IntroductionIchthyophonus hoferi is a cosmopolitan parasite infecting a wide range of fish species across marine, estuarine, brackish, and freshwater environments. Currently, diagnosis relies on destructive sampling methods, posing challenges for disease surveillance in aquaculture and wild populations. This study aimed to identify pre-clinical metabolic markers of I. hoferi infection in White stumpnose fish (Rhabdosargus globiceps) using non-lethal blood-based diagnostics.MethodsAn untargeted metabolomics approach employing comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GCxGC-TOFMS) was conducted on dried blood spot (DBS) samples. Multivariate (PCA, PLS-DA) and univariate analyses were used to evaluate metabolic differences between infected and control groups.ResultA total of 655 metabolites were detected, with 98 showing significant variation between groups. Infected fish exhibited elevated levels of xenobiotics and altered carbohydrate metabolism, with key downregulated metabolites including D-mannose, arabinofuranose, and pento-3-ulose. Several fatty acids, such as oleic acid and pentadecanoic acid, were also significantly altered. Pathway analysis revealed that propanoate metabolism, galactose metabolism, inositol phosphate metabolism, and the TCA cycle were significantly impacted by I. hoferi infection, suggesting disruption in energy production and anti-inflammatory pathways. Among the top discriminatory metabolites, D-mannose was notably downregulated and identified as a potential biomarker for I. hoferi infection.DiscussionThis study demonstrates that metabolomics, in combination with DBS sample collection, offers a promising non-destructive approach for detecting parasitic infections in fish. The findings contribute to the development of diagnostic tools for early detection and monitoring of I. hoferi infection, with implications for fish health management and conservation.

During live export, abalone are transported in air, exposing them to environmental hypoxia for extended periods. Hypoxia-inducible factor-1 (HIF-1), a fundamental moderator of hypoxia, activates a variety of genes in response to hypoxic stress, including inducible nitric oxide synthase (iNOS), which is involved in vasodilation, which ultimately can lead to improved perfusion. This study hypothesized that Haliotis midae, the South African abalone, recruits the under-perfused left gill during hypoxia via HIF-1 and iNOS induction whereas changing metabolic rates, transitions to anaerobic respiration and maintaining circulating oxygen by means of reverse Bohr shift that contribute to alleviating tissue hypoxia during 24-h air exposure. Haliotis midae were exposed to air for 24 h as a proxy for hypoxia and were reimmersed in water for 1 h for recovery before the HIF-1 alpha protein expression, iNOS gene expression and 14C inulin space were measured in the gills. We also measured oxygen consumption rates, hemolymph d-lactate levels, pH, and Po2. We found no evidence of HIF-mediated iNOS induction, but abalone hypometabolism and the presence of lactic acid in circulation during the short recovery indicate some reliance on anaerobiosis.

Type 2 diabetes is a non-communicable metabolic syndrome that is characterized by the dysfunction of pancreatic β-cells and insulin resistance. Both animal and human studies have been conducted, demonstrating that helminth infections are associated with a decreased prevalence of type 2 diabetes mellitus (T2DM). However, there is a paucity of information on the impact that helminths have on the metabolome of the host and how the infection ameliorates T2DM or its progression. Therefore, this study aimed at using a non-targeted metabolomics approach to systematically identify differentiating metabolites from serum samples of T2DM-induced Sprague Dawley (SD) rats infected with a tissue-dwelling nematode, Trichinella zimbabwensis, and determine the metabolic pathways impacted during comorbidity. Forty-five male SD rats with a body weight between 160 g and 180 g were used, and these were randomly selected into control (non-diabetic and not infected with T. zimbabwensis) (n = 15) and T2DM rats infected with T. zimbabwensis (TzDM) (n = 30). The results showed metabolic separation between the two groups, where d-mannitol, d-fructose, and glucose were upregulated in the TzDM group, when compared to the control group. L-tyrosine, glycine, diglycerol, L-lysine, and L-hydroxyproline were downregulated in the TzDM group when compared to the control group. Metabolic pathways which were highly impacted in the TzDM group include biotin metabolism, carnitine synthesis, and lactose degradation. We conclude from our study that infecting T2DM rats with a tissue-dwelling nematode, T. zimbabwensis, causes a shift in the metabolome, causing changes in different metabolic pathways. Additionally, the infection showed the potential to regulate or improve diabetes complications by causing a decrease in the amino acid concentration that results in metabolic syndrome.

Crustaceans inhabiting freshwater are more vulnerable to increased loading with dissolved trace metals than those from more saline conditions for both physicochemical and physiological reasons. Potamonautes warreni (Calman, 1918) is a true freshwater crab inhabiting rivers in proximity to mining activities in South Africa. In order to assess whether the uptake of zinc (Zn2+) and cadmium (Cd2+) were associated with Na+- and Ca2+-uptake routes, individuals of P. warreni were acclimated to different Na+ or Ca2+ concentrations before measuring trace-metal uptake into the haemolymph and tissues in an intermediate freshwater. Cd2+ uptake into the haemolymph was elevated following acclimation to ‘Na+ replete’ conditions, when compared with ‘Na+ deplete’ conditions, indicating that a physiological response to the acclimation salinity had influenced Cd2+ uptake. Zn2+ uptake by the whole body was elevated in P. warreni individuals from the ‘Na+ replete’ group when compared with crabs acclimated to Na+ free water. A general change in permeability may have occurred and indeed, the apparent water permeability (water efflux) of P. warreni was increased in elevated Na+ and Ca2+ concentrations compared with that in softer water. A comparison of the osmoregulatory ability between P. warreni and the catadromous crab Eriocheir sinensisH. Milne Edwards, 1853 highlighted the greater permeability of the latter. Lower Zn2+ and Cd2+ uptake rates were measured in P. warreni in comparison to those measured in E. sinensis by other authors, indicating that the more well adapted a species is to life in freshwater, the less susceptible they will be to metal uptake. Future investigations to understand further the mechanisms influencing Zn2+ and Cd2+ uptake are required to understand the role that salinity can play in protecting a species from trace-metals released into the environment.

Trichinella infections have been documented globally and have been detected in wild and/or domestic animals except Antarctica. There is paucity of information in the metabolic responses of hosts during Trichinella infections and biomarkers for infection that can be used in the diagnosis of the disease. The current study aimed to apply a non-targeted metabolomic approach to identify Trichinella zimbabwensis biomarkers including metabolic response from sera of infected Sprague-Dawley rats. Fifty-four male Sprague-Dawley rats were randomly assigned into T. zimbabwensis infected group (n = 36) and the non-infected control (n = 18). Results from the study showed that the metabolic signature of T. zimbabwensis infection consists of enriched methyl histidine metabolism, disturbance of the liver urea cycle, impeded TCA cycle, and upregulation of gluconeogenesis metabolism. The observed disturbance in the metabolic pathways was attributed to the effects caused by the parasite during its migration to the muscles resulting in downregulation of amino acids intermediates in the Trichinella-infected animals, and therefore affecting energy production and degradation of biomolecules. It was concluded that T. zimbabwensis infection caused an upregulation of amino acids; pipecolic acid, histidine, and urea, and upregulation of glucose and meso-Erythritol. Moreover, T. zimbabwensis infection caused upregulation of the fatty acids, retinoic acid, and acetic acid. These findings highlight the potential of metabolomics as a novel approach for fundamental investigations of host-pathogen interactions as well as for disease progression and prognosis.

Type 2 diabetes mellitus (T2DM) is an expanding global health concern, closely associated with the epidemic of obesity. Individuals with diabetes are at high risk for microvascular and macrovascular complications, which include retinopathy, neuropathy, and cardiovascular comorbidities. Despite the availability of diagnostic tools for T2DM, approximately 30–60% of people with T2DM in developed countries are never diagnosed or detected. Therefore, there is a strong need for a simpler and more reliable technique for the early detection of T2DM. This study aimed to use a non-targeted metabolomic approach to systematically identify novel biomarkers from the serum samples of T2DM-induced Sprague Dawley (SD) rats using a comprehensive two-dimensional gas chromatography coupled with a time-of-flight mass spectrometry (GCxGC-TOF/MS). Fifty-four male Sprague Dawley rats weighing between 160–180 g were randomly assigned into two experimental groups, namely the type 2 diabetes mellitus group (T2DM) (n = 36) and the non-diabetic control group (n = 18). Results from this study showed that the metabolite signature of the diabetic rats was different from that of the non-diabetic control group. The most significantly upregulated metabolic pathway was aminoacyl-t-RNA biosynthesis. Metabolite changes observed between the diabetic and non-diabetic control group was attributed to the increase in amino acids, such as glycine, L-asparagine, and L-serine. Aromatic amino acids, including L-tyrosine, were associated with the risk of future hyperglycemia and overt diabetes. The identified potential biomarkers depicted a good predictive value of more than 0.8. It was concluded from the results that amino acids that were associated with impaired insulin secretion were prospectively related to an increase in glucose levels. Moreover, amino acids that were associated with impaired insulin secretion were prospectively related to an increase in glucose levels.

Crustaceans inhabiting metal-contaminated freshwaters are susceptible to toxic insult to their osmoregulatory systems. The main osmoregulatory organs of decapod crustaceans, the gills, are continually bathed in freshwater and are therefore at risk from trace metal impacts. The effects of chronic (21 d) exposure to raised dissolved concentrations of Zn, Cd, Cu and Pb on aspects of hydromineral balance were investigated in Potamonautes warreni, a freshwater crab endemic to rivers in South Africa at potential risk from trace metal contamination from mining operations. Generally, hydromineral balance of P. warreni was tolerant to chronic metal exposures although sublethal cadmium exposure of 860 mu g.l- 1 for 21 days resulted in a reduced sodium concentration in the haemolymph. A chronic exposure to 43 mu g.l- 1 cadmium produced an elevated maximum unidirectional sodium uptake, possibly resulting from acclimation to the metal exposure. Branchial Na+/K+-ATPase and V-Type H+-ATPase activity were not affected by chronic in vivo Cd (43 mu g.l- 1) and Zn (500 mu g.l- 1) exposures. An important aspect of ameliorating metal toxicity may be through antioxidants and therefore the effects of applying a reducing agent were tested following in vitro metal treatment. Inhibition of Na+/K+-ATPase could be prevented by pre-incubation with a reducing agent, indicating the importance of antioxidants in reducing metal toxicity in this species. Although this study demonstrates the physiological resilience of P. warreni to dissolved trace metal impacts, the energetic consequences of long-term exposure are as yet not known.

The peritrich ciliate Epistylis sp. is commonly found attached to the dominant calanoid copepod Pseudodiaptomus stuhlmanni in the St Lucia Estuary, South Africa. A study was conducted to test if salinity interacted with temperature and turbidity to affect the survivorship of Epistylis sp. Two-way analysis of variance on ranks was conducted to test for the main and interaction effects of these parameters. There was no interaction between temperature and salinity, and temperature (15, 21 and 30 degrees C) did not affect Epistylis sp. The survivorship of Epistylis sp. was however significantly and negatively affected by high salinity. Post hoc analyses revealed that the highest salinity level (25) was responsible for most of the Epistylis sp. mortalities. The survivorship of Epistylis sp. was significantly affected by the interaction between turbidity and salinity. High turbidity (1000 nephelometric turbidity units [NTU]) negatively affected survivorship across the entire salinity gradient (0 to 25), and high salinity (25) also generally resulted in low survivorship across the turbidity gradient of 50 to 1000 NTU. As the host P. stuhlmanni is more tolerant of high salinity and high turbidity, these findings suggest that these conditions can modulate epibiosis by limiting the prevalence and cover of Epistylis sp. in the St Lucia Estuary, particularly when salinity is high.

Abalone is currently considered South Africa's most successfully produced aquaculture export product, with a 76% share of the total value generated by the aquaculture sector. A major risk factor for this sector is slow growth rates experienced during farming. Abalone feeds are often supplemented with amino acids in an attempt to enhance abalone growth. This is a first investigation of the effect of added proline to standard abalone feed, on the metabolite profile of slow-growing abalone. A targeted liquid chromatography tandem mass spectrometry metabolomics research approach was followed to recognise the metabolic response of abalone showing slower growth performance. The addition of proline to the standard abalone diet was found to serve as a substrate for amino acid catabolism in slower growing abalone, by means of proline breakdown to assist with energy production via the tricarboxylic acid cycle. Other amino acids and urea cycle intermediates, that is, arginine, asparagine, ornithine and creatine further support energy production via the action of protein catabolism in slow-growing abalone. Additionally, the importance of understanding how abalone respond metabolically to modified feed highlights the use of metabolomics to answer abalone aquaculture farming questions.

The South African abalone farming industry is entirely based on Haliotis midae, which has been commercially cultured with great success the last 20 years. Even though abalone are cultivated under identical farming conditions, originating from homogenous genetic stocks, variation is experienced in individual abalone growth rates, justifying further research into the mechanisms related to these growth differences. Insights into the biochemical processes of abalone would help to identify the various metabolic factors related to varied abalone growth rates. Metabolomics, aims to investigate the metabolism holistically, and is considered a powerful tool for better elucidation of observed phenotypical changes. A metabolomics approach including the use of untargeted gas chromatography-time of flight spectrometry, semi-targeted liquid chromatography-quadrupole time of flight mass spectrometry and targeted liquid chromatography-tandem mass spectrometry were used to indicate and better describe the metabolic variation associated with slow and fast growing abalone. The results obtained by metabolomics analysis of H. midae adductor muscle samples showed that faster growing individuals utilise energy pathways and reserves (via elevated insulin production) in such a way that they promote protein synthesis. In contrast the metabolic profile of slow growing individuals supports protein catabolism, where energy allocation for breakdown of metabolic products has priority over mechanisms utilised for abalone growth. Overall these results sets the stage for future work highlighting metabolic pathways which should be investigated in a qualitative manner ensuring reference values which can furthermore be monitored to assist with abalone growth predictions.

Introduction Oxygen is essential for metabolic processes and in the absence thereof alternative metabolic pathways are required for energy production, as seen in marine invertebrates like abalone. Even though hypoxia has been responsible for significant losses to the aquaculture industry, the overall metabolic adaptations of abalone in response to environmental hypoxia are as yet, not fully elucidated. Objective To use a multiplatform metabolomics approach to characterize the metabolic changes associated with energy production in abalone (Haliotis midae) when exposed to environmental hypoxia. Methods Metabolomics analysis of abalone adductor and foot muscle, left and right gill, hemolymph, and epipodial tissue samples were conducted using a multiplatform approach, which included untargeted NMR spectroscopy, untargeted and targeted LC-MS spectrometry, and untargeted and semi-targeted GC-MS spectrometric analyses. Results Increased levels of anaerobic end-products specific to marine animals were found which include alanopine, strombine, tauropine and octopine. These were accompanied by elevated lactate, succinate and arginine, of which the latter is a product of phosphoarginine breakdown in abalone. Primarily amino acid metabolism was affected, with carbohydrate and lipid metabolism assisting with anaerobic energy production to a lesser extent. Different tissues showed varied metabolic responses to hypoxia, with the largest metabolic changes in the adductor muscle. Conclusions From this investigation, it becomes evident that abalone have well-developed (yet understudied) metabolic mechanisms for surviving hypoxic periods. Furthermore, metabolomics serves as a powerful tool for investigating the altered metabolic processes in abalone.

Ciliate epibionts inhabit aquatic systems globally and may exert negative impacts on their hosts. Evidence of the environmental drivers of these epibionts is lacking. This study aimed to test the effects of turbidity on the attachment success of the ciliate Epistylis sp. on the copepod Pseudodiaptomus stuhlmanni in the turbid St Lucia Estuary, South Africa. Epistylis sp. was exposed to P. stuhlmanni individuals for 24 h under six turbidity treatments (range 8–1,500 NTU). The prevalence and density of Epistylis sp. exposed to inorganic silt decreased significantly across the turbidity range in both runs of this experiment. In the natural silt treatments, prevalence increased with turbidity up to 500 and 250 NTU in the first and second experiment, respectively. Beyond these peaks, prevalence decreased. Density peaked at 250 NTU in both experiments. High prevalence and density in the natural silt experiments is directly related to the high organic matter content in this treatment. Association of Epistylis sp. with turbidity may impact negatively P. stuhlmanni, as the longevity of this copepod is negatively related to heavy cover by Epistylis sp. and to turbidity. These findings show that environmental factors may play a key role in modulating epibiotic interactions.

Functional hypoxia is a stress condition caused by the abalone itself as a result of increased muscle activity, which generally necessitates the employment of anaerobic metabolism if the activity is sustained for prolonged periods. With that being said, abalone are highly reliant on anaerobic metabolism to provide partial compensation for energy production during oxygen-deprived episodes. However, current knowledge on the holistic metabolic response for energy metabolism during functional hypoxia, and the contribution of different metabolic pathways and various abalone tissues towards the overall accumulation of anaerobic end-products in abalone are scarce. Metabolomics analysis of adductor muscle, foot muscle, left gill, right gill, haemolymph and epipodial tissue samples indicated that South African abalone (Haliotis midae) subjected to functional hypoxia utilises predominantly anaerobic metabolism, and depends on all of the main metabolite classes (proteins, carbohydrates and lipids) for energy supply. Functional hypoxia caused increased levels of anaerobic end-products: lactate, alanopine, tauropine, succinate and alanine. Also, elevation in arginine levels was detected, confirming that abalone use phosphoarginine to generate energy during functional hypoxia. Different tissues showed varied metabolic responses to hypoxia, with functional hypoxia showing excessive changes in the adductor muscle and gills. From this metabolomics investigation, it becomes evident that abalone are metabolically able to produce sufficient amounts of energy when functional hypoxia is experienced. Also, tissue interplay enables the adjustment of H. midae energy requirements as their metabolism shifts from aerobic to anaerobic respiration during functional hypoxia.This article has an associated First Person interview with the first author of the paper.

Worldwide, there are approximately 100 Haliotis species, more commonly known as abalone or Paua' in New Zealand, Venus's-ears' in Greece, Awabi' in Japan, Perlemoen' in South Africa and Ormers' in Europe. Regardless of what they are called in any part of the world, a high monetary value is coupled to this animal, because it is largely considered a seafood delicacy. Subsequently, a great deal of research primarily focused on improving the health and growth rates of abalone were carried out to maximise productivity of the commercial farming efforts in various countries. In this review, we comprehensively describe the most recent available scientific literature on abalone biology, and those aspects related to the growth of this organism; more specifically, those factors related to the uptake and breakdown of metabolic products which ensures long-term growth. We subsequently discuss this in terms of basic animal design, farming outcomes, feeding, cellular growth mechanisms and the unique metabolic processes that exist in these species. Using this information and the knowledge of the metabolic processes in other organisms, we additionally make a number of new hypotheses regarding how these metabolic processes may function in terms of abalone growth. Based on the information presented in this review, we also identify major research opportunities and gaps in the existing knowledge of abalone metabolism, which when elucidated may not only serve the purpose of better understanding these organisms growth but also could potentially lead to increased productivity of the abalone commercial farming sector.

Hydrogen peroxide (H2O2) is used routinely by the abalone farming industry to induce spawning of adult abalone. The aim of the current study was to quantify DNA integrity of adult hemolymph cells as well as germ cells (oocytes and sperm) with different recovery times between spawning events. In addition, fertilization success of exposed germ cells was quantified. The results indicate that spawning events separated by short recovery (2-6 wk) will not provide sufficient recovery from H2O2-induced DNA fragmentation in adult hemocytes. It is recommended that the industry should ideally allow 6-8 wk between spawning events to prevent accumulation ofDNAstrand breaks in adults over time and to ensure adequate fertilization success to maximize farm production.

Abalone have a unique ability to use pyruvate, various amino acids and dehydrogenases, to produce opines as means to prevent the accumulation of NADH during anaerobic conditions. In this study, the theoretical masses, formulae and fragment patterns of butylated opines were used to predict which of these compounds could be found in the abalone adductor muscle using untargeted liquid chromatography quadrupole time-of flight-mass spectrometry. These findings were validated using synthesised opine standards. In essence alanopine, lysopine, strombine and tauropine produced in abalone adductor muscle could be characterised using the highest identification confidence levels.

The intensification of cattle production has raised concern for animal welfare due to the stress that is associated with farming practices. The welfare of an animal is determined by the animal’s ability to cope with or adapt to its continuously changing environment and the biological cost that is associated with this adaptation and maintenance. Stressors arise from various psychological, physiological and physical aspects of farming practices due to management and human–cattle interactions. Measuring the activity of the hypothalamopituitary-adrenocortical (HPA) axis with plasma cortisol levels is a useful method for determining the effects of stress on animals as it is stimulated at the onset of a perceived stress. The activation of the HPA axis affects various target tissues or systems and can result in suppression of the immune system, increased susceptibility to disease and adverse effects on reproductive success in prenatal and neonatal calves. Although some levels of stress associated with farming practices are unavoidable, improvements in farming methods need to be implemented in order to maintain or increase the efficiency of cattle production in a way that does not compromise the welfare of the animal.

Biomarkers require sensitivity and rapid turnover for health assessment of individuals and populations. Blood glucose in fish shows sensitive and measurable changes in response to environmental, physiological or nutritional stressors. Argyrosomus japonicus is currently the only commercially produced marine aquaculture finfish species in South Africa, yet knowledge about its responses to biological and environmental stressors is limited. Herein we confirm the utility of the Accu-Chek® Active diabetic glucometer under field conditions to provide accurate blood glucose data for A. japonicus, in comparison with laboratory-based enzymatic analyses using plasma, whole blood and dried blood spots (DBS). Glucometer, whole blood and DBS glucose was correlated with plasma glucose (R2=0.973, R2=0.955, R2=0.898 respectively). Whole blood glucose was consistently and significantly lower than plasma glucose, thus indicating that more complex sample preparation than storage on ice and freezing at −80°C are required to inhibit glycolysis in whole blood samples and generate accurate results. Diabetic glucometers offer a means to measure on-farm blood glucose with sufficient accuracy and rapidity. We include an analysis of a subsequent sampling from the farm, and demonstrate that through routine glucose measurements we were able to identify hypoglycaemia at the farm level, and that this corresponds to decreased condition of fish. The ease of DBS storage and stability of metabolites offer the opportunity of expanding fish health and condition monitoring by measuring multiple indicators in DBS.

Shallow coastal lakes are prone to large fluctuations in physico-chemical variables such as salinity and turbidity. This is now escalating in response to global change. A flood event in March 2014 resulted in a silt plume spreading through part of Lake St Lucia (South Africa). To determine the impact of this event on zooplankton, the Narrows region of St Lucia was sampled on a monthly basis from March to September 2014. For comparative purposes, data from samples collected prior to the flood event were included in the analyses. Analysis of similarity (ANOSIM) revealed dissimilarities in zooplankton community structure among the sampling occasions. The March 2014-May 2014 period was characterized by the highest abundance of freshwater species. Conversely, the abundance of the resident St Lucia copepods Acartiella natalensis and Oithona brevicornis was lowest during this time, and highest in September 2014. The other dominant copepod Pseudodiaptomus stuhlmanni prevailed in March 2014, but declined markedly in April. As of September 2014, P. stuhlmanni had yet to regain its pre-flood densities. The BIOENV procedure, which relates biological and environmental data, revealed that turbidity, salinity and dissolved oxygen were responsible for the observed changes in zooplankton community structure during the study period. Careful management of turbidity and salinity is stressed, as both factors are major drivers of the biota of St Lucia and similar systems worldwide.

Abalone meat is a delicacy worldwide, fetching high prices and a valuable source of income for the many countries farming and exporting this commodity. The quality of abalone is based on its unique sensory properties and an analytical metabolomics method for determining the compounds related to this would serve as a valuable tool for ensuring quality and consumer satisfaction. Metabolomics is a promising “omics” tool which can be applied towards this goal; however, widely applicable parameters for the evaluation of an untargeted gas chromatography mass spectrometry (GC-MS) metabolomic approach is still lacking. GC-MS is a popular and suitable metabolomics method due to its high separation power, reproducible retention times, and selective mass detection. The aim of this study was to establish a reliable untargeted GC-MS method for analyzing firstly a standard compound mixture consisting of 10 compounds representing various compound classes and secondly applying the method in an untargeted manner to abalone muscle samples. Using a standard compound mixture with a concentration range of 1 to 100 μg/mL, the limit of detection (LOD) ranged between 0.01 and 3.30 μg/mL, the limit of quantification (LOQ) resulted in values between 0.02 and 9.49 μg/mL, the accuracy determined was <1.5 μg/mL, and the precision displayed a coefficient of variance (CV) <25 %. When evaluating the method in terms of biological samples harvested, the repeatability and intermediate precision showed CV values <50 % for most compounds measured, allowing application of this method for metabolite profiling of abalone to answer important biological questions.