I work across broad disciplines in aquatic biology, aquaculture and fisheries, metabolomics, immunology, molecular biology, and ecotoxicology, among others. My current research focuses on molecular mechanisms involved during embryonic and larval development of marine invertebrates, and in response to environmental influences e.g. ocean acidification, pollution, pathogens. Gaining a better understanding of biochemical regulatory networks involved during early development and in response to environmental change is critical for the successful management of our oceans and the organisms which inhabit them. Other research interests include characterising mechanisms of disease and resistance, and development of new tools for assessing fish/shellfish health. Supervisors: Professor Dr Andrea C. Alfaro and Dr Silas Villas-Bôas Phone: 921 9999 Ext:8185 Address: Auckland University of Technology
Institute for Applied Ecology New Zealand
31-33 Symonds St, WL113
Auckland, New Zealand
Cultivation of the geoduck Panopea zelandica (Quoy & Gaimard, 1835) requires knowledge on embryon... more Cultivation of the geoduck Panopea zelandica (Quoy & Gaimard, 1835) requires knowledge on embryonic development to produce spat in hatcheries. This study investigated the development of P. zelandica embryos at 15°C and 35 ppt and the optimal sperm:egg ratios for fertilization under hatchery conditions. Panopea zelandica broodstock were induced to spawn by serotonin injection. Sperm and eggs were collected and fertilization was conducted at sperm:egg ratios of: 50:1, 100:1, 500:1, 1000:1 and 10,000:1 over 40 min. The optimal sperm:egg ratio was <500:1 and the normal embryo yield at 3 and 18 h post-fertilization (hpf) ranged from 83–96%. Panopea zelandica eggs (~80 μm diameter) developed the first and second polar bodies within 15–20 and 50–55 min post-fertilization, respectively. The blastula appeared at ~8 hpf, including the XR and XL cells and the presumptive shell field depression. Gastrulation occurred at 12–18 hpf with organic material apparent at the shell field depression. The mid-stage trochophore, which appeared at around 35 hpf had an apical plate with an apical tuft. The shell field spread to form the periostracum, which expanded and folded into right and left segments covering the late trochophore. The early D-stage veliger appeared at 45 hpf with the soft body being enclosed by two valves and the appearance of the velum. These observations will serve as the basis for future analyses of P. zelandica embryogenesis and for optimization of commercial production of D-veliger larvae.
We present here the first laboratory study on the effects of pharmacologically active compounds o... more We present here the first laboratory study on the effects of pharmacologically active compounds on the larval metamorphosis of the New Zealand geoduck, Panopea zelandica (Quoy and Gaimard, 1835). Two batches of competent hatchery-reared larvae were exposed to acetylcholine chloride, epinephrine hydrochloride and excess potassium ions in the form of KCl and K2SO4. None of the tested chemicals increased the proportion of metamorphosed geoducks, and in some cases, the chemical caused significant mortality, despite having been used extensively with other species, such as mussels and oysters. This might indicate that geoduck larval physiology and development differs from other bivalves. Geoducks may have evolved distinct chemoreceptor patterns that facilitate metamorphosis under environmentally favourable conditions for subtidal soft sediment habitats suitable for burrowing. Thus, further research is needed to identify alternative cues (e.g. conspecific adults, sediment characteristics and surface biofilm) and understand their role in settlement and metamorphosis. This information will aid the design of reseeding methods and contribute to the development of reliable hatchery production of geoduck spat.
Metabolomics is a fast-evolving field that provides qualitative and quantitative analyses of meta... more Metabolomics is a fast-evolving field that provides qualitative and quantitative analyses of metabolites within cells, tissues or biofluids. Recent applications of metabolomics approaches in aquaculture research have highlighted the huge potential for solving problems within all aspects of the production line, from hatchery production to post-harvest quality control. To assist with the growing application of metabolomics in aquaculture research, this contribution provides a review of techniques and steps necessary to conduct metabolomics research, from experimental design to data interpretation. Specifically, we target scientists who are new to the field of metabolomics, and we offer simple, but comprehensive steps and strategies to conduct this type of research. We conclude this primer with some advice on how to access relevant expertise and facilities for metabolomics-based aquaculture research.
Aquaculture production is currently challenged to meet the growing demands for seafood protein th... more Aquaculture production is currently challenged to meet the growing demands for seafood protein throughout the world. To achieve this growth in an efficient, safe and sustainable manner, novel tools and applications will need to be incorporated at each step of the production line. A variety of 'omics' (e.g. transcriptomics, pro-teomics, metabolomics) applications have already begun to emerge in aquaculture research with extreme success. A promising new 'omics' approach is metabolomics, which aims to use metabolite profiles to identify biomarkers indicative of physiological responses of living samples (e.g. whole organism, tissues, cells) to environmental or culture conditions. One of the benefits of this approach is that it uses a broad scan of biological conditions to identify often unexpected problem or risk areas upon which to focus management attention. In this contribution, we have selected relevant research examples to showcase the applications of metabolomics in aquaculture in four major areas: hatchery production, nutrition and diet, disease and immunology, and post-harvest quality control. The novelty of this approach is highlighted by the fact that we cite the majority of published papers in this field, and they are all recent (within the last decade) contributions.
Biotechnology has had a major impact on aquaculture production in the last decade or two, and con... more Biotechnology has had a major impact on aquaculture production in the last decade or two, and continues to grow with a wide range of applications. Innovative technologies, often borrowed from other disciplines, have allowed for enhanced growth rates of farmed species, increased nutritional value of aquafeeds, improved stock health and reduced environmental impacts. The overall management of cultivated species seems to be more than ever dependent on technological advances of increasing sophistication and complexity. To highlight this new technological thinking, a radiation of biological fields under the ‘omics’ suffix has revolutionized biological research. Metabolomics aims to use metabolite profiles to identify biomarkers indicative of physiological responses of living samples such as whole organisms, tissues and cells to environmental or culture conditions. We provide examples of recent metabolomics research in hatchery production, nutrition and diet, disease and immunology and post-harvest quality control to illustrate the power of this innovative approach.
Variability in the quantity and quality of larval yields limit aquaculture growth. However, new b... more Variability in the quantity and quality of larval yields limit aquaculture growth. However, new biotechnological advances promise to revolutionize the way we assess and solve bottlenecks. A study demonstrated the use of metabolomics, which provide a snapshot of larval physiology through metabolite profile analysis, to assess and classify mollusk larvae quality and identify biochemical pathways that may reveal further important insights. Analyses of metabolites and their ratios can be integrated with gene and protein expression data to provide new avenues for selective-breeding programs.
Gas chromatography mass spectroscopy was applied to characterize the metabolic profiles of hatche... more Gas chromatography mass spectroscopy was applied to characterize the metabolic profiles of hatchery-reared mussel (Perna canaliculus) larvae before and after a prolonged handling and water exchange process, and to investigate the effect of culture conditions. A decrease in succinate and an increase in alanine were observed after the water exchange, which indicated alterations in energy production and osmotic balance. However, these variations were subtle and it is unlikely that the water exchange practice had any lasting negative effects on larval physiology and performance. Multivariate pattern recognition tools (hierarchical clustering, principal component analysis and projection to latent squares discriminant analysis) were used to assess metabolite variations in larvae reared in low-density static and high-density flow through systems and to construct a culture condition classification model. Twelve metabolites contributed most towards the model, which indicated differences in energy, protein and lipid metabolism. The clear group separations were not represented by observable variations in morphological traits. This suggests that growth performance is metabolically buffered through an adaptive physiological mechanism to provide similar developmental characteristics under these conditions.
We investigated the effect on Mytilus galloprovincialis larval settlement, as well as the toxicit... more We investigated the effect on Mytilus galloprovincialis larval settlement, as well as the toxicity, of serial concentrations in filtered seawater of acetylcholine (AC), γ-aminobutiric acid (GABA); 3-isobutyl-1-methylxanthine (IBMX); and the potassium ion in the form of potassium chloride (KCl) and potassium sulfate (K2SO4). All the substances assayed induced larval settlement and peak responses were above 90% in exposures to 10− 2 mol L− 1 (M) AC, 10− 4 and 10− 5 M epinephrine, 10− 3 M GABA and 20, 30 and 40 mM KCl. The optimal concentration of K+ varied depending on the anionic component of the compound assayed, and peak settlement response to KCl was higher (100%) than that achieved with K2SO4 (69.7%). The estimated LC50 of the compounds assayed ranged from 9.4 × 10− 6 M (GABA) to 3.1 × 10− 2 M (KCl). GABA, IBMX and K2SO4 treatments displayed toxic effects in all the active concentrations. In contrast, AC 10− 5 M, epinephrine 10− 4 and 10− 5 M and KCl 20 mM treatments enhanced larval settlement without an acute short-term effect on mortality. These results provide new insights on the molecular mechanisms controlling settlement in M. galloprovincialis larvae, and yield promising outcomes for the mussel industry to find a reliable method to enhance larval settlement in hatcheries.
Larval settlement responses of the ribbed mussel, Aulacomya maoriana Iredale 1915, were investiga... more Larval settlement responses of the ribbed mussel, Aulacomya maoriana Iredale 1915, were investigated after exposure to various chemicals and mono-species bacteria. Identification of settlement inductive compounds assists in the elucidation of intermediary biochemical mechanisms involved in the neuronal control of settlement behaviour downstream from primary cue reception. Neuroactive compounds and amino acids (potassium ions, GABA, acetylcholine, l-Phenylalanine, l-Tyrosine, dopamine, epinephrine, l-Tryptophan, and 5-HTP) and planktonic bacteria, biofilms and biofilm exudates of Macrococcus sp. AMGM1, Bacillus sp. AMGB1, and Pseudoalteromonas sp. AMGP1 were tested for their abilities to induce larval settlement. Toxicity effects of each treatment also were simultaneously identified by recording larval mortalities. Results indicate that all chemicals used induced larvae to settle, with acetylcholine being the most effective (~ 24% at 10−6 M compared to < 2% in control assays). Toxicities of treatment compounds were low at optimal settlement inducing concentrations, except for l-Tryptophan (~ 32%) and GABA (~ 59%). Our data suggest that catecholamines (and their precursors) play an important role in the biochemical mechanisms of settlement for A. maoriana. While serotonin precursors did induce low levels of larval settlement at some concentrations, high toxicity responses to 5-HTP at 10−5 M, combined with complete settlement inhibition indicate that the mechanism of action may be more complex than can be elucidated in this study. Larval settlement responses to bacterial treatments were low for planktonic and biofilm phases across all three strains, and settlement inhibition was observed when larvae were exposed to biofilm exudates of all bacterial strains. Comparisons of A. maoriana responses to other endemic and worldwide distributed mussel species are provided as a means to highlight potential evolutionary differences in chemoreception mechanisms.
Herein, we present the first laboratory study on the effects of pharmacologically active compound... more Herein, we present the first laboratory study on the effects of pharmacologically active compounds on larval settlement of the green-lipped mussel, Perna canaliculus. Competent hatchery-reared larvae were exposed to seawater containing excess K+ in the form of KCl and K2SO4 and the neurotransmitters γ-aminobutyric acid (GABA) and acetylcholine. Both KCl and K2SO4 were identified as active inducers of larval settlement with maximum inductions occurring after exposures to 10 and 7.5 mM, respectively. Peak settlement response to KCl was higher (> 64%) than that achieved with K2SO4 (> 41%). GABA did not induce larval settlement and displayed toxic and settlement inhibitive effects at 10−4 and 10−3 M. Acetylcholine induced larval settlement (> 49%) at 10−4 M with minimal acute toxic effects (LC < 10%). To gain insight into the class of acetylcholine receptors involved, atropine was used to block the muscarinic-type receptors. Atropine treatment alone did not inhibit settlement compared to control assays, indicating that muscarinic-type receptors are not involved in settlement behavior. Furthermore, results showed that atropine did not significantly decrease acetylcholine induced settlement responses, which suggests an active role of the nicotinic-type receptors in the biochemical pathways of mussel settlement. Results of this study provide new insights on the mechanism of settlement behavior in P. canaliculus, which may have direct application to the growing New Zealand aquaculture industry.
Note: Presented by Sylvia G. Sander (University of Otago), numerous co-authors.
It is well est... more Note: Presented by Sylvia G. Sander (University of Otago), numerous co-authors.
It is well established that the availability of essential trace metals to organisms depends on their chemical form, called speciation. It is also known now that the speciation of most essential trace metals in natural aqueous systems is governed by strong ligands of predominantly unknown identity and origin. However, equally established is that the bioavailable form of these trace metals (with the exception of iron) can be approximated by its labile species. These are usually the inorganic ligands, such as H₂0, OH-, HCO₃-, CO₃²-, Cl-, SO₄²- etc., but also weak organic ligands. While the inorganic speciation of trace metals can accurately be calculated using thermodynamic equilibrium models at any given major ion concentration or salinity, the estimation of the organic metal binding ligands is more difficult. It usually involves electrochemical methods such as anodic or cathodic stripping voltammetry. Good progress has lately been made to more accurately determine both strong and weaker ligands, but also kinetically labile and inert complexes . As an alternative to electrochemical measurements passive samplers, such as DGT’s (Diffusive gradient in thin films) have found much attention, as they are also be able to distinguish between labile and inert species, and thus can provide a useful tool to estimate the bioavailable for of trace metals. We are currently comparing all of these analytical methods for New Zealand fresh and marine waters. Additionally we are correlating our results from speciation analysis with actual bioassays of Cu-toxicity on different estuarine and coastal species with two goals in
mind, 1) to accurately estimate the bioavailable form of essential and potentially toxic trace metals and 2) to equip guardians of fresh and marine water bodies, such as city and regional councils, with a simple method to sample for trace metal speciation that minimizes contamination and therefore false results.
The development of new tools for assessing the health of cultured shellfish larvae would be highl... more The development of new tools for assessing the health of cultured shellfish larvae would be highly valuable for the New Zealand aquaculture industry, which is seeking to develop and refine hatchery methodologies. A semi-commercial scale trial was established using embryos of the economically important Greenshell mussel, Perna canaliculus. We induced stress by exposing the embryos to various concentrations of copper dissolved in the incubation tank water. An integrated, multi-disciplinary analytical approach was used to assess key biological endpoints during embryonic and larval development over the first 72h of larval culture. Cellular viability and developmental abnormalities were evaluated via optical and scanning electron microscopy. To provide an instantaneous physiological snapshot of larvae throughout the trial and to identify potential new biomarkers for monitoring health, non-targeted metabolic profiling was performed via GC/MS-based metabolomics. As a key impact of copper exposure is the generation of reactive oxygen species (ROS), we also developed a high throughput assay to measure ROS levels in vivo using a fluorescent molecular probe. Additional targeted analyses for an array of ROS-induced stress biomarkers were also performed in vitro, including the quantification of enzymes involved in the regulation of glutathione and markers for DNA, protein and lipid damage. Copper speciation and determination of the bioavailable fractions were modelled through use of anodic stripping voltammetry, diffusive gradients in thin films and inductively-coupled mass spectrometry. At 3h post-fertilisation, bioavailable copper concentrations of around 50 µg L-1 resulted in large increases in oxidative DNA damage, lipid peroxidation and levels of protein carbonyls, and concomitant decreases in catalase and superoxide dismutase. By 18h post-fertilisation, initiation of cell death and decreased levels of glutathione regulatory enzymes were detected. At a low sub-lethal bioavailable copper concentration of approximately 1 µgL-1, the first observable effects became apparent at 18h post-fertilisation with significant increases in lipid peroxidation and protein damage. By 36h post-fertilization, ROS levels, DNA damage, and all other oxidative stress markers had increased. By 72h post-fertilisation, slowed development, structural abnormalities, and changes in behaviour were observed. Our results indicate that the early embryonic and larval stages of mussels are more sensitive to low level copper toxicity than previously thought.
Modern molecular biology has experienced huge shifts in the way that we approach and execute biol... more Modern molecular biology has experienced huge shifts in the way that we approach and execute biological research. With the advent of omics-based technologies, we now have the ability to analyse a vast array of genes, proteins and metabolites simultaneously which allows us to generate large amounts of data. This data can be mined using new bioinformatics processes to identify patterns, generate new hypotheses, and gain novel insights into the functioning of organisms on a systems-wide level. Using recent examples from our own group and that of others, we highlight some of the benefits which can be gained from such non-hypothesis driven research in marine science.
Future environmental and economic sustainability of aquaculture will depend to a large extent on ... more Future environmental and economic sustainability of aquaculture will depend to a large extent on improvements to large-scale hatchery production. Currently, high variability in quantity and quality of larval yields limits industry growth throughout the world. However, new biotechnological advances in -omics approaches promise to revolutionize the way we assess and solve bottlenecks in hatchery production and other aquaculture sectors. We illustrate this innovative approach using metabolomics, which provides an instantaneous snapshot of larval physiology through metabolite profile analysis. Using a variety of univariate and multivariate feature selection methods, we identified biomarkers that reflect physiological condition of mussel larvae and determine quality variation during hatchery production. Analysis of biochemical pathways may elucidate how slight modifications in culturing conditions may result in significantly different hatchery production outcomes.
Global mollusc production is rapidly increasing and is one of the largest aquaculture activities... more Global mollusc production is rapidly increasing and is one of the largest aquaculture activities in the world. Considerable attention is being placed in the production of hatchery-reared juveniles to preserve wild populations and provide industry with high-quality stock for grow-out. However, variations in larval health and growth is proving to be a significant bottleneck within the industry. In order to develop remedial strategies, it is crucial that we mature our understanding of endogenous regulatory mechanisms involved in early developmental timing, energy acquisition/allocation, sensory systems and immunological responses. Metabolomics is the non-targeted analysis of a broad range of metabolites within cells, tissues and organisms. Metabolites are end products of gene and protein expression and are exceptionally sensitive to genetic and environmental perturbations. Thus, metabolomics offers a revolutionary framework for phenotyping organisms at the molecular level. Examples of applications are widespread across diverse areas of research from human medicine to deep-sea microbial ecology. However, despite its wide applicability, metabolomics-based approaches for studying marine invertebrate larval development has not yet been realised. We report the first application of metabolomics to investigate developmental variation in marine invertebrate larvae.
Cultivation of the geoduck Panopea zelandica (Quoy & Gaimard, 1835) requires knowledge on embryon... more Cultivation of the geoduck Panopea zelandica (Quoy & Gaimard, 1835) requires knowledge on embryonic development to produce spat in hatcheries. This study investigated the development of P. zelandica embryos at 15°C and 35 ppt and the optimal sperm:egg ratios for fertilization under hatchery conditions. Panopea zelandica broodstock were induced to spawn by serotonin injection. Sperm and eggs were collected and fertilization was conducted at sperm:egg ratios of: 50:1, 100:1, 500:1, 1000:1 and 10,000:1 over 40 min. The optimal sperm:egg ratio was <500:1 and the normal embryo yield at 3 and 18 h post-fertilization (hpf) ranged from 83–96%. Panopea zelandica eggs (~80 μm diameter) developed the first and second polar bodies within 15–20 and 50–55 min post-fertilization, respectively. The blastula appeared at ~8 hpf, including the XR and XL cells and the presumptive shell field depression. Gastrulation occurred at 12–18 hpf with organic material apparent at the shell field depression. The mid-stage trochophore, which appeared at around 35 hpf had an apical plate with an apical tuft. The shell field spread to form the periostracum, which expanded and folded into right and left segments covering the late trochophore. The early D-stage veliger appeared at 45 hpf with the soft body being enclosed by two valves and the appearance of the velum. These observations will serve as the basis for future analyses of P. zelandica embryogenesis and for optimization of commercial production of D-veliger larvae.
We present here the first laboratory study on the effects of pharmacologically active compounds o... more We present here the first laboratory study on the effects of pharmacologically active compounds on the larval metamorphosis of the New Zealand geoduck, Panopea zelandica (Quoy and Gaimard, 1835). Two batches of competent hatchery-reared larvae were exposed to acetylcholine chloride, epinephrine hydrochloride and excess potassium ions in the form of KCl and K2SO4. None of the tested chemicals increased the proportion of metamorphosed geoducks, and in some cases, the chemical caused significant mortality, despite having been used extensively with other species, such as mussels and oysters. This might indicate that geoduck larval physiology and development differs from other bivalves. Geoducks may have evolved distinct chemoreceptor patterns that facilitate metamorphosis under environmentally favourable conditions for subtidal soft sediment habitats suitable for burrowing. Thus, further research is needed to identify alternative cues (e.g. conspecific adults, sediment characteristics and surface biofilm) and understand their role in settlement and metamorphosis. This information will aid the design of reseeding methods and contribute to the development of reliable hatchery production of geoduck spat.
Metabolomics is a fast-evolving field that provides qualitative and quantitative analyses of meta... more Metabolomics is a fast-evolving field that provides qualitative and quantitative analyses of metabolites within cells, tissues or biofluids. Recent applications of metabolomics approaches in aquaculture research have highlighted the huge potential for solving problems within all aspects of the production line, from hatchery production to post-harvest quality control. To assist with the growing application of metabolomics in aquaculture research, this contribution provides a review of techniques and steps necessary to conduct metabolomics research, from experimental design to data interpretation. Specifically, we target scientists who are new to the field of metabolomics, and we offer simple, but comprehensive steps and strategies to conduct this type of research. We conclude this primer with some advice on how to access relevant expertise and facilities for metabolomics-based aquaculture research.
Aquaculture production is currently challenged to meet the growing demands for seafood protein th... more Aquaculture production is currently challenged to meet the growing demands for seafood protein throughout the world. To achieve this growth in an efficient, safe and sustainable manner, novel tools and applications will need to be incorporated at each step of the production line. A variety of 'omics' (e.g. transcriptomics, pro-teomics, metabolomics) applications have already begun to emerge in aquaculture research with extreme success. A promising new 'omics' approach is metabolomics, which aims to use metabolite profiles to identify biomarkers indicative of physiological responses of living samples (e.g. whole organism, tissues, cells) to environmental or culture conditions. One of the benefits of this approach is that it uses a broad scan of biological conditions to identify often unexpected problem or risk areas upon which to focus management attention. In this contribution, we have selected relevant research examples to showcase the applications of metabolomics in aquaculture in four major areas: hatchery production, nutrition and diet, disease and immunology, and post-harvest quality control. The novelty of this approach is highlighted by the fact that we cite the majority of published papers in this field, and they are all recent (within the last decade) contributions.
Biotechnology has had a major impact on aquaculture production in the last decade or two, and con... more Biotechnology has had a major impact on aquaculture production in the last decade or two, and continues to grow with a wide range of applications. Innovative technologies, often borrowed from other disciplines, have allowed for enhanced growth rates of farmed species, increased nutritional value of aquafeeds, improved stock health and reduced environmental impacts. The overall management of cultivated species seems to be more than ever dependent on technological advances of increasing sophistication and complexity. To highlight this new technological thinking, a radiation of biological fields under the ‘omics’ suffix has revolutionized biological research. Metabolomics aims to use metabolite profiles to identify biomarkers indicative of physiological responses of living samples such as whole organisms, tissues and cells to environmental or culture conditions. We provide examples of recent metabolomics research in hatchery production, nutrition and diet, disease and immunology and post-harvest quality control to illustrate the power of this innovative approach.
Variability in the quantity and quality of larval yields limit aquaculture growth. However, new b... more Variability in the quantity and quality of larval yields limit aquaculture growth. However, new biotechnological advances promise to revolutionize the way we assess and solve bottlenecks. A study demonstrated the use of metabolomics, which provide a snapshot of larval physiology through metabolite profile analysis, to assess and classify mollusk larvae quality and identify biochemical pathways that may reveal further important insights. Analyses of metabolites and their ratios can be integrated with gene and protein expression data to provide new avenues for selective-breeding programs.
Gas chromatography mass spectroscopy was applied to characterize the metabolic profiles of hatche... more Gas chromatography mass spectroscopy was applied to characterize the metabolic profiles of hatchery-reared mussel (Perna canaliculus) larvae before and after a prolonged handling and water exchange process, and to investigate the effect of culture conditions. A decrease in succinate and an increase in alanine were observed after the water exchange, which indicated alterations in energy production and osmotic balance. However, these variations were subtle and it is unlikely that the water exchange practice had any lasting negative effects on larval physiology and performance. Multivariate pattern recognition tools (hierarchical clustering, principal component analysis and projection to latent squares discriminant analysis) were used to assess metabolite variations in larvae reared in low-density static and high-density flow through systems and to construct a culture condition classification model. Twelve metabolites contributed most towards the model, which indicated differences in energy, protein and lipid metabolism. The clear group separations were not represented by observable variations in morphological traits. This suggests that growth performance is metabolically buffered through an adaptive physiological mechanism to provide similar developmental characteristics under these conditions.
We investigated the effect on Mytilus galloprovincialis larval settlement, as well as the toxicit... more We investigated the effect on Mytilus galloprovincialis larval settlement, as well as the toxicity, of serial concentrations in filtered seawater of acetylcholine (AC), γ-aminobutiric acid (GABA); 3-isobutyl-1-methylxanthine (IBMX); and the potassium ion in the form of potassium chloride (KCl) and potassium sulfate (K2SO4). All the substances assayed induced larval settlement and peak responses were above 90% in exposures to 10− 2 mol L− 1 (M) AC, 10− 4 and 10− 5 M epinephrine, 10− 3 M GABA and 20, 30 and 40 mM KCl. The optimal concentration of K+ varied depending on the anionic component of the compound assayed, and peak settlement response to KCl was higher (100%) than that achieved with K2SO4 (69.7%). The estimated LC50 of the compounds assayed ranged from 9.4 × 10− 6 M (GABA) to 3.1 × 10− 2 M (KCl). GABA, IBMX and K2SO4 treatments displayed toxic effects in all the active concentrations. In contrast, AC 10− 5 M, epinephrine 10− 4 and 10− 5 M and KCl 20 mM treatments enhanced larval settlement without an acute short-term effect on mortality. These results provide new insights on the molecular mechanisms controlling settlement in M. galloprovincialis larvae, and yield promising outcomes for the mussel industry to find a reliable method to enhance larval settlement in hatcheries.
Larval settlement responses of the ribbed mussel, Aulacomya maoriana Iredale 1915, were investiga... more Larval settlement responses of the ribbed mussel, Aulacomya maoriana Iredale 1915, were investigated after exposure to various chemicals and mono-species bacteria. Identification of settlement inductive compounds assists in the elucidation of intermediary biochemical mechanisms involved in the neuronal control of settlement behaviour downstream from primary cue reception. Neuroactive compounds and amino acids (potassium ions, GABA, acetylcholine, l-Phenylalanine, l-Tyrosine, dopamine, epinephrine, l-Tryptophan, and 5-HTP) and planktonic bacteria, biofilms and biofilm exudates of Macrococcus sp. AMGM1, Bacillus sp. AMGB1, and Pseudoalteromonas sp. AMGP1 were tested for their abilities to induce larval settlement. Toxicity effects of each treatment also were simultaneously identified by recording larval mortalities. Results indicate that all chemicals used induced larvae to settle, with acetylcholine being the most effective (~ 24% at 10−6 M compared to < 2% in control assays). Toxicities of treatment compounds were low at optimal settlement inducing concentrations, except for l-Tryptophan (~ 32%) and GABA (~ 59%). Our data suggest that catecholamines (and their precursors) play an important role in the biochemical mechanisms of settlement for A. maoriana. While serotonin precursors did induce low levels of larval settlement at some concentrations, high toxicity responses to 5-HTP at 10−5 M, combined with complete settlement inhibition indicate that the mechanism of action may be more complex than can be elucidated in this study. Larval settlement responses to bacterial treatments were low for planktonic and biofilm phases across all three strains, and settlement inhibition was observed when larvae were exposed to biofilm exudates of all bacterial strains. Comparisons of A. maoriana responses to other endemic and worldwide distributed mussel species are provided as a means to highlight potential evolutionary differences in chemoreception mechanisms.
Herein, we present the first laboratory study on the effects of pharmacologically active compound... more Herein, we present the first laboratory study on the effects of pharmacologically active compounds on larval settlement of the green-lipped mussel, Perna canaliculus. Competent hatchery-reared larvae were exposed to seawater containing excess K+ in the form of KCl and K2SO4 and the neurotransmitters γ-aminobutyric acid (GABA) and acetylcholine. Both KCl and K2SO4 were identified as active inducers of larval settlement with maximum inductions occurring after exposures to 10 and 7.5 mM, respectively. Peak settlement response to KCl was higher (> 64%) than that achieved with K2SO4 (> 41%). GABA did not induce larval settlement and displayed toxic and settlement inhibitive effects at 10−4 and 10−3 M. Acetylcholine induced larval settlement (> 49%) at 10−4 M with minimal acute toxic effects (LC < 10%). To gain insight into the class of acetylcholine receptors involved, atropine was used to block the muscarinic-type receptors. Atropine treatment alone did not inhibit settlement compared to control assays, indicating that muscarinic-type receptors are not involved in settlement behavior. Furthermore, results showed that atropine did not significantly decrease acetylcholine induced settlement responses, which suggests an active role of the nicotinic-type receptors in the biochemical pathways of mussel settlement. Results of this study provide new insights on the mechanism of settlement behavior in P. canaliculus, which may have direct application to the growing New Zealand aquaculture industry.
Note: Presented by Sylvia G. Sander (University of Otago), numerous co-authors.
It is well est... more Note: Presented by Sylvia G. Sander (University of Otago), numerous co-authors.
It is well established that the availability of essential trace metals to organisms depends on their chemical form, called speciation. It is also known now that the speciation of most essential trace metals in natural aqueous systems is governed by strong ligands of predominantly unknown identity and origin. However, equally established is that the bioavailable form of these trace metals (with the exception of iron) can be approximated by its labile species. These are usually the inorganic ligands, such as H₂0, OH-, HCO₃-, CO₃²-, Cl-, SO₄²- etc., but also weak organic ligands. While the inorganic speciation of trace metals can accurately be calculated using thermodynamic equilibrium models at any given major ion concentration or salinity, the estimation of the organic metal binding ligands is more difficult. It usually involves electrochemical methods such as anodic or cathodic stripping voltammetry. Good progress has lately been made to more accurately determine both strong and weaker ligands, but also kinetically labile and inert complexes . As an alternative to electrochemical measurements passive samplers, such as DGT’s (Diffusive gradient in thin films) have found much attention, as they are also be able to distinguish between labile and inert species, and thus can provide a useful tool to estimate the bioavailable for of trace metals. We are currently comparing all of these analytical methods for New Zealand fresh and marine waters. Additionally we are correlating our results from speciation analysis with actual bioassays of Cu-toxicity on different estuarine and coastal species with two goals in
mind, 1) to accurately estimate the bioavailable form of essential and potentially toxic trace metals and 2) to equip guardians of fresh and marine water bodies, such as city and regional councils, with a simple method to sample for trace metal speciation that minimizes contamination and therefore false results.
The development of new tools for assessing the health of cultured shellfish larvae would be highl... more The development of new tools for assessing the health of cultured shellfish larvae would be highly valuable for the New Zealand aquaculture industry, which is seeking to develop and refine hatchery methodologies. A semi-commercial scale trial was established using embryos of the economically important Greenshell mussel, Perna canaliculus. We induced stress by exposing the embryos to various concentrations of copper dissolved in the incubation tank water. An integrated, multi-disciplinary analytical approach was used to assess key biological endpoints during embryonic and larval development over the first 72h of larval culture. Cellular viability and developmental abnormalities were evaluated via optical and scanning electron microscopy. To provide an instantaneous physiological snapshot of larvae throughout the trial and to identify potential new biomarkers for monitoring health, non-targeted metabolic profiling was performed via GC/MS-based metabolomics. As a key impact of copper exposure is the generation of reactive oxygen species (ROS), we also developed a high throughput assay to measure ROS levels in vivo using a fluorescent molecular probe. Additional targeted analyses for an array of ROS-induced stress biomarkers were also performed in vitro, including the quantification of enzymes involved in the regulation of glutathione and markers for DNA, protein and lipid damage. Copper speciation and determination of the bioavailable fractions were modelled through use of anodic stripping voltammetry, diffusive gradients in thin films and inductively-coupled mass spectrometry. At 3h post-fertilisation, bioavailable copper concentrations of around 50 µg L-1 resulted in large increases in oxidative DNA damage, lipid peroxidation and levels of protein carbonyls, and concomitant decreases in catalase and superoxide dismutase. By 18h post-fertilisation, initiation of cell death and decreased levels of glutathione regulatory enzymes were detected. At a low sub-lethal bioavailable copper concentration of approximately 1 µgL-1, the first observable effects became apparent at 18h post-fertilisation with significant increases in lipid peroxidation and protein damage. By 36h post-fertilization, ROS levels, DNA damage, and all other oxidative stress markers had increased. By 72h post-fertilisation, slowed development, structural abnormalities, and changes in behaviour were observed. Our results indicate that the early embryonic and larval stages of mussels are more sensitive to low level copper toxicity than previously thought.
Modern molecular biology has experienced huge shifts in the way that we approach and execute biol... more Modern molecular biology has experienced huge shifts in the way that we approach and execute biological research. With the advent of omics-based technologies, we now have the ability to analyse a vast array of genes, proteins and metabolites simultaneously which allows us to generate large amounts of data. This data can be mined using new bioinformatics processes to identify patterns, generate new hypotheses, and gain novel insights into the functioning of organisms on a systems-wide level. Using recent examples from our own group and that of others, we highlight some of the benefits which can be gained from such non-hypothesis driven research in marine science.
Future environmental and economic sustainability of aquaculture will depend to a large extent on ... more Future environmental and economic sustainability of aquaculture will depend to a large extent on improvements to large-scale hatchery production. Currently, high variability in quantity and quality of larval yields limits industry growth throughout the world. However, new biotechnological advances in -omics approaches promise to revolutionize the way we assess and solve bottlenecks in hatchery production and other aquaculture sectors. We illustrate this innovative approach using metabolomics, which provides an instantaneous snapshot of larval physiology through metabolite profile analysis. Using a variety of univariate and multivariate feature selection methods, we identified biomarkers that reflect physiological condition of mussel larvae and determine quality variation during hatchery production. Analysis of biochemical pathways may elucidate how slight modifications in culturing conditions may result in significantly different hatchery production outcomes.
Global mollusc production is rapidly increasing and is one of the largest aquaculture activities... more Global mollusc production is rapidly increasing and is one of the largest aquaculture activities in the world. Considerable attention is being placed in the production of hatchery-reared juveniles to preserve wild populations and provide industry with high-quality stock for grow-out. However, variations in larval health and growth is proving to be a significant bottleneck within the industry. In order to develop remedial strategies, it is crucial that we mature our understanding of endogenous regulatory mechanisms involved in early developmental timing, energy acquisition/allocation, sensory systems and immunological responses. Metabolomics is the non-targeted analysis of a broad range of metabolites within cells, tissues and organisms. Metabolites are end products of gene and protein expression and are exceptionally sensitive to genetic and environmental perturbations. Thus, metabolomics offers a revolutionary framework for phenotyping organisms at the molecular level. Examples of applications are widespread across diverse areas of research from human medicine to deep-sea microbial ecology. However, despite its wide applicability, metabolomics-based approaches for studying marine invertebrate larval development has not yet been realised. We report the first application of metabolomics to investigate developmental variation in marine invertebrate larvae.
Marine invertebrates have complex life histories and diverse behaviours, mediated by environmenta... more Marine invertebrates have complex life histories and diverse behaviours, mediated by environmental and biological stimuli. Approximately 80% of marine invertebrates produce microscopic larvae that develop in the plankton. These larvae, which have morphologies completely unlike those of their parents, may remain in their larval phases for minutes to months, depending on the species. During this time, larvae may drift great distances in the water column, being swept along by ocean currents, before settling on a suitable substratum and metamorphosing into their adult forms. Over the past forty years or so, considerable interest has been mounting into the processes which govern larval settlement behaviour. Gaining an understanding of these processes is important to enhance our knowledge of marine invertebrate ecology, and also may assist in the development of technologies with applications in aquaculture and marine biofouling. The transition of larvae from a pelagic environment to a benthic one is, at least partially, modulated by the presence of various chemicals in the environment. These chemical stimuli often are species-specific, and may be associated only with particular substrata. Unfortunately, the identities of natural compounds which have the ability to induce larval settlement are currently unknown for the majority of marine invertebrate species. There are many challenges facing researchers who undertake such tasks. Because of these difficulties, a common technique employed to investigate settlement behaviour is the application of pharmacological compounds in the laboratory, which have the ability to induce larval settlement. Although insight into the biochemical mechanisms controlling settlement behaviour can be gained through such methods, the lack of consistency and standardisation of the techniques employed often lead to ambiguous claims in the literature. Furthermore, results of such studies often are misinterpreted, and the progression of the field restricted. These problems are compounded by the often limited collaboration among larval biologists, natural product chemists, and biochemists. Because of these issues, comparing and contrasting results from the literature are fallible. This presentation aims to review the literature on marine invertebrate larval settlement, and identifies common sources for error. This review was motivated, in part, by the results gained from our own research on larval settlement behaviour, using the New Zealand green-lipped mussel, Perna canaliculus, as a model species. Our results are briefly explored in this presentation, with an emphasis on those which suggest a cautionary approach should be taken by researchers when reviewing the literature, and designing future larval settlement experiments in the laboratory.
In the modern age of scientific discovery and technological advancement, we have new tools availa... more In the modern age of scientific discovery and technological advancement, we have new tools available to apply innovative approaches toward questions which have remained unanswered for decades. Larval settlement of marine invertebrates is a complex and diverse biological process and the regulatory factors mediating this transitional phase are varied among species. It is this very species-specificity which is so fascinating, broadening the attention of researchers. As yet there exists no umbrella model nor any completed mechanism of settlement for any one key species. Although the isolation and identification of marine natural products that have the ability to induce larval settlement is vastly increasing, endogenous biochemical pathways and processes remain elusive. In a world of multidisciplinary research, enhanced communication among scientists, and breakthroughs in analytical instrumentation and computational software, new domains in science are emerging. With the rapid expansion of ‘omics’ subdisciplines, metabolomics (the comprehensive profiling of all metabolites in a biological system under given conditions) is the latest field gaining a stronghold in the various approaches towards molecular biology. Metabolomics provides a unique opportunity for biomarker discovery of particular events during larval development and compliments other ‘omics’ data. Furthermore, global metabolite profiling may identify unknown similarities in physiological processes involved in chemoreception of natural and synthetic cues within, and among, species. In this presentation we provide an overview of metabolomics, explain the application of new techniques available to larval biologists, introduce some of the challenges involved, and present possible strategies for future larval settlement research.
Marine invertebrates have complex life histories and diverse behaviours, mediated by environmenta... more Marine invertebrates have complex life histories and diverse behaviours, mediated by environmental and biological stimuli. More than 90,000 species produce microscopic larvae that develop in the plankton. Depending on species, they remain in their larval phases for minutes to months. During this period, larvae may be swept great distances along ocean currents before contacting a suitable substratum for settlement and metamorphosing into their adult forms. This transition is modulated by the chemoreception of chemical cues of various biological origins and most often has proven to be highly species-specific. Exogenous regulation of settlement may be mediated by surface-associated compounds and waterborne substances released from microbial biofilms, macroalgae, sediments, and conspecifics among others. The endogenous biochemical processes which occur after cue detection are of much interest to researchers. Our research has identified a number of monospecies biofilms which have the ability to induce larval settlement behaviour in the NZ green-lipped mussel. Furthermore, we have shown that agonisation of endogenous receptors by neuroactive compounds also result in larval settlement - giving important insights into the biochemical mechanisms of this critical life process. The relationship between microbial induction and direct receptor activation is currently unknown. To determine whether an association exists and in what capacity, we are undertaking a combined study involving proteomic and metabolomic approaches in an effort to unravel the mechanisms behind cue reception and marine invertebrate larval settlement.
Approximately 80% of marine invertebrates (roughly 90,000 species) produce microscopic larvae tha... more Approximately 80% of marine invertebrates (roughly 90,000 species) produce microscopic larvae that develop in the plankton. These larvae, which have morphologies completely unlike those of their parents, may remain in their larval phases for minutes to months, depending on the species. At the end of the early planktonic stage, the swimming larvae ‘settle’ out of the water column onto particular substrates. The preferences for these surfaces often are species-specific. This transition is partly modulated by the chemoreception of chemical cues of various biological origins. In all but a few species, the identities and locations of these receptors are currently unknown. The binding of these cues control specific biochemical pathways ultimately leading to behavioural and morphological transformations in the organism. Over the past twenty years or so, interest has been mounting as to the source and identity of naturally occurring settlement cues. However, very few cues have been characterised to date. Furthermore, the endogenous biochemical mechanisms responsible for translating external cue reception into behavioural responses are wholly unidentified. My previous research investigated these mechanisms using a pharmacological approach. With the New Zealand green-lipped mussel as the model organism of choice, I identified a number of neuroactive compounds which had the ability to induce larval settlement behaviour. These compounds are thought to act directly on the nervous system, bypassing the external chemoreceptors. The results of this research gave important insight into the identification of endogenous receptors likely involved in downstream cue reception. However, due to the high degree of complexity, the elucidation of specific biochemical mechanisms cannot be made using such an approach. Therefore, an alternative route of inquiry is needed. My current research incorporates a newly emerging field, metabolomics, to investigate relationships in the biochemical mechanisms among settlement inducing compounds, naturally and non-naturally occurring.
The green-lipped mussel industry is New Zealand’s principal aquaculture sector, and by value the ... more The green-lipped mussel industry is New Zealand’s principal aquaculture sector, and by value the largest single-species of seafood currently exported. Although this mussel is extensively cultivated, very little is known about its early larval behaviours. After a brief planktonic stage, the young mussels settle onto specific substrates before undergoing metamorphosis into their adult forms. However, the timing of this settlement, and preference for substrata, is poorly understood. Chemical stimuli of biological origin that modulate neuronal signalling are thought to mediate this process. In order gain insight into the endogenous biochemical mechanisms underlying settlement behaviour, mussel larvae were exposed to a range of neurotransmitters, their precursors and degradation products. Results demonstrate that activation of catecholamine, serotonin, and nicotinic acetylcholine receptors induce larval settlement in this species. Identification of such receptor involvement may aid in the discovery of novel inducer compounds, naturally occurring or synthetic, and assist in the elucidation of their action. Furthermore, the ability to regulate larval settlement of the green-lipped mussel in closed systems may assist in the development of protocols for cost effective large-scale hatchery rearing of this lucrative bivalve.
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Papers by Tim Young
Conference Presentations by Tim Young
It is well established that the availability of essential trace metals to organisms depends on their chemical form, called speciation. It is also known now that the speciation of most essential trace metals in natural aqueous systems is governed by strong ligands of predominantly unknown identity and origin. However, equally established is that the bioavailable form of these trace metals (with the exception of iron) can be approximated by its labile species. These are usually the inorganic ligands, such as H₂0, OH-, HCO₃-, CO₃²-, Cl-, SO₄²- etc., but also weak organic ligands. While the inorganic speciation of trace metals can accurately be calculated using thermodynamic equilibrium models at any given major ion concentration or salinity, the estimation of the organic metal binding ligands is more difficult. It usually involves electrochemical methods such as anodic or cathodic stripping voltammetry. Good progress has lately been made to more accurately determine both strong and weaker ligands, but also kinetically labile and inert complexes . As an alternative to electrochemical measurements passive samplers, such as DGT’s (Diffusive gradient in thin films) have found much attention, as they are also be able to distinguish between labile and inert species, and thus can provide a useful tool to estimate the bioavailable for of trace metals. We are currently comparing all of these analytical methods for New Zealand fresh and marine waters. Additionally we are correlating our results from speciation analysis with actual bioassays of Cu-toxicity on different estuarine and coastal species with two goals in
mind, 1) to accurately estimate the bioavailable form of essential and potentially toxic trace metals and 2) to equip guardians of fresh and marine water bodies, such as city and regional councils, with a simple method to sample for trace metal speciation that minimizes contamination and therefore false results.
It is well established that the availability of essential trace metals to organisms depends on their chemical form, called speciation. It is also known now that the speciation of most essential trace metals in natural aqueous systems is governed by strong ligands of predominantly unknown identity and origin. However, equally established is that the bioavailable form of these trace metals (with the exception of iron) can be approximated by its labile species. These are usually the inorganic ligands, such as H₂0, OH-, HCO₃-, CO₃²-, Cl-, SO₄²- etc., but also weak organic ligands. While the inorganic speciation of trace metals can accurately be calculated using thermodynamic equilibrium models at any given major ion concentration or salinity, the estimation of the organic metal binding ligands is more difficult. It usually involves electrochemical methods such as anodic or cathodic stripping voltammetry. Good progress has lately been made to more accurately determine both strong and weaker ligands, but also kinetically labile and inert complexes . As an alternative to electrochemical measurements passive samplers, such as DGT’s (Diffusive gradient in thin films) have found much attention, as they are also be able to distinguish between labile and inert species, and thus can provide a useful tool to estimate the bioavailable for of trace metals. We are currently comparing all of these analytical methods for New Zealand fresh and marine waters. Additionally we are correlating our results from speciation analysis with actual bioassays of Cu-toxicity on different estuarine and coastal species with two goals in
mind, 1) to accurately estimate the bioavailable form of essential and potentially toxic trace metals and 2) to equip guardians of fresh and marine water bodies, such as city and regional councils, with a simple method to sample for trace metal speciation that minimizes contamination and therefore false results.