards the stimulus-non-instinctual) and evaluated the effect of repeated stress exposures on their cognitive flexibility in a reversal task. We measured heart rate as a proxy for noradrenaline release, salivary cortisol and serum Brain Derived Neurotrophic Factor (BDNF) to infer possible neural correlates of the learning outcomes. The instinctual task which aligned with innate equine escape responses to aversive stimuli was acquired significantly faster than the non-instinctual task during both learning phases, however contrary to expectations, the repeated stress exposure did not impair the reversal learning. We report a preliminary finding that serum BDNF and salivary cortisol concentrations in horses are positively correlated. The ethological salience of training tasks and cognitive flexibility learning can significantly affect learning in horses and trainers should adapt their practices where such tasks challenge innate equine behaviour.

Bioaccessibility and Antioxidant Activity of Polyphenols from Pigmented Barley and Wheat.

Polyphenols in pigmented cereals are believed to enhance health outcomes through their antioxidant properties. This study aimed to characterise polyphenols from Hordeum vulgare (purple barley), Triticum turgidum (purple wheat) and Triticum aestivum (blue wheat) in order to evaluate their bioaccessibility and antioxidant activity. An ultra-high performance liquid chromatography mass spectrometry coupled with an online 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) system was used to identify the polyphenols and quantify their relative antioxidant levels. Simulated gastrointestinal digestion of the cereals allowed for the assessment of polyphenol bioaccessibility using benchtop assays. Between cereals, the bioaccessible phenolic content was similar following digestion, but the antioxidant activity was significantly different (purple barley > purple wheat > blue wheat; p < 0.01). Among the polyphenols identified, flavan-3-ols and anthocyanins were the least bioaccessible whereas flavones were the most bioaccessible after digestion. This study demonstrated that these pigmented cereal varieties are sources of bioaccessible polyphenols with antioxidant activity. These findings may aid in utilising these pigmented grains for the future design and development of novel functional food products with enhanced health properties.